E - Copy Manual of Lab Procedures in Haematology

Manual of
Laboratory Procedures in
Haematolog y
Sri Lanka College of Haematologists 1 st

Edition
Manual of
Haematolog y
Sri Lanka College of Haematologists

Manual of Laboratory Procedures in
HAEMATOLOGY
© Sri Lanka College of Haematologists
First Edition - 2022
ISBN 978-624-5776-02-3
Page layout & Cover design

Roshan Senarath
FIRSTNEED 077 860 4144
Illustrations by
Prof. Senani Williams
Published by
No. 6, Wijerama House
Wijerama Mawatha, Colombo 07 Sri Lanka
All rights reserved.

Parts of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any
means, without any modification or alteration to its contents, with acknowledgment of the source as Manual
of Laboratory Procedures in Haematology.
Comments and suggestions are invited to improve future editions. Please forward your comments and suggestions to the
following address by post or e-mail.
Wijerama Mawatha, Colombo 07 Sri Lanka
E-mail : slchaem@yahoo.com
Electronic version is available on www.slchaem.lk
ii
MESSAGE FROM DGHS
I
t is indeed a great pleasure to write this message to the Manual of Laboratory Procedures in
Haematology, compiled by the Sri Lanka College of Haematologists. There is a huge demand for
the tests in haematology, not only for the diagnosis of haematological disorders, but also, they
are used in the other fields of medicine. The reliable laboratory test results have become an asset
for patient management and control of diseases. Therefore, there is a necessity to provide high
quality laboratory services. One of the essential components in maintaining high quality laboratory
services is keeping SOPs (standard operating procedures).
This comprehensive manual will guide the technologists at the bench and enable to maintain a
uniformity in test procedures performed in any hospital in the country by conforming to accepted
methodology. It will also serve as a reference material to trainees in laboratory haematology.
I take this opportunity to thank the Sri Lanka College of Haematologists for taking this initiative and
congratulate them for a job well done.
Dr. Asela Gunawardena

Director General of Health Services,
Ministry of Healthcare & Nutrition,
Colombo 10.
iii
MESSAGE FROM DDGLS
S
tandard operating procedures (SOPs) are an essential component in good laboratory practice.
Since SOPs provide a uniform pattern of function for laboratory staff, it is considered that
using SOPs is the best way to maintain optimal quality of performance in laboratories.
Creating an effective and practical SOP is not a simple task. My sincere congratulations to the Sri
Lanka College of Haematologists (SLCH) as it marks a notable step forward by launching this Manual
of Laboratory Procedures in Haematology mainly composed of SOPs for a range of haematology
tests. Availability of this kind of manual at the bench side would assist maintenance of uniformity
in test performance at all levels of haematology laboratories within the country.
I appreciate the SLCH for taking this giant step forward by introducing this manual to the laboratory
service of Sri Lanka. I hope this manual would facilitate further improvement and maintenance of
high-quality performance in haematology laboratories in our country. I sincerely hope that this
would be the initial step in producing similar publications by the Sri Lanka College of Haematologists
and wish them strength to continue this good work.
Dr. G. Sudath K. Dharmaratne

Deputy Director General of Laboratory Services,
Ministry of Healthcare & Nutrition,
Colombo 10.
iv
PREFACE
A
manual of laboratory procedures in haematology was a long felt need for the haematology
laboratories in Sri Lanka, in helping with any level of haematology practice as a medical
laboratory technologist, a trainee in haematology or a consultant haematologist. Such a
manual is also an essential document in the way forward for accreditation process of haemtology
laboratories. Therefore, Sri Lanka College of Haematologists took the initiative in the project of
compiling this manual consisting of 6 sections, which include Standard Operating Procedures
(SOPs) for a spectrum of investigations ranging from the basic haematology tests to more advanced
tests in haematology, paving way for the use at any level of laboratory practice in the country. This
also contains some of the ancillary procedures related to haematology laboratory testing.
This task was initiated in 2020 and was completed within a period of three years by a group of
consultant haematologists and medical laboratory technologists who generously dedicated their
valuable time. The authors of this manuscript used reference material including standard text
books, journal articles, practical hand books and manuals of analyzers for writing the contents
which were then reviewed in number of rounds by several panels of reviewers. Then the content
editors undertook the tedious task of final reviewing of the contents and formatting of the manual.
Lastly, the manuscript went through the editorial board of Sri Lanka College of Haematologists
(SLCH) for proof reading and editing before going to the print.
First of all, I should thank Sri Lanka College of Haematologists for the trust placed when nominating
me as the overall coordinator of this mammoth project. As the coordinator of this project, I wish
to thank the presidents of the college Dr. Nipunika Senadheera (2020) for initiating the process
and Dr. Dammika Gunawardena (2021) and Dr. Anoma Weerawardhana (2022) for extending their
unstinted support during successive years to make this a reality. On behalf of the college, I wish
to express my sincere gratitude to all contributors including authors and reviewers of the manual
and especially the devoted content editors. The time spent by the editorial board of SLCH on proof
reading and correction is much appreciated. I also acknowledge Prof. Senani Williams for providing
us with nicely depicted illustrations for this book. I am very grateful to Dr. Asela Gunawardane,
Director General of Health Services and Dr. Sudath K. Dharmaratne, Deputy Director General of
Laboratory Services for the continued support extended to the college and agreeing to make this
publication available at state sector laboratories. My sincere thanks would also go to Mr. Roshan
Senerath for the attractive presentation of this manuscript and to CCL Pharmaceuticals (Pvt) Ltd for
providing the funds for printing it.
I hope this manual will be used by many, in their haematology practice and be a valuable resource
in the process of obtaining accreditation in haematology laboratories.
Dr. Thanuja Dissanayake

Coordinator of the project,
Sri Lanka College of Haematologists.
v
vi
CONTRIBUTORS
(In alphabetical order)
LIST OF AUTHORS
PANEL OF REVIEWERS
Ms. A.M. Hemalie CONTENT EDITORS
Kanchana Abeykoon Dr. Nadeeja Amarasinghe
Medical Laboratory Technologist Consultant Haematologist Dr. Nadeeja Amarasinghe
Consultant Haematologist
Dr. Nadeeja Amarasinghe Dr. Sunethra
Consultant Haematologist Bandaranayake Athauda Dr. Thanuja Dissanayake
Consultant Haematologist Consultant Haematologist
Dr. Seuwandi Basnayake
Consultant Haematologist Ms. L. Dhammika Dr. H.M.J. Priyanka Herath
Medical Laboratory Technologist Consultant Haematologist
Ms. L. Dhammika
Medical Laboratory Technologist Dr. Thanuja Dissanayake Dr. Nipunika Senadheera
Consultant Haematologist Consultant Haematologist
Consultant Haematologist Dr. H.M.J. Priyanka Herath
Dr. Bernadene Fernandopulle
Consultant Haematologist Dr. Chandima Kulathilake EDITORIAL BOARD OF SLCH
Dr. Swarna Gunathilake Prof. Lallindra Gooneratne
Consultant Haematologist Dr. Nishadya Ranasinghe Consultant Haematologist
Dr. H.M.J. Priyanka Herath Dr. Chamarika Moonesinghe
Consultant Haematologist Dr. Nipunika Senadheera Consultant Haematologist
Dr. Dilini Jayaratne Dr. Indika Somaratne
Consultant Haematologist Dr. Dinuka De Silva Consultant Haematologist
Mr. B.B. Isuru Namal Priyankara Prof. Senani Williams
Medical Laboratory Technologist Dr. Sasikala Suresh Consultant Haematologist
Dr. Nipunika Senadheera
Consultant Haematologist Dr. Chandima Thevarapperuma
Mr. L.H.S. Sujeewa COORDINATOR
Medical Laboratory Technologist Dr. Mala Tudawe
Consultant Haematologist Dr. Thanuja Dissanayake
Mr. M.D.C. Tharanga Consultant Haematologist
Medical Laboratory Technologist Dr. Deepthi Vidyarathna
Dr. Anoma Weerawardana
Consultant Haematologist Dr. Sudharma Vidyatilake
Dr. Chandana Wickramaratne
Consultant Haematologist Dr. Chandana Wickramaratne
Dr. Indira Wijesiriwardena

vii
CONTENTS
SECTION 01 SECTION 05
SAMPLE COLLECTION, TRANSPORT AND STANDARD OPERATING PROCEDURES FOR
RECEPTION AT THE HAEMATOLOGY ROUTINE COAGULATION TESTS
LABORATORY
..................................................................................... 11 5.1 ADJUSTMENT OF
ANTICOAGULANT FOR HAEMATOCRIT.......... 75
SECTION 02
GENERAL SAFETY IN THE 5.2 PREPARATION OF
HAEMATOLOGY LABORATORY PLATELET POOR PLASMA (PPP)........................ 77
..................................................................................... 17
5.3 PREPARATION OF
POOLED NORMAL PLASMA (PNP)....................... 79
SECTION 03
CALIBRATION AND MAINTENANCE OF 5.4 ESTABLISHMENT OF
EQUIPMENT, AND REAGENT REFERENCE INTERVALS AND
CALCULATION OF MEAN NORMAL
MANAGEMENT IN HAEMATOLOGY
PROTHROMBIN TIME (MNPT) ............................. 81
..................................................................................... 23
5.5 PROTHROMBIN TIME (PT) .................................... 83
5.6 ACTIVATED PARTIAL

THROMBOPLASTIN TIME (APTT) ......................... 87
SECTION 04
STANDARD OPERATING PROCEDURES FOR 5.7 THROMBIN TIME (TT)............................................. 91
ROUTINE HAEMATOLOGY TESTS
5.8 FIBRINOGEN ASSAY
4. 1 FULL BLOOD COUNT (MODIFIED CLAUSS METHOD).............................. 95
BY AUTOMATED ANALYZER (THREE PART)....... 33
5.9 BLEEDING TIME (BT)............................................... 99
4. 2 FULL BLOOD COUNT BY AUTOMATED ANALYZER
(FIVE PART/SEVEN PART)..................................... 39
4. 3 HAEMOGLOBIN CONCENTRATION BY
HAEMOGLOBINCYANIDE
(CYANMETHAEMOGLOBIN METHOD)................. 47 SECTION 06
STANDARD OPERATING PROCEDURES FOR
4. 4 MANUAL PLATELET COUNT................................... 51 SPECIAL HAEMATOLOGY TESTS
4. 5 PACKED CELL VOLUME 6. 1 HAEMOGLOBIN H INCLUSIONS............................ 105

(MICROHAEMATOCRIT METHOD).................... 55
6. 2 HEINZ BODY PREPARATION................................... 107
4. 6 MANUAL RETICULOCYTE COUNT......................... 59
4. 7 BLOOD FILM AND DIFFERENTIAL COUNT......... 63 6. 3 SICKLING TEST.......................................................... 109
4. 8 ERYTHROCYTE SEDIMENTATION RATE (ESR) 6. 4 HAEMOGLOBIN S SOLUBILITY TEST..................... 113

(WESTERGREN METHOD)...................................... 69
6. 5 OSMOTIC FRAGILITY TEST..................................... 115
6. 6 CRYOHAEMOLYSIS TEST........................................ 119
viii
6. 7 BREWER’S SECTION 08
METHAEMOGLOBIN REDUCTION TEST.............. 123 STANDARD OPERATING PROCEDURES FOR
HIGHLY SPECIALIZED HAEMATOLOGY
6. 8 ACIDIFIED SERUM LYSIS TEST (HAM TEST)........ 127
TESTS
6. 9 DONATH-LANDSTEINER TEST............................... 131 8. 1 BONE MARROW EXAMINATION..................... 203
6. 10 KLEIHAUER TEST (ACID ELUTION TEST).............. 133 8. 2 IDENTIFICATION AND QUANTIFICATION OF

HAEMOGLOBIN BY HIGH PERFORMANCE
6. 11 HAEMOSIDERIN IN URINE.................................... 137 LIQUID CHROMATOGRAPHY (HPLC) ............. 213
8. 3 IDENTIFICATION AND QUANTIFICATION OF

HAEMOGLOBIN BY
CAPILLARY ELECTROPHORESIS........................ 219
8. 4 FLOW-CYTOMETRIC
SECTION 07 IMMUNOPHENOTYPING (FCM) FOR
STANDARD OPERATING PROCEDURES FOR LEUKAEMIA/LYMPHOMA/MYELOMA................ 225
SPECIAL COAGULATION TESTS
8. 5 FLOW-CYTOMETRIC IMMUNOPHENOTYPING
7. 1 CLOT SOLUBILITY TEST........................................... 143 FOR PAROXYSMAL NOCTURNAL
HAEMOGLOBINURIA (PNH).................................. 235
7. 2 COAGULATION MIXING STUDIES........................ 147
7. 3 COAGULATION FACTOR ASSAY........................ 153
7. 4 VON WILLEBRAND DISEASE TEST PROFILE........ 161 SECTION 09

STANDARD OPERATING PROCEDURES FOR
7. 5 PLATELET AGGREGATION BY STAINS USED IN HAEMATOLOGY
LIGHT TRANSMISSION PLATELET
AGGREGOMETRY..................................................... 165 9. 1 LEISHMAN ............................................................... 245
7. 6 INHIBITOR SCREENING (BASED ON APTT)......... 171 9. 2 MAY GRUNWALD - GIEMSA ............................... 249
7. 7 QUANTITATIVE INHIBITOR ASSAY 9. 3 NEW MODIFIED GIEMSA (NEW MGS) .............. 253
(BETHESDA ASSAY).................................................. 175
9. 4 PERLS (PRUSSIAN BLUE) ..................................... 257
7. 8 DILUTE RUSSELL’S VIPER VENOM TIME
(DRVVT) .................................................................... 181
9. 5 SUDAN BLACK B .................................................... 261
7. 9 KAOLIN CLOTTING TIME (KCT)............................. 187
9. 6 PERIODIC ACID SCHIFF (PAS) ............................. 265
7. 10 ROTATIONAL THROMBOELASTOMETRY
9. 7 NON-SPECIFIC ESTERASE (NSE) ......................... 269
(ROTEM).................................................................... 191
9. 8 DOUBLE ESTERASE (DE) ...................................... 273
7. 11 ANTI FACTOR Xa LEVEL.......................................... 197
9. 9 TOLUIDINE BLUE .................................................. 277
9. 10 NEUTROPHIL ALKALINE PHOSPHATASE SCORE

(NAP SCORE)............................................................ 281
ix
Manual of
Haematolog y
SECTION01
SAMPLE COLLECTION,
TRANSPORT AND RECEPTION
AT THE HAEMATOLOGY
LABORATORY
11
SAMPLE COLLECTION,
TRANSPORT AND RECEPTION
AT THE HAEMATOLOGY LABORATORY
A properly collected specimen of blood is essential for generation of correct results in the laboratory. This includes a
range of activities which need strict adherence to quality and standard practices.
Information regarding sample collection, transport, and sample reception at the laboratory should be available as written
procedures /manuals and these documents should be readily available both in the laboratory and in wards/ clinics/OPD.
If the laboratory is providing sample referring facilities to reference institutions/laboratories, detailed information should
be available to the clinical teams.
The laboratory should provide sample collection and transport training to the relevant staff.
PATIENT IDENTIFICATION
 Before collecting the sample, the patient should be positively identified by asking minimum of two unique identifiers
such as full name, age and date of birth. Compare these data with the information on the request form and confirm
the identity.
 The request form for laboratory tests should contain the following:
 Patient’s full name
 Patient’s unique identifier- BHT/Clinic Number/ ID
 Gender of the patient
 Date of birth /age of the patient
 Ward/Clinic number
 Type of sample
 Test requested
 Name / signature / initials of the requester
 Contact details of the requester (optional)
 Clinically relevant information
 Date and time of sample collection
SAMPLE COLLECTION, TRANSPORT AND RECEPTION AT THE HAEMATOLOGY LABORATORY 13

SAMPLE COLLECTION
 Venous blood is preferred for most haematological examinations. Peripheral capillary samples may be satisfactory
for some purposes, but in general the use of capillary blood should be restricted to children and some point of care
screening tests.
 Before venepuncture, the appropriate sample collection tubes required for the requested tests should be selected
and kept ready. The expiry date of the sample collection tubes should be checked.
 Patients should avoid strenuous exercise and stress immediately prior to blood draw and rest for fifteen minutes in
a seated position prior to phlebotomy.
 The best site for obtaining blood is veins of the antecubital fossa.
 Limbs with intravenous lines and sites of haematomas should be best avoided.
 A tourniquet is applied to the upper arm. It should not be too tight and should not remain in place for more than
2 minutes as this can cause haemoconcentration and changes in test results. The patient can be asked to make a fist
to make the veins more prominent.
 The puncture site is cleansed with 70% ethanol and allowed to dry.
 Sterile, dry, disposable needles and syringes are used for the collection of blood. Needle size should be 22 – 23 gauge
in children and 19 – 21 gauge in adults.
 Venepuncture is performed in the direction of the long axis of the vein and with the bevel of the needle facing up.
 Blood is withdrawn slowly. Pulling the piston quickly can cause haemolysis and collapse the vein.
 The tourniquet should be released as soon as the blood begins to flow into the syringe. This will prevent haematoma
formation at site of venepuncture.
 When the required volume of blood is collected, the patient is asked to open the fist. The needle is removed from
the vein. A sterile alcohol swab is pressed over the puncture site till the bleeding ends.
 The needle is removed from the syringe and the required amount of blood is transferred in to the tube containing
anticoagulant.
 Most tests in haematology require whole blood. Therefore, as soon as a specimen of blood is withdrawn from a
patient, it is necessary to mix it with the correct anticoagulant to prevent clot formation. This is done by 10 gentle
and complete inversions of the tube after replacing its cap. Avoid shaking the tube as it can cause haemolysis.
 Needle and syringe are disposed into a biohazard box. Avoid recapping the needle as this may cause needle stick
injuries.
Sample labelling
 Blood samples should be labelled immediately before or immediately after blood collection.
 The label should not cover the tube wall completely and a space should be available to check the sample quality.
 The label should contain patient’s full name, patient’s unique identifier (BHT/ Clinic number/ ID), sample type and
test, date and time of collection and ward number or clinic.
 This should be completed before leaving the side of the patient.
Figure 1.1 Order of draw
Order of draw
When multiple types of blood collection tubes are filled from
the same venepuncture or when the blood in a single syringe
is transferred to multiple tubes, the order of filling should be
as shown in figure 1.1
14 MANUAL OF LABORATORY PROCEDURES IN HAEMATOLOGY

Anticoagulant in sample container
Test Anticoagulant
FBC, blood film, reticulocyte count and K2EDTA-preferred

most special haematology tests (refer section 06) K3EDTA
All coagulation tests Eg: PT, APTT, TT, plasma fibrinogen Trisodium citrate (32 g/L, 3.2%)

ESR Trisodium citrate (32 g/L, 3.2%)
Trisodium citrate (38 g/L, 3.8%)
Ratio of blood to anticoagulant
 Critical ratios between anticoagulant and blood should be maintained.

 ESR – maintain 1:4 ratio with anticoagulant: blood.
 Coagulation tests- maintain 1:9 ratio with the anticoagulant: blood
 FBC/blood film- generally, unless specified otherwise, EDTA in a tube is adequate to anticoagulate 2 mL of
blood.
 If using commercially prepared tubes, volume of blood needed should to be verified with manufacturer’s instructions
for the type of tube.
SAMPLE TRANSPORT
 Samples should be sent to the laboratory without delay together with a properly filled request form.
 Samples should be transported upright in a leak proof container; all possible care must be taken not to contaminate
the request forms and exterior surfaces of collection tubes with blood.
 Recommended environment conditions should be maintained (e.g. time and temperature).
SAMPLE RECEPTION AT THE LABORATORY

 The laboratory should have written sample acceptance/rejection criteria for both re-collectable and non re-
collectable samples.
 Rejected samples should be recorded with the reason for rejection mentioning the actions taken.
 Rejected samples should be informed to the clinician/ unit and a fresh sample should be requested.
 The average monthly rejection rate should be assessed periodically (e.g. last 6 months) and action should be taken
to improve the system. e.g. arrange relevant staff training.
 There should be an identified procedure to receive and process urgent samples
 Accepted samples should be given a unique identification laboratory number for sample tracking; this number
should be put on the sample label, request form and in the sample registry.
SAMPLE ACCEPTANCE AND REJECTION CRITERIA

Sample acceptance criteria
 Samples should be collected into the appropriate tube for the test.
 Sample collection tubes should not have expired.
 Should have correct sample volume; up to the fill line printed on the tube label and should not be overfilled or under
filled.
SAMPLE COLLECTION, TRANSPORT AND RECEPTION AT THE HAEMATOLOGY LABORATORY 15

 The sample label should have all required information.
 Sample should be accompanied by a relevant request form containing the required
information.
 Information on the label of the tube should tally with the infomation on the
accompanying request form.
 Samples should have required quality; not clotted, non-haemolyzed, not
decomposed.
 Samples should be transported in the recommended condition for the individual
test (time and temperature).
Sample rejection criteria
 Samples collected into improper containers/ anticoagulant.

 Wrong sample identification (unlabeled/ wrongly labeled/ mismatched).
 Underfilled/ overfilled samples.
 Samples not complying with the required quality; clotted, haemolysed, deteriorated,
transported/stored in wrong conditions (time or temperature).
 Damaged packages/ leaking tubes / outer surface of the tubes contaminated with
blood.
 References
1. CLSI guideline – Procedures for the Handling and Processing of Blood
Specimens for Common Laboratory Tests; Approved Guideline – Fourth
Edition H18 – A4, Vol.30 No.10.
2. Kitchen, S, Adcock, DM, Dauer, R, et al. International Council for
Standardisation in Haematology (ICSH) recommendations for collection of
blood samples for coagulation testing. Int J Lab Hematol. 2021; 43: 571-580.
3. De la Salle, B. Pre- and postanalytical errors in haematology. Int J Lab
Hematol. 2019; 41(Suppl. 1): 170- 176.

SECTION 02
GENERAL SAFETY IN THE
HAEMATOLOGY LABORATORY
17
18 Laboratory Manual of Test Procedures in HAEMATOLOGY
GENERAL SAFETY IN THE
HAEMATOLOGY LABORATORY
Laboratory safety can be addressed under different topics. Laboratories are considered potentially hazardous and
therefore unauthorized entry should be restricted. Laboratories should ensure the safety of its employees and users,
safety of the environment and safety of patients.
With COVID - 19 pandemic, safety requirements became a major concern. Generally, a haematology laboratory falls
under biosafety level 2 (BSL 2).
DESIGN REQUIREMENTS
The current concept is to minimize partitioned and separated rooms. Instead, more open, ventilation ensured systems
are recommended. If the laboratory is large, there should be separate entry and emergency exit doors. Entry doors
can have electronic systems and auto doors to minimize handling. It is recommended to locate hand washing or hand
sanitization facilities at entry and exit doors of laboratories.
Floor space should be appropriately determined considering work tops (minimum height 76 cm), minimum clearance in
walking areas (112 cm), and minimum clearance between work benches when people work back-to-back (152 cm) etc.
The laboratory layout should always consider controlled entry and exit as well as fire exit or emergency exits. Ergonomics
should be considered for a healthy working environment. Height and knee space should be designed in countertops
using standard recommendations such as sit-down countertops with 76 cm and stand-up counter tops at 91 cm. Similarly
sinks used for procedures should be installed at 91 cm.
Any non-technical areas (e.g. documentation, record keeping) should be clearly separated from sampling/technical area.
Power supply for critical equipment should be given through UPS systems to ensure uninterrupted operation of the
equipment.
Floors should be non-slip, cleanable, with no cracks or crevices, free of carpets or rugs, bench tops smooth and free of
cracks or crevices, impervious to water & resistant to chemicals used to decontaminate the surfaces and equipment,
chairs covered with a nonporous material that can be easily cleaned and decontaminated.
Ambient and task lighting should be available as per the tasks. Good ventilation and air flow should be ensured. Each test
function and activity should be located in a manner ensuring minimal cross over and repeat movements of laboratory
staff (“spaghetti diagram”).
General cleaning of surfaces can be performed using 1% hypochlorite and other surfaces using 70% alcohol. This should
be done at the end of the day or in a frequency suitable for the laboratory. The 1% hypochlorite should be prepared
freshly. For this 10% hypochlorite can be prepared and kept with the expiry date written on the label.
GENERAL SAFETY IN THE HAEMATOLOGY LABORATORY 19

Separate sinks should be there for hand washing. Emergency eye wash station should be located within 100 feetIf
several sections and many doors are present, it is better to have at least one eye wash station in each laboratory. Use
of portable hand-held eyewash bottles is an alternative. However, if corrosive substances such as Dithiothreitol (DTT) is
used, eye wash stations should be located by the work bench. Emergency shower is not a must. However, if fire or major
chemical spillage is identified as a potential major risk in a laboratory, it is advisable to fix emergency showers. In routine
haematology it is not required.
STORAGE
Chemicals and samples should be stored appropriately to ensure safety of staff handling them.
WASTE MANAGEMENT
There should be separate colour-coded containers for collecting the different categories of waste (e.g infectious,
noninfectious). Containers with infectious material should have lids and appropriate labels on them (e.g.-biohazard sign).
Clinical waste generated in haematology laboratory are left over samples of blood and body fluids or, reagents mixed
with blood or body fluids following test completion. Most of the automated analyzers have closed waste collection
systems. Disposal of such chemical contaminated waste should be done as per the manufacturer’s instructions or as per
central environment authority recommendations (CEA). For this, all chemicals used and their risk category should be
known by the laboratory. When laboratories are planned, drainage systems of work tops should go to separate tanks for
pretreatment. The CEA recommend release of such water, chemicals and concentration levels acceptable for release in
to common drainage systems.
DISPOSAL OF SAMPLE TUBES

Autoclaving or incineration is the ideal. Failing to do so, decontamination by overnight soaking in 10% hypochlorite
would be an alternative. However, when this is performed, should ensure tubes are fully immersed and tubes are filled
with hypochlorite to avoid air trapping and prevention of decontamination. However, removal of blood contaminated
hypochlorite and cleaning of tubes should be done as per the CEA or MoH recommendations. These tubes can be
handed over for proper disposal, if external service providers are available. Such service providers should have CEA
certification for clinical waste transport, storage and disposal.
HANDLING OF SAMPLE SPILLS

Spills should be cleaned using hypochlorite and absorbent material. Pouring of adequate volume of hypochlorite and
allowing adequate contact time ( 20 minutes) is important. Final cleaning of surface can be done by 70% alcohol. If a
patient sample is spilled before testing, arrangements should be made to collect a fresh sample and root cause should
be identified for the mishap and all possible preventive actions should be taken to prevent future occurrence of such
incidents.
PERSONAL PROTECTIVE EQUIPMENT AND GENERAL HYGIENE

In a haematology laboratory, laboratory overcoat and gloves are sufficient to assure safety. If splashes are expected to
occur, goggles should be worn. Ideally foot ware should cover toes. Hair should be neatly combed. Use and storage of
personal belongings within laboratory should be avoided. Laboratory coats should be removed when going out of the
laboratory thus there should be a suitable place/ hanger to keep lab coats at exit / entry doors. Gloves worn for personal
protection should be removed when handling commonly used items such as telephone, door handles etc.
ELECTRIC HAZARD
Check circuit breakers, wiring, plug bases and sockets for failures or loosened parts. When servicing or cleaning,
equipment, they should be disconnected from the electricity supply. Grounding should be done as appropriate.

FIRE HAZARD
Fire extinguishers and fire suppression systems such as sprinklers should be considered as per the complexity of the
laboratory and fire risk. Fire and smoke alarms, gas leak sensors would provide more security related to fire if needed.
The fire extinguishers should be functional and staff training on utilization of the fire extinguisher should be provided.
CHEMICAL SAFETY
Chemical safety is not a major issue in a haematology laboratory due to use of closed systems and due to use of
minimally hazardous chemicals as per regulations. However, materials safety data sheets (MSDS) for chemicals used in
the laboratory should be available. The MSDS sheets must contain following key contents:
1. Name of the chemical
2. Manufacturer’s information
3. Hazardous ingredients/identity information
4. Physical/chemical characteristics
5. Fire and explosion hazard data
6. Reactivity data
7. Health hazard data
8. Precautions for safe handling and use
9. Control measures
As per the MSDS information, required antidotes, personal protective equipment (eg: non porous aprons) and safety
gadgets (eg: bottle carriers) should be made available. Labelling of chemicals identified as hazardous should be
appropriately done as per hazard information and standard color code.
OTHER EMERGENCIES AND RISK ESTIMATION

Risk assessment should be done in each key process and appropriate safety measures should be made available for the
risks identified. Risk assessment should be done using standard risk assessment matrix.
TRAINING AND DOCUMENTATION

There should be a designated safety officer/ focal point with a job description and deputies as per the complexity of the
laboratory. They should have appropriate training and should be able to report directly to relevant authorities regarding
safety issues.
All new staff should receive safety training before they begin working and there should be periodic safety training
program for all staff (e.g. annually)
Safety policy procedures and processes should be documented appropriately in a safety manual. A regular safety check
should be done using a checklist at regular intervals. Safety drills and rehearsals can provide good input on actions and
expected behavior in case of a real emergency in laboratories.
Staff should refrain from using cellphone and bringing personal items (purses, backpacks, books, magazines etc.) in
technical areas.
 References
1. Clinical and Laboratory Standards Institute QMS 04 Laboratory design 3rd edition 2016.
2. ISO 15189:2012.
3. Laboratory safety guidance OSHA 2011.
GENERAL SAFETY IN THE HAEMATOLOGY LABORATORY 21

SECTION 03
CALIBRATION AND
MAINTENANCE OF EQUIPMENT,
AND REAGENT MANAGEMENT
IN HAEMATOLOGY
23
CALIBRATION AND
MAINTENANCE OF EQUIPMENT,
AND REAGENT MANAGEMENT
IN HAEMATOLOGY
Preventive maintenance is the secret of a trouble-free performance of any equipment. For instruments having measuring
functions, calibration is a mandatory requirement to assure accuracy of measurements. Haematology laboratories use
different instruments ranging from pipettes, glassware to fully automated high throughput systems. Any test system or
item has its own maintenance and calibration requirements unique to it. Aim of this section is to provide guidance on
appropriate maintenance and calibration requirements to ensure quality performance in haematology laboratories.
GENERAL REQUIREMENTS FOR CALIBRATION

For calibration of test equipment other than automated analyzers and coagulometers, following should be adhered to.
1. The calibration services should be obtained by an institute accredited for calibration.
2. Most importantly, calibration should have unbroken chain of comparisons up to international standard.
3. Calibration should ideally be performed under similar conditions the laboratory is having.
4. Calibration should be done at the same (or closer) working conditions such as working temperature
(eg: water baths), working volumes (eg: pipettes) or working speed (eg: centrifuge).
MAINTENANCE OF EQUIPMENT
All equipment and each item or part should be maintained properly. The minimum maintenance that should be adhered
to or implemented by the laboratory is the use of manufacturer’s instructions.
It is strongly recommended to have a copy of the original manual at work benches for each test item or equipment to
refer and understand requirements.
1. All the specific maintenance requirements stated in the manufacturer’s manual should be copied in to a
standard operating procedure (SOP).
2. To ensure appropriate implementation, a check list of maintenance can be prepared. This can be displayed
as a flowchart at the work station, next to the analyzer.
3. A copy of the SOP on maintenance should always be available at the work bench.
CALIBRATION AND MAINTENANCE OF EQUIPMENT, AND REAGENT MANAGEMENT IN HAEMATOLOGY 25

4. Maintenance should be implemented as specified in the SOP without any alterations.
5. Maintenance frequency of equipment should be given as daily, weekly, monthly or “when needed”.
“Maintenance when needed” is ignored most of the time. Due to cost issues or lack of knowledge of service personnel,
maintenance procedures are delayed and that can lead to sub optimal function of the analyzer and generation of error
results.
In addition, providing appropriate conditions for each equipment is important. Those are given in the manufacturer’s
manual.
- Room temperature should be 21-25 0C.
- Environment should be dust free.
- Standard air conditioning will provide relatively dry air and less humidity which are appropriate for
diagnostic equipment in haematology. Humidity recommended for laboratories is 40%-50%.
Laboratories should have 100% exhaust or ‘single pass’ air flow. Specifically, air recirculation cannot be accommodated
when considering handling of significant biohazardous material in laboratories.
Generally, all the equipment should be located away from direct sunlight, direct wind currents and vibrations. Work
benches with flat, strong, stable surfaces with adequate height and width should be selected for the positioning
of equipment. When positioning any equipment, allowable space around it for adequate ventilation and prevention
of heating should be provided as per the manufacturer’s instructions.
MAINTENANCE OF GLASSWARE/ GLASS PIPETTES

Class A glassware does not need calibration. Plastic or Class C glassware need calibration verification. When plastic
pipettes are reused after washing, drying at high temperatures can distort internal caliber, linearity etc. Therefore, single
use disposable plastic labware should not be reused unless volume and performance are verified in each.
Use of hypochlorite solution to decontaminate glassware used for handling blood or plasma can ensure adequate safety.
However, amount of blood in each glassware would require frequent change of hypochlorite solution for appropriate
decontamination. Glassware can be washed using detergents and then with tap water or distilled water as per the
requirement.
As air drying is time consuming, glassware can be easily dried in a hot air oven. Glassware should be stored appropriately
to prevent contamination, breakage and formation of fungi if infrequently used.
MAINTENANCE OF THERMOMETERS
Different types of thermometers are available in laboratories.
a. Mercury column thermometers

Mercury column thermometers are more reliable. However, they are less available due to regulatory restrictions.
If available, most are total immersion or partial immersion types. Check this in your thermometer. The depth of immersion
is indicated by a black line across the tube. When measuring the temperature in liquids such as in water baths, we should
immerse the thermometer up to the mark.
Mercury column thermometers can be verified for its readings at two points, using melting ice for 0 0C and boiling water
for 100 0C. Once calibration verification is done, no need to recalibrate. No specific maintenance is required. Careful
storage and handling to prevent breakage and wipe cleaning to remove dampness or dirt is sufficient.
Most of the laboratories use mercury column thermometers to monitor room temperature and they are continuously
fixed to the wall. In such instances, when readings are taken, it is advisable to remove and expose to a different
temperature for a while and then check the temperature at the required site.

b. Digital thermometers
Digital thermometers should be purchased with a calibration certificate from the manufacturer. If the certificate is with
complete traceability, simple verification in the laboratory is sufficient up to one year. However, digital thermometers can
deteriorate due to mishandling, accidental dropping and due to discharged batteries. Therefore, intermediate checks at
regular intervals are mandatory. Digital thermometers should be calibrated. Calibration can be verified in the laboratory
intermittently. Annual recalibration is safer due to electronic systems in it. Verification of temperature can be performed
using a calibrated water bath or equivalent.
MAINTENANCE OF CENTRIFUGE
The latest WHO recommendation is to use no-aerosol forming centrifuges in laboratories. Noise level should be below
WHO recommended levels. Centrifuge should be located on a solid, leveled surface in a damp and dust free area.
Centrifuges can be of different types: fixed angle rotor, fixed rotor, removable rotor, swing out bucket, refrigerated,
microcentrifuge etc.
Maintenance of centrifuge needs
1. regular checking for wear and tear
2. cleaning of any spills
3. cleaning exterior
4. calibration of speed
5. calibration verification
6. if centrifuge is used for platelet poor plasma preparation (PPP) for coagulation testing, RPM for PPP
preparation should be verified. Literature show G force of 1500- 2500 for PPP preparation. Laboratory can
apply different G forces and verify PPP preparation.
Ideally, RPM calibration should be carried out onsite. Verification of calibration can be done intermittently using a
calibrated tachometer or using indirect methods eg: if your laboratory has verified platelet poor plasma preparation and
verified platelet count, maintaining almost similar platelet counts indirectly verify the achieved RPM.
Generally, cleaning of exterior can be done by a mild detergent. Cleaning of metal surfaces using hypochlorite is not
recommended thus, mild detergent or 70% alcohol should be used.
Interior of centrifuge and buckets should be cleaned either with 1% hypochlorite or 70% alcohol at regular intervals. Care
should be taken when cleaning is performed following breakage of tubes. Heavy duty gloves and forceps should be used
to remove all sharps.
MAINTENANCE OF WATER BATH

Water baths are of many different types. However, commonly used ones in haematology laboratories are of the
water circulating type. These need temperature calibration. Calibrated water baths need calibration verification and
intermediate checks using a calibrated thermometer. Water in water baths should be regularly replaced with fresh water.
Temperature in water baths should be verified frequently when performing tests especially, manual coagulation tests as,
variations in temperature affect results of these tests.
MAINTENANCE OF MICROSCOPES
Microscopes need regular cleaning to remove dirt and dust. Leaving them in air-conditioned rooms even without a cover
is adequate to prevent most of the storage problems. However, if the laboratory is not air conditioned, they should be
stored in a separate storage area to prevent fungal growth. Surface of body can be cleaned with damp cloth using mild
detergent soap. However, cleaning of lenses and condensers should be done carefully using special lens tissue. As per the
manufacturer’s instructions, either ether, alcohol or similar material can be used to clean lenses.

If humidity is a concern, the microscopes can be stored in wooden boxes fixed with 100-watt bulbs or they should be
stored in a dehumidifier room or under continuous air conditioning.
Some microscopes need regular verification of light path axis. This should be done using the manufacturer’s instructions.
Start with scanning power, condenser aperture turned to the smallest and set the light point exactly at the centre of the
field using two screws.
The novel microscopes which have fixed systems do not need light path axis adjustments.
When purchasing microscopes, specifying illumination requirement (LED), its half-life, quality of objectives, achromatic
lenses, absence of jutting out parts and ergonomic design to minimize back pain would enable purchasing a good quality
microscope.
There are other microscopes such as phase contrast, fluorescent, dissection, confocal etc. Each need dust free, low
humidity environment for storage. Some, such as phase contrast microscope need calibration check intermittently.
MAINTENANCE OF AUTOMATED FULL BLOOD COUNT ANALYZER

Upon installation, calibration and calibration verification of FBC analyzer should be done by the supplier. Calibration
schedule/ requirements of automated FBC analyzers are given below.
- Upon installation
- Following major repair
- When IQC deviations indicate systematic error or when significant deviations are present in EQA results
- As per the regulatory requirement. (In Sri Lanka there is no such regulation.)
Calibrator should have comparison data to international standard or method and assigned value and measurement
uncertainty (MU). The MU of a calibrator is expected to be lower than those of quality control materials. Quality control
materials are not recommended for calibration.
Manufacturer’s recommendations for installation and verification are given in the user manual. We should refer that
and compare installation report submitted by the supplier. Any deviations noted in installation or verification process
compared to the manufacturer’s recommendations should be officially clarified before commissioning.
Once above process is completed satisfactorily, laboratory should plan verification of performance. The verification
experiment can take even longer than one month if properly done. Minimum verification process is given below.
1. Verification of accuracy and performance is specified as CV% for each parameter by the manufacturer: Using a
third-party QC with target value specified for the machine can be used to perform this task. Repeated analysis of
same QC sample 20 times will provide data for comparison. The acceptance criteria for the deviation from target
mean should be defined using international norms. Use of common 10% rule is not satisfactory for this purpose. All
the calculated CV% should be either equal or low compared to the manufacturer specified CV %. If CV% is deviated,
clinical significance should be assessed and considering that, the laboratory can accept the machine or request the
supplier to rectify it. In most instances, incorrect handling of QC samples can generate incorrect performance data.
thus greater attention should be paid on every step followed when carrying out the task.
2. Verification of precision – this can be done by either using QC or patient samples. The target values need not be
known. Inter-assay precision can be calculated between batches, machines and shifts. When patient samples are
used, ensure that samples are of good quality and freshly collected from the laboratory.
3. Carry-over verification: this needs samples with very high platelet count, very high WBC, RBC etc. If the laboratory
cannot find such samples, spiked samples can be used.
4. Flagging verification: this experiment needs patient samples with platelet clumps, blasts, reactive lymphocytes,
agglutination etc. and can take a few days to weeks
5. DC verification – this should be done by manual DC
6. Verification of manual platelet count for the microscope in use.

Verification of performance of sample collection tubes in FBC and coagulation tests in relation to sample stability, test
performance, usability in analyzer etc. need to be done before using them for patient testing. When compare different
brands of collection tubes are compared, verification study should be performed using sufficient number of blood
samples (e.g. 100) collected using standard technique by the best trained phlebotomist to minimize collection errors
and freshly analyzed. Stability can be verified by repeated analysis of same sample set collected at regular intervals
while stored in relevant conditions. The analyzers used should be calibrated, appropriately maintained with accuracy and
precision of results generated verified.
Maintenance of automated FBC analyser should be done as per the manufacturer’s instructions. Maintaining a file with
all the records related to maintenance, services and repairs carried out in equipment in chronological order is required.
Intermediate checks for the performance of the machine can be verified through planned experiments using spiked
samples, separate QC samples or by using known patient samples.
MAINTENANCE OF AUTOMATED COAGULOMETER

Installation and upon installation verification of automated coagulometer as well should follow the same processes and
procedures described for automated FBC analyzers. However, calibration is required only for quantitative measurements
such as fibrinogen. Otherwise, the term calibration is used for verification of ISI values and INR of coagulometers.
More importantly, upon installation of a coagulometer, laboratory should verify following;

1. Verify ISI specified for the machine and the reagent using reference plasma. If generic ISI is given, ISI should be
calculated for the machine and for the specific reagent. Every time when reagent lot is changed, ISI verification
should be done.
2. Verify PT and INR results using reference plasma.
3. Manual verification of automated INR should be done every time when reagent lot is changed.
4. Onboard stability of reagents. (Vial volumes/ capping and storage stability). This need attention especially when
onboard temperature in not controlled.
5. Probe alignment is very important as fault alignment leads to probe damage.
6. Verify carryover specially if single probe analyzer,
7. Performance data (CV%) can be verified by repeat analysis of IQC or patient samples.
Records of all the steps carried out, service and repair should be filed separately in chronological order similar to FBC
automated analyzer.
REAGENT MANAGEMENT
Storage of chemicals and samples should ensure prevention of deterioration. Most of the haematology analytical systems
use closed containers of reagents and room temperature is preferred. However, if refrigerated or lower temperatures
are needed, depending on turnover of lots, either refrigerators or cold room or both should be available. Reagents in
the refrigerator or store area should be stored according to first-to-expire, first-out (FEFO) practice. This is facilitated by
maintaining lot numbers, expiry date and date of receipt of the reagent.
Reagents which are very sensitive to temperature variations should be appropriately aliquoted to prevent wastage.
Procedures should be implemented to return such reagent vials as soon as possible when the test process is completed.
Leaving such sensitive reagent vials longer at room temperature deteriorate reagent rapidly and thus causing error
results generation.
Reagent performance verification is very important for coagulation tests and for some specialized tests. Reagent
performance can vary based on test method, and analyzer in use. Documentation of condition of reagent upon receipt
will ensure suppliers maintaining appropriate cold chain when transporting reagents.
Some reagents such as reticulocyte reagent, Leishman stains etc need verification of timing for good positive staining.
Storage of such reagents should prevent deterioration and contamination. Some staining reagents mature during storage,
thus there can be changes in time required for staining. Absence of a proper lid and evaporation can cause deterioration
of such reagents. Storage at higher temperatures and filtration when required would enable Leishman stain to work
properly.

 References
1. Clinical and Laboratory Standards Institute QMS 04 Laboratory design 3rd
edition 2016.
2. ISO 15189:2012.
3. Laboratory safety guidance OSHA 2011.
4. Clinical and Laboratory Standards Institute (CLSI), Procedures for the
Collection of Diagnostic Blood Specimens by Venipuncture, CLSI H3-A6
document, Clinical and Laboratory Standards Institute, Wayne, Pa, USA, 6th
edition, 2007.
5. Lima-Oliveira G, Lippi G, Salvagno GL, Montagnana M, Picheth G, Guidi
GC. Preanalytical management: serum vacuum tubes validation for routine
clinical chemistry. Biochemia Medica. 2012 Jun 15;22(2):180-6.

SECTION 04
Standard Operating Procedures for
ROUTINE
HAEMATOLOGY TESTS
4. 1 Standard Operating Procedure

FULL BLOOD COUNT
BY AUTOMATED ANALYZER (THREE PART)

FULL BLOOD COUNT
BY AUTOMATED ANALYZER (FIVE PART/ SEVEN PART)

HAEMOGLOBIN CONCENTRATION
BY HAEMOGLOBINCYANIDE (CYANMETHAEMOGLOBIN METHOD)

MANUAL PLATELET COUNT

PACKED CELL VOLUME (MICROHAEMATOCRIT METHOD)

MANUAL RETICULOCYTE COUNT

BLOOD FILM AND DIFFERENTIAL COUNT

ERYTHROCYTE SEDIMENTATION RATE (ESR)
(WESTERGREN METHOD)
4.1
Standard Operating Procedure
FULL BLOOD COUNT
BY AUTOMATED ANALYZER
(THREE PART)
Dr. Nadeeja Amarasinghe
Scope
The scope of this SOP is to guide technical personnel on proper evaluation of full blood count (FBC) by three-part
automated analyzer by providing the protocol and requirements for its correct performance.
Responsibility
Evaluation of full blood count by three-part automated analyzer as per the procedure stated in this SOP is the responsibility
of all medical laboratory technologists who are assigned to perform it in the haematology laboratory.
Principle
The three-part (differentiating white cells in to three categories) analyzer is capable of measuring haematological
parameters on EDTA-anticoagulated whole blood.
Principles of measurement of blood cell parameters are as follows. Some of the principles may vary depending on the
make of the analyzer.
 Cell counting (RBC, WBC and Platelets) is based on impedance counting or by light scattering technology.
Impedance counting – Is based on the fact that blood cells are poor conductors of electricity whereas certain
diluents are good conductors. As a blood cell is carried through the aperture, it displaces some of the conducting
fluid and increases the electrical resistance which is proportional to cell volume enabling cell counting and measuring
cell volume.
Light scattering - Diluted cell suspension is allowed to flow through an aperture so that the cells pass in single file in
front of a light source. The amount of light scattered is proportional to the surface area and therefore the volume of
the cell.
 Measurement of haemoglobin is based on the colorimetric method.
Both impedance counters and light-scattering instruments are capable of producing three-part differential counts from
a single channel and the categorization is based on the different volume of various types of cells following partial lysis
and cytoplasmic shrinkage.
STANDARD OPERATING PROCEDURES FOR ROUTINE HAEMATOLOGY TESTS 33

A three-part differential count assigns cells to categories usually designated (1) ‘granulocytes’ or ‘large cells’; (2)
‘lymphocytes’ or ‘small cells’; and (3) ‘monocytes’, ‘mononuclear cells’, or ‘middle cells’. In theory, the granulocyte
category includes eosinophils and basophils, but in practice it is common for a proportion of cells to be excluded from
the granulocyte category and to be counted instead in the monocyte category.
 Analysis of red cell parameters

1. RBC (x1012 /L) – Direct measurement by counting the number of erythrocytes passing through the aperture.
2. Hb (g/L) – Direct measurement by colorimetric method
3. MCV (fL) – Direct measurement using the height of the electrical pulses generated.*
4. Hct (%) – Derived measurement by multiplying RBC and MCV obtained as above.*
5. MCH (pg) – Derived from the Hb divided by RBC
6. MCHC – MCHC is derived from the Hb, MCV and RBC according to the given formula*
MCHC (g/L) = Hb (g/L) x 1000
MCV (fL) x RBC x 1012/L
7. RDW (SD and CV) - The RDW is derived from pulse height analysis and can be expressed either as the standard
deviation (SD) in fL or as the coefficient of variation (CV) (as a percentage) of the measurements of the red cell
volume.
The RDW SD is measured by calculating the width in fL at the 20% height level of the red cell size
distribution histogram, and the RDW CV is calculated mathematically as the coefficient of variation; that is,
RDW (CV) % = (1 SD/MCV) x 100.
*Note:
Sysmex analyzers use a different principle in producing Hct, MCV and MCHC.
 Hct is obtained using the cumulative pulse height method.
 MCV is calculated from Hct divided by RBC.
 MCHC is calculated from Hb divided by Hct.
 Analysis of white cell parameters

1. WBC (x 109 / L) – Directly measured by counting the number of white cells passing through
the aperture after complete lysis of red cells
2. Differential count – 3 part analyzers classify cells as ‘granulocytes’ or ‘large cells’,
‘lymphocytes’ or ‘small cells’, and ‘monocytes’, ‘mononuclear cells’,
or ‘middle cells’
 Analysis of platelet parameters

1. Platelet count (x 109 /L) – Directly measured by the analyzer by counting the number of platelets
passing through the aperture.
It is important to differentiate platelets from red cells and smaller particles such as debris and pulses generated
by electronic noise. One of the basic techniques is to count platelets between two fixed thresholds (e.g. between
2 and 20 fL)
2. MPV (Mean Platelet Volume) (fL) – Derived from impedance platelet size distribution curve.
3. PDW (Platelet Distribution Width) – Measure of platelet anisocytosis
4. PCT (Plateletcrit) – Derived from MPV and platelet count
5. P-LCR (Platelet Large Cell Ratio) - The number of platelets falling above the 12 fL threshold on the platelet
size histogram divided by the total number of platelets.

Clinical significance
FBC is the assessment of the blood cell parameters (Red cells, White cells and Platelets) including the number, size and
the proportions of the blood cells and Hb concentration. It is one of the basic tests which is used to assess the general
health status of an individual. It is affected not only by the diseases related to blood cells but also by systemic illnesses
ranging from benign to malignant conditions. FBC is used for the diagnosis as well as for the monitoring of the diseases.
Automated FBC usually have a high level of precision, for cell-counting and cell-sizing techniques, and is greatly superior
to that achievable with manual techniques provided these instruments are carefully calibrated and their correct operation
is ensured by quality control procedures.
Sample
 Whole blood is collected by venipuncture into a container with EDTA anticoagulant (K2 EDTA, K3 EDTA)
 FBC should preferably be performed within 4 hours of collection if kept at room temperature (15 -25 0C). If delayed,
blood sample should preferably be refrigerated at 2 – 8 0C and analyzed within 24 hours. Before processing refrigerated
samples should be allowed to warm up to room temperature (minimum 15 minutes), then mix preferable by rotation
for at least 2 minutes.
Equipment and reagents

Different models of 3-part haematology analyzers use the instrument specific reagents.
Please refer the manufacturer instructions.
Method
Each step in the operation of fully automated 3-part haematology analyzer and analysis of FBC should be done in
accordance with the manufacturer’s instructions. The main steps are as follows.
Ensure that the analyzer is kept at the recommended room temperature while in operation
1. Start up
 Turn on the power supply in accordance with the instructions provided by the manufacturer
 Check for technical factors ie. reagents/ diluents, their expiry dates, electricity supply, instrument pressure
gauges and vacuum etc.
 Background count check (ie electronic noise and particulate material in diluent solution)
Modern day analyzers are programed to perform these steps automatically at the start up and be ready to
operate only when they are in proper order.
 Check the date and time.
2. Running IQC samples to check whether they are within control limits.
If QC fails (according to the Westgard rules set by laboratory), halt the analysis, perform corrective actions
and run QC again.
3. Running whole blood samples
 Sample should be checked for the correct (EDTA) container, presence of clots, correct volume etc.
(Refer rejection criteria)
 Must be mixed well (leaving for 3 - 5 min on a mechanical mixture or manually by inverting 20 times) before
analyzing. (only open vial mode –OV mode, needs manual mixing, auto loader mode –AL mode, mixing is done
by analyzer).
 Load the samples ensuring the correct identity.
 If there is flagging of abnormal results microscopic examination of a stained blood film should be undertaken

 Three- part differential count should be issued with a differential count performed on a stained blood film to
make it more informative in the clinical settings.
 When NRBCs are present in significant numbers (>10 per 100 WBC), they will be included in WBC count giving
erroneously high count. In such situations, NRBCs should be counted on a blood smear and corrected WBC
count should be done using the following formula and reported together with the NRBC percentage.
Corrected WBC = Uncorrected WBC X 100
NRBCs per 100 WBC + 100
 Each laboratory should set up its own criteria for validation of the report before issuing.
4. Shut down at the end of daily work
 Instructions displayed on the screen should be followed by the operator.
 Before the instrument is turned off, cleaning of the tubing system should be done to ensure that no protein
clogs exist inside.
 The cleanser provided by the manufacturer should be set to the sample probe so that the analyzer performs
this step automatically before shutting down.
5. Maintenance schedule
Please refer the manufacturer’s instruction for the daily, weekly, and periodical maintenance schedule. Maintain
Good Laboratory Practice (GLP) charts.
In a special logbook record, the dates of all maintenance checks, replacement of components, down time and
identifiable causes of malfunction, servicing by manufacturer’s agent, recalibrations, traceable batch numbers
of calibrators and controls.
Reporting the results
Parameter Unit Parameter Unit
WBC … x 103/ uL RBC … x 106/ uL

LYM% …% Hb … g/ dL
MXD% …% HCT …%
NEUT% …% MCV … fL
LYM# … x 103/uL MCH … pg
MXD# … x 103/uL MCHC … g/dL
NEUT# … x 103/ uL RDW-CV … %
RDW-SD … fL
WBC Differential Count Platelet … x 103/ uL
Neutrophils ….% PCT …%
Lymphocytes ….% MPV … fL
Monocytes …..% PDW … fL
Eosinophils ..…% P-LCR …%
Basophils …..%
Quality control procedures

Internal Quality Control (IQC)
Tri level commercial QC material, with reference ranges for the specific model should be used.

Quality control should be performed,
 daily after startup. (ideally 8 hourly if there are 2 shifts)
 following corrective actions
 after reagent replacement
 after calibration.
 after maintenance, repair or part replacement.
 as required by clinical or laboratory QC regulations
If IQC material is not available for a particular analyzer, a comparison can be performed with results of another analyzer
performing regular IQC.
Calibration
Calibration is a procedure to standardize the analyzer by determining its deviation, if any, from calibration references and
to apply any necessary correction factors.
This procedure has to be done using calibrators with assigned values, in accordance with the manufacturer’s instructions.
Calibration of the analyzer is indicated,
 at installation.
 at least every six months
 after major preventive maintenance, a major repair or critical part replacement.
 when analyzer is to be reused after a long-term storage.
 when control materials reflect an unusual trend or shift of analyzer performance.
External Quality Assurance (EQA)

Analyzer performances should be compared in the national (and international if available) EQA program at least once in
3 months.
Sources of error
 Inadequate mixing of specimens or diluted suspensions before counting

 Air bubbles from vigorous shaking or dispensing
 Electric interference or voltage fluctuations
 External noise contamination e.g. from other electrical equipment
 Aspiration failure due to aperture blockage, leaks or loss of vacuum
 Residual traces of lytic agent or detergent cleaner causing haemolysis or artefactual red cell volume changes
Precautions and hazards
 All parts and surfaces of the analyzer should be considered as potentially infectious since the instrument handles
patient specimens.
 All clinical samples should be treated as possibly infectious and handled as per guidelines on sample handling
available in the laboratory.
 Appropriate personal protective gear should be worn when handling samples and reagents.
 Disposal of waste should be done according to local, provincial or national regulations.

 References
1. WHO Guidelines for Standard Operating Procedures for Haematology, January 2000.
2. Dacie and Lewis Practical Haematology, 12th Edition, 2017.
3. Blood cells, A practical guide, 5th Edition.
4. Sysmex XP 100, Instructions for use; 2012.
5. CLSI guideline - Validation, Verification and Quality Assurance of Automated Hematology Analyzers; Approved
Standards – Second Edition, 2011.
6. SEED Haematology – Overview of the benefits of switching from a 3-part differential to a 5- Part differential
haematology analyser, Sysmex Educational Enhancement and Development, March 2012.
7. SEED Haematology - The red blood cell indices; Sysmex Educational Enhancement and Development, October
2012.
8. Methods recommended for essential clinical chemical and haematological tests for intermediate hospital
laboratories, WHO/LAB/86.3.

4.2
FULL BLOOD COUNT
BY AUTOMATED ANALYZER
(FIVE PART/ SEVEN PART)
Scope
The scope of this SOP is to guide technical personnel on proper evaluation of full blood count by automated analyzer by
providing the protocol and requirements for its correct performance.
Responsibility
Evaluation of full blood count by automated analyzer as per the procedure stated in this SOP is the responsibility of all
medical laboratory technologists who are assigned to perform it in the haematology laboratory.
Principle
The five-part (differentiating white cells in to five categories) and seven part (extended differential count which
includes immature granulocytes or large immature cells and atypical lymphocytes including small blasts in addition
to the parameters of the five part analyzer) analyzers are capable of measuring haematological parameters on EDTA-
anticoagulated whole blood.
Principles of measurement of blood cell parameters are as follows.
 Cellular counting (RBC, WBC and Platelets) is based on impedance counting, by light scattering technology or
fluorescence methods.
Impedance counting – Is based on the fact that blood cells are poor conductors of electricity whereas certain
diluents are good conductors. As a blood cell is carried through the aperture, it displaces some of the conducting
fluid and increases the electrical resistance which is proportional to cell volume enabling cell counting and measuring
cell volume.
Light scattering - Diluted cell suspension is allowed to flow through an aperture so that the cells pass in single file in
front of a light source. The amount of light scattered is proportional to the surface area and therefore the volume of
the cell.
 Measurement of haemoglobin is based on the colorimetric method.
 WBC differential count is based on study of cells by number of modalities e.g. flow cytometry, impedance technology
with current of various frequencies, light scattering and light absorbance.
 Automated full blood count analyzers use different technologies to assess differential count. (Table 4.2.1)

Table 4.2.1 Technology used for differential count in haematology analyzers
Instrument and manufacturer Technology used for Differential Count

 Impedance with low-frequency electromagnetic current
Beckman Coulter GEN-S series, LH series, DxH  Impedance with high-frequency electromagnetic current
 Laser light scattering
 Impedance with low-frequency direct current
Sysmex SE, X-series (XE, XT, XN)  Impedance with radiofrequency current
 Fluorescence flow cytometry
Mindray BC series  Impedance with low-frequency direct current
 Electrical impedance with intact cells and following
Horiba Medical Pentra series differential cytoplasmic stripping
 Light absorbance
Nihon-Kohden MEK series  Impedance with low-frequency direct current
The analyzer relies on a multi-dimensional analysis (cell complexity, DNA/ RNA content, cellular content, cell size) for cell
identification for WBC differentiation. Most analyzers contain more than two channels.
In one channel a diluent is added, and red cells are counted and sized. In another channel a lytic agent is added, together
with diluent, to reduce red cells to stroma, leaving the white cells intact for counting and also producing a solution in
which Hb can be measured. Further channels are required for a differential WBC which depends on study of cells by
different modalities mentioned already.
 Analysis of red cell parameters

1. RBC (x1012 /L) – Direct measurement by counting the number of erythrocytes passing through the aperture.
2. Hb (g/L) – Direct measurement by colorimetric method
3. MCV (fL) – Direct measurement using the height of the electrical pulses generated. *
4. Hct (%) – Derived measurement by multiplying RBC and MCV obtained as above. *
5. MCH (pg) – Derived from the Hb divided by RBC
6. MCHC – MCHC is derived from the Hb, MCV and RBC according to the given formula*
MCHC (g/L) = Hb (g/L) x 1000
MCV (fL) x RBC x 1012/L
7. RDW (SD and CV) - The RDW is derived from pulse height analysis and can be expressed either as the standard
deviation (SD) in fL or as the coefficient of variation (CV) (as a percentage) of the measurements of the red cell
volume.
The RDW SD is measured by calculating the width in fL at the 20% height level of the red cell size distribution
histogram, and the RDW CV is calculated mathematically as the coefficient of variation; that is, RDW (CV) % =
(1 SD/MCV) x 100.
*Note:
Sysmex analyzers use a different principle in producing Hct, MCV and MCHC.
 Hct is obtained using the cumulative pulse height method.
 MCV is calculated from Hct divided by RBC.
 MCHC is calculated from Hb divided by Hct.

 Analysis of white cell parameters
1. WBC (x 109 / L) – Directly measured by counting the number of white cells passing
through the aperture after complete lysis of red cells
2. Differential count – Basic five-part analyzers classify cells as Neutrophils, Eosinophils,
Basophils, Lymphocytes and Monocytes.
Seven-part analyzers provide an extended differential count which
includes immature granulocytes or large immature cells and atypical
lymphocytes including small blasts
 Analysis of platelet parameters

1. Platelet count (x 109 /L) – Directly measured by the analyzer by counting the number of platelets
passing through the aperture.
It is important to differentiate platelets from red cells and smaller
particles such as debris and pulses generated by electronic noise. One
of the basic techniques is to count platelets between two fixed
thresholds (e.g.,between 2 and 20 fL);
Advanced technologies for more accurate platelet counting include
optical fluorescence and immunofluorescence methods.
2. MPV (Mean Platelet Volume) (fL) – Derived from impedance platelet size distribution curve.
3. PDW (Platelet Distribution Width) – Measure of platelet anisocytosis
4. PCT (Plateletcrit) – Derived from MPV and platelet count
5. P-LCR (The Platelet Large Cell Ratio) - The number of platelets falling above the 12 fL threshold on the
platelet size histogram divided by the total number of platelets.
Novel haematological parameters
Many modern automated haematology analyzers offer number of novel parameters which are summarized
below. (Table 4.2.2)

Table 4.2.2 Novel parameters in haematology analysers and their clinical utility
Novel parameter Description Clinical utility

Nucleated red blood cell Measure of red cell Increased in,
(NRBC) count precursors. - Prematurity and infants of diabetic mothers
Modern analyzers can
- Haemolysis
correctly quantify
NRBCs and hence - Haemorrhage
provide a - Hypoxia, Shock, Sepsis
corrected WBC count
Markers of Sensitive indicators - Differentiate functional iron deficiency (FID) from Iron
iron restrictive of Functional Iron deficiency anaemia (IDA)
erythropoiesis (IRE) deficiency states - Useful in deciding the need for iron supplements in
1.Percentage (Chronic infection/ anaemia of chronic disease
hypochromic red cells inflammation, - To decide the need for parenteral iron in CKD
(%HRC) malignancy, Chronic
[Equivalent - Low kidney disease on
Haemoglobin Density dialysis)
(LHD%)]
2. Mean reticulocyte
haemoglobin content
(CHr)
[Equivalent -
Reticulocyte
Haemoglobin
Concentration (Ret-He)]
Immature Reticulocyte Measure the - Sensitive indicator of successful engraftment

Fraction (IRF) proportion of most following stem cell transplant
immature reticulocytes - Reliable indicator of erythropoietic regeneration after
to the total number of myeloablative chemotherapy
reticulocytes - Reticulocytopenia with high IRF is indicative of a
regenerating marrow
- Reticulocytopenia with low IRF is indicative of marrow
aplasia
- Reticulocytosis with high IRF is indicative of haemolysis
or haemorrhage
- Normal or low reticulocyte count with high IRF is
indicative of dyserythropoiesis and early phase of
haematinic therapy
Reticulated platelets These parameters - Increased in patients with immune thrombocytopenia/

and Immature Platelet detect the immature thrombotic thrombocytopenia
platelets using their - Normal/ decreased in marrow failure
high level of RNA
- Sensitive indicator of successful engraftment after
content
stem cell transplant

FBC is the assessment of the blood cell parameters (Red cells, White cells and Platelets) including the number, size and
the proportions of the blood cells and Hb concentration. It is one of the basic tests which is used to assess the general
health status of an individual. It is affected not only by the diseases related to blood cells but also by systemic illnesses
ranging from benign to malignant conditions. FBC is used for the diagnosis as well as for the monitoring of the diseases.
Automated FBC usually have a high level of precision, for cell-counting and cell-sizing techniques, and is greatly superior
to that achievable with manual techniques provided these instruments are carefully calibrated and their correct operation
is ensured by quality control procedures.
Five-to-seven-part differential is more informative than 3-part differentials in the clinical settings.
Sample
 Whole blood is collected by venipuncture into a container with EDTA anticoagulant (K2 EDTA, K3 EDTA)
 FBC should preferably be performed within 4 hours of collection if kept at room temperature (15-25 0C). If delayed,
blood sample should preferably be refrigerated at 2 – 80C and analyzed within 24 hours. Before processing refrigerated
samples should be allowed to warm up to room temperature (minimum 15 minutes), then mix preferable by rotation
for at least 2 minutes.

Different models of five to seven-part haematology analyzers use the instrument specific reagents.
Please refer the manufacturer instructions.
Method
Each step in the operation of fully automated five to seven-part haematology analyzer and analysis of FBC should be
done in accordance with the manufacturer’s instructions. The main steps are as follows.
Ensure that the analyzer is kept at the recommended room temperature while in operation.
1. Start up
 Turn on the power supply in accordance with the instructions provided by the manufacturer
 Check for technical factors ie. reagents/ diluents, their expiry dates, electricity supply, instrument pressure
gauges and vacuum etc.
 Background count check (ie electronic noise and particulate material in diluent solution)
Modern day analyzers are programed to perform these steps automatically at the start up and be ready to
operate only when they are in proper order.
 Check the date and time.
2. Running IQC samples to check whether they are within control limits.
If QC fails (according to the Westgard rules set by laboratory), halt the analysis, perform corrective actions and
run QC again.
3. Running whole blood samples
 Sample should be checked for the correct (EDTA) container, presence of clots, correct volume etc. (Refer
rejection criteria)
 Must be mixed well (leaving for 3- 5 minutes on a mechanical mixture or manually by inverting 20 times) before
analyzing. (only open vial mode –OV mode, needs manual mixing, auto loader mode –AL mode, mixing is done
by analyzer).
 Load the samples ensuring the correct identity.
 If there is flagging of abnormal results microscopic examination of a stained blood film should be undertaken.

 In analyzers where NRBCs are not counted and indicated separately, they will be included in the WBC. When
NRBCs are present in significant numbers (>10 per 100 WBC), they will give erroneously high WBC count. In
such analyzers, NRBCs should be counted on a blood smear and corrected WBC count should be done using
the following formula and reported together with the NRBC percentage.
Corrected WBC = Uncorrected WBC X 100
NRBCs per 100 WBC + 100
 Each laboratory should set up its own criteria for validation of the report before issuing.
4. Shut down at the end of daily work
 Instructions displayed on the screen should be followed by the operator.
 Before the instrument is turned off, cleaning of the tubing system should be done to ensure that no protein
clogs exist inside.
 The cleanser provided by the manufacturer should be set to the sample probe so that the analyzer performs
this step automatically before shutting down.
5. Maintenance schedule
Please refer the manufacturer’s instruction for the daily, weekly, and periodical maintenance schedule.
Maintain Good Laboratory Practice (GLP) charts.
In a special logbook record, the dates of all maintenance checks, replacement of components, down time and
identifiable causes of malfunction, servicing by manufacturer’s agent, recalibrations, traceable batch numbers
of calibrators and controls.
Parameter Unit Parameter Unit
WBC … x 103/ uL RBC … x 106/ uL

Neutrophils … x 103/ uL Hb … g/ dL
Lymphocytes … x 103/ uL HCT …%
Monocytes … x 103/ uL MCV … fL
Eosinophils … x 103/ uL MCH … pg
Basophils … x 103/ uL MCHC … g/dL
Neu% …% RDW-CV …%
Lym% …% RDW-SD … fL
Mon% …% Platelet … x 103/ uL
Eos% …% MPV … fL
Bas% …% PDW …fL
PCT …%
IMG# … x 103/ uL P-LCR …%
IMG% …% RET# … x 1012/ L
IPF …% RET% …%
RHE … pg IRF …%
LFR …%
MFR …%
HFR …%

Reference intervals
Parameter Birth 3-6 1 2–6 6 – 12 Adult
months year years years Male Female
RBC (x1012/L) 6.0 ± 1.0 4.7 ± 0.6 4.5 ± 0.6 4.6 ± 0.6 4.6 ± 0.6 5.0 ± 0.5 4.3 ± 0.5
Hb (g/L) 180 ± 40 126 ± 15 126 ± 15 125 ± 15 135 ± 20 150 ± 20 135 ± 15
HCT (L/L) 0.60±0.15 0.35±0.05 0.34±0.04 0.37±0.03 0.40±0.05 0.45±0.05 0.41±0.05
MCV (fL) 110 ± 10 76 ± 8 78 ± 6 81 ± 6 86 ± 9 92.0 ± 9
MCH (pg) 34 ± 3 27 ± 3 27 ± 2 27 ± 3 29 ± 4 29.5 ± 2.5
MCHC (g/L) 330 ± 30 330 ± 30 340 ± 20 340 ± 30 340 ± 30 330 ± 15
WBC (x109/L) 18 ± 8 12 ± 6 11 ± 5 10 ± 5 9±4 4 - 10
Neutrophils (x109/L) 4 - 14 1–6 1- 7 1.5 - 8 2- 8 2 – 7 (40-80%)
Lymphocytes (x109/L) 3- 8 4 – 12 3.5 - 11 6- 9 1–5 1 – 3 (20-40%)
Monocytes (x109/L) 0.5 – 2.0 0.2 – 1.2 0.2 – 1.0 0.2 – 1.0 0.2 – 1.0 0.2 – 1 (2 -10%)
Eosinophils (x109/L) 0.1 – 1.0 0.1 - 1 0.1 – 1.0 0.1 – 1.0 0.1 – 1.0 0.02 - 0.5 (1- 6%)
Platelets (x109/L) 100 - 450 200 - 550 200 - 550 200 - 490 170 - 450 280 ± 130
(Source: Dacie and Lewis Practical Haematology, 12th Edition)

Internal Quality Control (IQC)
Tri level commercial QC material, with reference ranges for the specific model should be used.
Quality control should be performed,
 daily after startup. (ideally 8 hourly if there are 2 shifts)
 following corrective actions
 after reagent replacement
 after calibration.
 after maintenance, repair or part replacement.
 as required by clinical or laboratory QC regulations
If IQC material is not available for a particular analyzer, a comparison can be performed with results of another analyzer
performing regular IQC.
Calibration
Calibration is a procedure to standardize the analyzer by determining its deviation, if any, from calibration references and
to apply any necessary correction factors.
This procedure has to be done using calibrators with assigned values, in accordance with the manufacturer’s instructions.
Calibration of the analyzer is indicated,
 at installation.
 at least every six months
 after major preventive maintenance, a major repair or critical part replacement.
 when analyzer is to be reused after a long-term storage.
 when control materials reflect an unusual trend or shift of analyzer performance.
External Quality Assurance (EQA)

Analyzer performances should be compared in the national (and international if available) EQA program at least once in
3 months.

Sources of error
 Inadequate mixing of specimens or diluted suspensions before counting
 Air bubbles from vigorous shaking or dispensing
 Electric interference or voltage fluctuations
 External noise contamination e.g. from other electrical equipment
 Aspiration failure due to aperture blockage, leaks or loss of vacuum
 Residual traces of lytic agent or detergent cleaner causing haemolysis or artefactual red cell volume changes

 All parts and surfaces of the analyzer should be considered as potentially infectious since the instrument
handles patient specimens.
 All clinical samples should be treated as possibly infectious and handled as per guidelines on sample
handling available in the laboratory.
 References
2. Dacie and Lewis Practical Haematology,12th edition, 2017.
3. Blood cells, A practical guide (5th Edition).
4. SEED Haematology – Overview of the benefits of switching from a 3-part differential to a 5-Part differential
haematology analyser, Sysmex Educational Enhancement and =Development March 2012.
5. SEED Haematology - The red blood cell indices; Sysmex Educational Enhancement and Development October
2012.
6. SOP of Beckman Coulter DxH 500 Heamatology analyzer.
7. Thomas DW, Hinchliffe RF, Briggs C, Macdougall IC, Littlewood T, Cavill I; Laboratory diagnosis of functional iron
deficiency; British Journal of Haematology, 2013, 161, 639–648.
8. Rastogi P, Bhatia P, Varma N; Novel Automated Hematology Parameters in Clinical Pediatric Practice; Indian
Pediatr 2017; 54: 395 – 401.

4.3
HAEMOGLOBIN CONCENTRATION
BY HAEMOGLOBINCYANIDE
(CYANMETHAEMOGLOBIN METHOD)
Scope
The scope of this SOP is to guide technical personnel on correct performance of haemoglobin (Hb) concentration testing
by providing the protocol and requirements for this test.
Responsibility
Performance of Hb concentration testing as per the procedure stated in this SOP is the responsibility of all medical
laboratory technologists who are assigned to perform this test in the haematology laboratory.
Principle
Blood is diluted in a solution containing potassium cyanide and potassium ferricyanide, potassium dihydrogen phosphate
& nonionic detergent. Red cells get lysed and releases haemoglobin. Ferrous ions (Fe2+) of the haemoglobin are oxidized
to the ferric (Fe3+) state by potassium ferricyanide. K3Fe(CN)6, to form methaemoglobin, (Hi). Hi in turn, reacts with
cyanide ions (CN) provided by potassium cyanide to form cyanmethaemoglobin HiCN. The reaction is generally carried
out at room temperature (25 0C), and time necessary for full color development is five minutes or less at a pH 7.0 -7.5.
The absorbance of this solution is measured in a spectrometer at a wavelength of 540 nm.
Hb is decreased in haemorrhage, nutritional deficiencies and haemolytic reactions. Hb is increased in dehydration,
hypoxic conditions and polycythemia vera.
Sample
2 mL fresh venous blood collected in to an EDTA tube.

1. Spectrophotometer
In general, dual beam spectrophotometers are the most accurate instruments for the measurement of haemoglobin
concentration. However, other varieties of spectrophotometers and colorimeters may also be acceptable.

2. Glassware
Glassware used in the measurement of haemoglobin as HiCN must meet rigid standards of accuracy (Class A) and
must be chemically clean.
3. Pipettes required include the following:
 Microliter or Sahli type pipettes, 20 +/- 0.1 µL
 Measuring pipettes, calibrated to deliver 100 +/- 0.5 µL or positive displacement micropipettes
 Transfer pipettes, 5.0 +/- 0.005 mL and 10 +/- 0.03 mL
4. Drabkin-type solution - Only solutions in accordance with specifications published by the International Council
for Standardization in Haematology (ICSH) are acceptable.
Preparation
 Following reagents are mixed to get the Drabkin type solution which has a pH of 7 –7.4.
KCN, (0.768 mmol/L) 0.050 g
K3 Fe (CN)6, (0.607 mmol/L) 0.200 g
KH2PO4 (Anhydrous), (1.029 mmol/L) 0.140 g
Nonionic detergent 0.5 to 1.0 mL
Distilled water 1L
 The solution should be clear and pale yellow.
 Can be stored for several months at room temperature in a brown borosilicate glass bottle.
 If ambient temperature is >300C, the solution should be stored in the refrigerator (not allowed to freeze) but
brought to the room temperature before use.
 The solution should be discarded;
if it becomes turbid
pH is outside the 7.0-7.4 range
has an absorbance other than zero at 540 nm against a water blank.
Method
 Add 20 µL of well mixed EDTA blood to 4 mL of diluent to make a 1 in 201 dilution of blood. Stopper the tube
containing the solution and invert it several times.
 Allow to stand at room temperature for at least 5 minutes to ensure complete conversion of haemoglobin to
cyanmethaemoglobin.
 Read the absorbance in a spectrometer at 540 nm against a reagent blank.
 A semi-automated biochemistry analyzer may be used as the spectrophotometer. The Hb concentration is directly
obtained from the machine.
 Calculate the haemoglobin concentration using the following formula:

Hb (g/L) = (A540 test / A540 standard) x Conc. of standard (mg/L)* x (201 / 1000)
*Given on the label of the vial
 Preparation of the standard graph

 A reference standard with a concentration of about 800 mg/L from a reference laboratory, is used.
 The standard graph is prepared in order to check linearity of the measurements of the instrument.
 Set up a series of five tubes. Into the tubes pipette sequentially 6, 4.5, 3.0, 1.5 and 0 mL of HiCN reference
standard. Make the volumes up to 6 mL in each tube by adding, in the sequence, 0, 1.5, 3.0, 4.5 and 6 mL of
reagent, giving, respectively, 100%, 75%, 50% and 25% of the original concentration of the reference standard.
Measure the absorbance at 540 nm of each solution. Plot on arithmetic graph paper with Hb concentration on
the X axis and absorbance on the Y axis.

 All points should fall on a line passing through zero. If there is irregularity in the graph, check the function of the
spectrophotometer.
 This check should be carried out whenever a spectrophotometer is newly installed. serviced or repaired, and
also routinely approximately every six months.

Haemoglobin ……………………..g/L or g/dL
Reference intervals (g/L) - (Source: Dacie and Lewis Practical Haematology, 12th Edition)
Birth Day 3 1 2 3-6 1 2-6 6-12 Adult Adult

months months months years years years male female
180±40 180±30 140±25 112±18 126±15 126±15 125±15 135±20 150±20 135±15

Control preparations of whole blood should be used alongside the batch of tests in order to check that the test is being
performed reliably by means of a control chart. A QC sample is run daily.
Sources of error
 Measurement of absorbance of the test sample after 6 hours of its initial dilution
 Inadequate mixing of specimen before sampling
 Pipetting or dilution errors
 Deterioration of the quality of the Drabkin solution
 Reference preparation out of date or deteriorating, especially if it has been left standing on the bench for some time
after opening the vial.
 Improper instrument calibration
 Instrument fault
 Insufficient warm – up time
 Lamp failing or overheating
 Failing photocell
 Mains voltage variation
 Non-linearity
 Incorrectly positioned cuvettes
 Dirty or scratched cuvettes
 Potassium cyanide is highly poisonous. Great care must be taken when handling this reagent and to ensure safe
storage.
 References
2. Dacie and Lewis Practical Haematology, 12th edition, 2017.

4.4
MANUAL PLATELET COUNT
Scope
The scope of this SOP is to guide technical personnel on proper performance of manual platelet count testing by providing
the protocol and requirements for this test.
Responsibility
Performance of manual platelet count as per the procedure stated in this SOP is the responsibility of all medical laboratory
technologists who are assigned to perform this test in the haematology laboratory.
Principle
Whole blood is mixed with a diluent which lyses red cells and leaves the platelets intact. Platelets are counted in a
counting chamber (improved Neubauer) under high power with reduced light.
Low platelet count – Acquired thrombocytopenia (viral infections, DIC, ITP, liver disease, bone marrow failure
syndromes)
– Congenital and inherited thrombocytopenia
High platelet count – Reactive conditions (bleeding, iron deficiency, chronic wound etc.)
– Myeloproliferative disorders
Sample
2 mL of EDTA venous blood

1. Microscope
2. Improved Neubauer counting chamber (Haemocytometer)
3. Moist chamber

4. Mechanical mixture
5. 20 µL pipette
6. 2 mL/ 5 mL graduated pipette
7. 100 mL pipettes
8. Pasture pipette
9. 75x12 mm plastic tubes
10. 1% ammonium oxalate
Preparation -
 Dissolve ammonium oxalate 1 g in 100 mL distilled water and filter using Whatman No.42.
 Distributed into 2 mL aliquots or into small screw capped glass bottles.
 If autoclaved, can be stored at room temperature, if not refrigerate at 4 0C.
 Always filter the solution before using.
Method
 Take 1.9 mL of 1% ammonium oxalate into a clean and grease free plastic tube.
 Add 100 µL of blood.

 Mix well and leave for 15 minutes.
 Fix the well cleaned cover glass to the counting chamber.
 Look for Newton’s rings.
 Charge the counting chamber by using a pasture pipette.
 Leave it inside the moist chamber without any disturbance for 15 minutes.
 Count under the high power of the microscope with lowered condenser and with reduced light.
 Platelets will appear as highly refractile well separated bodies.
Counting
Method 1 (Figure 4.4.1)
Count the platelets in the central heavily ruled area (25 squares)
Figure 4.4.1
Method 1 for
counting platelets
using improved
Neubauer
counting
chamber
Calculation
Platelet count = Number of platelets counted x 10 (depth 0.1 mm) x 20 (dilution factor) x109/L
=N x 200 x 109 / L

Method 2 (Figure 4.4.2)
Count the platelets in the 5 squares only as shown below.
Figure 4.4.2
Method 2 for counting platelets using improved
Neubauer counting chamber
Calculation
Platelet count = Number of platelets counted x 10 (depth 0.1 mm) x5x 20 (dilution factor) x 109/L
= N x 1000 x 109 / L.

Platelet count …………….............……… x109/L
Reference intervals (x109/L) - (Source: Dacie and Lewis Practical Haematology, 12th Edition)
Table 4.4.1
Birth 3 1 2 3-6 1 2-6 6-12 Adult

Day months months months years years years
100-450 210-500 200-500 210-650 200-550 200-550 200-490 170-450 280±130
 Two diluents must be made and the mean of the two counts taken. The two counts should agree within 10%.
 The platelet count on the chamber should be correlated with the platelets seen on the smear.
 Slide correction should be done -1 platelet is equal to 10,000 platelets in oil immersion field.
 Blank count should be done to check the platelet dilution fluid.
Sources of error
 Poor technique in obtaining a blood sample including sample from a drip arm
 Partially clotted sample
 Platelet clumps
 Presence of red cell fragments and other debris
 Insufficient mixing of specimen, cell suspension mixture
 Inaccurate pipetting and the use of badly calibrated pipettes or counting chambers
 Faulty filling of the counting chamber (flooding of the chamber)
 Air bubbles or debris in chamber

 Careless counting of cells within the chamber (failing to count all the platelets in the square or including
‘non- platelet’ elements in the count)
 Bacterial / particle contamination of diluent
 Wrong calculations
 References
1. WHO guidelines on standard procedures for Haematology, January 2000.
2. Dacie and Lewis-Practical Haematology 12th edition, 2017.

4.5
PACKED CELL VOLUME
(MICROHAEMATOCRIT METHOD)
Scope
The scope of this SOP is to guide technical personnel on correct performance of packed cell volume by providing the
protocol and requirements for this test.
Responsibility
Performance of packed cell volume as per the procedure stated in this SOP is the responsibility of all medical laboratory
technologists who are assigned to perform this test in the haematology laboratory.
Principle
The total volume of erythrocytes in a given volume of blood divided by the whole volume of blood is called the packed
cell volume.
High packed cell volume - dehydration
- dengue fever
- polycythaemia vera
Low packed cell volume - anaemia of any etiology
Sample
 EDTA venous blood (K2-EDTA is recommended)

*Free air space above the sample should be >20% of the container volume to ensure adequate oxygenation and
proper mixing
*The test should be performed at least within 6 h of collecting the blood sample if kept at room temperature, or
within 24 h if kept at 4°C.
 Capillary blood (alternative sample)

1. Microhaematocrit centrifuge
2. Haematocrit reader
3. Capillary tubes
 Plain non-heparinized glass capillary tubes (75 ± 0.5 mm long with internal diameter 1.07-1.25 mm; wall thickness
0.18–0.23 mm; and bore taper not exceeding 2% of the internal diameter over the entire length of the tube) for
EDTA blood
 Heparinized glass capillary tubes should be used for blood collected directly from a skin puncture (1 IU of heparin
is coated inside the tube).
4. Magnifying glass
5. Sealing material. e.g. Plastic clay – like tube sealant
These materials must be kept in a sealed container or plastic bag when not being used, as it should not be allowed
to dry.
6. Arithmetic graph paper or a ruler – To take readings, if a special haematocrit reading device is not available.
Method
 If using capillary blood -
 Deeply prick (1.5 – 2.4 mm) the fingertip and wipeout the first drop of blood with sterile cotton.
 Apply the tip of the capillary tube (heparinized and circled with red) to the second drop of blood. Through
the capillary action the blood will flow into the tube.
 If using EDTA blood –
 Thoroughly mix the sample and apply the tip of the capillary tube to the blood and let it fill by capillarity.
 Fill about 2/3 of the tube leaving at least 15 mm unfilled and wipeout the outer layer of the tube.
 Seal the dry end that has not come into contact with blood with sealing material (check whether it is completely
sealed with 4 cm depth and a flat seal).
 Place the tubes in the microhaematocrit centrifuge in numbered slots with the sealed end pointing outwards.
Note the position and identity of each tube.
 Ensure both lids are closed properly. Centrifuge at 12,000 g (high speed) for 5 minutes (or the minimum time taken
for complete packing of the red cells determined by the lab).
 When the centrifuge has stopped automatically, remove the tubes, stand them upright and measure the proportion
of cells to the whole column (i.e. the PCV) using the reading device. (Figure 4.5.1).
After centrifugation the tubes will show 3 layers;
Top layer - a column of plasma
Middle layer - a very thin layer of white cells and platelets (Buffy coat)
Bottom layer - a column of red cells
 White cells and platelets (the buffy coat) must be excluded as far as possible from the reading of the PCV.
Therefore, a magnifying glass should be used to take the reading
 If a special reading device is not available, the ratio of red cell column to whole column can be calculated
from measurements obtained by placing the tube against an arithmetic graph paper or against a ruler.

Figure 4.5.1
Measuring haematocrit using haematocrit reader

 Results are expressed as a decimal fraction or red cell volume as a percentage of the total volume of blood.
Reference intervals ( L/L) - (Source: Dacie and Lewis Practical Haematology, 12th Edition)
Adults – Male – 0.45 ± 0.05
Female – 0.41 ± 0.05
Children - Newborn – 0.60 ± 0.15
1 year – 0.34 ± 0.04
2 – 6 yrs – 0.37 ± 0.03
6 – 12 yrs – 0.40 ± 0.05
Quality control procedure

 Check with the haemoglobin level.
 Test to be done in duplicate and must agree within 0.01.
 Centrifuges should be checked at intervals (at least annually) by a tachometer for speed and by a stopwatch
for timer accuracy.
 Efficiency of packing should be periodically tested by centrifuging samples of normal and polycythaemic
blood for varying times from 5 to 10 minutes to determine the minimum time for complete packing of the red cells.
Sources of error
 Faulty venepuncture technique e.g prolonged application of tourniquet (> 1 min) may result in falsely high PCV
 Squeezing the finger which causes tissue fluid coming to the tube causes dilution of the sample and hasten
clotting
 Use of K3-EDTA as the anticoagulant which causes shrinking of the red cells, reducing the PCV by about 2%
 Incorrect concentration of anticoagulant e.g anticoagulant in excess of 2.2 mg/ml may cause a falsely low PCV as a
result of cell shrinkage
 Clotted sample due to inadequate mixing
 Inadequate oxygenation of sample due to overfilling of EDTA tube resulting insufficient free air space above the
sample – results falsely low PCV
 Variation of the bore of the capillary tubes
 Insufficient centrifugation – too little time or too low speed- this will cause excess trapped plasma
 Storing blood beyond 6 hours at room temperature or > 24 hours at 4 0C results in an artifactual increase in PCV,
especially in hot climates
 Continuous use of centrifuge for several hours causes over heating leading the samples to lyse
 Evaporation of plasma during centrifugation or when left for a time before being read
 Slanting of the cell layer if the tubes are kept horizontal
 Including the buffy-coat layer in the reading of the red cell level
 Delay in taking readings after centrifugation - because the red cells begin to swell, and the interface becomes
progressively more indistinct making difficulties in accurate reading
 Inapparent leakage from the capillary tube

 References
1. WHO guidelines on standard procedures for Haematology, January 2000.
2. Dacie and Lewis-Practical Haematology 12th edition, 2017.

4.6
MANUAL RETICULOCYTE COUNT
Scope
The scope of this SOP is to guide technical personnel on proper performance of manual reticulocyte count by providing
Responsibility
Performance of reticulocyte count as per the procedure stated in this SOP is the responsibility of all medical laboratory
technologists who are assigned to perform this test in the haematology laboratory. Each laboratory should set up its own
criteria for validation of the report before issuing. Clinical validation when required, is the responsibility of consultant
haematologist.
Principle
Ribosomal RNA has the property of reacting with certain dyes (e.g. New methylene blue or Brilliant cresyl blue) to form
a blue or purple precipitate of granules or filaments. This reaction will only take place in supravitally stained, unfixed
preparations. Different stages of reticulocyte maturation can be identified by their morphological features. The most
immature reticulocytes are those with the largest amount of precipitable ribosomal material (Group 1), whilst in the least
immature only a few dots or short strands (Group IV) are seen. The number of reticulocytes is expressed as a percentage
of the total number of erythrocytes counted.
Reticulocytes are juvenile red cells. Since the number of reticulocytes in the peripheral blood is a fairly accurate reflection
of erythropoietic activity, a reticulocyte count is one of the essential procedures in diagnostic haematology.
 The reticulocyte count is high in blood loss, haemolytic conditions and during effective treatment of anaemia.
 The reticulocyte count is low in aplastic anaemia, bone marrow malignancies, problems of erythropoietin production
and vitamin or mineral deficiencies (B12, iron, folic acid).
Sample
 2 mL fresh venous blood collected in to an EDTA tube.
 Satisfactory counts may be made on blood that has been allowed to stand (unstained) for as long as 24 h, although
the count will tend to fall slightly after 6 – 8 h unless the blood is kept at 4 0C.
 Heel prick samples collected in to microtainers are used in neonates.

1. Microscope
2. 25 x 75 mm glass slides
3. Spreader slide
4. Pasteur pipettes
5. Miller ocular disc
6. 12 x 75 mm tubes
7. Hand counter
8. 37 0C water bath
9. New methylene blue – (NMB –Basic blue 24, color index- CI-52030) (most often used and is preferred) → 1 g of
New methylene blue is dissolved in 100 mL diluent (citrate – saline: 20 mL of 30 g/L sodium citrate + 80 mL of 9 g/L
sodium chloride)
or
Brilliant cresyl blue → 1 g of Brilliant cresyl blue is dissolved in 0.85% Sodium chloride
 After dissolving the dye, the solution is filtered and is then ready for use.
 Keep in room temperature (22 0C – 25 0C).
 Keep the bottles tightly closed at all times.
 Unopened reagents may be used until the expiry date on the label.
 Staining solution should be filtered through Whatman No. 1 filter paper prior to use to remove any
precipitated dye, and any other particulate matter.
Method
 Staining method
 With a Pasteur pipette deliver one drop of stain into a tube and add approximately two volume of patient's
blood. (for normal Hb, use equal volumes, and for low Hb, use blood 2 volumes and stain 1 volume, for high
Hb, increase stain volume)
 Mix and leave in water bath or incubator at 37 0C for 15 
– 20 minutes.
 At the end of this time, re-suspend the red cells by gentle mixing and make a thin film in the usual way using
the spreader. Allow the film to dry in the air.
 When dry, the films are examined without fixing or counter-staining. The reticular material should be stained
deep blue and the non-reticulated cells stained diffusely with shades of pale greenish-blue.
 Counting
 Choose an area of the field where the cells are undistorted, cells are not overlapped, and the staining is
good.
 Using the x100 oil-immersion lens, count the number of reticulocytes seen per 1000 red cells.
 Counting the red cells can be helped by inserting into the eye piece a paper or cardboard diaphragm in the
center of which has been cut a small square to reduce the optical field. An easier labor-saving method is
to use a Miller ocular insert; this consists of a large square inside which in one corner is a smaller square
of one-ninth the area of the large square. Provided that the red cells are evenly distributed, the red cells
need to be counted only in the small square as there will be approximately nine times that number in the
complete large square.
 Calculation
Number of reticulocytes in ‘N’ fields =X
Average number of red cells per field =Y
Total number of red cells in ‘N’ fields =NxY
 Reticulocyte percentage = [ X / ( N x Y) ] x 100%
 Absolute reticulocyte count = % Reticulocytes x RBC (x1012 /L)
Usually more convenient to report as a percentage.

E.g- if there were 18 reticulocytes per 1000 red cells, the percentage of reticulocytes is:
18/1000 x 100% = 1.8%
If the RBC count = 4.5 x1012 /L
Absolute reticulocyte count = 1.8 x 4.5 x 1012 = 81 x 109 /L
100
Misinterpretation may occur when reporting only the percentage of reticulocytes present in the peripheral blood,
because it is dependent on the total number of erythrocytes present in the peripheral blood. If the total erythrocyte
count is decreased, the reticulocyte percentage does not accurately reflect the bone marrow's production of new
erythrocytes. The correction formulas (e.g. Corrected reticulocyte count or reticulocyte production index) are used to
avoid interpretation errors due to the total erythrocyte count and increased bone marrow stimulation.
Reticulocyte Production Index is calculated as follows:

 Retic index = Percentage reticulocyte count X Haematocrit of the patient
Normal haematocrit
(A value of 45 is usually used as a normal haematocrit)
 The next step is to correct for the longer life span of prematurely released reticulocytes in the blood- a phenomenon
of increased red blood cell production. This relies on a table: (Table 4.6.1)
Table 4.6.1 Reticulocyte maturation correction
Haematocrit (%) Retic survival (days) = maturation correction

36 - 45 1.0
26 - 35 1.5
16 - 25 2.0
15 2.5
 Reticulocyte production index (RPI) = Retic index

Maturation correction
E.g. In a person whose reticulocyte count is 5%, haemoglobin 7.5 g/dL, haematocrit 25%,
the RPI would be: - 5 x (25/45) = 1.4
2
 Interpretation
 The RPI > 3 with anaemia indicates an effective erythropoietic response (e.g. haemolytic anaemia, recent
haemorrhage, response to therapy)
 The RPI < 2 with anemia indicates hypoproliferative erythropoiesis (e.g. aplastic anaemia, ineffective
erythropoiesis seen in megaloblastic anaemia)

Reticulocyte percentage……………………..%
Absolute reticulocyte count ……….………/L
Reference intervals - (Source: Dacie and Lewis Practical Haematology, 12th Edition)
Reticulocyte percentage (%)

Adults & Children 0.5 – 2.5
Infants (Full term, cord blood) 2.0 – 5.0
Absolute reticulocyte count (×109/L)
Table 4.6.3
Birth Day 3 1 2 3-6 1 2-6 6-12 Adult
months months months years years years
120-400 50-350 20-60 30-50 40-100 30-100 30-100 30-100 50-100

 All normal blood specimens have a few reticulocytes. Thus, a normal blood film should be included, and it should
be checked to determine whether the reticulocytes have stained. If a film with high reticulocyte count, for example
a film from a patient with haemolytic anaemia, can be obtained, it should also be included. For better results, three
slides should be made for each retic performed. Final calculated reticulocyte percentage from two slides counts
should agree within 15%. If this agreement is not reached, count the third slide.
 Quality of stain shall be verified with positve staining of white blood cells and platelets in the smear.
 When reticulocyte count appears very low, exended counting up to 5,000 or 10,000 red cells is recommended.
Sources of error
 Allowing the incubation time to exceed 15 – 20 minutes increases the possibility of erroneous results due to the dye
adhering to mature erythrocytes.
 Other RBC inclusions (Pappenheimer bodies, Howell-Jolly bodies, and Heinz bodies, Hb H inclusions) will be stained
with new methylene blue and misinterpreted as reticulocytes.
 Howell-Jolly bodies and Heinz bodies may be distinguished from precipitated reticulum by their shape and
staining characteristics.
 Heinz bodies appear as light blue – green inclusions located at the periphery of the erythrocyte.
 Howell-Jolly bodies are usually one or two round, deep purple staining inclusions and are also visible on
Romanosky stains.
 Pappenheimer bodies are indistinguishable from reticulum of reticulocytes. If Pappenheimer bodies are
suspected, a Prussian blue iron stain should be performed to verify their presence.
 Haemoglobin H inclusions will appear as multiple pale-staining greenish blue, almost spherical, bodies of
varying size.
 In splenectomized patients, presence of other inclusions in the red cells may be confused with reticulocytes.
 The whole blood-stain mixture should be re-suspended prior to making the smears to prevent counting and
calculation errors, because reticulocytes have a lower density than mature erythrocytes, and therefore will be
located near the top during incubation.
 Poor drying or moisture may result in the presence of refractile artifact on the smears. This refractile artifact may be
confused with precipitated reticulum. However, precipitated reticulum is not refractile, and fine focus adjustment
will reveal the difference.
 High glucose levels can cause reticulocytes to stain poorly.
 Use of non filtered stain can result in presence of precipitated material which can resemble a reticulocyte.

 Disposal of waste should be done according to local, provincial or national regulations
 References
2. Guidelines on Standard Operating Procedures for Haematology – WHO Regional Office for South-East Asia,
Last update 27 April 2006.

4.7
BLOOD FILM AND
DIFFERENTIAL COUNT
Scope
The scope of this SOP is to guide technical personnel on preparing smears for blood picture and performing differential
count by providing the protocol and requirements for this test.
Responsibility
Preparing smears for blood picture and performing differential count as per the procedure stated in this SOP is the
responsibility of all medical laboratory technologists who are assigned to perform this test in the haematology laboratory.
Interpretation and recommendations on blood picture should be made by consultant haematologist or a trained medical
officer.
Principle
Romanowsky stains are mixtures of acidic and basic dyes that give a differential staining of the different cellular
components of blood. Peripheral blood smears thus stained are used to identify the morphology of these cells in normal
and disease states.
The morphology of cellular components of peripheral blood can identify many haematological as well as non
haematological diseases. Blood smear reports (blood picture) are useful for diagnosis of diseases, assessment of
treatment outcomes and in follow up.
Differential count of white cells is done to verify the analyzer differential count or for five part differentiating when Full
blood count is done using a 3-part analyzer.
Sample
1. Capillary blood from finger prick / heel prick (direct smear) or
2. Venous blood collected in to EDTA tube
 Correct anticoagulant and blood ratio should be maintained. Excess anticoagulant can result in changes in cell
morphology.

 The blood film should be prepared within 2 hours of collecting blood. Delay can lead to morphological
changes or degeneration of cells.
 If a delay is anticipated, it is advisable to prepare a blood film at the time of collection preferably prior to
mixing with EDTA.
1. Microscopic slides – Need clean grease-free slides which might be available commercially.
If not, it is advisable to clean the available slides as below prior to use;
Cleaning of new slides – Leave overnight in a detergent solution and then wash thoroughly in running tap-water.
Rinse in distilled or deionized water and wipe dry with a clean linen cloth. Keep the prepared slides covered to avoid
having dust settle on the surface. Before use, wipe the surface with methylated spirits (95% ethanol) or methanol
and dry with a clean cloth.
Cleaning of used slides – Put the slides in a detergent solution and heat at 60 °C for 20 min. Then wash the slides in
running tap water. Finally, rinse in distilled or deionised water and dry with a clean cloth.
2. Spreader
Select a glass microscope slide with at least one smooth end. With a glass cutter, break off one corner of this end,
leaving width of 15 mm. The edge must be wiped carefully and dried before and after each use, and the slide must
be discarded if the spreading edge becomes chipped.
3. Pipette/capillary tube
4. Staining rack
5. Stains
Method
1. Preparation of blood film
 Blood film preparation should be done as soon as the sample arrives in the lab as delay will result in changes
in blood cell morphology.
 Place a drop of well mixed blood (minimum 10 gentle inversions) on the base of a slide close to one end
(about 1 cm from the edge) with a pipette/ capillary tube.
 Place a spreader in front of the drop at an angle of about 30o to the slide; move it backwards to make contact
with the drop. The blood should run quickly along the contact line.
 Now draw the film with one action by moving the spreader forward at an angle of 30- 45 degrees to the base
slide. The film should be about 3 cm in length covering approximately two-thirds of the base slide length and
should have an oval feathered end.
 Blood film should be one cell layer thick. With anaemic blood the correct thickness is achieved by using a
wider angle and, conversely with polycythaemic blood, the angle should be narrower.
 Care should be taken not to apply excessive pressure on the spreader slide when smearing. This can lead to
slide breaks and laboratory accidents.
 Label the slide with grease pencil or marker pen on the frosted end of the slide or the head end.
 Fix the dried smear with absolute methanol or ethyl alcohol. A properly airdried smear should be fixed within
4 hours of preparation but preferably within one hour. Improper fixation causes artefactual burr cells (crenated
red cells with refractile borders).
NOTE: Commonly used staining reagents include methanol which fixes cells. Therefore, a separate fixing
step is not necessary
 Stain with a Romanowsky stain (refer SOPs on stains).
 Wipe the underside of the slide with cotton wool to remove excess stain.
 Finally, the slide is placed on a rack with the feathered end sloping upwards to dry.
 Two or more slides should be made per specimen and the quality of the slide should be assessed immediately.
Poor quality slides should be discarded, and new ones prepared.

2. Microscopic examination of blood films
 Every film should first be inspected at low power (x10). This power is used for checking the approximate
differential cell distribution and also features like the presence of unusual cells or cell clusters suggestive of
malignancy. platelet clumps, rouleaux, fibrin strands suggestive of clotting of sample etc.
 Thereafter find an area where the red cells are evenly distributed, just touching but not overlapping, and study
their morphology at x 40. This should be done systematically taking note of the number and morphology of red
cells, white cells and platelets.
 The x100 oil-immersion lens should generally be reserved for examining unusual cells and for looking for fine
cellular details such as red cell inclusions, cytoplasmic granules, parasites, etc. Visual confirmation of platelet
count is also done using this power.
3. Method of performing differential leucocyte count (DLC)
 Check the film macroscopically to confirm its identity and assess the quality.
 Using a low-power lens (x10 objective) on the microscope, check the approximate differential cell distribution
and the presence of unusual cells or cells in bunches suggestive of malignancy.
 Using the x40 objective high-power lens, perform a 200 cell DLC. The x100 objective oil immersion lens should
be reserved for examining fine intracellular details and when searching for parasites.
 Move the slide along the stage of the microscope in a broad battlement track, running transversely across the
body of the film, avoiding the edges completely (Figure 4.7.1).
Figure 4.7.1
Technique for differential count
 Record the numbers of each type of white cell. It helps to have a mechanical or electronic differential counter.
 Calculate the percentage of each of the five basic leucocytes (neutrophils, eosinophils, basophils, lymphocytes,
monocytes).
 Report the DLC as percentage.
 Note the presence of any immature cells, especially blast cells, and report these alongside the DLC.
 Note the presence of normoblasts – if there is a significant number (>10 per 100 WBC), do not include in the
DLC but record the number per 100 WBC.

1. Reporting on blood film – The morphology of red cells, white cell and platelets should be reported separately.
Rouleaux need to be mentioned if present.
Conclusion should be made after correlating with clinical findings.
2. Reporting on the differential count – The percentage and the number of all five white cell types should be given.
3. Reporting on presence of nucleated red blood cells (NRBC) –Reported as number of NRBC per 100 WBC.

Reference intervals
Total Leukocytes Neutrophils Lymphocytes Monocytes Eosinophils
Age Mean Range Mean Range % Mean Range % Mean % Mean %
Birth 18.1 9.0 - 30.0 11.0 6.0 - 26.0 61 5.5 2.0 - 11.0 31 1.1 6 0.4 2
12 h 22.8 13. - 38.0 15.5 6.0 - 28.0 68 5.5 2.0 - 11.0 24 1.2 5 0.5 2
24 h 18.9 9.4 - 34.0 11.5 5.0 - 21.0 61 5.8 2.0 - 11.5 31 1.1 6 0.5 2
1 wk 12.2 5.0 - 21.0 5.5 1.5 - 10.0 45 5.0 2.0 - 17.0 41 1.1 9 0.5 4
2 wk 11.4 5.0 - 20.0 4.5 1.0 - 9.5 40 5.5 2.0 - 17.0 48 1.0 9 0.4 3
1 mo 10.8 5.0 - 19.5 3.8 1.0 - 9.0 35 6.0 2.5 - 16.5 56 0.7 7 0.3 3
6 mo 11.9 6.0 - 17.5 3.8 1.0 - 8.5 32 7.3 4.0 - 13.5 61 0.6 5 0.3 3
1y 11.4 6.0 - 17.5 3.5 1.5 - 8.5 31 7.0 4.0 - 10.5 61 0.6 5 0.3 3
2y 10.6 6.0 - 17.0 3.5 1.5 - 8.5 33 6.3 3.0 - 9.5 59 0.5 5 0.3 3
4y 9.1 5.5 - 15.5 3.8 1.5 - 8.5 42 4.5 2.0 - 8.0 50 0.5 5 0.3 3
6y 8.5 5.0 - 14.5 4.3 1.5 - 8.0 51 3.5 1.5 - 7.0 42 0.4 5 0.2 3
8y 8.3 4.5 - 13.5 4.4 1.5 - 8.0 53 3.3 1.5 - 6.8 39 0.4 4 0.2 2
10 y 8.1 4.5 - 13.5 4.4 1.8 - 8.0 54 3.1 1.5 - 6.5 38 0.4 4 0.2 2
16 y 7.8 4.5 - 13.0 4.4 1.8 - 8.0 57 2.8 1.2 - 5.2 35 0.4 5 0.2 3
21 y 7.4 4.5 - 11.0 4.4 1.8 - 7.7 59 2.5 1.0 - 4.8 34 0.3 4 0.2 3
(Source: Interpretation of the Complete Blood Count. Walters, Mark C.et al. Pediatric Clinics, Volume 43, Issue 3, 599 – 622)
 The stain quality should be compared with a well-made, normal, cover-slipped slide on day-to-day basis to detect
deterioration in stain quality.
 The intensity of the staining varies with the duration of stain contact time and concentration of the stain. It is
important to determine the adequate contact time with each new batch of stain made or procured.
Sources of error
Error Source of error
Irregular spread Spreader edge not even, dirt on slides

Film too short/thick Error in spreading technique-incorrect angle
Film extending to end of slide Blood drop too large
Film short/thin Blood drop too small
Film extending to edge of slide Spreader too wide/ not positioned correctly
Holes in film Slide contaminated with fat or grease
Irregular distribution of cells Error in spreading technique
Cellular degeneration changes Delay in fixing/ inadequate fixing time/ Methanol
contaminated with water
Sharp refractile border around
area of central pallor Inadequate drying

 Disposal of waste should be done according to local, provincial or national regulations

 References
1. WHO guidelines on standard procedures for haematology, January, 2000.
2. Dacie and Lewis-Practical haematology,12th edition, 2017.
3. Adewoyin AS, Nwogoh B. Peripheral blood film - a review Ann Ibd. Med 2014. Vol.12, No.2 Pg.71-79.
4. Walters, Mark C.et al. Interpretation of the Complete Blood Count. Pediatric Clinics, Volume 43, Issue 3, 599 –
622.

4.8
ERYTHROCYTE SEDIMENTATION RATE (ESR)
(WESTERGREN METHOD)
Scope
The scope of this SOP is to guide technical personnel on proper performance of ESR by providing the protocol and
requirements for this test.
Responsibility
Performance of ESR as per the procedure stated in this SOP is the responsibility of all medical laboratory technologists
who are assigned to perform this test in the haematology laboratory
Principle
The ESR expresses rate at which red blood cells (RBC) settle in mm per hour when anti-coagulated and diluted blood is
allowed to stand in a narrow tube (Westergren). The settling rate of the RBC is directly proportionate to the weight of the
RBC (rouleaux / RBC piling formation) and RBC surface area. eg surface area to weight ratio.
ESR is a screening test for all diseases that are associated with alterations of the plasma proteins like globulin, albumin
and fibrinogen. A raised ESR reflects an increased production of acute-phase proteins. When ESR is very high it can
indicate diseases like tuberculosis, multiple myeloma, severe sepsis and severe autoimmune diseases, malignancy etc.
ESR can be used to help in the diagnosis and also monitor response to therapy in such diseases.
Sample
 Fresh venous blood collected in to 3.2% (or 3.8%) trisodium citrate solution tube in 4:1 ratio or 2 mL fresh venous
blood collected in to an EDTA tube. (EDTA collected blood need to be further diluted as stated below before setting
up for ESR).
 If a commercially available ESR tube is used for sample collection, blood should be filled up to the mark depicted on
the tube by the manufacturer.
 If in-house tubes/ bottles are prepared, as per the volume of citrate added, it is necessary to specify the required
blood volume. [e.g.: 1.6 mL blood: 0.4 mL citrate]. If EDTA blood is used it should be diluted with citrate or normal
saline at 4:1 ratio for testing.

1. Westergren rack – stable with tightly sealing rubber stoppers.
2. Westergren tubes – clean and dry with no cracks- glass, has a length of about 30 cm and a bore of 2.5 mm, graduated
scale in mm from 0 to 190 mm. These can be re-used after thorough washing in tap water and rinsing with deionized
or distilled water, followed by adequate drying.
3. Filling device or a syringe with rubber/plastic tube attached.
4. Timer
5. 3.2% tri-sodium citrate solution (if dihydrated salt- 3.2g%, if pentahydrted salt- 3.8g%)
Method
 The test should be carried out within four hours of sample collection at room temperature. A delay of up to six hours
is permissible provided the blood is kept at 40C. If EDTA is used, EDTA blood can be kept refrigerated upto 12 - 24 hrs,
but dilution for ESR should be done only when performing the test. (The advantage of EDTA blood is when delay in
transportation >6 hrs is anticipated or in a neonate or infant less volume of blood is available so that FBC and ESR is
done from the same sample).
 Blood sample should be homogenized by at least ten gentle complete inversions (without frothing) immediately
before testing.
 Using a filling device or syringe and a tube, fill a clean and dry Westergren tube with the well mixed blood up to the
0 mark.
 Make sure no air bubbles enter the tube.
 Recheck that the tube is filled up to the 0 mark, exactly.
 Place the tube into the stand, taking care that the base is firmly positioned on the base pad to prevent leakage.
 Adjust the rack so that the tube rests in an exactly vertical position.
 The place to set up the ESR is very important for a correct ESR value. It should be in a place, away from the wind,
without direct sunlight falling on it, without any/ away from vibrations.
 Immediately set your timer for 1 hour or write down the time on a sheet of paper and with a patient number. Leave
undisturbed for 60 minutes.
 Exactly after 1 hour read how far the red cell layer has fallen (i.e: read the height of clear plasma above the upper
margin of the column of sedimenting cells to the nearest millimetre.)
Special Note:
While taking the result, should pay attention to the following:
 Colour of the plasma: Yellow/ Icteric - lipaemic, pink colour – haemolysis.
 The layer of white blood cells just above the red cells - If increased, indicates a leucocytosis Eg: Leukaemia.

…………………….. mm/1 hour (Time/ date of collection and time of test)
Reference intervals
Age (years) Men 95% upper limit Women 95% upper limit
17-50 10 12
51-60 12 19
61-70 14 20
>70 30 35

(Source: Dacie and Lewis Practical Haematology (12th Edition)

 Correct dilution of blood and tri-sodium citrate solution [1:4]
 Store tri-sodium citrate solution in the refrigerator
 Tri-sodium citrate solution should not be turbid
 Avoid air-bubbles in the Westergren tube
 Place the Westergren tube exactly vertical
 Avoid all the sources of error
Sources of error
 Specimen older than 6 hours
 Incorrect proportion of anticoagulant
 Incorrect type of anticoagulant
 Haemolysed sample
 Contaminated Westergren tubes
 Tubes tilted during sedimentation
 Test set up near central heating or direct sunshine
 Test set up adjacent to centrifuge, direction of fan or blower of air conditioner or other instrument causing vibration
 Failure to read at exactly one hour

 Disposal of waste should be done according to local, provincial or national regulation.
 References
1. WHO guidelines on standard procedures for haematology, January, 2000.
2. Dacie and Lewis-Practical haematology, 12th edition, 2017.

SECTION 05
ROUTINE
COAGULATION TESTS
5.1
ADJUSTMENT OF
ANTICOAGULANT FOR HAEMATOCRIT
5.2
PREPARATION OF
PLATELET POOR PLASMA (PPP)
5.3 PREPARATION OF
POOLED NORMAL PLASMA (PNP)
5.4 ESTABLISHMENT OF
REFERENCE INTERVALS AND CALCULATION OF
MEAN NORMAL PROTHROMBIN TIME (MNPT)
5.5 Standard Operating Procedure

PROTHROMBIN TIME (PT)

ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT)

THROMBIN TIME (TT)

FIBRINOGEN ASSAY (MODIFIED CLAUSS METHOD)

BLEEDING TIME (BT)
73
STANDARDOPERATINGPROCEDURESFORROUTINECOAGULATIONTESTS
5.1
ADJUSTMENT OF
ANTICOAGULANT FOR HAEMATOCRIT
When a patient’s haematocrit (Hct) is high, the blood sample will contain less plasma. As a result, when the blood is
centrifuged the plasma fraction will contain an increased concentration of anticoagulant. This can cause prolongation of
clotting times. Therefore, it is recommended that when the Hct is greater than 55% the citrate volume in the collection
tube should be adjusted.
If a patient is severely anaemic, there is an increased plasma volume so that there may be sufficient residual calcium
after mixing with trisodium citrate in the tube for coagulation to proceed in the sample, leading to activation and
possible shortening of APTT alongside consumption of clotting factors, including fibrinogen. However, it seems that low
haematocrit has less effect on results so there is usually no requirement to adjust the citrate-to-blood ratio for samples
from subjects with anaemia.
 A formula can be used to calculate the volume of sodium citrate when adjustment is needed.
C = (1.85 × 10-3)(100 – Hct) x V
C - volume of citrate remaining in the tube
Hct - haematocrit of the patient (a Hct performed within 24 hours is acceptable)
V - volume of blood to be added (If a 2.0 mL tube is used, the volume is 1.8 mL)
Eg: Patient has a haematocrit of 60% and blood is to be drawn to a 2.0 mL blue-top tube containing 0.2 mL of citrate.
Adjust the citrate level in the tube as follows;
C = (1.85x 10-3)(100-60)(1.8 mL)
= 0.13 mL
(Remove 0.07 mL of citrate, leaving 0.13 mL in the tube)
STANDARD OPERATING PROCEDURES FOR ROUTINE COAGULATION TESTS 75

 A nomogram can also be used to find the volume directly (Table 5.1.1)
Table 5.1.1 Nomogram for citrate volume adjustment
Correction chart – 2.0 mL tube

Hct % Citrate volume needed (mL) mL of citrate to be removed
55 0.15 0.05
60 0.13 0.07
65 0.12 0.08
70 0.10 0.10
Correction chart – 5.0 mL tube

Hct % Citrate volume needed (mL) mL of citrate to be removed
55 0.39 0.11
60 0.35 0.15
65 0.30 0.20
70 0.26 0.24
 Add the correct amount of blood to the tube containing the adjusted citrate concentration.
 Mix and process the sample in the same manner as the other coagulation samples.
 A note should be added to the laboratory record and patient record stating that the haematocrit value was
elevated and the citrate concentration adjusted.
 References
1. Dacie and Lewis Practical Haematology,12th edition, 2017.
2. Effect on Routine and Special Coagulation Testing Values of Citrate Anticoagulant Adjustment in patients with
high Hematocrit values. Am J Clin Pathol 2006;126:400-405.
3. CLSI guidelines – H21-A5 (2012).
4. Kitchen et al. International Council for Standardisation in Haematology (ICSH) recommendations for collection
of blood samples for coagulation testing. Int J Lab Hematol. 2021; 43:571–580.

5.2
PREPARATION OF
PLATELET POOR PLASMA (PPP)
Introduction
Most routine coagulation tests are performed on platelet poor plasma.
Sample
Blood collected as per standard procedure to collection tube with 3.8% trisodium citrate anticoagulant.
Method
 Check the whole blood specimen for clot formation by gentle inversion and observation.
 For basic coagulation tests, centrifuge samples at 2000 g for 15 minutes at room temperature. If the room temperature
of the laboratory is above 29 0C ( non- airconditioned laboratories), it is recommended to used refrigerated centrifuge
for PPP preparation.
 For special coagulation tests a refrigerated centrifugation is preferred. However, samples for platelet function testing,
lupus anticoagulant (LA) and activated PC resistance (APCR) tests should not be centrifuged at 4 0C. These samples
should be prepared by centrifugation at room temperature.
 For LA and APCR testing platelet count in the sample should be less than 10x109 / L. This is best achieved by double
centrifugation. Transfer PPP to polypropylene tubes and perform a platelet count to verify that it is less than
10x109 / L. If not, the plasma must be re-spun until the desired platelet count is obtained.
 If the sample is not processed immediately, remove 1 mL of plasma using a plastic pipette, and transfer to a plastic
aliquot tube and label with patient's name and a second patient identifier (BHT number, age). Care must be taken
not to disturb the buffy coat layer when removing the PPP.
Sample storage
 If a sample is not analyzed immediately or is to be transported, FREEZE it immediately
(SHOULD NOT BE REFRIGERATED).
 Quick freezing should be done. Avoid frost free freezers.
 At the time of testing, the frozen sample should be quickly thawed at 37 0C for 10 minutes, and mixed gently but
thoroughly.
 Once thawed, the sample should not be frozen again.
Specimen stability
Frozen: -20 0C for 2 weeks, -70 0C for 6 months.

 References

5.3
PREPARATION OF
POOLED NORMAL PLASMA (PNP)
Introduction
Pooled normal plasma (PNP) has the following advantages;
1. Can be used in coagulation mixing studies.
2. Can be used as the internal quality control (QC) sample for coagulation tests in the laboratory on a daily basis, to
confirm the integrity of reagents, coagulation instruments, operator techniques and all other test system variables.
Sample collection
 Samples should be taken from a minimum of 20 normal healthy individuals who are not taking medications which
interfere with clotting factors and coagulation reaction.
 An approximately equal number of males & females.
 The age range should be 20 – 50 years.
 Donors should preferably be bled between 9.00 and 11.00 a.m.
 Blood is collected as per standard procedure to collection tubes with 3.2% trisodium citrate anticoagulant.
 The volume of the sample should be decided by the laboratory depending on work load and the available storage
facilities.
 If the laboratory expects to use these samples for establishment of normal reference ranges for coagulation tests,
sample collection should be done according to the instructions given in the relevant section.
Method
 Store samples on melting ice during preparation of the pool.

 Prepare platelet poor plasma following the procedure in the respective SOP (5.2). Plasma separation by refrigerated
centrifugation is preferred.
 Verify the platelet count of few random samples of platelet poor plasma obtained. Platelet count should be less than
10 x 109/L.
 Test individual sample for prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT).

 Selection of samples for pooling –
 Preferably, calculate the mean and the standard deviation (SD) of the group statistically (ref 5.4) and identify the
outlying samples. Remove these samples and pool the rest in a plastic non-contact container.
 If statistical calculation is not feasible immediately, carefully go through the PT /APTT / TT results of individual
samples. Select the samples without extremely prolonged or shortened readings and pool them.
 Dispense 0.5 mL aliquots of pooled plasma using a plastic pipette into 1.5 mL plastic vials.
 Snap freeze on dry ice or solid CO2 if available. Alternatively place immediately on an open shelf at -70 0C.
 Complete the above procedure within 4 hours.
Sample storage and stability

Plasma can be stored at -20 0C for 2 weeks, -70 0C for 6 months
Calculation of the target PT/APTT/TT ranges for PNP

 Analyse the prepared PNP for PT/APTT /TT repeatedly, minimum of 10 times for each test on different days and
calculate the target ranges for the PNP (see below) to be used as an internal control.
 Statistically calculate the arithmetic mean & standard deviation (SD) for PT/APTT/TT separately using individual
readings obtained for each test type.
 Use the following formula to calculate the standard deviation;
SD =(x- x )2
N-1
where,
SD - the standard deviation
X - each value in the sample
X̄ - the mean of the values
N - the number of the values (the sample size)
 The target ranges for PNP = Mean ± 2SD.
 References
1. Diagnosis of Hemophilia and other bleeding Disorders – A Laboratory Manual, Second Edition (2010).
2. Dacie and Lewis Practical Haematology – 12th Edition, 2017.
3. Ministry of Healthcare & Nutrition National Guidelines,2007 (ISBN 978-955-9093-46-6).
4. Collection, transport and processing of blood specimens for testing plasma-based coagulation assays and
molecular hemostasis assays; Approved Guideline – 5th Edition (CLSI Guideline - H21-A5 Vol.28, No.5).

5.4
ESTABLISHMENT OF
REFERENCE INTERVALS AND
CALCULATION OF
MEAN NORMAL PROTHROMBIN TIME (MNPT)
Introduction
In order to interpret the results of any laboratory test, it is important to compare with relevant data related to the
particular test in healthy normal subjects, which is referred as “reference interval”. Ideally each laboratory should
establish its own reference intervals for each coagulation test type.
Important points to be considered in establishment of reference intervals for coagulation tests;
 Should ideally be established each time a brand/ batch of reagent is changed, the method is modified or when a
coagulometer is replaced or after a major repair.
 Samples should be collected, processed, and analysed using as near as possible identical techniques to that for
patient samples.
 Data from samples taken and analysed on different days can be used for calculation.
Mean Normal Prothrombin Time (MNPT), is used for calculation of prothrombin ratio and International Normalised
Ratio (INR). It should ideally be established each time a brand/ batch of thromboplastin is changed or the method is
modified or when a coagulometer is replaced or after a major repair.
Sample collection
 Samples should be taken from more than 30 normal healthy individuals (allowing minimum of 30 readings left for
calculation after excluding the outliers) who are not taking medications which interfere with clotting factors and
coagulation reaction.
 An approximately equal number of males & females (non-pregnant and not on oral contraceptives) over a wide age
range (20 – 80 years) is desirable.
 Should use an environment where physical and mental stress are lessened.
 Donors should abstain from intense physical exercise for 24 hours prior to venipuncture.
 Donors should abstain from fatty foods and smoking on the morning of venipuncture.
 Obtain samples early in the morning (7 am to 9 am), after the subject has sat in a relaxed position for 20 to 30 minutes.
 Use plastic blood collection tubes with 3.2% trisodium citrate dihydrate solution. Final blood: anticoagulant ratio
should be 9:1.

Method
a) Analysis of individual samples
 Prepare platelet poor plasma following the procedure in the respective SOP.
 Verify the platelet count of few random samples of platelet poor plasma obtained. Platelet count should be less than
10,000/ µL.
 Test individual sample for prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT)
following the relevant SOPs used for analysis of patients’ samples.
b) Calculation of reference intervals for the laboratory
 The results obtained for each test should ideally show normal or Gaussian distribution.
 Clear outliers that stand unexpectedly far from most other reference values are probably aberrant results and it is
acceptable to exclude these from further calculations.
 If the distribution of the results differ markedly from a Gaussian distribution (e,g skewed in one direction) additional
normal samples may be required.
 Statistically calculate the arithmetic mean & standard deviation (SD) for PT/APTT/TT separately using individual
readings obtained for each test type.
 Use the following formula to calculate the standard deviation;
SD =(x- x )2
N-1
where,
SD - the standard deviation
X - each value in the sample
X̄ - the mean of the values
N - the number of the values (the sample size)
 Reference interval for the laboratory = Mean ± 2SD

 After calculating the reference interval for the laboratory, see whether all individual coagulation test results (PT/
APTT/TT) included for calculation of the reference interval fall within this range.
 If there are any outliers, re-calculation should be done after removing those readings. This re - calculation has
to be repeated until there are no > 2 outliers. (because of this reason, it is better to take samples from at least
35 individuals, allowing minimum of 30 readings left at the end, for calculation of the reference interval for the
laboratory)
 If test results do not show normal distribution, non parametric method and 2.5th and 97.5th centiles should be
taken as the reference interval.
C) Calculation of Mean Normal Prothrombin Time (MNPT)

 Ideally, Geometric Mean of Prothrombin Time values (GMNPT) of at least 20 healthy individuals should be taken as
the MNPT. GMNPT of 20 healthy individuals is the 20th root of the product of 20 individual PT values.
 However, for convenience, usually the arithmetic mean of the ≥20 individual PT values is taken as the MNPT in the
laboratory for calculation of prothrombin ratio and International Normalised Ratio (INR).
 References
1. Diagnosis of Heaemophilia and other bleeding Disorders – A Laboratory Manual, Second Edition (2010).

5.5
PROTHROMBIN TIME (PT)
Scope
The scope of this SOP is to guide technical personnel on proper performance of PT testing by providing the protocol and
requirements for this test.
Responsibility
Performance of PT as per the procedure stated in this SOP is the responsibility of all medical laboratory technologists who
are assigned to perform this test in the haematology laboratory. Each laboratory should set up its own criteria for clinical
validation of the report before issuing.
Principle
The prothrombin time (PT) and its derived measures, prothrombin ratio (PR) and international normalized ratio (INR) are
measures of the extrinsic pathway of coagulation. PT measures factors I, II, V, VII, and X. The INR is the ratio of a patient's
prothrombin time to a normal (control) sample, raised to the power of the ISI (International Sensitivity Index) value for
the analytical system used.
INR = (PTTest/PTMean Normal PT*)ISI

* Mean Normal PT(MNPT) = GMNPT (Geometric Mean Normal Prothrombin Time)
A prolonged PT may be caused by conditions such as liver disease, vitamin K deficiency, effects of some anticoagulants
and an inherited deficiency of a factor in the extrinsic pathway (eg. Factor VII deficiency). PT is used along with activated
partial thromboplastin time (APTT) which measures the intrinsic pathway to evaluate the function of all coagulation
factors. PT can also be used to screen patients for any previously undetected bleeding problems prior to surgical
procedures. INR is used to monitor the effectiveness of anticoagulant warfarin.
Sample
 Platelet poor plasma prepared as per standard protocol.

 The request form should indicate if patient is on Warfarin.
 The sample should be kept at room temperature prior to processing.

1. Normal control plasma
2. Glass test tubes
3. Pipettes (adjustable or fixed 0.1 mL)
4. Centrifuge
5. Automated coagulometer/ semi-automated coagulometer/ water bath at 370C
6. Stopwatch (for manual method)
7. Thromboplastin, with calcium, stored at 4 0C. (this reagent can be commercially obtained)
Method
Method 1 – Manual
 Reconstitute a vial of thromboplastin in accordance with the manufacturer's instructions. Mix in a mechanical mixer
for 5 minutes and incubate the reagent in a water-bath at 370C for 10 minutes.
 Dispense 0.1mL plasma into a glass tube and pre warm for 1-2 minutes.
 Add 0.2 mL of the pre warmed thromboplastin-calcium reagent and start the stopwatch.
(PT reagent should always be mixed prior to adding plasma to the tube)
 Tilt the tube gently every other second, (tilt 3 times per 5 seconds) keeping it as much as possible under water to
maintain the temperature. Record the time for appearance of a fibrin clot as the endpoint.
 Perform the test on the patient's plasma and normal control plasma in duplicate. Repeat the test if duplicate
measurements differ by more than 5%.
Method 2 – Analysis with semi-automated analyzer (analyzer has to be calibrated by inserting ISI value and MNPT. These
values have to be changed whenever the reagent or the reagent lot number changes)
 Place test plasma 0.05 mL in the reaction cuvette.

 Incubate for 2 minutes.
 Add 0.1 mL of PT reagent to the above solution (PT reagent should be mixed before adding)
 Record the time on display at end of clotting.
(Method may vary according to the reagent and analyzer manufacturer’s guidelines)
Method 3 – Analysis with fully automated analyzer (analyzer has to be calibrated by inserting ISI value and MNPT.
These values have to be changed whenever the reagent or the reagent lot number changes)
 Switch on the analyzer as it will take standard time to stabilize.

 Load reagents to reagent positions.
 Do probe cleaning step by placing cleaning agents in their positions. Then on touch screen.
Diagnostics Cleaning Start
 Once probe cleaning is complete a beep sound will be heard.

 Run control samples.
 Check if controls are within range.
 If controls are within range, enter patient details and load patient samples (in primary tube after centrifugation or
plasma transferred to a secondary tube) in relevant positions in the sample rack.
 Click start button.
 Once analysis is complete a beep sound is heard.
 The report will be automatically printed.
 Once the day’s run is completed, remove reagents and keep at 40C. However, reagents can be kept within its on-
board stability time, loaded in the automated analyzer with a peltier which keeps the reagents at 10-150C, as long as
the analyzer is kept switched on.

PT ……………………........... seconds
PT Control……………….... seconds
PT ratio …………....……….
INR………….....................
Reference range………… seconds
Clinical validation of the test results should be done where appropriate.
Reference interval
 The reference interval for prothrombin time is usually 12-15 seconds.

 However, this depends on the thromboplastin. Age-appropriate reference intervals should be established by testing
a group of healthy subjects whenever a new reagent is introduced.
 Reference interval for the INR is 0.8 -1.2.

 Check quality of sample on reception and apply rejection criteria.
 Check appearance and storage temperature of tri-sodium citrate solution. (if in house preparation of tubes)
 Daily run of normal and abnormal IQC samples (prepared from pooled plasma or commercial IQC material) and
plotting on a L-J chart.
 Highest quality ISI verification and INR verification should be done using reference plasma with new reagents or
machines
 Participation in an EQA programme.
Sources of error
 Inappropriate sample
 Collection of blood through a line that has at some stage been in contact with heparin
 Inaccurate pipettes and techniques
 Machine malfunction
 Incorrect water bath temperature
 In automated analyzers,
 temperature variations
 inadequate stirring in analyzer “stir” position
 higher onboard temperature
 carryover in single probe analyzers
 end point detection error
 non dispensing of stirrer bead ( in automated electromechanical/magnetic method)
 extreme turbidity of plasma e.g. lipemic samples (in photoelectric method)
patient specimens.
85
 References
1. Dacie and Lewis Practical Haematology – 12th Edition, Churchill Livingstone, 2017.
2. Guidelines on Standard Operating Procedures for Haematology – WHO Regional Office for South-East Asia, Last
update 27 April 2006.
3. NCCLS guidelines: How to define and determine reference intervals in the clinical laboratory:C28-A2.
4. CLSI guidelines – Procedures for Validation of INR and Local Calibration of PT/INR Systems; Approved Guideline
H54-A, August 2005.

5.6
ACTIVATED PARTIAL THROMBOPLASTIN TIME
(APTT)
Scope
The scope of this SOP is to guide technical personnel on proper performance of APTT testing by providing the protocol
and requirements for this test.
Responsibility
Performance of APTT as per the procedure stated in this SOP is the responsibility of all medical laboratory technologists
who are assigned to perform this test in the haematology laboratory. Each laboratory should set up its own criteria for
clinical validation of the report before issuing.
Principle
The test measures the clotting of plasma after the activation of contact factors but without added tissue thromboplastin.
Thus, it indicates the overall efficiency of the intrinsic pathway. To standardize the activation of contact factors, the
plasma is first pre-incubated for a set period of time with a contact activator such as kaolin, ellagic acid or micronized
silica. During this stage of the test, factor XIIa is produced which cleaves the factor XI to factor XIa but coagulation does
not proceed beyond this in the absence of calcium. After calcification, factor XIa activates factor IX and coagulation
follows.
A standardized phospholipid is provided to allow the test to be performed on platelet poor plasma (PPP). The test
depends not only on the contact factors, factor VIII and IX, but also on the reactions of factor X, V, prothrombin and
fibrinogen.
APTT is useful for the detection of deficiencies of factors in the intrinsic pathway such as factors VIII, IX, XI and XII. It is also
useful in the monitoring of heparin therapy. APTT is prolonged in conditions where there is a combined factor deficiency
such as liver disease, disseminated intravascular coagulation. It is also sensitive to inhibitors of clotting factors and lupus
anticoagulant.
Sample
Platelet poor plasma prepared as per standard protocol.

2. Glass test tubes
4. Centrifuge
7. APTT Reagent – select lupus sensitive/ insensitive reagent as appropriate. Available commercially. Refer manufactures
instructions for storage.
8. Calcium chloride solution, 0.025 mol/L (i.e. M/40); stored at 40C
Method
Method 1 – Manual
 Pre-incubate the calcium chloride 0.02 mol/L to 370 C for at least 10 minutes.
 Pipette 0.1mL of test plasma and control plasma to the tubes. Incubate at 37 0C for 1 minute.
 Add 0.1 mL of APTT reagent to the tubes containing plasma.
 Incubate the mixture at 370C for 3 minutes.
 Rapidly add 0.1mL of pre-incubated calcium chloride and simultaneously start the timer.
 Tilt the tube gently keeping it as much as possible under water to maintain the temperature. Record the time for
appearance of a fibrin clot as the endpoint.
measurements differ by more than 5%
Method 2 – Analysis with semi-automated analyzer
 Place 0.1 mL of test plasma in the reaction cuvette.

 Add 0.1 mL of APTT reagent.
 Incubate for 3 minutes.
 Add 0.1 mL of CaCl2.
(method may vary according to the reagent and analyzer. Refer manufacturers’ guidelines)
Method 3 – Analysis with fully Automated analyzer
 Switch on the analyzer and it will take standard time to stabilize.

 Load reagents to reagent positions
 Do probe cleaning step by placing cleaning agents in their positions. Then on touch screen

 Run control samples.
 If controls are within range, enter patient details and load patient samples ( in primary tube after centrifugation or
 Once analysis is completed a beep sound is heard.

 Once the day’s run is completed remove reagents and keep at 40C. However, reagents can be kept within its on-board
stability time, loaded in the automated analyzer with a peltier which keeps the reagents at 10-150C, as long as the
analyzer is kept switched on.

Patient’s APPT…………………….seconds
Control APTT ……………………seconds
*Reference interval ………………seconds
*This depends on the reagents used and age-appropriate reference intervals should be established by testing a
group of healthy subjects whenever a new reagent is introduced.

 Participate in an EQA programme.
Sources of error
 Inappropriate sample (Incorrect volume).
 Collection of blood through a line that has at some stage been in contact with heparin.
 Contamination of the kaolin/platelet substitute reagent with a trace of thromboplastin. (reagent carryover is a risk in
automated coagulometer with a single probe, if inadequate washing of the probe occurs)
 Delay in sample analysis

patient specimens.
89
 References
2. Guidelines on Standard Operating Procedures for Haematology – WHO Regional Office for South-East Asia,
Last update 27 April 2006.
3. NCCLS guidelines: How to define and determine reference intervals in the clinical laboratory:C28.

5.7
THROMBIN TIME (TT)
Scope
The scope of this SOP is to guide technical personnel on proper performance of thrombin time by providing the protocol
Responsibility
Performance of thrombin time as per procedure stated in this SOP is the responsibility of all medical laboratory
criteria for clinical validation of the report before issuing.
Principle
Thrombin time (TT) is a screening test designed to assess fibrin formation from fibrinogen in plasma. Thrombin is added
to plasma and the clotting time is measured. The TT is affected by the concentration and reaction of fibrinogen, and by
the presence of inhibitory substances, including fibrinogen /fibrin degradation products and heparin. Both the clotting
time and the appearance of the clot are informative.
TT is prolonged in hypofibrinogenaemia seen in DIC or in congenital deficiency and in dysfibrinogenaemia either inherited
or acquired e.g in liver disease. Extreme prolongation of TT is nearly always due to the presence of heparin, which
interferes with the thrombin-fibrinogen reaction. Hypoalbuminaemia and raised fibrinogen degradation products, will
also prolong it. A gross elevation of the plasma fibrinogen concentration may prolong TT and correction can be obtained
by diluting patient’s plasma with saline. Shortening of the TT occurs in conditions of coagulation activation.
A transparent bulky clot is found if fibrin polymerization is abnormal as in the case of liver disease and some congenital
dysfibrinogenaemias.
Sample

2 Glass test tubes
4. Centrifuge
7. Thrombin solution: human thrombin 8-10 IU/mL
8. Buffer
9. Sodium azide <0.01%
(the above is the currently used commercially available solution and concentration may vary according to the
manufacturer)
Method
Method 1 – Manual
 Reconstitute the thrombin reagent according to the manufacturer’s instructions.

 Bring reagents to room temperature before use.
 Pipette 0.2 mL of PPP to a glass tube at 370C.
 Add 0.1 mL of thrombin reagent to PPP and start the stop watch.
 Tilt the tube gently every other second, keeping it as much as possible under water to maintain the temperature.
 Record the clotting time and observe the nature of the clot. eg. whether transparent, opaque, firm, wispy etc.
measurements differ by more than 5%
 Note - (please refer the reagent pack insert as the reagent volume vary with the type of reagent)
Method 2 – Analysis with semi-automated analyzer
 Place test plasma 0.05 mL in the reaction cuvette.

 Incubate for 3 minutes at 370C.
 Add 0.05 mL of TT reagent to the above solution
(method may vary according to the reagent manufacturer’s guidelines)
Method 3 – Analysis with fully Automated analyzer
 Switch on the analyzer and it will take standard time to stabilize.

 Load reagents to reagent positions
 Do probe cleaning step by placing cleaning agents in their positions. Then on touch screen

 Now run control samples.
 If controls are within range, enter patient details and load patient samples (in primary tube after centrifugation or

 Once analysis is complete a beep sound is heard.
 Once the day’s run is completed remove reagents and keep at 40C. However, reagents can be kept within its on-board
stability time, loaded in the automated analyzer with a peltier which keeps the reagents at 10-150C, as long as the
analyzer is kept switched on.

Thrombin Time ……………………. seconds
Normal control …………………...... seconds
*Reference interval ………………… seconds
Clinical validation of the test results where appropriate.
*This depends on the reagents used and age-appropriate reference intervals should be established by testing a group of
healthy subjects whenever a new reagent is introduced.

Sources of error
 Inappropriate sample (Incorrect volume).

 Collection of blood through a line that has at some stage been in contact with heparin.
 Contamination of the kaolin/platelet substitute reagent with a trace of thromboplastin. (reagent carryover is a risk in
automated coagulometer with a single probe, if inadequate washing of the probe occurs)
patient specimens.
93
 References
1. Dacie and Lewis Practical Haematology 12th Edition, Churchill Livingstone, 2017.
2. Manufacturer’s guidelines-Human Biochemica and Diagnostica, Germany.

5.8
FIBRINOGEN ASSAY
(MODIFIED CLAUSS METHOD)
Ms. L. Dhammika
Scope
The scope of this SOP is to guide technical personnel on proper performance of the fibrinogen assay (modified Clauss
method) by providing the protocol and requirements for this test.
Responsibility
Performance of fibrinogen assay as per the procedure stated in this SOP is the responsibility of all medical laboratory
criteria for clinical validation of the report before issuing.
Principle
Dilutions of standard normal (calibration) plasma with known amount of fibrinogen are prepared in glyoxalin (imidazole)
buffer. The clotting time is measured after the addition of high concentration of thrombin (to make sure that the clotting
time is independent of the thrombin concentration) and a graph is constructed. Test plasma is diluted to give a low level
of any inhibitors if present [e.g. fibrinogen degradation products (FDPs) and heparin]
The clotting time is proportional to the concentration of fibrinogen, and the 1/10 dilution is taken to represent the value
in the standard preparation. The test plasma is diluted 1/10, and the result read from the standard line.
The clauss fibrinogen is low in both hypofibrinogenaemia and dysfibrinogenaemia. Being a functional assay, this test gives
an indication of fibrinogen function in plasma. When dysfibrinogenaemia is suspected, a physico-chemical estimation of
fibrinogen (antigen assay) should be done and it will reveal a discrepancy between the functional clauss assay and the
physical amount of fibrinogen present.
Sample

3. Pipette to deliver 200 µL
4. 75 x 15mm glass tubes
5. Stopwatch
6. Centrifuge
7. Log/log or log linear graph paper
8. Calibration plasma (standard normal plasma) with a known level of fibrinogen calibrated against an International
Reference Standard
9. Thrombin reagent: 30 IU / mL – 100 IU/mL (concentration may vary according to source)
Either commercial or non-commercial
*The stability of frozen thrombin.
If thrombin is stored in a frozen state, then it is extremely important to aliquot the working thrombin solution in
volumes appropriate for individual assay runs, because repeated freeze-thaw cycles will deteriorate the enzyme.
Method
Method 1 – Manual
 Dilute calibration plasma in required volume of distilled water, mix by swirling, and leave for 20-30 minutes.
 Prepare 1/5, 1/10, 1/20, 1/25 and 1/40, dilutions of standard plasma in imidazole buffer.
Preparation of Imidazole (glyoxaline) or Owren’s buffer pH 7.4
 Dissolve 2.72 g of glyoxaline (Imidazole) and 4.68 g of NaCl in 650 mL of distilled water.
 Add 148.8 mL of 0.1 mol/L HCl and adjust pH to 7.4.
 Adjust volume to 1 L with distilled water.
Pipette 0.2 mL of volumes of each dilution into glass tubes.
 Warm to 370C for 2 minutes.
 Add 0.2 mL of thrombin (30 IU/mL – 100 IU/mL) and time the clot formation.
(If 100 IU/mL thrombin is used, 0.1 mL of thrombin solution should be added)
 Thrombin (bovine or human) of known National Institute of Health (NIH) units prepared in glyoxaline buffer,
should be kept in a lyophilized form or frozen in aliquots in non-activating surface containers.
 A frozen thrombin stock solution of 1000 NIH units/mL is stable for at least 1 year at – 700C.
 For manual technique, test in duplicate. This is not necessary for most coagulometers when the test is automated.
 Plot the mean clotting time versus fibrinogen concentration on log/log or log/linear graph paper, taking the 1/10
dilution to represent the standard value.
 The 1/10 dilution is considered to be 100% and there should be a straight-line correlation between 5 -50 seconds.
 Dilute the test plasma 1/10 and add 0.2 mL of the dilution with 0.1 mL of 100 IU/mL thrombin to determine the
clotting time. Read the fibrinogen result off the calibration curve.
 For most Clauss techniques, the relationship between clotting time and fibrinogen is linear over a limited range
of clotting times.
 For normal test plasma, a 1/10 dilution can be used.
 For lower concentrations, (for example, 0.75 -1.5 g/L) the plasma should be diluted 1/5 (and the value read from
the graph and multiplied by 5/10).

 For levels < 0.75g/L, the test plasma should be diluted 1/2 (and the value read from the graph and multiplied
by 2/10).
 For higher levels (>4g/L), the test plasma should be diluted 1/20 (and the value read from the graph and
multiplied by 20/10).
Method 2 – Analysis with semi-automated / automated analyzer
 This test can be performed using non activating surface containers in semi-automated or automated end point
determination systems.
 Analyzers should be calibrated according to manufactures guidelines and appropriate quality control measures
should be undertaken.

Fibrinogen level ……...... g/L
Reference interval ( 1.5 - 3.5 g/L )

plotting on a L-J chart. Prepare a calibration curve each time the thrombin reagent is changed and when there is a
drift in control results.
Sources of error
 Inappropriate sample (Incorrect volume)
 Inappropriate thrombin preparation
 Contaminated thrombin reagent or buffer
 Reconstitution with incorrect volume of buffer
 Use of thrombin working solution after freezing
 Defects in the commercial thrombin reagent
 Storage in glass containers after reconstitution
 Inappropriate storage of diluted working solution – e.g. Prolonged (>1 hour) storage or storage at > 80C
97
 Para proteins -high levels of some para proteins may interfere with polymerization of fibrin monomers.
 High levels of FDPs (>190 µg/mL), may interfere with assay.
 High levels of heparin
The Clauss fibrinogen assay is insensitive to heparin at the levels usually used for the treatment of venous
thromboembolism, but higher levels of heparin >0.8 u/ml (e.g. used for cardiopulmonary bypass) can prolong
clotting times, leading to an underestimation of fibrinogen.
patient specimens.
 References
2. Manufacturer’s guide lines -Siemens Health Care Diagnostics Products Germany.
3. Diagnosis of Hemophilia and other bleeding disorders, A Laboratory Manual, World Federation of Hemophilia,
2010.
4. CLSI H30-A2, Vol.21 No.18.

5.9
BLEEDING TIME (BT)
Scope
The scope of this SOP is to guide technical personnel on proper performance of bleeding time by providing the protocol
Responsibility
Bleeding time as per the procedure stated in this SOP is the responsibility of all medical laboratory technologists who
are assigned to perform this test in the haematology laboratory. Each laboratory should set up its own criteria for clinical
validation of the report before issuing.
Principle
A standard incision is made on the volar surface of the forearm and the time the incision bleeds is measured. Cessation of
bleeding indicates the formation of haemostatic plugs which are in turn dependent on an adequate number of platelets
and on the ability of the platelets to adhere to the sub endothelium and to form aggregates.
 Bleeding time will be prolonged in platelet function defects, defects of von Willebrand factor, diseases of the vessel wall
collagen and when there is thrombocytopenia. Occasionally, severe deficiency of factor V or XI or afibrinogenaemia
will prolong bleeding time.
 Bleeding time is not performed if there is thrombocytopenia as this will prolong the test anyway. There is insufficient
data to support the appropriateness or clinical usefulness of performing a bleeding time on patients with platelet
counts of 100 x 109/L or less.3 Therefore, it is important to check the platelet count before performing this test.
 Drugs that interfere with platelet function (E.g. aspirin, clopidogrel), may give abnormal test results.
 Bleeding time is subjected to a large number of variables and confounding factors such as performer bias, difficulty
to standardizing the skin cut and blotting technique.

1) Ivy method
 Sphygmomanometer (blood pressure cuff).

 Cleaning swabs 70% alcohol, sterile gauze and adhesive plaster.
 Standard blood lancets made for Ivy method (to produce 1mm deep cut).
 Whatman filter paper (1 mm thick).
 Stopwatch
2) Standard template method
 Sphygmomanometer (blood pressure cuff).

 Cleaning swabs, 70% alcohol, sterile gauze and adhesive plaster.
 Standard blood lancets made for template method (to produce 1mm deep two cuts parallel to each other).
 Whatman filter paper (1 mm thick).
 Stopwatch
3) Duke method (ear lobe method) – obsolete and not recommended.
Method
Method 1 – Ivy method
 Place the sphygmomanometer cuff around upper arm and inflate to 40 mmHg. Keep at 40mmHg for 30 to 60
seconds before the incision is made and make certain that the pressure is maintained steadily at 40 mmHg during
the procedure.
 Select an area of skin on the volar surface of forearm 2-3 cm distal to the antecubital crease.
 Observe for superficial visible veins and avoid any scars, fresh wounds, infected pustules or scars.
 Clean the area with 70% alcohol and allow to dry for at least 30 seconds.
 Two separate punctures are made 5-10 mm apart in quick succession using a microlancet with a cutting depth of
2.5 mm and width of just over 1mm.
 Start the stopwatch as soon as the incision is made.
 Gently touch the edge of a filter paper, to the drop of blood. Repeat the same for every 30 second intervals,
changing site of the filter paper.
 Avoid contact with the wound during procedure because this may disturb the formation of platelet plug.
 Stop the stopwatch when the bleeding has ceased. Bleeding time is reported when no blood stain is seen on the
filter paper after a gentle touch.
 Stop the procedure if bleeding continues for more than 20 minutes.
 Apply a sterile gauze and put a plaster. If bleeding continued more than 20 minutes, apply firm pressure with the
gauze pack.
Method 2 – Standard template method

Steps introduced to standardize the conditions so that performer bias is minimized compared to Ivy method.
 Follow all the steps 1 to 4 as given above.
 Apply the appropriate template lengthwise to the forearm and press firmly but without undue pressure. Place the
lancet parallel to the length of the arm (cuts that are perpendicular bleed longer). The standard incision should be
6mm in length and 1mm in depth in adults.
 Start the stopwatch. Without touching the cuts, gently blot the drops of blood with filter paper every 30 seconds,
until the bleeding stops.

 Report the bleeding time to the nearest half a minute.
 Mention the reference interval for the method used for assessing the bleeding time.
Bleeding time …………. minutes
Reference interval -…………......……minutes
Clinical validation of the test results where appropriate
Reference interval * : lvy method 2 – 7 minutes
Standard template method 2.5 – 9.5 minutes
*It is recommended to define reference interval for individual laboratories whenever appropriate.

Bleeding time is a performer biased test. Therefore, adhere to proper technique when performing incision and blotting.
Sources of error
 Selection of wrong area in the forearm. (e.g., superficial vein, scars)
 Improper incision (variation in the depth of the incision).
 Improper blotting technique (disturbing the wound).
 Improper maintenance of the timing.
 Improper maintenance of pressure (40 mm Hg) throughout the test.
 Improper documentation of results.

 When the platelet count is below 50 x 109/L it may be difficult to arrest the bleeding. Therefore, it is advisable to
check the platelet count before carrying out the bleeding time test.
 If bleeding persists for more than 20 minutes, record the bleeding time as more than 20 minutes. Do not continue
the test until bleeding stops. Bleeding should be stopped by placing a dry sterile gauze pack and keeping pressure
over the site for five minutes.
 Sterility should be maintained during the procedure.
 Appropriate personal protective gear should be worn when performing the procedure.
 References
1. WHO Guidelines for Standard Operating Procedures for Haematology.
2. Dacie and Lewis Practical Haematology, 9th Edition (2001).
3. Clinical and Laboratory Standards Institute H45-A2 - Performance of the Bleeding Time Test; Approved
Guideline - Second Edition H45 A2.
4. https://practical-haemostasis.com/index.html - Bleeding Time.
101
SECTION 06
SPECIAL
HAEMATOLOGY TESTS

HAEMOGLOBIN H INCLUSIONS

HEINZ BODY PREPARATION

SICKLING TEST

HAEMOGLOBIN S SOLUBILITY TEST

OSMOTIC FRAGILITY TEST

CRYOHAEMOLYSIS TEST

BREWER’S METHAEMOGLOBIN REDUCTION TEST

ACIDIFIED SERUM LYSIS TEST (HAM TEST)

DONATH-LANDSTEINER TEST

KLEIHAUER TEST (ACID ELUTION TEST)

HAEMOSIDERIN IN URINE
STANDARD OPERATING PROCEDURES FOR SPECIAL HAEMATOLOGY TESTS 103

6.1
HAEMOGLOBIN H INCLUSIONS
Mr. L.H.S. Sujeewa
Scope
The scope of this SOP is to guide technical personnel on correct performance of the test to demonstrate haemoglobin H
(Hb H) inclusions by providing the protocol and requirements of this test.
Responsibility
Demonstration of haemoglobin H (Hb H) inclusions as per the procedure stated in this SOP is the responsibility of all
medical laboratory technologists who are assigned to perform this test in the haematology laboratory. Interpretation and
recommendations should be made by the consultant haematologist.
Principle
Tetramers of β chains precipitate within red cells in patients with α thalassemia. These tetramers are named as
Hb H inclusions. When stained with brilliant cresyl blue or new methylene blue, they form blue green spherical inclusions
giving the red cell the appearance of a “golf ball” under the microscope.
This test is used as a screening test for detection of alpha thalassemia in patients and carriers.
Sample
2 ml fresh EDTA anticoagulated venous blood. Sample should be tested within 24 hrs of collection.

1. Staining solution - 1.0% brilliant cresyl blue or 2% new methylene blue
Preparation of staining solution; 1.0% brilliant cresyl blue or new methylene blue in iso-osmotic phosphate buffer
pH 7.4.
2. Khan tube
3. Pasteur pipette
4. Water bath
5. Test tube racks

6. Slide spreader
7. Immersion oil
8. Cover slip
9. Glass slides
10. Light microscope
Method
 Mix 2 volumes of EDTA anticoagulated blood with 1 volume of 1% brilliant cresyl blue or 2% new methylene blue.
 Incubate at 370C for 2 hrs or at room temperature for 4 hours.
 Resuspend the cells and spread a thin blood film.
 Observe under the microscope (high power / oil immersion).
 Hb H inclusions will be seen as greenish blue spherical bodies of varying sizes (“Golf -ball” appearance).

Hb H inclusion bodies – positive/negative

 If available, positive and negative controls should be included in the test.
 New batches of stain must preferably be tested with a known positive control because the redox action of the dyes
may vary from batch to batch.
Sources of error
 Inadequate mixing of sample before preparation of smear
 Deterioration of reagents
 Inadequate incubation time

 References

6.2
HEINZ BODY PREPARATION
Mr. L.H.S. Sujeewa
Scope
The scope of this SOP is to guide technical personnel on the correct performance of Heinz body preparation by providing
Responsibility
Performance of Heinz body preparation as per the procedure stated in this SOP is the responsibility of all medical
laboratory technologists who are assigned to perform this test in the haematology laboratory. Interpretation and
Principle
Heinz bodies are denatured haemoglobin that are precipitated inside red cells. They can be seen as single or multiple
inclusions, usually close to the cell membrane.
When a solution of supravital stain such as methyl violet is incubated with a few drops of blood, the Heinz bodies
are stained. A thin preparation is made and Heinz bodies are observed microscopically. They are recognized as purple-
coloured granules inside red cells.
Heinz bodies are found in blood in the presence of oxidative damage, chemical poisoning, and in the presence of an
unstable haemoglobin. Heinz bodies are demonstrated during an acute haemolytic episode in G6PD deficiency.
Sample
2 mL fresh venous blood collected into an EDTA tube or any anticoagulant.

1. 0.9% (0.9 g /l00 mL) NaCl solution.
2. Methyl violet

Preparation of the mixture of methyl violet and 0.9% NaCl:
Dissolve approximately 0.5 g of methyl violet in 100 mL of 0.9% NaCl and filter. (Always filter the solution just
before use).
3. Khan tube
4. Pasteur pipette
5. Test tube racks
6. Slide spreader
7. Immersion oil
8. Cover slip
9. Glass slides
10. Light microscope
Method
 Add one volume of blood (1 drop) and 4 volumes (4 drops) of filtered methyl violet solution into a khan tube.
 The mixture is allowed to stand for 10 minutes at room temperature.
 Prepare films and allow to dry.
 A wet preparation can be prepared by adding a drop of mixture onto a slide and placing a cover glass on it.
 Observe both slides (dried film and wet preparation) under a microscope. Heinz-bodies appear as purple colour
granules. The illumination should be reduced by lowering the microscope condenser, when observing the wet
preparation, then the Heinz bodies may be seen as refractile objects that may move around within the cells in a slow
Brownian movement.
Reporting of results
Heinz bodies - positive/negative

A positive and a negative control should be included in the test.
Sources of error
 Inadequate mixing of sample before preparation of smear
 Inadequate incubation time

 References

6.3
SICKLING TEST
Mr. L.H.S. Sujeewa
Scope
The scope of this SOP is to guide technical personnel on correct performance of sickling test by providing the protocol
Responsibility
Performance of sickling test as per the procedure stated in this SOP is the responsibility of all medical laboratory
technologists who are assigned to perform this test in the haematology laboratory. Interpretation and recommendations
should be made by the consultant haematologist.
Principle
Red cells in sickle cell disease contain Hb S, which is abnormal compared to normal Hb A. When red cells are exposed to a
hypoxic environment, hydrophobic Hb S forms polymers within red cells. The polymerized Hb S distort the red cell shape
from a smooth round shape to the shape of a sickle. The hypoxic environment can be created on a glass slide with a wet
blood smear. Sealing of all four sides of the coverslip by a non-porous material make oxygen diffusion impermeable. The
amount of sickling and the time needed for its appearance depend on the amount of haemoglobin S present and rate
of deoxygenation. This process may take up to 12 hours in Hb S trait, whereas changes are apparent in homozygous or
compound heterozygous state even after 1 hour at 37°C. These changes can be hastened by the addition of a reducing
agent such as sodium dithionite or sodium metabisulfite.
Used for screening of patients with sickle cell disease or sickle cell trait.
Sample
2 mL fresh venous blood collected into an EDTA tube. A finger prick sample can also be used. Perform test within one hour.

1. Slides
2. Cover slips
3. Wax/ vaseline or suitable non porous material for sealing
4. Hot air oven/ incubator
5. Microscope

Method
Method 1 – Without using reducing agents
 Place a fresh anticoagulated drop of blood on a glass slide and place a coverslip.
 Take care to avoid air bubbles or spaces under the coverslip.
 Seal all four sides of the coverslip with a suitable non-porous material.
 Keep the slide at room temperature or at 37 0C in a hot air oven/ incubator.
 Examine after 15 minutes and 60 minutes. In positive samples, the typical sickle-shaped red blood cells will appear.
However, if still negative, the preparation should be kept upto 24 hours and re-examined. In this case keep the slides
in a moist Petri dish to prevent drying.
Method 2 – Using sodium metabisulfite

Reagents:
1. Sodium metabisulfite 2g
2. Distilled water 100 mL
 Freshly prepare the sodium metabisulfite reagent.
 With a pasteur pipette add 2 drops of freshly prepared metabisulfite reagent to the middle of a glass slide.
 Dip an applicator stick into the blood and transfer a very small amount of blood to the metabisulfite reagent on the
slide.
 Mix evenly.
 Put a glass cover slip. Ensure no air bubbles or spaces remain under the coverslip.
 Using an applicator stick, seal edges between slide and cover slip with vaseline/ petroleum jelly/ paraffin wax or nail
varnish.
 If facilities are available the slide can be stored at 37 0C in a hot air oven/ incubator.
 If facilities are not available, keep at room temperature.
 Observe the slide immediately, after 15 minutes and 60 minutes under light microscope x 40.
 In positive samples, the typical sickle-shaped red blood cells will appear. However, if still negative, the preparation
needs to stand for upto 24 hours and examined prior to reporting. In this case keep the slides in a moist Petri dish to
prevent drying.
Method 3 – Dithionate method

Reagents:
1. Solution A Na2HPO4  1.62 g and up to 100 mL distilled water.
2. Solution B Sodium dithionite (Na2S2O4)  1.985 g and up to 100 mL distilled water.
 Prepare solutions A and B freshly just before use.
 Working solution should be freshly prepared by mixing 3 volumes of solution A with 2 volumes of solution B to get
final pH of 6.8.
 Perform the sickling test using the working solution immediately after preparation, following the same technique as
for the sodium metabisulfite method.

Report as Sickling test – Positive or Negative.

A sample from a known sickle cell carrier or a sample containing Hb A + Hb S can be used as the positive control.

Sources of error
 False negative results may occur if reagents are not freshly prepared.
 Faulty reagent concentration: Hypertonic reagent solution may cause crenation of erythrocytes which may be
mistaken for sickling. Hypotonic reagent solution may cause formation of spherocytes and give rise to false negative
results.
 Drying of the preparation before examination.
 Results reported without waiting up to 24 hours.
 If the test is performed after a recent blood transfusion- it will give unreliable results (need to wait for 3 months after
blood transfusion).
 Cover slip not sealed properly.

 References
2. Old J, Harteveld CL, Traeger-Synodinos J, et al. Prevention of Thalassaemias and Other Haemoglobin Disorders:
Volume 2: Laboratory Protocols, 2nd edition (2012.): Thalassaemia International Federation.

6.4
HAEMOGLOBIN S SOLUBILITY TEST
Mr. L.H.S. Sujeewa
Scope
The scope of this SOP is to guide technical personnel on the correct performance of haemoglobin S solubility test by
providing the protocol and requirements for this test.
Responsibility
Performance of haemoglobin S solubility test as per the procedure stated in this SOP is the responsibility of all medical
Principle
Red cells in sickle cell disease contain Hb S, which is abnormal compared to normal Hb A. Sickle cell haemoglobin is
insoluble in the deoxygenated state in high molarity phosphate buffer. The crystals that form refract light and cause the
solution to be turbid.
Used for screening of patients with sickle cell disease or sickle cell trait.
Sample
2 mL of fresh venous blood collected into an EDTA tube. Perform the test within one hour.

1. Glass test tubes
2. Tube rack
3. Pipette
4. Centrifuge
5. Phosphate buffer

Preparation:
Anhydrous dipotassium hydrogen phosphate 215 g
Anhydrous potassium dihydrogen phosphate 169 g
Sodium dithionite 5g
Saponin 1g
Distilled water to 1 litre.
Note: Dissolve the K2HPO4 in water before adding the KH2PO4, then add the dithionite and finally the saponin. This
solution is stable for 7 days when refrigerated.
Method
 Pipette 2 mL of reagent into three 12 x 75 mm test tubes.
 Allow the reagent to warm to room temperature.
 Add 10 μL of packed cells (from EDTA-anticoagulated blood) to one tube, 10 μL of packed cells from a known sickle
cell trait subject as a positive control to the second tube, and add 10 μL of packed cells from a normal subject as a
negative control to the final tube.
 Mix well and leave to stand for 5 minutes.
 Note: The blood reagent mixture should be light pink or red. A light orange colour indicates that the reagent has
deteriorated.
 Hold tube 2.5 cm in front of a white card with narrow black lines and read for turbidity, in comparison with the
positive and negative control samples.
 If the test appears to be positive, centrifuge at 1200 g for 5 minutes. A positive test will show a dark red band at the
top, whereas the solution below will be pink or colourless.

Haemoglobin S solubility test – Positive or Negative

A normal control is done with the test. If available, a known positive sample is run as the positive control in parallel.
Sources of error
 False negative results may be obtained if reagents are not freshly prepared.
 If the test is performed after a recent blood transfusion- it will give unreliable results (need to wait for 4 months after
blood transfusion).
 False positive results may be seen in severe leukocytosis, in hyperproteinaemia (such as multiple myeloma) and in
the presence of unstable hemoglobin, especially after splenectomy. The use of packed cells will minimize this error.
 False negative results can occur in patients with a low Hb and the use of packed cells will overcome this problem.

 All clinical samples should be treated as possibly infectious and handled as per guidelines on sample
handling available in the laboratory.
 References

6.5
OSMOTIC FRAGILITY TEST
Mr. L.H.S. Sujeewa
Scope
The scope of this SOP is to guide technical personnel on correct performance of osmotic fragility test (OFT) by providing
Responsibility
Performance of osmotic fragility test as per the procedure stated in this SOP is the responsibility of all medical laboratory
technologists who are assigned to this test in the haematology laboratory. Interpretation, conclusions and comments
Principle
When red blood cells are suspended in isotonic (normal) saline (0.9% NaCl), there is no change in net intake of ions or
water in or out from red cells thus no change in their size or shape. When red cells are exposed to decreasing strengths of
saline (hypotonic solutions), they take up water and swell until a critical volume is reached, following which they rupture.
The cells, which are already spherical, reach the critical volume early when exposed to hypotonic solutions thus rupture
early. In osmotic fragility test, unit volume of blood is mixed with a large excess of buffered saline solutions of varying
concentration (tonicity) (0.9% to 0.2%). The fraction of red cells lysed at each saline concentration is determined using
standard colorimetry.
Increased osmotic fragility is seen in the presence of spherocytic red cells. The main clinical use of this test is as a
screening test for hereditary spherocytosis. It is also increased when spherocytes are present due to other causes such
as warm autoimmune haemolytic anemia. Other conditions leading to increased OFT include hereditary elliptocytosis
and hereditary stomatocytosis.
Sample
 2 mL heparinized or EDTA anticoagulated venous blood from the patient
(Use of EDTA as an anticoagulant increases the osmotic fragility of red blood cells as compared with heparin. However,
the laboratory can use EDTA as the anticoagulant for sample collection, after determining its own reference values
for EDTA samples, which would reflect the local environmental and technical factors).
 The test should be carried out within 2 hours of collection with blood stored at room temperature. If the blood has
been kept at 4 0C, the test should be carried out within 6 hours of collection.

1. 26 centrifuge tubes in a rack (13 test tubes for ‘test sample set’ and 13 test tubes for ‘control sample set’)
2. 10% buffered NaCl stock solution
Preparation of stock solution
 Dissolve NaCl 9 g, Na2HPO4 1.365 g (or Na2HPO4.2H2O 1.7115 g) and NaH2PO4.2H2O 0.234 g in
distilled water. Adjust the final volume to 100 mL.
 This solution can be kept for months at 4 °C in a well-stoppered bottle. Salt crystals may form on
storage and must be thoroughly redissolved before use.
3. Glass pipettes (10 mL)
4. Measuring cylinder (100 mL)
5. Automated adjustable pipettes (1000 µL and 100 µL) with tips for each.
6. Vortex mixer
7. Spectrophotometer with cuvettes
8. Tissue papers
9. Graph papers with a pencils and eraser
Method
 Prepare the 1% buffered NaCl working solution by diluting 10 mL of stock solution in 90 mL of distilled water.
 Arrange the tube rack and mark the tubes from 1 - 13 separately for test and control samples (two sets of tubes
should be prepared. One set is for test blood sample and other set is for control blood sample).
 Make the dilutions with 1% buffered NaCl to the concentrations given in the table below to prepare solutions of
decreasing strengths of saline.
 Add 50 μL (0.05 mL) of well mixed control blood to each tube of ‘control’ set and 50 μL of well mixed patient blood
to each ‘test’ tube set.* Mix well.
 Incubate the two sets of tubes at room temperature for 30 minutes (after 30 minutes, RBC lysis in tubes can be
visualized).
 Mix the tubes again and centrifuge at 1200 g for 5 minutes. The haemolysis should be clearly visualized in the
supernatant.
 Take the spectrophotometer readings at 540 nm after setting zero with buffered saline solution.
 Readings of control sample should be taken first. 13th tube reading is taken as 100% lysis.
 Calculate the percentage lysis of blood with different concentrations as indicated below and enter the results in a
table.
 Then a graph is prepared on a graph paper using the percentage of lysis against concentration (Figure 6.5.1).
 First, the graph of the normal range (which should have been established for the laboratory) should be plotted.
 Then the readings of control and test samples should be plotted separately on the graph.
 Thereafter, take the following readings relevant for control and test sample from the graph;
 Highest concentration of saline at which lysis is just detectable (initial lysis)
 Highest concentration of saline at which lysis appears to be complete (complete lysis)
 Concentration of saline causing 50% lysis (i.e. the median corpuscular fragility, MCF)
 Also inspect the entire fragility curves of control and test samples as a whole and compare.
*Note:
The blood must be delivered into the 13 tubes very carefully. The amount of blood added to each tube must be the
same. Two methods can be used:
1) Using automated pipette, after aspirating the blood, gently wipe out the outside with tissue paper, taking care not
to suck out any blood from the tip by capillary action. The blood is then delivered into saline solution and pipette is
rinsed in and out several times until no blood is visible inside its tip.

The tip has to be changed before moving on to the next tube. This procedure takes time and may result in an
increased exposure for the first few tubes. It is therefore advisable to start the timing only after the addition of the
sample to the first tube.
2) When amount of blood available is limited (e.g. from babies), and the spectrophotometer takes 1 mL cuvettes,
the volumes can be scaled down to 1 mL of saline solution and 10 μL of blood. However, delivery of 10 μL of blood
reproducibly is not easy. Using a Pasteur pipette or capillary pipette with a much smaller diameter, calibrated to give
10 μL drops of blood, would have to be used. It is more difficult to maintain accuracy by using this method.
A proportion of 1 volume of blood to 100 volumes of saline is chosen because the concentration of blood is so small
that the effect of the plasma on the final tonicity of the suspension is negligible.
Table 6.5.1 Preparation of different dilutions in osmotic fragility test
Tube No. 1 2 3 4 5 6 7 8 9 10 11 12 13
Conc. of NaCl g/dL (%) 0.9 0.8 0.7 0.6 0.55 0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.0
1% buffered NaCl (mL) 4.5 4.0 3.5 3.0 2.75 2.5 2.25 2.0 1.75 1.5 1.25 1.0 0.0
Distilled water (mL) 0.5 1.0 1.5 2.0 2.25 2.5 2.75 3.0 3.25 3.5 3.75 4.0 5.0
Calculation of percentage lysis

Reading of a tube =X
Reading of the 13 tube
th
=Y
As Y is 100% lysis,
Percentage lysis of the tube = X ×100
Y
Table 6.5.2 Recording the readings of osmotic fragility test

Tube No. 1 2 3 4 5 6 7 8 9 10 11 12 13
% lysis of control
% lysis of test
Figure 6.5.1
Osmotic fragility curves
TEST LOW NORMAL HIGH NORMAL CONTROL

Following readings are to be taken from the graph and recorded for both control and test;
 Concentration of saline at the initial lysis
 Concentration of saline at complete lysis
 Concentration of saline causing 50% lysis (median corpuscular fragility /MCF)
Depending on the above readings, the results should be reported as ''Osmotic fragility is increased/ not increased''.

Reference interval
Initial lysis – At 0.5 g /dL NaCl
Complete lysis – At 0.3 g /dL NaCl
MCF (50% lysis) – At 0.4 – 0.445 g /dL NaCl
(It is recommended that individual laboratories establish their own reference intervals for the test)

A control blood sample from a normal person must be included along with the test sample to check the procedure and
the saline solution, even though the lab has already established, its own normal range for the test.
Sources of error
 Inaccurate volumes of blood and saline.
 The final pH of the blood in saline suspension.
When weak suspensions of blood in saline are used, it is necessary to control the pH of the hypotonic solutions and
it is for this reason that phosphate buffer is added to the saline. The effect of pH is more important: a shift of 0.1 of
a pH unit is equivalent to altering the saline concentration by 0.1 g/L, the fragility of the red cells being increased by
a decrease in pH.
 The temperature at which the tests are carried out.
An increase in temperature decreases the fragility, an increase of 50C being equivalent to an increase in saline
concentration of about 0.1 g/L.
 Malfunctioning of the spectrophotometer

 References
2. Kafka M, Yermiahu T. The effect of EDTA as an anticoagulant on the osmotic fragility of erythrocytes. Clin Lab
Haematol. 1998 Ag; 20 (4) : 213-6.

6.6
CRYOHAEMOLYSIS TEST
Mr. L.H.S. Sujeewa
Scope
The scope of this SOP is to guide technical personnel on correct performance of cryohaemolysis test by providing the
Responsibility
Performance of the cryohaemolysis test as per the procedure stated in this SOP is the responsibility of all medical
Principle
Spherocytic red blood cells in hereditary spherocytosis (HS) are specifically susceptible to temperature changes. The
cryohaemolysis test is based on the observation that red blood cells of HS patients are particularly susceptible to cold
(4 0C) temperature.
Cryohaemolysis test is done to detect defects of the red cell membrane. Unlike osmotic fragility which is increased
in spherocytosis caused by any condition (because it depends on changes in the surface area to volume ratio), the
cryohaemolysis test is specific for membrane defects causing spherocytosis.
Sample
2 mL EDTA blood. The test should be performed within 24 hours of sample collection.

1. Phosphate buffer (iso-osmotic) pH 7.4
Reagent A – Dissolve 0.78 g of NaH2PO4.2H2O in 100 mL of distilled water
Reagent B – Dissolve 0.71 g of Na2HPO4 in 100 mL of distilled water
Mix 18 mL of reagent A with 82 mL of reagent B and prepare the phosphate buffer (50 mmol/L)

2. Buffered 0.7 mol/L sucrose
23.96 g of sucrose in 100 mL of 50 mmol/L phosphate buffer, pH 7.4
Keep in the refrigerator.
2 mL aliquots of buffered sucrose reagent can be stored at - 20 0C for 3 months.
3. 9 g/L NaCl
Dissolve 0.9 g of NaCl in 100 mL of distilled water. Keep in the refrigerator at 4 0C.
4. Distilled water (1000 mL)
5. Freezer
6. Centrifuge
7. Pasteur pipette
8. 10 – 100 µL micropipettes with tips
9. 100 – 1000 μL micropipettes with tips
10. Beaker (100 mL) to keep ice
11. Water bath (37 0C)
12. Ice bath
13. Khan tubes
14. Centrifuge tubes
16. Vortex mixer
17. Ice cubes and ice packs
Method
 Centrifuge the patient’s blood and control blood in separate centrifuge tubes.
 Wash patient’s and control red cells separately three times with cold (4 0C) 9 g/L NaCl.
 Make patient and control 50 – 70% cell suspensions in 0.9 % NaCl.
 Keep both tubes on ice until tested.
 Take 4 centrifuge tubes (two for test and two for control) and add 2 mL buffered 0.7 mol/L sucrose reagent to each
tube and keep them in a 37 0C water bath for 10 minutes.
 Into the first two tubes (1st and 2nd tubes) add 50 μL of prepared patient’s red cell suspension.
 Add 50 μL of prepared control cell suspension into the other two tubes (3rd and 4th tubes).
 Vortex all 4 tubes immediately for a few seconds.
 Then incubate all tubes at 37 0C for exactly 10 minutes.
 Without delay, transfer the tubes to an ice bath for another 10 minutes.
 Vortex all tubes for a few seconds.
 Centrifuge all 4 tubes to sediment remaining cells (1000 – 1500 g for 5 minutes).
 Transfer some of the supernatants of each tube to new clean centrifuge tubes (and label the tubes as patient’s blood
and control).
 Prepare 100% haemolysate samples of patient’s and control blood samples as follows;
 Prepare a 100% haemolysate solution of patient’s blood by pipetting 50 μL (0.05 mL) of the original sample
(EDTA) into 2 mL of distilled water in a test tube. Mix, centrifuge and dilute 200 μL of the supernatant in 4 mL
of distilled water.
 Prepare a 100% haemolysate solution for control also by pipetting 50 μL (0.05 mL) of the original control
sample (EDTA) into 2 mL of distilled water in a test tube. Mix, centrifuge and dilute 200 μL of the supernatant in
4 mL of distilled water.
 Using the spectrophotometer, take absorbance at 540 nm (A540) of the test and control and the 100% lysis samples
of test and control.

 Calculation is done as follows;
A 540 of Test
% Cryohaemolysis (Test) = x 100
A 540
of haemolysate of test x 21
A 540 of Control
% Cryohaemolysis (Control) = x 100
A 540
of haemolysate of Control x 21
Take the average absorbance of the two test samples for A540 test and average absorbance of the two control samples
for A540 control.

Reference interval of cryohaemolysis 3- 15 %.
In hereditary spherocytosis > 20% lysis*.
*Increased lysis is not exclusive to HS and may be observed in hereditary stomatocytosis also.
(Individual laboratories are recommended to establish their own reference intervals for the test)

A normal control is performed with the test.
Sources of error
 Pipetting errors
 Errors when preparing the solutions
 Errors occurring due to not adhering to proper time durations
 Calculation errors

 References
2. Streichman S, Gesheidt Y, Tatarsky I. Hypertonic cryohemolysis: a diagnostic test for hereditary spherocytosis.
American Journal of Hematology. 1990 Oct;35(2): 104 – 9.
3. Ledesma AME, Haro C, Terán MM, Mónaco ME, Issé BAl, Sandra SL. Cryohemolysis, erythrocyte osmotic
fragility, and supplementary hematimetric indices in the diagnosis of hereditary spherocytosis. Blood Res
2018; 53(1): 10-7.

6.7
BREWER’S
METHAEMOGLOBIN REDUCTION TEST
Scope
The scope of this SOP is to guide technical personnel on the correct performance of Brewer’s test by providing the
Responsibility
Performance of Brewer’s test as per the procedure stated in this SOP is the responsibility of all medical laboratory
Principle
Sodium nitrite (NaNO2) is an oxidant that convert oxyhaemoglobin (Hb) to methaemoglobin (Hi). Methylene blue
activates the pentose phosphate pathway, resulting in enzymatic conversion of Hi back to oxy Hb in those red cells with
normal Glucose 6-phosphate dehydrogenase (G6PD) activity. In G6PD deficient cells there is no enzymatic reconversion
to oxy Hb. This method describes a semi quantitative assay of G6PD enzyme activity in red cells.
Reduced G6PD activity in red cells can cause acute intravascular haemolysis following exposure to oxidant agents Eg.
chloroquine, certain antibiotics, certain plants eg “Kuppameniya”
Sample
 EDTA anticoagulated venous blood, 2 mL
 The blood must preferably be tested within one hour of collection if kept at room temperature or within 6 hours if
kept at 4 0C.
 Blood should not be collected during a haemolytic crisis because reticulocytes contain higher levels of G6PD enzymes
(even in deficient persons) and may mask low enzyme activity of mature cells. Therefore, do the reticulocyte count
simultaneously on the day of the Brewer's test as a counter check, to eliminate haemolysis.
 When patient is anaemic use a plasma reduced blood sample (Remove sufficient plasma until the PCV is about 0.4).

1. Water bath (37 0C)
2. Test tube rack
3. Thermometer
4. Test tubes (should be glass tubes in equal size)
5. Pipettes (1 mL, 2 mL, 10 mL)
6. Sodium nitrite – 180 mmol/L
7. Dextrose - 280 mmol/L
Preparation of sodium nitrite- dextrose solution:
 Sodium nitrite 1.25 g and dextrose 5.0 g dissolved in distilled (deionised) water up to100 mL
 This reagent should be prepared fresh on the day of use.
8. Methylene blue solution -methylene blue 0.15 g in distilled water up to 1000 mL
(Nile blue sulphate – 22 mg in 100 mL of distilled water. This may be used as an alternative to methylene blue)
Method
 Dispense the samples and reagents according to the following table;
Table 6.7.1 Guide for dispensing samples and reagents
Reagent Test Positive control Negative control

Sodium nitrite - dextrose solution 0.1 mL 0.1 mL 0.1 mL
Methylene blue solution 0.1 mL --- 0.1 mL
Patient’s blood 2.0 mL 2.0 mL ---
Control blood* --- --- 2.0 mL
*Control blood sample should be from a healthy individual with comparable Hb content.
 Mix well, close the tube with a stopper and incubate at 37 0C for 1.5 hours.
 After incubation transfer 0.1 mL of well mixed sample from each tube to large tubes with 10 mL distilled water and
mix well.
 Examine the colour of the solution in each tube.

 Normal G6PD activity - colour of the test is similar to the colour of the negative control (cherry pink to cherry red
colour).
 Reduced G6PD activity (G6PD deficiency in homozygotes) - colour of the test is similar to the colour of the positive
control (brown colour).
 Heterozygotes give intermediate colours between normal G6PD activity and G6PD activity of homozygotes.

 Follow the technique exactly.
 The positive reference must be a brown colour. The negative reference must have a cherry pink to cherry red colour.
These colours must be achieved to validate the test run.
 If the positive and negative reference tubes have a different colour than expected the test must be repeated.
 It must be noted however that the test reference will show varying colours from red to brown depending upon the
degree of G6PD deficiency in the sample.

Sources of error
 Testing blood with high reticulocyte count
 Testing blood with a very low haemoglobin concentration
 Not using freshly made sodium nitrite
 Delay in testing
 Vitamin C supplements or a large dietary intake of vitamin C may interfere with the reaction.

 References
2. District laboratory practice in tropical countries by Monica Cheesbrough (1998).

6.8
ACIDIFIED SERUM LYSIS TEST
(HAM TEST)
Mr. L.H.S. Sujeewa
Scope
The scope of this SOP is to guide technical personnel on the correct performance of acidified serum lysis test (Ham test)
Responsibility
Performance of acidified serum lysis test (Ham test) as per the procedure stated in this SOP is the responsibility of all
medical laboratory technologists who are assigned to perform this test in the haematology laboratory. Interpretation,
conclusions and comments should be made by the consultant haematologist.
Principle
At 37 0C, Patient’s red blood cells are exposed to the action of normal (donor/control) and patient’s own serum,
acidified to the optimum pH for lysis (pH 6.5 - 7.0). Addition of acid adjusts the pH of the serum- cell mixture to the
optimum for the activity of the complement system. Complement in the serum is activated via the alternative pathway.
Red cells in healthy individuals can withstand complement mediated lysis in acidified serum. In paroxysmal nocturnal
haemoglobinuria (PNH) cells are unusually susceptible to lysis by the activated complement in the acidified serum. That
is due to acquired genetic defect/mutations in the proteins which anchor protective molecules on to cell membrane.
The sensitivity of the HAM test can be improved by the addition of magnesium chloride to optimize the activation of
complement.
This test is used for the screening of patients with PNH.
Sample
Freshly collected venous blood samples from patient and control as follows;
 From patient – 2 mL EDTA blood (heparinized / citrated / oxalated blood can also be used)
– 10 mL blood in to plain tube
 From control (Donor should be ABO compatible with patient or AB positive)
– 2 mL EDTA blood (heparinized / citrated / oxalated blood can also be used)
– 12 mL blood in to plain tube

1. Clean Khan tubes
2. Tube racks
3. Centrifuge
4. Water bath
6. Conical defibrinating flask with central glass rod or glass beads (if serum is obtained by defibrination)
7. Isotonic saline
8. 0.2 M HCl
9. MgCl2 (23.7 g/L)
10. Drabkin’s solution
Method
 Wash red cells of patient and control samples separately, twice in isotonic saline and prepare 50% cell suspensions
in isotonic saline.
 Centrifuge them separately and obtain serum.
(If facilities are available obtain patient’s serum by defibrination, because in PNH if serum is obtained from blood
allowed to clot in the ordinary way at 37 0C or at room temperature, it will almost always be markedly lysed. However,
control serum can be derived from blood allowed to clot spontaneously at room temperature or at 37 0C).
 Prepare heat inactivated serum (to inactivate complement activity) by incubating the required amount of patient/
control serum at 56 0C for 30 minutes.
 Arrange 9 clean Khan tubes in a rack, 3 tubes for test (T1, T2, T3), 3 tubes for control (C1, C2, C3) and last 3 tubes
(X, Y, Z) for excluding congenital dyserythropoietic anaemia - CDA type II or HEMPAS ( Hereditary erythroblastic multi
nuclearity with positive acidified serum lysis test)
 Reagents and samples are mixed according to the table below;
Table 6.8.1 Guide for dispensing samples and reagents
Test Control To exclude HEMPAS
Sample/ Test
T1 tube T2 T3 C1 C2 C3 X Y Z
Reagent added
Fresh control serum (mL) 0.5 0.5 - 0.5 0.5 - - - -

Patients own serum (mL) - - - - - - 0.5 0.5 -
Heat inactivated patient’s own serum (mL) - - - - - - - - 0.5
Heat inactivated control serum (mL) - - 0.5 - - 0.5 - - -
0.2 M HCL (mL) - 0.05 0.05 - 0.05 0.05 - 0.05 0.05
50% Suspension of patient RBC (mL) 0.05 0.05 0.05 - - - 0.05 0.05 0.05
50% Suspension of control RBC (mL) - - - 0.05 0.05 0.05 - - -
MgCl2 (mL) 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
After careful mixing, all tubes are capped and kept at 37 0C for about 1 hour.
Centrifuge in a low speed for 5 minutes and observe supernatant for lysis.
 Preparation of standards for quantitative measurement of lysis

 Add 0.05 mL of patient cell suspension to 0.55 mL of distilled water (100% lysis – test standard).
 Add 0.05 mL of control cell suspension to 0.55 mL of distilled water (100% lysis – control standard).
 Prepare blank by adding 0.3 mL of normal serum into 5 mL of Drabkin’s solution (0% lysis).
 Observe each tube for presence of lysis (Table 6.8.2).

Table 6.8.2 Pattern of lysis seen in PNH and HEMPAS
Test Control To exclude HEMPAS

Test tube
T1 T2 T3 C1 C2 C3 X Y Z
Lysis in PNH trace +++ - - - - trace +++ -

Lysis in HEMPAS - ++ - - - - - - -
 For measurement of lysis of each tube,

 Deliver 0.3 mL volumes of supernatant of each tube into 5 mL volumes of Drabkin’s solution separately.
 Zero the spectrophotometer with blank solution.
 Take absorbance readings (“A”) of all tubes of test and control series and two standard solutions at
540 nm in a spectrophotometer.
 Calculation
"A" of test / Control
Percentage of Lysis = x 100
"A" of Standard

 If the patient has PNH;
Obvious haemolysis (10 - 50%) - in the tubes T2 & Y
Trace of haemolysis (2%)/ even no lysis - in the tubes T1 & X
No haemolysis - in the tubes T3, Z (due to lack of complement) and C1, C2 and C3 (as normal red blood cells
without PNH are used)
The test is reported as: HAM Test – Positive (results are suggestive of PNH)
 If the patient has HEMPAS;

Red cells undergo lysis in only a proportion (about 30%) in normal sera (tube T2) and no lysis is seen in patient’s
own acidified serum (tube Y)
The test is reported as: HAM Test – results are suggestive of HEMPAS
 If the patient does not have either PNH or HEMPAS;

None of the tubes will show haemolysis.
The test is reported as HAM Test – Negative

 Controls are incorporated into the test for all variables.
 Maintaining the specific temperatures required and using correct volumes are critical in this test. Therefore,
a properly calibrated water bath and pipettes should be used.
Sources of error
 Temperature and timing errors – lead to lack of inactivation of complement
 Incubation errors
 Volume errors
 Errors in observation
 Labeling errors
 DAT positivity can give false positive results

 HCl can be corrosive. Therefore, it should be handled carefully.
 References

6.9
DONATH - LANDSTEINER TEST
Mr. L.H.S. Sujeewa
Scope
The scope of this SOP is to guide technical personnel on correct performance of Donath-Landsteiner test by providing the
Responsibility
Performance of Donath-Landsteiner test as per the procedure stated in this SOP is the responsibility of all medical
Principle
Donath-Landsteiner Ig G autoantibody binds to patient’s red cells at low temperatures and fixes complement at warmer
temperatures. Sensitized red cells undergo complement mediated intravascular haemolysis (due to formation of
membrane attack complex) when the body temperature rises to 37 0C.
This test is done to screen for the presence of biphasic antibody with specificity for the P blood group antigens. This
antibody causes an autoimmune haemolytic anaemia with paroxysmal haemoglobinuria when exposed to cold, termed
as paroxysmal cold haemoglobinuria (PCH).
Sample
2 mL fresh venous blood samples collected into pre warmed (37 0C) plain tubes

1. Khan tubes
2. Test tube racks
3. Water bath
4. Labelling tapes
5. Ice cubes
6. Pasteur pipettes

Method
 Warm 2 plain khan tubes at 37 0C for 5 minutes.
 Transfer the samples of venous blood into pre warmed (37 0C) tubes containing no anticoagulant.
 Incubate the first sample at 37 0C for 1 ½ hour.
 Place the second sample in a beaker packed with ice and allow to stand for 1 hour.
 Incubate the second tube at 37 0C for further 20 minutes after taking out from ice.
 Centrifuge both tubes at 37 0C at 1000 - 1500 g for 5 minutes and observe the supernatant for lysis.
 A positive result is indicated by lysis in the sample which had been kept in ice first and then at 37 0C.

 Positive for the Donath-Landsteiner antibody
 Negative for the Donath-Landsteiner antibody

A negative normal control is performed with the test and when possible, a positive control can also be done with the test.
Sources of error
 Low antibody level.
 Low complement level (complement is consumed during the haemolytic process).

 References

6.10
KLEIHAUER TEST
(ACID ELUTION TEST)
Scope
The scope of this SOP is to guide technical personnel on correct performance of the semiquantitative estimation of
fetomaternal haemorrhage by Kleihauer test by providing the protocol and requirements for this test.
Responsibility
Performance of Kleihauer test as per the procedure stated in this SOP is the responsibility of all medical laboratory
Principle
Fetal haemoglobin (Hb F) is more resistant than adult haemoglobin (Hb A) to elution at acid pH. When dry blood films
are fixed and then immersed in an acid buffer solution, Hb A is denatured and eluted, leaving red cell ghosts. Red cells
containing Hb F are resistant and the haemoglobin is stained; these fetal cells stand out in a sea of ghost maternal cells.
Detection of Hb F in the maternal circulation is an indicator of fetomaternal haemorrhage (FMH) and provides valuable
information on the pathogenesis of haemolytic disease of the new born. Kleihauer test provides an estimation of a volume
of fetal cells in the maternal circulation following a potentially sensitizing event in a Rh D negative pregnant woman such as
amniocentesis, cordocentesis, antepartum haemorrhage, external cephalic version, fall/ abdominal trauma, intrauterine
death, still birth, in-utero therapeutic interventions (transfusion, surgery), miscarriage and therapeutic termination of
pregnancy. Usually, an anti-D immunoglobulin injection is given in these situations in a standard dose according to local
protocols. Kleihauer test is helpful to determine whether an additional dose of anti-D immunoglobulin is required.
Sample
2 mL maternal EDTA blood

1. Microscopic slides
2. Coplin jar
3. Fixative - 80% ethanol
4. Elution solution
Solution A - 7.5 g/L haematoxylin in 90% ethanol
Solution B - FeCl3 24 g, 2.5 mol/L HCl 20 mL, doubly distilled water to 1L
 For use, mix well 5 volumes of A and 1 volume of B
 The pH of the solution should be approximately 1.5
 When prepared, the working solution can be kept and used for about 4 weeks at room temperature. The date
of preparation of the solution should be written on the container. If a precipitate forms, the solution should be
filtered.
5. Counter stain - 1 g/L aqueous erythrosine or 2.5 g/L aqueous eosin
Method
 Prepare fresh air-dried thin films on clean dry slides (the thickness of the blood film is important and should result in
red cells touching but not overlapping when examined under a microscope). Thin films can be achieved by diluting
an aliquot of the maternal whole blood sample at 1:2 or 1:3 dilution with phosphate buffered saline before making
the film. The diluted sample should be well mixed before making the film.
 Immediately after drying, fix the films for 5 minutes in 80% ethanol (in a coplin jar)
 Rinse the slides rapidly in water and stand them vertically on a blotting paper for 10 minutes to dry.
 Place slides in a coplin jar containing elution solution for 20 seconds.
 Wash slides thoroughly in water and place them in counter stain for 2 minutes.
 The staining process should be continued without any delays to avoid fixation of haemoglobin.
 Rinse the slides in tap water and allow them to dry in air. (washing stages should be thorough and clearer films maybe
obtained using de-ionized water or distilled water rather than tap water)
 Wipe the back of each slide thoroughly to remove any excess stain deposit, and dried in an upright position.
 When observed under the microscope fetal cells appear deep red and adult cells appear as ghost cells (pale pink/
only margins are visible).
 Stain controls are run as follows alongside the test maternal films;
 Positive control - fresh EDTA cord blood added to fresh adult whole blood to give a dilution of 1:100
 Negative control - fresh EDTA blood (from a suitable adult subject, not likely to have raised Hb F)
Note:
The control samples should be ABO compatible and should be mixed well before film preparation.
The control samples may be stored at 4 0C for a maximum of 4 days, but fresh slides should be made each time when a
batch of tests is performed.
 Screening method
 Screen the test slides (and positive control) under low power x10 eyepiece and a x10 objective, to check for
adequate staining and even distribution of fetal cells.
 Select an area of the film where the adult cells are touching but not overlapping each other and count adult cells
under high power.
 If there are more than 100 adult cells in one high power field, proceed to count fetal cells.
 If there are fewer than 100 adult cells in one high power field, either remake the film or proceed to full
quantification if any fetal cells are seen in 25 low power fields.

 Semi-quantitative screen
 Go back to low power and examine 25 fields, counting fetal cells.
 If a total of 10 or more fetal cells are seen, then quantification must be performed.
 If fewer than 10 cells are seen in total then it can be assumed that the volume of fetal cells are less than 2 mL.
This will be covered by the standard anti-D immunoglobulin dose and no further action is required.
 Quantification of fetomaternal haemorrhage

 If semi-quantitative screen showed 10 or more fetal cells in 25 low power fields, quantification should be
performed.
 For accurate quantification minimum of 10,000 maternal red cell ghosts are examined using x 40 objective.
 Miller square or an indexed grid is necessary to obtain an accurate ratio of fetal to maternal cells in each blood
film.
 Using x 10 eye piece and x 40 objective, select an area of the film where cells are touching but not overlapping.
 Count adult cells (A) in the small square, including all cells which overlap the left hand or upper edges but not
those overlapping the right hand or lower edges.
 Count fetal cells (F) in the large square, taking cells overlapping the edges as 3 above.
 Move across the slide so that the next area to fall within the counting grid is contiguous with the preceding one
and repeat the procedure.
 Assume the total adult cells scanned = A x 9 and total fetal cells counted = F
 Calculation of fetomaternal haemorrhage

 This is calculated using the following Mollison formula, which assumes that:
 The maternal red cell volume is 1800 mL
 Fetal cells are 22% larger than maternal cells
 Only 92% of fetal cells stain darkly
 The FMH should be calculated as follows:
Number of fetal cells per high power field x 1800 x 122 x 100
Number of maternal cells per high power field 100 92
or can be simplified to:
Number of fetal cells per high power field x 2400 mL packed fetal red cells
Number of maternal cells per high power field

 It is important to issue FMH results urgently and effectively.
 The report format needs to communicate the following;
 The reason for the sample (e.g. post-delivery, sensitizing event, follow-up of significant FMH etc.)
 The result of the FMH test in ‘mL fetal red cells’ rounded to the nearest mL
 Whether any supplementary anti - D immunoglobulin is required if a standard dose of anti - D immunoglobulin
has already been administered
 Advice about the anti - D immunoglobulin dose required to cover the reported bleed
 Advice regarding follow - up samples to check for clearance of fetal cells

Films prepared from a mixture of cord blood and adult blood and from normal adult blood are stained alongside the test
films as positive and negative controls, respectively.

Sources of error
 If films are too thick, overlapping cells may prevent proper elution resulting in adult cells taking up some of the
counterstain.
 Aged reagents affect quality of staining
 Improper pH of stain
 Lengthy time delays in staining process can cause fixation of haemoglobin
 Inadequate washing stages
 Inappropriate storage temperature of reagents
 Physiological increase of Hb F during pregnancy and genetic disorders with increased Hb F affect quantification of
FMH.

 HCl can be corrosive and should be handled with care.
 References
1. Dacie and Lewis Practical Haematology – 12th Edition (2017).
2. Austin E, Bates S, Silva M, Howarth D, Lubenko A, Rowley M, Scott M, Thomas E, White J, Williams M.
Guideline for the estimation of fetomaternal haemorrhage, BCSH FMH Guidelines. 2009, 1 - 23.

6.11
HAEMOSIDERIN IN URINE
Mr. L.H.S. Sujeewa
Scope
The scope of this SOP is to guide technical personnel on correct performance of demonstration of haemosiderin in urine
Responsibility
Demonstration of haemosiderin in urine as per the procedure stated in this SOP is the responsibility of all medical
Principle
Haemosiderin is found in urine as a result of chronic intravascular haemolysis. The haemoglobin that has been liberated
into the blood, filters through glomeruli and passes through renal tubules. Renal tubules absorb this haemoglobin in the
filtrate. Within renal tubular cells, breakdown of Hb releases iron and excess iron is converted to ferritin. Continuous and
surplus availability of ferritin leads to formation of water insoluble deposits of iron called haemosiderin in renal tubular
cells. These tubular epithelial cells slough off and appear in urine regularly. Therefore, haemosiderin deposits within
tubular cells in urine deposits can be stained with Perls stain.
Presence of haemosiderin in urine is an indicator of chronic intravascular haemolysis. Appearance of haemosiderin in
urine needs prolonged persistent intravascular haemolysis for at least two weeks.
Sample
Freshly collected urine (10 – 20 mL)
 Urine must be collected in to an iron free bottle*.
 Early morning first voided sample is preferable and performance of test on three consecutive days can yield good
results.

1. Light microscope for smear examination oil immersion x100
2. Cytospin
3. Centrifuge (In the absence of cytospin)
4. Conical tubes*
5. Glass slides*
6. Methanol
7. Distilled water
8. Freshly prepared Perls stain (refer SOP 9.4)
9. Eosin 1 g/L or neutral red 1 g/L (counter stain) in distilled water
*Preparation of glassware : prepare glassware (slides/glass tubes/iron free containers) by soaking in 3 mol/L HCl before
washing to avoid iron contamination.
Method
 Mix sample of urine gently to make cells suspended appropriately.
 If cytospin is available prepare a direct smear using cytospin. If cytospin is not available, follow ordinary centrifugation.
 Centrifuge 10 mL of well mixed sample of urine in an iron free conical tube at 1200 g for 10 – 15 minutes.
 Decant supernatant. Break sediment by gentle tapping at the conical tip of tube.
 Transfer sediment onto iron free glass slide, spread out to about 1 – 2 cm using a clean, dirt free applicator and allow
it to dry in air.
 Make sure that during air drying, no contaminants fall on to the smear while allowing natural drying, may be
overnight.
 Fix slides in absolute methanol and allow to air dry.
 Cover slides with freshly prepared Perls stain and leave for 25 minutes. (stain can be put in a staining jar and slides
can be immersed in it).
 Pour off solution and wash with distilled water.
 Counter stain with eosin for 30 seconds.
 Wash with tap water and air dry.
 Examine using x100 oil immersion in a light microscope.
 First examine the positive control slide to confirm the reagent quality.
 Then examine the slide with urine sediment. If present, haemosiderin appears in the form of isolated or grouped
blue-staining granules, both intracellularly and extracellularly.

Urine for Haemosiderin - Positive or Negative

Should use a film made from a previously known iron positive bone marrow aspirate/ known iron positive tissue section
(e.g. splenectomy specimen of a thalassaemia patient) as a positive control to confirm the quality of the stain. The
control slide should always be stained together with the test slide/s.
Sources of error
 Sample contamination due to use of iron contaminated tubes, pipettes etc.
 Inadequate fixation and removal of cells.
 Not mixing the original sample before taking aliquot.

 Sample is not fresh and not the first voided sample.
 Use of previously prepared Perls stain.
 Use of expired/ decomposed potassium ferrocyanide for preparation of Perls stain due to exposure to light.

 HCl can be corrosive and should be handled with care.
 References
1. Dacie and Lewis Practical Haematology. 12th Edition (2017).

SECTION 07
SPECIAL
COAGULATION TESTS

CLOT SOLUBILITY TEST

COAGULATION MIXING STUDIES

COAGULATION FACTOR ASSAY

VON WILLEBRAND DISEASE TEST PROFILE

PLATELET AGGREGATION BY
LIGHT TRANSMISSION PLATELET AGGREGOMETRY

INHIBITOR SCREENING (BASED ON APTT)

QUANTITATIVE INHIBITOR ASSAY (BETHESDA ASSAY)

DILUTE RUSSELL’S VIPER VENOM TIME (DRVVT)

KAOLIN CLOTTING TIME (KCT)

ROTATIONAL THROMBOELASTOMETRY (ROTEM)

ANTI FACTOR Xa LEVEL
7.1
CLOT SOLUBILITY TEST
Ms. L. Dhammika
Scope
The scope of this SOP is to guide technical personnel on correct performance of clot solubility test by providing the
protocol and requirements of this test.
Responsibility
Performance of clot solubility test as per the procedure stated in this SOP is the responsibility of all medical laboratory
technologists who are assigned to perform this test in haematology laboratory.
Principle
Fibrin clots formed in the presence of factor XIII and thrombin (or calcium) are stable (due to cross linking) for at least
1 hr in 5 mol/L (5M) urea. Whereas, clots formed in the absence of factor XIII dissolves rapidly.
 Factor XIII crosslinks fibrin and stabilizes the clot formed during haemostasis. Deficiency of Factor XIII leads to
formation of weak clots and is associated with bleeding. This can be caused by inherited deficiency or abnormality,
consumption (DIC) or lack of synthesis (liver failure). Clot solubility test is a screening test for FXIII deficiency.
Screening for Factor XIII deficiency is indicated in a patient with bleeding whose platelets and other baseline clotting
tests are normal.
 Use of 5M urea will only detect FXIII deficiency of < 0.05 IU/mL, whereas use of 2% acetic acid as the lysing solution
may detect a deficiency of <0.1 IU/mL of FXIII. Therefore, the United Kingdom Haemophilia Centre Doctor’s
Organization (UKHCDO) guidelines recommend the use of thrombin/ acetic acid technique for clot solubility test,
as it gives the highest proportion of abnormal results in marked FXIII deficiency. However, mild factor XIII deficiency
may not be detected by this test.
Sample
Platelet poor plasma (PPP) prepared as per standard protocol.
STANDARD OPERATING PROCEDURES FOR SPECIAL COAGULATION TESTS 143

1. Thrombin 10 NIH unit solution or 10 U/mL solution
2. CaCl2 0.025 mol/L solution
3. Urea 5 mol/L or 2% Acetic acid (BDH) in isotonic saline (depending on the method used)
4. Water bath
5. Centrifuge
6 Glass tubes
7. Pipettes
Method
There are 3 methods which are described below. The test has to be performed in duplicate and positive and negative
controls should be performed in parallel.
The test is more sensitive when the sample is clotted with thrombin than with calcium. When thrombin is used
preparations containing calcium should be avoided. In the place of urea, 2% acetate can be used and this has shown to
increase the sensitivity of the test.
Method:1
 Place 0.2 mL of plasma in glass tubes.
 Add 0.2 mL of 10 NIH thrombin solution to each tube and incubate at 37 0C for 20 minutes.
 Fill tubes with approximately 3 mL of 5 mol/L urea solution.
 Carefully dislodge the clots and leave undisturbed at 37 0C for 24 hrs.
 Inspect test and control tubes for presence of clots.
Method:2
 Place 0.5 mL of plasma in glass tubes.
 Add 0.5 mL of CaCl2 and incubate for 30 minutes at 37 0C.
 Add 3 mL of 5 mol/L urea and suspend the clot in the solution by lightly tapping and loosening from the wall.
 Leave at room temperature for 24 hours.
 Inspect test and control tubes for presence of clots.
Method:3
 Place 0.2 mL of plasma in to a glass tube. Keep at 37 0C.
 Add 0.1 mL of thrombin and 0.2 mL of saline into tubes, mix and incubate at 370C for 30 minutes.
 Add 5 mL of 2% acetic acid to the tubes and suspend the clot in solution by lightly tapping and loosening from wall.
 Incubate at 37 0C for 24 hours.
 Inspect the test and control tubes for the presence of clots.

 If clot is absent, it is reported as “Clot solubility test – Positive”
 If clot is present, it is reported as “Clot solubility test – Negative”

Internal quality control can be done by using normal citrated plasma as negative control and normal EDTA plasma as
positive control.
Sources of error
 Small friable clots produced in fibrinogen abnormalities may be easily missed on inspection after 24 hours.

 References
1. Dacie and Lewis Practical Haematology 12th Edition, Churchill Livingstone (2017).
2. Bolton-Maggs PHB et al. The rare coagulation disorders – review with guidelines for management from the
United Kingdom Haemophilia Centre Doctor’s Organization. Haemophilia (2004), 10, 593 – 628.

7.2
COAGULATION MIXING STUDIES
Mr. M.D.C. Tharanga
Scope
The scope of this SOP is to guide technical personnel on proper performance of coagulation mixing studies by providing
the protocol and requirements of this test.
Responsibility
Performance of mixing studies as per the procedure stated in this SOP is the responsibility of all medical laboratory
technologists who are assigned to perform this test in the haematology laboratory. Interpretation, conclusions and
comments should be made by the consultant haematologist.
Principle
A prolonged clotting time can be due to deficiency of one or more clotting factors or due to the presence of inhibitors to
clotting factors. In factor deficiency, mixing the test plasma 1:1 with plasma containing 100% of the factor level (normal
control) results in a level ≥50% of the factor in the mixture. Correction of the clotting time following mixing indicates a
factor deficiency, while failure to correct indicates the presence of an inhibitor.
There are several methods available to interpret mixing studies as having either “corrected” (suggesting factor deficiency)
vs “not corrected” (suggesting factor inhibitor)
1. Mixed test result within the normal reference range

If the test/normal mixture provides a test result that is within the normal reference range, “correction” is said to
occur.

2. The inter-quartile rule
Normal plasma / test plasma (1:1) mix result (s)
Normal plasma Test
result (s) result (s)
+ 25% 50% -25%
‘Correction’ ‘Partial correction’ ‘Non correction’

(Factor deficiency) (‘Weak / moderate’ inhibitor) (‘Strong’ inhibitor)
3. Index of circulating anticoagulant (ICA)- “Rosner Index”
The ICA is identified by the following formula:

ICA = [(1: 1mix CT−PNPCT)/patient CT] x 100
where CT = clotting time of the test under investigation, and PNP = pooled normal plasma.
In general, the cut-off value to consider the mixing studies as corrected will range from 10 to 15%.
4. Percent correction method (Chang method)
The percent correction is identified by the following formula:

% correction = (patient CT−1: 1mix CT)/ (patient CT−PNPCT) x 100
where CT is again the clotting time of the test under investigation, and PNP again reflects the pooled normal plasma.
In general, the cut-off value to consider the mixing studies as corrected will range from 65 to 80%
Mixing studies are tests performed on plasma of patients with high PT, APTT or TT values, and used as the initial step
in determining the cause of prolonged clotting times. Mixing studies with normal plasma distinguish factor deficiencies
from factor inhibitors. Further mixing with factor deficient plasma identifies the exact deficient factor, which should be
confirmed by specific factor assay testing.
Sample

1. Normal control plasma (commercially available) or pooled normal plasma (PNP)
2. In house prepared factor deficient plasma/ serum (adsorbed plasma, aged plasma and aged serum*) or commercially
available factor deficient plasma
3. APTT, PT, TT reagents available commercially
4. Calcium chloride solution (0.025 mol/L)
5. Centrifuge
6. Water bath at 37 0C / semi-automated coagulometer/ fully automated coagulometer.
7. Glass test tubes
8. Stop watch (for manual method)
9. Pipettes (adjustable or fixed 0.1 mL and 0.2 mL pipettes)
10. Mechanical mixer
Pooled normal plasma (PNP)

PNP (prepared as per protocol) is rich in all the coagulation factors.

*Adsorbed plasma
Adsorbed plasma provides fibrinogen and factors V, VIII, XI, XII and XIII; adsorption removes the vitamin K dependent
factors II, VII, IX and X.
Preparation:
With aluminum hydroxide
 Aluminum hydroxide gel (alumina) is prepared by thoroughly mixing 1 g of moist gel with 4 mL of distilled water
to a smooth suspension.
 Citrated platelet-poor plasma is collected from five normal donors and pooled.
 A 1/10th volume of aluminum hydroxide is added to pre-warmed plasma, mixed and incubated for three minutes
at 37 0C.
 The mixture is then centrifuged immediately (at 1700 g for 3 minutes at room temperature) to sediment the gel.
 The supernatant plasma is put in to plastic containers and may be stored at -350C for several weeks.
 This should have a prothrombin time (PT) of >60 seconds with a sensitive reagent.
With barium sulphate
 Barium sulphate (100 mg) is mixed with 1 mL control plasma and keep it at 37 0C for 3 minutes.
 The mixture is then centrifuged immediately (at 1700 g for 3 mins at room temperature)
 Take the supernatant plasma and check PT.
 This adsorbed plasma should have a PT of >60 secs with a sensitive reagent.
 Care must be taken in the adsorption time, as over-adsorption will result in loss of other clotting factors.
*Aged Plasma
Aged plasma is deficient in factor V and factor VIII.
Preparation
 Normal venous blood is added to 0.1 M sodium oxalate at a ratio of 9:1.
 The blood is centrifuged to obtain platelet poor plasma, separated under sterile conditions and incubated at 37 0C
for two to three days.
 The PT at the end of this time should exceed 90 seconds with a sensitive reagent.
 The plasma is then aliquoted in plastic containers and stored at -35 0C (or lower).
*Aged serum
 Aged serum is deficient in fibrinogen, FII, FV, FVIII.
 Blood is allowed to clot and serum separated.
 Serum is kept at room temperature for 48 hours and then 4 hours at 37 0C.
 During clotting and aging, fibrinogen, FII, FV, FVIII are consumed.
 The serum is then aliquoted in plastic containers and stored at -35 0C (or lower)
Method
1. Test plasma detected to have isolated high PT values: Perform PT based mixing studies
 Mix equal volumes of test plasma and normal plasma.
 Then perform PT testing.
 Results interpretation:
 If PT is corrected, it indicates factor VII deficiency.

2. Test plasma detected to have isolated high APTT values: Perform APTT based mixing studies
Step 1- using PNP
 Mix equal volumes of test plasma and normal plasma.
 Then perform APTT testing.
 Results interpretation:
 If APTT is corrected, it indicates factor VIII / IX / XII or XI deficiencies.
 If APTT is not corrected, it indicates that sample contains an immediate acting inhibitor such as factor IX
inhibitor or lupus anticoagulant.
 If APTT is corrected, proceed to step 2
Step 2- Mixing with factor deficient plasma
Table 7.2.1 Use of adsorbed and aged plasma
APTT corrected by mixing with

Factor Deficiency
Normal Adsorbed plasma Aged plasma
plasma (Deficient in II.VII. IX. X) (Deficient in V, VIII)
Factor VIII Yes Yes No
Factor IX Yes No Yes
Factor XI / XII Yes Yes Yes
If APTT is not corrected by addition of aged plasma it indicates deficiency of factor VIII and if it is not corrected by the
addition of adsorbed plasma, it indicates deficiency of factor IX.
Table 7.2.2 Use of commercially available factor deficient plasma

APTT corrected by mixing with
Factor Deficiency
Factor VIII Factor IX Factor XI Factor XII
deficient plasma deficient plasma deficient plasma deficient plasma
Factor VIII No Yes Yes Yes

Factor IX Yes No Yes Yes
Factor XI Yes Yes No Yes
Factor XII Yes Yes Yes No
If the APTT is not corrected by addition of a specific factor deficient plasma, it indicates deficiency of that particular
factor.
To confirm the finding, proceed to assay that particular factor.
3. Test plasma detected to have high PT and APTT values (with normal TT): Perform PT based mixing studies
 Above steps for the PT based mixing studies should be followed.
 If it is corrected, possible factor deficiencies are common pathway factors (factor X, V, II)
 Use adsorbed plasma correction and aged plasma correction to distinguish actual factor deficiency (Test plasma is
separately mixed in equal volumes with adsorbed plasma and aged plasma and perform PT testing)

Table 7.2.3 Use of adsorbed plasma and aged plasma
Prothrombin time corrected by mixing with

Factor Deficiency
Normal Adsorbed Plasma Aged Plasma Aged serum
Plasma (Deficient in II.VII. IX. X) (Deficient in V, VIII) (Deficient in I,II,V,VIII)
Factor II (prothrombin) Yes No Yes No
Factor V Yes Yes No No
Factor X Yes No Yes Yes
If PT is not corrected by both adsorbed plasma and aged serum, it can be prothrombin deficiency. If PT is not corrected
by both aged plasma and aged serum, it is likely to be FV deficiency. If PT is not corrected only by adsorbed plasma, it
indicates possibility of FX deficiency.
Table 7.2.4 Use of commercially available factor deficient plasma
Prothrombin time corrected by mixing with
Factor Deficiency
Factor II Factor V Factor X
deficient plasma deficient plasma deficient plasma
Factor II No Yes Yes

Factor V Yes No Yes
Factor X Yes Yes No
If the PT is not corrected by addition of a specific factor deficient plasma, it indicates deficiency of that particular factor.
To confirm the finding, proceed to assay of that particular factor.

 If done up to correction with PNP-
Prolonged PT/APTT corrected by normal plasma indicates deficiency of clotting factors.

Prolonged PT/APTT not corrected by normal plasma indicates presence of an immediately acting inhibitor.
 If done up to correcting with factor deficient plasma-
Prolonged APTT/PT corrected by …… deficient plasma indicates possible deficiency of … factor. Need factor assay for
confirmation.

Laboratory should follow generally accepted QC protocols and fulfill quality control requirements.
Sources of error
 Time dependent inhibitors (e.g. Factor VIII inhibitors) may show correction and show the inhibitory effect only if
incubated for 2 hours.
 Lupus-like anticoagulants are relatively weak and may be overcome by dilution in normal plasma. Therefore, they
may appear like a factor deficiency.
 When the test is only slightly prolonged it may be difficult to detect correction accurately and specific factor and
inhibitor tests should be performed from the outset.


 All parts and surfaces of the coagulometer should be considered as potentially infectious since the instrument
 References
1. Dacie and Lewis Practical Haematology12th Edition, Churchill Livingstone, (2017).
2. Diagnosis of Heamophilia and other bleeding Disorders – A Laboratory Manual, Second Edition (2010).
3. Favaloro E J, Coagulation mixing studies: Utility, algorithmic strategies and limitations for lupus anticoagulant
testing or follow up of abnormal coagulation tests, American Journal of Hematology. 2020;95:117–128.

7.3
COAGULATION FACTOR ASSAY
Ms. L. Dhammika
Scope
The scope of this SOP is to guide technical personnel on proper performance of coagulation factor assays based on
prothrombin time (factors II, V, VII or X) and factor assays based on activated partial thromboplastin time (factors VIII,
IX, XI or XII).
Responsibility
Performance of coagulation factor assay in the procedure stated in this SOP is the responsibility of all medical laboratory
comments should be made by the Consultant Haematologist.
Principle
The one-stage clotting assay (OSCA) is the predominant method used in clinical laboratories to measure factor levels.
This method of factor assay compares the ability of dilutions of a standard or reference plasma and test plasma to correct
the clotting times (PT or APTT) of a plasma known to be totally deficient in the clotting factor being measured.
NOTE:
In automated systems also factor assay results are dependent on obtaining parallel lines for the test and reference plasmas.
It is recommended that in automated analyzers at least three dilutions are measured. This improves the accuracy of the
result (interference of an inhibitor can be detected).
Reduced levels of clotting factors are seen in bleeding disorders such as haemophilia. In the workup of patients with
a history of bleeding, when the initial clotting tests show prolonged PT or APTT and if the mixing studies suggest a
possibility of factor deficiency, specific factor assays are done to confirm it.
Sample
Platelet poor plasma (PPP) prepared as per standard protocol

1 Owren’s buffered saline or Imidazole buffer (Glyoxaline buffer)
Imidazole buffer: 2.72 g imidazole
4.68 g sodium chloride
Place in volumetric flask and dissolve in approximately 650 mL of distilled water. Add 148.8 mL of 0.1M HCl, adjust
pH to 7.3. Adjust volume to 1 L with distilled water.
2 Standard plasma
The standard plasma used should be either a locally prepared pooled normal plasma kept at -700 C or lower, or a
commercial standard plasma. The reference plasma must be calibrated against an international standard for relevant
factor. It is not acceptable to assume that a pooled normal plasma has 100 U/dL of the factor to be assayed.
3. Factor deficient plasma / substrate plasma (relevant factor should be <1.0% - artificially deficient commercial plasma
or patient derived - from a patient who has not received treatment for 2 weeks and does not have inhibitors to the
factor)
(e.g FVIII deficient plasma for FVIII assay)
4. PT/APTT reagent
5. Coagulometer
6. Centrifuge
Method
I) Factor assays based on prothrombin time (for factors II, V, VII, or X)
The assay of an extrinsic pathway factor is based on PT. The assay compares the ability of dilutions of the patient’s
plasma and of a standard plasma (with a known concentration of the factor being assayed) to correct the PT of
a substrate plasma (deficient in the factor that is being assayed but contain other factors of that pathway).
This one-stage assay can be used for assays of factors II, V, VII and X.
An assay for FV is described below, but this can be adapted to FII, VII, X by substituting the relevant factor-deficient
plasma.
 Prepare dilutions for test and standard plasma in plastic tubes, as shown in table below (Table 7.3.1)
Table 7.3.1 Preparation of plasma dilutions

Dilution Plasma (mL) Buffer (mL)
1/5 0.1 0.4
1/10 0.1 0.9
1/20 0.5 (1/10) 0.5
1/40 0.5 (1/20) 0.5
Note: Mix the previous dilution well, before using it to prepare the next dilution. Plasma dilutions should be tested
immediately after preparation. If room temperature exceeds 25 ⁰C, it may be necessary to keep dilutions in wet ice prior
to analysis.
 Test each dilution of standard plasma as follows:

 Pipette 0.1 mL of each dilution into a 75 x 10 mm glass tube and add 0.1 mL factor V deficient plasma.
 Warm to 37 0C for 2 minutes and then add 0.2 mL of pre-warmed PT reagent. Start stop watch and record
clotting time.
 Repeat in the same manner for dilutions of tests plasma.
 For test plasmas expected to be normal, test 1/10, 1/20, and 1/40 dilutions.
 For test plasmas expected to have reduced levels, test 1/5, 1/10, and 1/20 dilutions.
 Duplicates for each dilution should be tested and duplicate values should agree within 5%.
Note: Duplicates in coagulation assays should always be tested in balanced order (e.g. ABCCBA)

 A “blank” containing following should also be tested:
 0.1 mL buffer + 0.1 mL factor V deficient plasma + 0.2 mL PT reagent
 The clotting time of the blank should be longer than the time of 1% FV activity of the standard from the
calibration graph. If the time is shorter, this indicates that the substrate plasma is not totally deficient of
factor V and thus not a suitable substrate plasma.
 The clotting time of the “blank” reflects the quality of the deficient plasma and should be equivalent to
less than 1 %.
 Calculation of results
 Plot clotting times of the test and standard against the concentration of factor V on log/ log graph paper or
log/ linear (plot concentration on logarithmic scale and clotting time on a linear scale).
 The 1/10 dilution is arbitrarily assigned as 100%, thus 1/5 dilution is equivalent to 200% etc.
 The relative amount of factor V in the patient’s plasma is extrapolated from the graph (Figure 7.3.1). An example
of this is shown below.*
Clotting Time (sec)
200 Test
Standard
100
60 1/40 1/20 1/10
40
20
5 10 20 30 50 100
% Factor V Activity
Figure 7.3.1
Parallel line bio-assay of factor V
 The clotting time equivalent to 100% test (the place where the test line passes through the 100% activity) is
read from the standard line (the concentration of standard that could give that particular clotting time).
 This gives the concentration of the test in percentage of the standard.
 This percentage is multiplied by the concentration of clotting factor in standard.
*Example: According to the above graph,
 Concentration of factor V in test plasma is 7.5% of that in the standard.
 The factor V concentration of standard plasma is 85 IU/dL.
 The final concentration of factor V in test plasma is equal to 7.5% x 85 IU/dL= 6.3 IU/dL
II) Factor assays based on APTT (Factors VIII: C, IX, XI, or XII)
An assay for FVIII is described below, but this can be adapted to FIX, FXI or FXII by substituting the relevant factor-
deficient plasma.

a) One - stage assay of factor VIII
Method
 Make dilutions of standard and test plasma in plastic tubes as in the table given below
(If the test plasma is expected to have a very low level of factor VIII, start at 1/5 dilution, if not can start with 1/10
dilution).
Table 7.3.2 Preparation of plasma dilutions

Dilution Plasma (mL) Buffer (mL)
1/5 0.1 0.4
1/10 0.1 0.9
1/20 0.5 (1/10) 0.5
1/40 0.5 (1/20) 0.5
 Mix each dilution well before transferring into next tube (without making froth).
Plasma dilutions should be tested immediately after preparation. If room temperature exceeds 25 0C, it may be
necessary to keep dilutions on wet ice prior to testing.
 Pipette 0.1 mL of first dilution of standard plasma into a 75 x 10 mm glass tube.
 Add 0.1 mL of factor VIII deficient plasma and transfer into 37 0C water bath.
 Add 0.1 mL of APTT reagent and incubate for 5 minutes.
 At 5 minutes add 0.1 mL CaCl2 and record the clotting time.
 Test in duplicate and perform the other dilutions in the same way.
 Repeat in the same manner for dilutions of test plasma.
 Duplicates for each dilution should be tested and duplicate values should agree within 5%.
Note: Duplicates in coagulation assays should always be tested in balanced order (e.g. ABCCBA)
 A “Blank “should also be set up

 0.1 mL buffer + 0.1 mL factor VIII deficient plasma + 0.1 mL APTT reagent.
 Incubate for 5 minutes, then add 0.1 mL CaCl2 and record the clotting time.
 The clotting time of the blank should be longer than the time of 1% FVIII activity of the standard from the
calibration graph. If the time is shorter, this indicates that the substrate plasma is not totally deficient of
factor VIII and thus not a suitable substrate plasma.
 The clotting time of the “blank” reflects the quality of the deficient plasma and should be equivalent to less
than 1 %.
 Plot the clotting times of the test and the standard against the concentration of factor VIII on log-linear paper or
log-log paper. (Log-log is used when higher dilutions are done e,g, FVIII assay in cryoprecipitate. For patient’s
samples usually log-linear is used)
 The 1/10 dilution is arbitrarily assigned a value of 100%, the 1/20 dilution is a value of 50%, the 1/40 a value of
25%.
 Read the concentration as shown in Figure 7.3.2

Clotting Time (sec)
200 Test
Standard
100
60 1/40 1/20 1/10
40
20
5 10 20 30 50 100
% Factor VIII Activity Figure 7.3.2
Parallel line bio assay of factor VIII
 The clotting time equivalent to 100% test (the place where the test line passes through the 100% activity) is
read from the standard line (the concentration of standard that could give that particular clotting time).
 This gives the concentration of the test in percentage of standard.
 This percentage multiplied by the concentration of clotting factor in in standard (% or IU/dL) to give the
concentration in the test (in % or IU/dL).
 In this example, the concentration in the test sample is 7% of that in the standard. If the standard has a
concentration of 85 IU/dL, test sample has a concentration of 85 IU/dl x 7% = 6 IU/dL.
NOTE: It is important to obtain straight and parallel lines if the results to be accurate. If the lines are not parallel, the
assay should be repeated.
Reasons for non-parallelism

 Non-parallel lines may occur due to technical error. If technical error has been identified, repeat the assay with fresh
dilutions.
 Pre-activation of the test plasma by poor collection: A new sample should be collected.
 A low concentration of FVIII in the test plasma giving rise to non-parallelism: Stronger concentration of plasma
should be prepared and tested.
 The presence of an inhibitor: Inhibitor screen and if inhibitor screen is positive, inhibitor assay should be carried out.
Limitations of one-stage FVIII assay

 The one-stage FVIII assay is sensitive to pre-activation of coagulation factors in the patient sample.
 The two-stage and chromogenic assays overcome this problem by allowing sufficient time for activation of all the
available FVIII and for generation of FXa which is then assayed in a separate step.
 Interference if lupus anticoagulant is present.
 Mild haemophilia A is not excluded by finding a normal FVIII:C level by one-stage assay. If there is strong clinical
suspicion of mild FVIII deficiency, FVIII:C level should be measured using different types of assays.
Based on these results, it is advantageous for all haemophilia centers to have a chromogenic or two-stage clotting assay
available. These tests should be performed on subjects with normal APTT and one-stage FVIII activity in the presence of a
personal or family history consistent with mild haemophilia.

b) Chromogenic assay
In some (but not all) chromogenic assays, all the FVIII in the sample is activated by thrombin. Activated FVIII then
accelerates the conversion of FX to FXa in the presence of activated FIX, phospholipids, and calcium ions. The FXa activity
is assessed by hydrolysis of a p-nitro alanine substrate specific to FXa. The initial rate of release of p-nitro alanine (yellow
colour) measured at 405 nm is proportional to the FXa activity and thus the FVIII activity in the sample.
Commercial chromogenic assays are available for measuring FIX but have not established a specific role to date. However,
they may prove to be important in assaying modified FIX molecules. FIX is activated to FIXa which in turn proportionately
activates FX to FXa. The FXa activity is then measured using a chromogenic substrate.
c) Two - stage clotting factor assay for Factor VIII: C

The two-stage FVIII: C assay is based on the principle that the amount of FVIII present in the system is rate-limiting
during clotting of a test mixture containing FX, activated FIX, phospholipid, calcium and FV in excess. Adsorption of
plasma by aluminum hydroxide removes activated factors and vitamin K-dependent factors. This is necessary to remove
prothrombin so that none is present in the initial incubation mixture. Without this step, the mixture would clot.
The dilutions of adsorbed standard and test plasma are incubated with the combined reagent in the 1st step. This
generates the FXa. A source of prothrombin and fibrinogen from pooled normal plasma is added in the 2nd stage, which
allows a clot to form and for which the resulting clotting time is dependent on the initial amount of FVIII.

Factor …… level - ………IU/dL
Reference interval: should be established locally.

 A normal and abnormal control plasma should be assayed with each run. A statistically valid mean and SD should be
established.
 Laboratory should follow generally accepted QC protocols and fulfill quality control requirements.
 There should be periodic review of QC data to look for long-term changes on analytic system.
 Each laboratory should enroll in a proficiency testing program acceptable to the relevant inspecting or accrediting
agencies.
 Automated coagulometers have to be calibrated whenever the deficient plasma or APTT reagents are changed.
 The analyzer has to be validated for accuracy and precision using standard reference material.
Sources of error
 The results of factor assays may show considerable variability because of the many dilutions of patient/ test and
reference specimens and also the assay involves the multiple enzyme reactions. Therefore, CVs of 5 – 10 % are
considered acceptable for the “normal control plasma”.
 If the test plasma FVIII (or FIX, FXI or FXII) concentration is close to zero (i.e. clotting times of all dilutions are similar
to the blank), non- parallel lines may occur.


 References
2. Diagnosis of Haemophilia and other bleeding Disorders – A Laboratory Manual, World Federation of
Haemophilia, Second Edition (2010).
3. Clinical and Laboratory Standards Institute – 2011/ H-48-A.
4. Baker P, Sean P, Gibson C, Gray E, Jennings I, Murphy P, Laffan M and On behalf of British Society for Haematology,
Haemostasis and Thrombosis Task Force. Guidelines on the laboratory aspects of assays used in haemostasis
and thrombosis. British Journal of Haematology, 2020, 191, 347–36.

7.4
VON WILLEBRAND DISEASE
TEST PROFILE
Ms. L. Dhammika
Scope
The scope of this SOP is to guide technical personnel on correct performance of tests for diagnosis of von Willebrand
disease (VWD) by providing the protocol and requirements of these tests.
Responsibility
Performance of tests for diagnosis of VWD as per the procedure stated in this SOP is the responsibility of all medical
laboratory technologists who are assigned to perform these tests in haematology laboratory. Interpretation, conclusions
and comments should be made by the Consultant Haematologist.
Principle
This will be stated under individual tests.
VWD results in a bleeding phenotype which range from mild to severe and is due to quantitative or qualitative defects
of von Willebrand Factor (VWF). VWD testing profile is used to investigate excessive or recurrent bleeding episodes or
a personal or family history of bleeding. These tests help to diagnose and distinguish between various types of VWD.
Sample
Food, especially fatty meals should be avoided after 10 pm, the previous night.
Tests done for diagnosis of VWD

1. VWF antigen assay
2. VWF: Ricof
3. VWF: activity
1. von Willebrand factor antigen assay (VWF: Ag)

This test determines the quantity of VWF: Ag which is the first step in the diagnosis of VWD. The amount of VWF
molecules present in the sample is measured by this test and not the activity.

Principle
When the latex reagent, reaction buffer and sample are mixed in the reaction cell of the analyzer, the micro particle
agglutinate proportionately to the VWF: Ag present in the citrated sample. A monochromatic light illuminates the reaction
mixture, which scatters out some of the light. The quantity of light scattered, measured as absorbance, is proportional to
the degree of agglutination, and therefore to the VWF: Ag concentration.

1. Coagulometer
2. Centrifuge
3. Reagents*
 Latex reagent: Latex particles coated with a rabbit polyclonal antibody specific for human vWF, suspended in a
buffer containing bovine serum albumin, stabilizer and preservative.
 Reaction buffer: HEPES buffer containing bovine serum albumin, stabilizers and preservative.
Note: Once opened and after being used, it is advisable to store the reagents at 2-80C, in their original vials
tightly capped to prevent evaporation.
4 . Calibration plasma, in which the VWF: Ag value is determined against the National institute for biological standards
and control (NIBSC) international standard for FVIII and VWF: Ag.
Note: The calibration curve is stored in the instrument’s memory. Its stability is 3 months. Therefore, a calibration
cycle only needs to be performed every 3 months, or when the reagent lot number changes.
5. Controls - normal and abnormal controls.
Method
Fully automated latex particle immunoturbidimetric assay
This method is a fully automated latex micro particle enhanced turbidimetric immunoassay for the determination of
VWF: Ag in human citrated plasma. The analysis time for a single test is less than seven minutes.
Refer to the instrument’s operator’s manual and /or application manual for the complete assay procedure instructions.
2. Von Willebrand Factor Activity
Principle
This VWF Activity test is an automated latex enhanced immunoassay for the quantitative determination of VWF Activity
in human citrated plasma.
The activity of VWF is determined by measuring the increase of turbidity produced by the agglutination of the latex
reagent. A specific anti-VWF monoclonal antibody adsorbed into the latex reagent, directed against the platelet binding
site of VWF (Glycoprotein 1b receptor), reacts with the VWF of patient plasma. The degree of agglutination is directly
proportional to the activity of VWF in the sample and is determined by measuring the decrease of transmitted light
caused by the aggregate.

1. Coagulation analyser
2. Centrifuge
3. Reagents
 Latex Reagent
Lyophilized suspension of polystyrene latex particles coated with purified anti-VWF mouse monoclonal antibody
directed against a functional epitope of VWF, containing bovine albumin, stabilizers and preservatives.
 Buffer
Tris buffer containing bovine serum albumin, stabilizers and preservatives.

Method
Refer to the instrument’s operator’s manual and/ or application manual for the complete assay procedure instructions.
Limitations /interfering substances

 Presence of rheumatoid factor, haemolysis and sample turbidity due to lipaemia and bilirubin can interfere the
results. (Refer the manufacturers recommendations.)
 Specimens from patients who have received preparation of mouse monoclonal antibody for diagnosis or therapy
may contain human anti- mouse antibody (HAMA). The presence of HAMA may cause an over-estimation of results
in immunoassays that utilize mouse monoclonal antibodies. (Reagent buffer contains blocking agent against HAMA).
 The presence of increased levels of human anti-bovine Ig G antibodies (HABIA) in some patients may lead to an
overestimation of results.
3) VWF ristocetin cofactor (VWF:RCo) assay

The VWF:RCo assay using platelets reproduces the in vitro ability of VWF to interact with one of its platelet receptors,
the glycoprotein GP1b-IX-V complex in the presence of ristocetin. The classical method is based on the property of the
antibiotic ristocetin to agglutinate formalin-fixed normal platelets in the presence of VWF; however, it has high inter-
laboratory variability due to the complexity of the procedure.
Principle
This test kit is a latex particle enhanced assay to quantify VWF: RCo activity in citrated plasma. The activity is determined
by measuring the increase of turbidity produced by the agglutination of the latex reagent. A recombinant fragment of
glycoprotein platelet receptor of VWF (r GP 1b alpha) is coated with latex particles by means of a specific monoclonal
antibody which orientates the GP1 alpha fragment in the proper way to interact with the VWF of patient sample in the
presence of ristocetin. The degree of agglutination is directly proportional to the activity in the sample and is determined
by measuring the decrease of transmitted light caused by the aggregates.

1. Coagulation analyser
2. Centrifuge
3. Reagents
 Latex reagent
Suspension of polystyrene latex particles coated with a recombinant fragment of glycoprotein platelet receptor
of VWF (r GP 1b alpha) by means of a specific monoclonal antibody. Contains bovine serum albumin, buffer and
preservative.
 Ristocetin
Ristocetin sulphate, a glycopeptide antibiotic isolated from Nocardia lurida in aqueous acidic solution.
 Buffer
Tris buffer containing bovine serum albumin, stabilizers, and preservative.
 Diluent
Saline solution containing bovine serum albumin and preservative.
Method
Immunoturbidimetric assay
The method described below is HemosIL von Willebrand factor Ristocetin Cofactor Activity (HemosIL VWF: RCo)
Refer to the appropriate operator’s manual of the instrument for the complete assay procedure instructions.
Limitations/ interfering substances

 HemosIL VWF: RCo results on ACL TOP family are not affected by haemoglobin up to 0.5 g/dL, bilirubin up to 18 mg/dL
and lipids up to 1300 mg/dL. The presence of rheumatoid factor was shown to produce an overestimate of the test
result.

 Specimens from patients who have received preparation of mouse monoclonal antibody for diagnosis or therapy
may contain human anti-mouse (HAMA). The presence of HAMA may cause an over-estimation of results in
immunoassays that utilize mouse monoclonal antibodies. The HemosIL VWF: RCo contains a blocking agent against
HAMA to minimize this interference on the assay results.

 Report should include;
- VWF :Ag ……..........….IU/mL
- VWF : Activity …………IU/mL
- VWF : RCo ……........….%
- VWF : Activity / VWF :Ag ratio …….
- FVIII : C level ………….IU/mL
Comment : ……………….
Recommendations : …………………
 The results of these assays should be interpreted with other information, including the clinical context and FVIII level
of the patient in making a diagnosis.
 Lower detection limit will depend on the analyzer used. Therefore, refer the manufactures instructions.
 Consider the CRP value and patient’s blood group when interpreting the results of VWF: Ag and activity assays.
Reference intervals
Reference intervals for blood groups A, B, AB and O are given in the reagent pack insert. However, it is advisable to
establish laboratory’s own reference intervals.

 Normal level and low-level controls are recommended for each batch of samples as IQC. Laboratory should establish
its own mean and standard deviation.
 Normal and abnormal controls should be analyzed during every 8-hour shift.
 Ideally laboratory should participate in EQA programme or should at least do periodical inter-laboratory comparison.
Inter- laboratory comparison should be done at a frequency determined by the individual laboratory, according to
their quality guide.
 Analyzer should be calibrated according to the manufacture recommendations.
Sources of error
 Falsely elevated results may be obtained in the presence of rheumatoid factor or in acquired von Willebrand
syndrome.
 Mild VWD can be masked due to physiological elevation of VWF in conditions such as pregnancy, OCP, infections,
inflammatory diseases, exercise, etc.

 References
2. Manufacturer’s guidelines-Siemens Health Care Diagnostics Products Germany.
3. Diagnosis of Hemophilia and other bleeding disorders, A Laboratory Manual, World Federation of Hemophilia, 2010.

7.5
PLATELET AGGREGATION BY
LIGHT TRANSMISSION
PLATELET AGGREGOMETRY
Dr. H.M.J. Priyanka Herath
Scope
This SOP is for platelet aggregation studies (PAS) using light transmission platelet aggregometry (LTA) method. This
could be used as a guide for other methods/analyzers of platelet aggregation studies with the specific manufacture
recommendations given for the analyzers.
Responsibility
The medical laboratory technologists performing the test will be responsible for the technical procedures of the pre-
analytical, analytical, and post analytical phases mentioned in this SOP, while the consultant haematologist will be
responsible for overall supervision, data analysis and test reporting.
Principle
The light transmission platelet aggregometry or low sheer aggregometry is considered as the gold standard for platelet
function testing. The principle is monitoring and detecting changes in light transmission through stirred platelet rich
plasma (PRP) within cuvettes incubated at 37 0C. Upon addition of a panel of agonists (e.g. Collagen, ADP, Ristocetin,
Arachidonic acid) at a range of different concentrations, the platelets begin to aggregate and light transmission increases
with time. Platelet aggregation with time can be monitored, including any lag-phase, shape change, primary (reversible)
and secondary (irreversible) aggregation. Parameters measured include the concentration of agonist inducing the second
wave aggregation, the percentage of final aggregation, the maximum amplitude and/or percentage of aggregation after
a fixed time (10 minutes). Additionally, area under the slope or area under the curve could be measured.
There are two clinical indications for platelet aggregation studies.
1. To diagnose congenital or acquired platelet function disorders in patients with significant bleeding symptoms
(bleeders).
2. To evaluate the therapeutic effect of antiplatelet drugs.
Sample
 Pretest requirements
 Platelet aggregation occurs through GPIIb-IIIa receptors via fibrinogen. Normal plasma fibrinogen function
should be confirmed with plasma fibrinogen assay (clauss method) and thrombin time prior to the test.

 If a patient has a transient acquired condition, which can give rise to platelet functional defects (e.g. eosinophilia,
drugs), it is recommended to defer the test until the transient cause is corrected. However, in urgent clinical
conditions, where testing cannot be delayed, proceeding with the testing should be decided by the consultant
haematologist and results should be interpreted accordingly.
 Platelet count should be checked prior to the test and spurious thrombocytopenia should be excluded. Ideally
the platelet count has to be normal (≥ 150 x109/L) to assess the function optimally, but a platelet count of
>100 x109/L is usually acceptable. However, platelet function disorders associated with thrombocytopenia
(e.g. Bernard Soulier Syndrome-BSS, Gray platelet syndrome) can be diagnosed even with lower platelet counts.
 When there is thrombocytosis, platelet count should be adjusted during the test process.
(refer to sample preparation).
 Patient preparation
 To diagnose platelet function disorders, patients have to be off all antiplatelet drugs as well as all medication that
can affect platelet function for 14 days*
 To evaluate efficacy of an antiplatelet drug/s, the particular drug should be continued for the specified period,
which will be adequate to have the expected therapeutic drug effect, while all other drugs that can affect platelet
function should to be deferred for 14 days*.
 Food which can affect platelet function should be deferred for 14 days (Table 7.5.1).
 If ristocetin Induced Platelet Agglutination (RIPA) is done as a part of vWD test profile, the test should be deferred
for 2 weeks after blood/ blood products /coagulation factor treatment.
(Platelet aggregation studies sometimes give inconclusive results immediately after blood/blood product
transfusion. It is preferred to defer the test for 2 weeks after blood/blood product transfusion).
 Food, especially fatty meals should be avoided after 10 pm the previous night. Advise on adequate water intake
to avoid dehydration and difficult venepuncture.
 Patients should refrain from smoking and ingestion of caffeine on the day of testing.
 Patient should rest for 15-30 minutes before sample collection.
* Should discuss with the clinical team.
 Sample collection
 Tube – siliconized glass tubes or polypropylene plastic tubes.
 Syringes – plastic syringes.
 Anticoagulant – 3.2% trisodium citrate
 Blood anticoagulant ratio – should be (9: 1)*
* if trisodium citrate is in excess, platelet response could be lower: ADP and Collagen could be most sensitive. Citrate
volume should be adjusted if HCT > 55%. (Refer the procedure in 5.1)
 Sample volume – 9 mL blood (Citrate 1 mL: blood 9 mL)

In patients with difficult venepuncture or unable to take large volume of blood (e.g. very small children), half
volume samples could be taken (Citrate 0.5 mL: blood 4.5 mL).
 Venepuncture
 Blood should be collected using standardized, atraumatic, clean venepuncture procedure.
 Blood is added to the containers in following order;
i. Sample for platelet aggregation study (PAS) - 9 mL
ii. Sample for FBC - 2 mL
iii. Non EDTA blood picture is prepared with the remaining blood in the syringe
 Specimen for PAS should be capped immediately and gently mixed by six complete inversions.

 Sample requirements
 Clotted, icteric, lipaemic, red cell contaminated and haemolysed samples should not be processed as these
factors can interfere with platelet aggregation.
 All procedures like sample transport, processing and testing should be done at room temperature -RT
(20 - 25 °C) and samples should not be refrigerated or kept in ice.
 All sample tubes should be kept capped.
 All samples should be handled with minimum disturbances.
 Sample testing procedure should be completed between 30 minutes- 4 hours from time of blood collection.
 Sample preparation
 After bleeding, samples should be kept undisturbed for 20 minutes at RT before processing.
 To prepare PRP- centrifuge the samples at 170 g for 15 minutes in a swing out rotor bucket at room
temperature (20 - 25 0C) without applying break at the end. Remove the top 2/3 of PRP carefully without
disturbing the buffy coat & red cells using a plastic pipette. PRP is collected to polypropylene tubes, with
limited surface area –to-volume ratio and should be kept well capped.
 To prepare platelet poor plasma (PPP)- centrifuge the remaining sample at 1500 g for not less than
15 minutes at RT. Remove the top 2/3 of PPP carefully without disturbing the buffy coat and red cells
using a plastic pipette, collect into polypropylene tubes and keep capped. PPP should be platelet free
(platelets < 10 x 10 9/L).
 For those samples with a high platelet count in the FBC- a platelet count should be done on the PRP.
 If the PRP platelet count > 600 x 109/L, should adjust to a platelet count of 200 - 250 x 109/L using physiologic
buffer.
 If platelet count of the test sample is < 150 x 10 9/L, adjust the control sample with physiological buffer to
obtain a comparable platelet count.
 Once prepared, PRP tubes should be left undisturbed at RT for at least 30 minutes prior to testing.
 PRP samples which are icteric/ lipaemic / red cell contaminated / haemolysed with visual inspection should
not be tested.
 Maintaining physiological pH of PRP is important as it is essential for platelet aggregation. If CO2 diffuse into
the ambient environment the pH can change. (Should be kept capped, collected into limited surface area-
to-volume ratio tubes, avoid frequent mixing and agitation during testing, plasma should be introduced
directly into the bottom of the tube)

1. Platelet aggregometer (this SOP is for the AggRAM-Helena platelet aggregometer)
2. Centrifuge
3. Agonists – should refer to manufacturer recommendations.
 ADP low dose: 0.5 µmol/L.
 ADP high dose: 10 µmol/ L (this is called the threshold dose. individual labs should adjust their own ADP high dose
according to the response. Normal response should show primary and secondary waves clearly which indicate
dense granule release).
 Collagen – 2 µg/mL.
 Arachidonic acid- 2 µmol/L.
 Ristocetin high dose - 1.2 mg/mL.
 Ristocetin low dose - ≤ 0.5 mg/mL (this is used if vWD type 2b is suspected).

Method
 Clean cuvettes should be used. During the procedures, cuvettes should be handled by holding the upper part
avoiding contamination of the optical pathway.
 Check the aggregometer- optical channel using distilled water.
 Put the cuvettes in the ambient section of the module.
 Use the PPP cuvette blank to set 100% aggregation and PRP cuvette for 0% aggregation.
 Test samples- Pipette 200 µL of each sample of PRP into a cuvette with a stir bar. Incubate the PRP cuvettes at 37 0C
for 2 minutes. Reverse pipetting is recommended. Remove any air bubbles by tapping.
 Check for spontaneous platelet aggregation.
 Adding agonists- pipette 20 µL of agonist into the cuvette directly to the PRP and press the channel button to begin
timing of the final reaction.
 It is important not to introduce any air bubbles at any stage of the procedure.
 Aggregation response should be observed initially for 5 minutes and if there is a response, should observe for
10 minutes.
 The response curves are observed for lag phase, shape change (negative deflection), primary and secondary
aggregation, delayed platelet responses and disaggregation. Each sample is continuously measured and plotted on
the chart, displaying each channel in a different color.
Interpretation / normal curve patterns

(If there is spontaneous platelet aggregation, it can interfere with interpretation of results)
 Individual laboratory should derive own reference values of maximum amplitudes for each agonist.
 Ristocetin low dose – should be 0% response (absent response).
 Collagen – lag period should be <60 seconds.
 ADP low dose - primary wave, which could be completely reversible.
 ADP high dose- biphasic wave showing primary and secondary response.
 Mixing studies (spontaneous platelet aggregation may interfere with mixing studies). Mixing studies are done in two
instances.
i. If the platelet aggregation curve pattern is suggestive of either vWD or BSS (absent response to ristocetin-high
dose with normal response to all the other agonists).
 Make a 50:50 mixture of patient PRP and cryoprecipitate (if cryoprecipitate is not available, may use PPP of
the normal control) and repeat the test with ristocetin-high dose.
 If there is corrected response (curve showing improved response from the earlier zero response), it is
suggestive of vWD. However, final diagnosis of vWD is by vWD test profile.
 If there is zero response with the mixing study also, it is suggestive of BSS. This should be correlated with
other features +/- confirmed by flow-cytometry.
ii. If platelet aggregation curve pattern is suggestive of either vWD- type2b or vWD-platelet type (exaggerated
response to low dose ristocetin with normal response to all the other agonists)
 Make a 50:50 mixture of patient PPP and washed normal control platelets and repeat the test with low dose
ristocetin. If exaggerated response is still noted, it is suggestive of vWD type2b.
Control procedure- Testing of 50:50 mixture of normal control PPP and washed normal control platelets with
low dose ristocetin should not show an exaggerated response.
 Cryoprecipitate challenging test- cryoprecipitate is directly added to the washed platelets of the patient and
if there is spontaneous platelet agglutination, this is suggestive of vWD- platelet type.
Control procedure- cryoprecipitate is directly added to the washed platelets of the normal control.
There should not be any spontaneous platelet agglutination.

 Further confirmatory tests
 Flow-cytometry – to study absence of a certain antigen on platelet membranes (e.g. GPllb/llla glycoprotein
receptors in Glanzmann thombasthenia)
 ATP release studies
 Assessment of ADP:ATP ratio
 Electron microscopic studies of platelets
 Genetic & molecular testing

Normal results - reported as “Normal platelet aggregation studies”.
Abnormal results – reported as “Abnormal platelet aggregation study”.
Further comments will be made by the consultant haematologist considering the platelet aggregation curve pattern and
the clinical details.

 There is no commercial IQC available. A normal control should be tested with each test batch. This normal control
should be treated the same way as the test samples/procedures.
 Abnormal results should be repeated with the same sample.
 All unexpected results should be repeated on a fresh date with strict patient preparation.
 Check whether the analyzer is actually standardized to PPP and PRP. Set 100% transmission with PPP and the baseline
(0%) with PRP. Remove the PRP tube and insert a second PPP tube. Check whether the chart deflect to 100% .
 Test linearity, by setting 0% transmission (PRP) and 100% transmission (PPP). Insert a tube of 1:1 mixture of PRP / PPP
and the chart should deflect to 50% transmission.
 The aggregation response should not exceed 100%. If curve goes beyond 100% check whether platelet count in PPP
> 10 x103 /µL or calibration failure.
 Analyzer should be calibrated according to the manufacture recommendations.
Sources of error
 Poor patient preparation
 Excessive handling of platelets
 Change in recommended temperature and pH
 Delay in processing

 All parts and surfaces of the platelet aggregometer should be considered as potentially infectious since the instrument

Table 7.5.1 List of drugs, and dietary components/ herbs that can affect platelet function.
Cyclo-oxygenase (COX)-1 aspirin and other preparations containing

inhibitors (irreversible) acetylsalicylic acid.

COX-1 and COX-2 inhibitors ibuprofen, indomethacin, naproxen, mefenamic acid
(reversible) NSAIDs
Inhibitors of platelet receptors abciximab, tirofiban, eptifibatide (αIIb3), ticlopidine,

clopidogrel, prasugrel (irreversible), cangrelor
(reversible), ticagrelor, (reversible) (P2Y12)
Phosphodiesterase inhibitors dipyridamole, cilostazole
Anticoagulants heparinoids, vitamin K antagonists and direct

Drugs thrombin inhibitors may indirectly influence platelet
function due to inhibition of thrombin.
Cardiovascular agents β-adrenergic blockers (propranolol), vasodilators

(nitroprusside, nitroglycerin), diuretics (furosemide),
calcium channel blockers
Antimicrobials β-lactams (penicillins, cephalosporins), amphotericin

(antifungal) hydroxychloroquine (antimalarial),
nitrofurantoin
Chemotherapeutic agents asparaginase, plicamycin, vincristine
Psychotropics and anaesthetics tricyclic antidepressants (imipramine),

phenothiazines (chloropromazine), local and general
anaesthesia (halothane)
Thrombolytic agents streptokinase, urokinase, tissue plasminogen

activator (TPA)
Food/herbs alcohol, caffeine (methylxanthine), cumin, dong quai, fenugreek, garlic, onion, ginger,
(at high ginseng, fish oil, tamarind, turmeric, willow, vitamins C and E, black tree fungus (“chinese
concentrations) mushroom”)
Miscellaneous clofibrate, dextrans, guaifenesin (expectorant), radiographic contrast media
N.B.: This is not a complete list and many other agents are also known to affect platelet function. A full drug and relevant
dietary history should always be taken for each subject tested for platelet function. If abnormal results are obtained,
then retesting can confirm if any defect is transiently acquired or not.
 References
1. CLSI guidelines- Platelet Function Testing by Aggregometry; Approved guideline, H58-A, Vol 28 No 31.
2. Dacie and Lewis Practical Haematology, 12th edition.
3. Harrison P, Mackie I, Mumford A, Briggs C, Liesner Ri, Winter M, Machin S and British Committee for Standards
in Haematology. Guidelines for the laboratory investigation of heritable disorders of platelet function, British
Journal of Haematology, 2011, 155, 30-44.

7.6
INHIBITOR SCREENING
(BASED ON APTT)
Scope
The scope of this SOP is to guide technical personnel on proper performance of inhibitor screening based on APTT by
providing the protocol and requirements of this test.
Responsibility
Inhibitor screening based on APTT as per the procedure stated in this SOP is the responsibility of all medical laboratory
comments should be made by the consultant haematologist.
Principle
Inhibitors are antibodies that inhibit the action or increase clearance of coagulation proteins. Prolonged APTT could be
due to either factor deficiency or inhibitors. Depending on the time taken for inhibition in vitro studies, inhibitors are
classified as immediate acting or late/delayed acting.
When plasma with an immediate acting inhibitor is mixed with normal plasma with 1:1 ratio, APTT will not be corrected.
However, when the inhibitor is late/delayed acting APTT will be corrected. But, after 1 or 2 hours this correction will be
lost and the APTT will become prolonged again. Therefore, to detect both types of inhibition, normal plasma and test
plasma samples are tested immediately after mixing and also after incubation at 37 0C for 120 minutes.
Antibodies or inhibitors against coagulation factors can be formed in individuals with haemophilia on factor replacement
therapy or as acquired inhibitors in patients with underlying malignancies, immune disorders etc. These inhibitors will act
against the relevant factor and cause prolonged APTT (or PT), depending on factor/s involved.
Usually, inhibitors developed against FVIII are time dependent (late/delayed acting), while inhibitors against FIX and
lupus anticoagulant are immediate acting.
Sample
Fresh platelet poor plasma (PPP) prepared as per standard protocol.

1. Coagulometer
2. Centrifuge
3. APTT reagent
4. CaCl2 0.025 M pre warmed to 37 0C
5. Control/ pooled normal plasma (PNP)
6. Khan tubes (plastics)
7. Stop watch (for manual method)
8. Water bath
9. Automated micropipettes (100 μL)
Method
 Perform APTT as per protocol on patient’s plasma, control plasma and 50:50 mix of patient and control plasma.
 Take three plastic Khan tubes and label.
 Place 0.5 mL of normal plasma in one tube (tube 1), 0.5 mL of the patient’s plasma in a second tube (tube 2) and a
mixture of 0.25 mL of normal and 0.25 mL of patient’s plasma in a third tube. (tube 3 - for incubated mix)
 Incubate the tubes for 120 min at 37 0C.
 Make a 50:50 mixture of the contents of tubes 1 and 2 into a fourth tube. (tube 4 - immediate mix)
 Perform APTT of the samples in the order of tube 1, 3, 4 and 2.
 Duplicate tests and get the mean values.
Note- FIX inhibitors can be measured in a system identical to that described above. Because FIX inhibitors act immediately,
there is no need for prolonged incubation; the mixtures can be assayed after 10 min at 37 0C.

More than 10 second difference between APTT values of incubated mix and immediate mix (APTT of tube 3 – APTT of
tube 4 >10 seconds) is taken as presence of a late/delayed acting inhibitor.
In the presence of an immediate acting inhibitor, both APTT values of immediate mix (tube 4) and incubated mix (tube 3)
will be prolonged.
Reporting format is as follows:
At 0 hour
APTT of test plasma............. seconds
APTT of contol plasma......... seconds
APTT of 50:50 mix of patient and control plasma......... seconds
2 hours after incubation
APTT of test plasma............. seconds
APTT of contol plasma......... seconds
APTT of 50:50 mix of patient and control plasma-immediate mix......... seconds
-incubated mix......... seconds
Comment:
Short acting/ long acting inhibitors are detected/ not detected.

 Check quality of sample on reception and apply rejection criteria. Monitor appearance and storage temperature of
tri-sodium citrate solution (if in house preparation of tubes).
 Daily run of normal and abnormal QC samples (prepared from pooled plasma or commercial IQA material) and
 Participation in an external QC programme.

Sources of error
 Improper labeling of tubes and mixing of samples
 Water bath temperature errors
 Timing errors

 References
2. Diagnosis of Heamophilia and other bleeding Disorders – A Laboratory Manual, Second Edition (2010).

7.7
QUANTITATIVE INHIBITOR ASSAY
(BETHESDA ASSAY)
Ms. L. Dhammika
Scope
The scope of this SOP is to guide technical personnel on proper performance of quantitative measurement of factor VIII
and IX inhibitors by providing the protocol and requirements of this test.
The description hereafter is for F VIII inhibitor assay. Modification of the method for FIX inhibitor assay is given at the
end of the SOP.
Responsibility
Performance of quantitative measurement of factor VIII and IX inhibitors in the procedure stated in this SOP is the
responsibility of all medical laboratory technologists who are assigned to perform this test in the haematology laboratory.
Interpretation, conclusions and comments should be made by the consultant haematologist.
Principle
When FVIII is added to plasma containing an inhibitor to FVIII and the mixture is incubated, FVIII will be progressively
neutralized. If amount of FVIII and incubation period are standardized, strength of the inhibitor may be defined in units
based on amount of added FVIII that is neutralized.
Dilutions of test plasma are incubated with an equal volume of plasma with known amount of factor VIII at 37 0C for
2 hours. At the end of the incubation period, residual FVIII is assayed and inhibitor strength is calculated from a standard
graph of residual FVIII activity versus inhibitor units.
In the Bethesda method, the unit is defined as amount of inhibitor that will neutralize 50% of 1 unit of FVIII in normal
plasma after 2 hours of incubation at 37 0C.
At low inhibitor titres (<1 BU) the classical Bethesda assay can result in false positives. The Nijmegen modified assay is
useful in this situation as it could detect zero levels of inhibition. The modifications done in this method prevents shifts in
pH and protein concentrations which can lead to changes in FVIII stability and inactivation. Here 0.1 M imidazole buffer
is used at pH 7.4 for buffering PNP to increase stability of FVIII and immune depleted FVIII-deficient plasma in the control
mixture for diluting test plasma instead of the glyoxalin buffer. The Nijmegen modified Bethesda assay is only required
when inhibitor value is low.

Antibodies (inhibitors) are formed against FVIII when individuals with deficiency of FVIII (haemophilia A) are treated with
FVIII. When inhibitors are formed, patients become refractory to standard treatment. Measurement of inhibitor level is
useful for confirming presence of inhibitors, to plan management and to monitor response to treatment of inhibitors.
This test can also be used for quantification of inhibitors in acquired haemophilia and monitoring efficacy of treatment.
Sample
Fresh platelet poor plasma (PPP) prepared as per standard protocol.

Manufacturer’s instructions for reagent and equipment should be strictly followed.
1. Water bath
2. Tubes
3. Coagulometer
4. Centrifuge
5. Standard plasma
The standard plasma used can be either a locally prepared PNP (FVIII level should be > 80%) kept at - 70 0C or lower,
or a commercial standard plasma.
The factor VIII level of the PNP should be available. It is not acceptable to assume that a PNP has 100 U/dL FVIII: C.
6. FVIII- deficient plasma
Commercially available or from a donor whose factor level is <1.0%, has no anti-factor VIII antibodies, has not
received treatment for two weeks, and has normal liver function. It is preferable to use commercially available
immune depleted factor VIII-deficient plasma.
7. Reagents for APTT
8. Owren’s buffered saline or glyoxalin buffer – pH - 7.4 (0.1 M imidazole buffer)
Method
It is always better to perform an inhibitor screening before an inhibitor assay. According to the difference between
incubated mix and fresh mix, the test plasma dilution series can be set up. But it is preferable to put up too many
dilutions than too few. They can always be kept on ice after 2-hour incubation for a second run of assays.
 Prepare doubling dilutions of test plasma starting from 1/2, using factor assay buffer (imidazole or glyoxalin buffer)
in plastic tubes.
 Standard plasma is added to each dilution of test plasma.
Patient plasma doubling dilutions + standard plasma
1 /2 1 /4 1 /8 1/16 1/32 1/64 1/128 1/256 1/512 1/1024

Note: -FVIII is already 1:2 diluted
 A control tube with equal amount of standard plasma and buffer (or FVIII deficient plasma) is set up.
Note- FVIII levels in the 1st tube (standard plasma + Buffer) should not be less than 80%.
 A tube with equal volumes of test plasma and standard plasma is also included.

Standard plasma + Buffer (or FVIII deficient plasma) Neat test plasma+ standard plasma
 All tubes are well mixed without frothing, capped and incubated at 37 0C for 2 hours in the water bath.
 After 2 hours FVIII assay should be performed without delay. If delayed all tubes should be kept at 4 0C until assay.
 In the assay of FVIII, the mix with standard plasma and buffer (or FVIII deficient plasma) is used as the standard
reference, and the FVIII concentrations of other mixtures are calculated against this. This is used as the 100%
reference in the assay.
 The residual FVIII level is measured in each tube, and the inhibitor level is calculated from a graph of residual FVIII vs.
inhibitor units (Bethesda chart).
 Calculation of results and interpretation

 The dilution of test plasma that gives a residual FVIII concentration nearest to 50%, but within the range of
30 – 60 % is chosen for calculation of the inhibitor.
Alternatively, calculate the result from each dilution and take average. Any residual FVIII of <25% or > 75%
should not be used for calculations of inhibitor level.
 Correct for assigned FVIII value of standard plasma.
 Correct for loss of FVIII:C over 2 hours of incubation.
 Read off inhibitor level corresponding to residual FVIII level obtained after the corrections using the graph or
table. (Figure 7.7.1 and Table 7.7.1)
 Then correct for the dilution by multiplying with dilution factor.
100 100% Residual = No inhibitor

90
80
50% RESIDUAL FVIII ACTIVITY (%)
70
60
50 50% Residual FVIII = 1.0 BU/mL
40
30
20
10
0 5 1.0 1.5 2.0 2.5 Figure 7.7.1
BETHESDA UNITS /mL Graph of % residual FVIII versus
inhibitor units

Table 7.7.1 Residual FVIII activity and corresponding Bethesda units
Residual Bethesda Residual Bethesda Residual Bethesda Residual Bethesda Residual Bethesda
FVIII(%) units FVIII(%) units FVIII(%) units FVIII(%) units FVIII(%) units
97 0.05 73 0.45 55 0.85 42 1.25 32 1.65
93 0.10 70 0.50 53 0.90 41 1.30 30 1.70
90 0.15 68 0.55 51 0.95 40 1.35 29 1.75
87 0.20 66 0.60 50 1.0 38 1.40 28 1.80
84 0.25 64 0.65 48 1.05 37 1.45 27 1.85
81 0.30 61 0.70 46 1.10 35 1.50 24 1.90
78 0.35 59 0.75 45 1.15 34 1.55 23 2.0
75 0.40 57 0.80 43 1.20 33 1.60
Example -
PNP is used as standard plasma. The results of FVIII measurement after 2 hour incubation are given below.
PNP + Buffer = 82%
PNP + Undiluted test plasma = 1.2% PNP + Test plasma1:32 dilution = 17.5%
PNP + Test plasma1:2 dilution = 2.7% PNP + Test plasma1:64 dilution = 33.9%
PNP + Test plasma1:16 dilution =11.1% PNP + Test plasma1:512 dilution = 72.2%
Note the dilution that gave a factor VIII assay closest to 50% is 1:128
Calculation of residual FVIII:C
 Select the dilution which is closest to 50% of FVIII.
1 / 128 dilution (52.8%)
 Correction for assigned FVIII value of PNP / commercial normal control plasma (89%)- 52.8 x 0.89 = 42.28%
(This correction is for manual factor assay. For automated analyzers, the assigned FVIII value of control plasma is
entered before calibration)
 Correction for loss of FVIII:C over 2 hrs. of incubation:-(100% → 82% - 1st tube)
Corrected residual FVIII activity(RA) = FVIII(TEST) x 100
FVIII(PNP)
= 42.28% x 100
82%
= 51.56 %
 Read corresponding Bethesda value for this residual FVIII activity - 51.56% RA = 0.95 BU
 Correct for the dilution

The dilution which has this Residual FVIII activity is 1/128,
Correction for the dilution factor - 128 X 0.95 BU 121.6 BU
Note:
For determination of anti-FVIII inhibitors in a sample containing greater than 5 IU/dL FVIII activity, the WFH recommends
that prior to testing, the sample be heated to 56 °C for 30 minutes and centrifuged at ambient temperature for a
minimum of 1700 g for at least 5 minutes.

Chromogenic Bethesda assay
This is recommended for,
 Quantification of low-titre anti-FVIII inhibitors
 Determination of FVIII inhibitor levels in patients receiving emicizumab. (engineered bispecific antibody used for the
treatment of haemophilia A that binds both human FIX/FIXa and FX/FXa and acts as a FVIII mimetic.). Emicizumab
affects all APTT-based laboratory tests and assays.
The chromogenic Bethesda assay is very similar to the standard Bethesda assay except the residual factor VIII is measured
using a chromogenic FVIII assay. For this follow manufacturer’s recommendations.
One chromogenic Bethesda unit is defined as the level of inhibitor/mL in the patient plasma sample that inactivates 50%
FVIII in 1 mL of normal plasma.
Tests for other factor inhibitors

FIX inhibitors can be measured in a system identical to FVIII inhibitor assay. Because FIX inhibitors act immediately, there
is no need for 2 hours incubation. The mixtures can be assayed after 5 or 10 minutes at 37 0 C.
The activity of an inhibitor against porcine FVIII concentrate can be measured by substituting porcine FVIII concentrate,
appropriately diluted in FVIII deficient plasma, for PNP.

Inhibitor level - ………BU

 FVIII assay should be accurate and precise.
 Normal and abnormal quality control plasma should be used.
 Participation in an EQA programme for inhibitor assays will ensure the accuracy of results.
 Plasma with known inhibitor level can be frozen (-40 0C for 3 months /-70 0C for 6 months) and can be used to
monitor precision.
Sources of error
 Errors during sample collection and preparation
 FVIII deficient plasma
 This should contain normal levels of von Willebrand factor (vWF). It has been shown that inhibitor titres are 30-50%
lower if the FVIII deficient plasma does not contain vWF.
 Errors in preparing the PNP and dilutions of plasma
 Incorrect calculation

 All clinical samples should be treated as possibly infectious and handled as per guidelines available in the laboratory
on sample handling.

 References
2. Diagnosis of Hemophilia and other bleeding disorders, A Laboratory Manual, World Federation of Hemophilia,
2010.
3. Clinical and Laboratory Standards Institute – 2011/ H-48-A.
4. Srivastava, A, Santagostino, E, Dougall, A, et al. WFH Guidelines for the Management of Hemophilia,
3rd edition. Haemophilia. 2020: 26 (Suppl 6):1- 158.

7.8
DILUTE RUSSELL’S VIPER VENOM TIME
(DRVVT)
Ms. L. Dhammika
Scope
The scope of this SOP is to guide technical personnel on proper performance of Dilute Russell’s Viper Venom Time
(DRVVT) by providing the protocol and requirements of this test.
Responsibility
Performance of DRVVT as per the procedure stated in this SOP is the responsibility of all medical laboratory technologists
who are assigned to perform this test in the haematology laboratory. Interpretation, conclusions and comments should
be made by the consultant haematologist.
Principle
Russell’s viper venom (RVV) present in lupus anticoagulant screening reagent (LA1) activates factor X, leading to a fibrin
clot in the presence of factor V, prothombin, phospholipid and calcium ions.
Lupus anticoagulant (LAC) prolongs clotting time by binding to phospholipid, and prevents action of RVV. Since RVV
activates factor X directly, defects in the contact system and factor VIII, IX or XI deficiencies will not influence the test.
Further, DRVVT bypass factor VII of the extrinsic pathway.
Lupus anticoagulant confirmation reagent (LA2) is similar to LA1, but contains a high phospholipid concentration.
The extra phospholipid counteracts the LAC and significantly corrects the clotting time.
Mixing tests may be useful to exclude factor II, V, X and fibrinogen deficiencies, which may prolong LA1 screening reagent
and LA2 confirmation reagent results. Mixing normal plasma with test plasma, replenishes any factors deficient in the
test plasma. If mixing test with LA1 is still prolonged, it indicates that an inhibitor (such as LAC) is present in the test
plasma.
Lupus anticoagulant are auto antibodies against negatively charged phospholipids, or complexes of phospholipids with
either beta 2 glycoprotein 1 or clotting factors such as prothrombin. They occur in various clinical conditions, especially
auto immune diseases and are now considered to be a significant risk factor in patients with otherwise unexplained
thrombosis. They are often present in women who have recurrent fetal losses or pregnancy morbidity. They have
traditionally been detected using phospholipids responsive clotting tests such as APTT, KCT, and DRVVT.

A rational approach to use LA1 and LA2 is shown below:
Suspected LA
Normal
LA screen (LA1) LAC not detected
Prolonged LA1
Corrected
Mixing study Factor deficiency
Not corrected
Corrected Not corrected
LA present LA Confirm (LA2) Other inhibitor
LA 1 ratio: LA2 LA 1 ratio: LA2
ratio>1.2 ratio<1.2
Sample
Blood should be collected before start of any anticoagulant drug or a sufficient period after its discontinuation.
Platelet count on PPP must be <10 x 109 /L. Recommend double centrifugation during preparation.

1. Coagulometer
2. Centrifuge
3. Water bath – 37 0C
4. Stop watch
5. Glass tubes -10 mm x 75 mm
6. Pipettes to deliver 200 µL, 1 mL, 2 mL or 5 mL
7. Pipette tips
8. Distilled water
9. Pooled normal plasma (Make platelet poor by double centrifugation)
9. Control plasma (Normal and abnormal)
10. Glyoxalin buffer- 0.05 mol /L, pH 7.4
11. LA1 screening reagent (contains: Russell’s viper venom, phospholipid, anti-heparin agents, calcium, buffers,
stabilizers, sodium azide and green dye.)
13. LA2 confirm reagent (contains: in addition to above, phospholipids in higher concentration and red dye.)
Reagent reconstitution
Check if there is evidence of vacuum when the vial is opened and the reagent appears dry. Reconstitute with the volume
of distilled water stated on the vial. Mix well by inversion to ensure complete re-suspension of the lyophilized material.
Storage instructions
Lyophilized reagents are stable at least until the expiry date stated on the label of vial when stored at 2 - 8 0C. Following
reconstitution, reagents can be stored for 8 hours at 37 0C, 24 hours at 20 - 25 0C, 48 hours at 2- 8 0C, and 1 month at -20 0C.
Method
DRVVT test can be performed by manual tilt tube method, using semi-automated coagulometers and by using fully
automated coagulometers.
Whatever the method used reference interval has to be verified for that method.

a) Manual tilt tube method
 Pre warm a slight excess of LA1 at 37 0C +/-0.5 0C in a reagent reservoir, allowing for 200 µL per test.
 Dispense 200 µL of test plasma into a glass tube and warm for 1 min at 37 0C.
 Add 200 µL of pre- warmed LA1 reagent to the plasma and record time, from the moment of addition of reagent to
a clotting end point.
 Perform LA1 of PNP in the same manner.
 If LA1 clotting time is within normal range, no further testing for LAC may be necessary.
 If LA1 clotting time result is equal or more than upper limit of reference range, the results should be considered
abnormal and investigated further by performing 50:50 mixing test with PNP.
 If LA1 clotting time of 50:50 mixture is not corrected (not within the reference range), proceed to LA2.
 Perform LA2 of test plasma in the same method as for LA1. If LA2 becomes normal within the LA2 reference range,
stop further testing.
 If LA2 is beyond upper limit of LA2 reference range, perform LA2 with 50:50 mixture of PNP and test plasma.
 Thrombin time has to be within normal range for result interpretation.
b) DRVVT by semi-automated and fully automated analyzers

 Protocols for LAC on most automated kits are available on request.
 Fully automated and semi-automated analyzers should be programmed for DRVVT screen and DRVVT confirm,
according to the reagent used.
 Appropriate quality control measures should be undertaken.
 Fully automated analyzers can be programmed to calculate the screen, confirm and normalized ratios, by putting the
values of DRVVT screen and confirm test values of PNP to the analyzer.
Calculate the screen ratio (LA1 ratio) : Test LA1
PNP LA1
Calculate the confirm ratio (LA2 ratio): Test LA2
PNP LA2
Calculate the normalized ratio : Screen ratio (LA1 ratio)
Confirm ratio (LA2 ratio)
When mixing tests done for BOTH LA1 and LA2:

Calculate the screen ratio : LA1 50: 50 mix
PNP LA1
Calculate the confirm ratio : LA2 50:50 mix
PNP LA2
Calculate the Normalized Ratio: Screen Ratio
Confirm Ratio
 Interpretation of results is done as in tables 7.8.1 and 7.8.2

Table 7.8.1 Interpretation of DRVVT
LA 1 LA2
Patient’s plasma Patient + PNP Patient’s plasma Patient +PNP Diagnosis
Normal LAC not detected
Abnormal Abnormal Normal LAC present (Ratio >1.2)
Abnormal Normal Factor deficiency
Abnormal Abnormal Abnormal Normal LAC present
(Ratio>1.2) + Factor deficiency
Abnormal Abnormal Abnormal Abnormal Other inhibitor
 With uncomplicated LAC, clotting times obtained with LA1 on mixtures and on neat patient plasma are abnormal,
while LA2 results are normal.
 Prolonged LA1 and LA2 results are obtained in patients with factor II, V and X deficiencies as well as those on warfarin
therapy. These defects correct with the addition of PNP.
 Plasma that contains both LAC and factor deficiencies remains abnormal in LA1 tests with neat patient plasma and
mixing studies with PNP whereas the mixing study with LA2 is normal due to the addition of factors in PNP.
 If addition of PNP fails to correct LA1 and LA2, an inhibitor directed against any of the factors II, V, or X may be
suspected and should show an abnormal PT result.
Table 7.8.2 Interpretation of normalized ratio
Normalized Ratio (NR) Interpretation

<1.10 Absence of LAC
1.10 – 1.19 Borderline results. Should be repeated
1.20 – 1.49 Weak LAC
1.50 – 1.99 Moderate LAC
≥ 2.00 Strong LAC
Each laboratory must determine its own reference intervals for the method (manual or automated) and the kit used. This
can be done by performing the test on 20 – 25 healthy normal individuals aged 18 – 55 years.

 DRVVT Test - Negative
Lupus anticoagulant not detected.
 DRVVT Test – Weakly / Moderately / Strongly positive
Lupus anticoagulant detected.

 Known normal and abnormal platelet depleted plasmas are used as controls and run with each batch of test plasmas.
Most kits supply positive control plasma samples (mild positive and strong positive).
 Each laboratory must determine its own range of normal and abnormal control values.
 The patient’s plasma must be treated exactly as the control plasma and LA1 and LA2 must be run at the same time.
 Ensure platelet count < 10,000/ µL in the sample.

Sources of error
 Blood collected while on anticoagulants.
 Inadequate platelet depletion in the test plasma.
 A delay in freezing plasma, if LAC testing is performed later.
 Inadequate thawing of frozen plasma before testing.

 References
2. Manufacturer’s guidelines -Siemens Health Care Diagnostics Products Germany.
3. Diagnosis of Thrombotic Disorders, International Society of Thrombosis and Haemostasis, Christian Medical
College, Vellore, India -2002.

7.9
KAOLIN CLOTTING TIME (KCT)
Ms. L. Dhammika
Scope
The scope of this SOP is to guide technical personnel on proper performance of Kaolin Clotting Time (KCT) by providing
the protocol and requirements of this test.
Responsibility
Performance of KCT as per the procedure stated in this SOP is the responsibility of all medical laboratory technologists
who are assigned to perform this test in the haematology laboratory. Interpretation, conclusions and comments should
be made by the consultant haematologist.
Principle
KCT is basically an APTT based test with kaolin used as a contact activator, without addition of phospholipid in the
reagent.
When APTT is performed in the absence of platelet substitute reagent, it is particularly sensitive to lupus anticoagulant
(LAC). If the test is performed on a range of mixtures of normal and patient’s plasma, different patterns of response are
obtained, indicating the presence of LAC, deficiency of one or more of the coagulation factors or the ‘lupus cofactor
effect’. It is a sensitive test to detect low titre LAC.
Lupus anticoagulant are auto antibodies against negatively charged phospholipids, or complexes of phospholipids with
either beta 2 glycoprotein 1 or clotting factors such as prothrombin. They occur in various clinical conditions, especially
auto immune diseases and are now considered to be a significant risk factor in patients with otherwise unexplained
thrombosis. They are often present in women who have recurrent fetal losses or pregnancy morbidity. Lupus anticoagulant
have traditionally been detected using phospholipids responsive clotting tests such as APTT, KCT and DRVVT.

1. Kaolin 20 mg/mL in Tris buffer, pH 7.4.
This may need to be reduced upto 5 mg/mL in some automated analyzers.
2. Normal platelet poor plasma (NPPP) - Make platelet poor by double centrifugation
3. CaCl2 -0.025 mol/L
4. Water bath – 37 0C
5. Stop watch
6. Glass tubes - 10 mm x 75 mm
7. Pipettes to deliver 100 µL, 200µL, 1 mL, 2 mL or 5 mL
8. Pipette tips
9. Distilled water
Sample
 Platelet poor plasma (PPP) prepared as per standard protocol.
 Blood should be collected before start of any anticoagulant drug or a sufficient period after its discontinuation.
 Platelet count on PPP must be <10 x 109 /L. Double centrifugation during preparation is recommended.
Method
 This test is typically performed by a manual method.
 There are commercially available kits based on the KCT such as Kaoclot. These show high sensitivity to LAC, but not
suitable for testing patients receiving heparin. It has high sensitivity for prothrombin dependent LAC.
 Only the manual method is described here.
 Keep 2% kaolin suspension and CaCl2 in the water bath (cap closed). Kaolin has to be well mixed prior to use as kaolin
gets sedimented on standing.
 Add 200 µL of patient’s plasma (TPPP) to a khan tube and incubate for 1 minute.
 Add 100 µL of well mixed kaolin suspension and start the timer. Mix and incubate for 3 minutes, mixing gently twice
during this time.
 Add 200 µL of CaCl2 and mix again and start the timer.
 After 1 minute, start observing for clot formation.
 Stop the timer when the clot forms.
 If the clot has formed before 1 minute, that indicates platelet contamination or sample pre-activation. In that case
re-centrifugation or obtaining a fresh sample has to be done.
 Perform KCT of NPPP the same way. NPPP KCT should be within normal range (60 – 100 seconds).
 If the patient’s KCT is > 60 seconds, proceed for mixing tests with NPPP.
 Mix NPPP and TPPP in plastic tubes in the following ratios of NPPP to TPPP- 10:0 (neat TPPP, already done), 9:1, 8:2,
5:5, 2:8, 1:9, 0:10 (NPPP, already done).
 Perform KCT of above mixtures.
 Plot the clotting times against the proportion of normal to patient’s plasma on linear graph paper. (Figure 7.9.1)
 The pattern obtained for each patient should be critically assessed.

KCT (s) KCT (s)
200 200
100 100
Type 1 Type 2
0 0
0 10 20 50 80 100% 0 10 20 50 80 100%
200 200
100 100
Type 3 Type 4
0 0
0 10 20 50 80 100% 0 10 20 50 80 100%
Patient's plasma in normal plasma
Figure 7.9.1
Curves obtained using KCT
 Interpretation of different patterns of graphs in Figure 7.9.1
Pattern 1 (convex pattern) – indicates presence of LAC (positive result).
Pattern 2 – indicates presence of a coagulation factor deficiency and LAC.
Pattern 3 – indicates presence of LAC + deficiency of a cofactor necessary for the full inhibitory effect.
Pattern 4 (concave pattern) - indicates a negative result.
 The initial rate of slope in the graph is important because a steep slope indicates a positive result. This allows the test
to be simplified so that only the tests of 100% normal and of 80% normal 20% test plasmas need be performed.
 The slope can be calculated using the ratio of KCT at 20% test plasma and KCT at 100% normal control plasma.
Calculate the KCT ratio by - KCT (80% NPPP: 20% TPPP)

KCT (100% NPPP)

 Interpretation of results
KCT Ratio:
1.1 - 1.2: Equivocal, need repeat testing
1.2 - 1.5: Weakly positive
1.5 - 2.0: Moderately positive
>2 : Strongly positive

 KCT Test - Negative
Lupus anticoagulant not detected.
 KCT Test – Weakly / Moderately / Strongly positive
Lupus anticoagulant detected.

 Ensure platelet count < 10,000/ µL in the sample.
Sources of error
 Blood collected while on anticoagulants.
 Inadequate platelet depletion in the test plasma.
 A delay in freezing plasma, if LAC testing is performed later.
 Inadequate thawing of frozen plasma before testing.
 Alteration of pH of the buffer (buffer should be freshly prepared if KCT of NPPP >100 sec)

 All parts and surfaces of the equipment should be considered as potentially infectious since the instrument handles
patient specimens.
 References
2. Manufacturer’s guidelines -Siemens Health Care Diagnostics Products Germany.
3. Diagnosis of Thrombotic Disorders, International Society of Thrombosis and Haemostasis, Christian Medical
College, Vellore, India -2002.

7.10
ROTATIONAL THROMBOELASTOMETRY
(ROTEM)
Dr. Seuwandi Basnayake
Scope
The scope of this SOP is to guide technical personnel on correct performance of ROTEM test by providing the protocol
and requirements of this test.
Responsibility
Performance of ROTEM as per the procedure stated in this SOP is the responsibility of all medical laboratory technologists
who are assigned to this test in the haematology laboratory under the supervision of consultant haematologist.
Interpretation of results is the responsibility of the consultant haematologist.
Principle
A cup and pin method where a pin oscillates through a known arc and when the whole blood in the cup clots, the
resistance to movement that is created is picked up by changes in light transmission and a graphical representation is
made. (Figures 7.10.1 and 7.10.2)

Figure 7.10.1
ROTEM thromboelastometry detection method
In ROTEM,various activators or inhibitors are added to the sample, in order to examine different aspects of the
haemostatic system. (Table 7.10.1)

The main advantage of this test is to provide an immediate global assessment of clot formation, including the kinetics of
clotting, clot strength and fibrinolysis, guiding the targeted treatment of bleeding events. This is widely used in trauma
with major haemorrhage, cardiac surgeries, liver transplant and obstetric haemorrhages requiring massive transfusion.
This also helps in the rational use of blood and blood products, reducing cost and avoiding potentially harmful adverse
effects.
Sample
Citrate anticoagulated whole blood – should ideally be analyzed as soon as received at the laboratory.
Equipment and reagent

1. ROTEM machine
2. Pipette tips provided by the manufacturer
3. Reagents;
- Startem (recalcification reagent)
- Activator and inhibitor reagents*
Table 7.10.1 Reagents and functions of ROTEM tests
Test *Reagent Function of the reagent
INTEM Ellagic acid Standard clot formation - activating the intrinsic pathway
EXTEM Tissue factor Standard clot formation - activating the extrinsic pathway
HEPTEM Ellagic acid + Heparinase Heparinase degrades heparin. Shorter CT time compared to
INTEM suggests presence of heparin
FIBTEM Tissue factor + Cytochalasin C Platelet inhibitor added: Shows the contribution of
fibrinogen to clot formation independent of platelet effect.
APTEM Tissue factor + Aprotinin Fibrinolysis inhibitor added: When compared to EXTEM, higher
MCF and correction of ML indicates hyperfibrinolysis.
Method
The method described below is for the ROTEM delta with liquid reagents analyzer.
 Reagent should be brought to room temperature.
 Switch on the machine.
 Properly attach the pin.
 Insert cup and bring to position.
 Select test and enter patient data.
 Follow the pipetting steps displayed on the screen.
 Insert the cup holder in measurement position.
 The TEMograms and numeric parameters will be displayed on the screen.
 Print the TEMogram (Complete analysis may take up to a maximum of one hour).

ROTEM tracing
Figure 7.10.2
ROTEM tracing
ROTEM parameters
Following are the 4 main parameters used in the interpretation:
CT (Clotting time) – Time from start of measurement until initiation of clotting.
Initiation of clotting, thrombin formation and start of clot polymerization.
CT is broadly similar to the PT (in EXTEM) or APTT (in INTEM)
CFT (Clot formation time) – Time from initiation of clotting until a clot firmness of 20 mm is detected.
Fibrin polymerization, stabilization of the clot with platelets and FXIII.
MCF (Maximum clot firmness) – Measures the clot strength often demarcated at 5 min intervals
Increasing stabilization of the clot by polymerized fibrin, platelets and FXIII.
ML (Maximum lysis) – Reduction of the clot strength after MCF in relation to MCF
Stability of the clot (ML <15%) or fibrinolysis (ML>15% within one hour).
ROTEM tests
EXTEM : Activation of clot formation by thromboplastin (tissue factor).
Assessment of factors VII, X, V, II, I, platelets, fibrinolysis
INTEM: Activation of clot formation via the contact phase.

Assessment of factors XII, XI, IX, VIII, X, V, II, I, platelets, fibrinolysis
FIBTEM: Activation as in EXTEM with the addition of cytochalasin D, a platelet blocking substance. In the FIBTEM
assay, fibrinogen levels and fibrin polymerization can be assessed in a functional way.
APTEM: Activation as in EXTEM with the addition of aprotinin or tranexamic acid, fibrinolysis inhibitors. In an
assay comparing APTEM to EXTEM, fulminant hyperfibrinolysis can be recognized within 10-20 minutes.
HEPTEM: Activation as in INTEM with the addition of heparinase. Heparinase degrades heparin. When HEPTEM
results are compared to INTEM, heparin related coagulation disturbances can be specifically detected.
Reference interval
 Viscoelastic haemostatic assays are poorly standardized.
 Ideally, hospitals should determine local reference intervals.
 In the absence of local reference intervals, manufacturers’ reported reference intervals are currently used.

Examples:
1. Normal ROTEM traces
2. Abnormal ROTEM traces

2.1 CTINTEM Prolonged – Suggests heparin influence or intrinsic pathway factor deficiency
CTHEPTEM Normal – Confirms the heparin influence
CTHEPTEM Prolonged – Confirms the intrinsic pathway factor deficiency (F XII, XI, IX, VIII, X, V, II)
2.2 A10/A20/MCFINTEM/EXTEM reduced – Suggest inadequate clot firmness as a result of decreased fibrinogen, platelets and
platelet dysfunction. Small reduction can be seen in F XIII deficiency
MCFFIBTEM normal – Thrombocytopenia/ platelet dysfunction, normal fibrinogen
2.3 MCFFIBTEM reduced – Suggests inadequate fibrinogen contribution to clot firmness

2.4 MLINTEM/EXTEM/FIBTEM > 15% - Suggests hyperfibrinolysis
MLAPTEM normal – Confirms hyperfibrinolysis as APTEM has a fibrinolytic inhibitor.
2.5 CFTINTEM/EXTEM shortened/ MCFINTEM/EXTEM elevated – Suggest a hypercoagulable state

Report should include the ROTEM tracing and interpretation with recommendations for correction of coagulopathy.

 Follow machine operating procedure to prevent operator variation.
 Staff should be trained and have good pipetting technique. Training and competency should be documented.
 Reagents should be stored according to manufacturer’s recommendations.
 Internal Quality Control (IQC) -
Frequency of IQC should be decided according to the number of tests performed. Manufacturers recommend weekly
quality control checks.
 External Quality Assurance (EQA) -
Participation in an accredited external quality assurance (EQA) programme is recommended
Sources of error
 Specimen older than 4 hours
 Incorrect proportion of anticoagulant
 Incorrect type of anticoagulant
 Haemolysed / partially clotted sample
 Interference from vibration

 All parts and surfaces of the ROTEM machine and other equipment should be considered as potentially infectious
since the instrument handles patient specimens.
 References
1. The use of viscoelastic haemostatic assays in the management of major bleeding, A British Society for
Haematology Guideline; 2018; British Society for Haematology and John Wiley & Sons Ltd: 10.1111/bjh.15524.

7.11
ANTI FACTOR Xa LEVEL
Dr. Anoma Weerawardana
Scope
The scope of this SOP is to guide technical personnel on performance of anti-factor Xa (anti-Xa) level and to indicate
requirements for its correct performance by providing the protocol and requirements of this test.
Responsibility
Performance of anti-Xa level measurement as per the procedure stated in this SOP is the responsibility of all medical
laboratory technologists who are assigned to perform this test in the haematology laboratory. Interpretation, conclusions
and comments should be made by the consultant haematologist.
Principle
Note: This SOP is prepared for anti-Xa assay for monitoring of heparin using Elite Pro analyzer. While this can be used as
a general guide, users of other analyzers should follow manufacturer instructions for the test procedure.
This is a one stage chromogenic assay based on a synthetic chromogenic substrate and on factor Xa inactivation. Heparin
levels in patient plasma are measured automatically on IL Coagulation Systems. Heparin is analyzed as a complex with
antithrombin present in the sample. The concentration of this complex is dependent on the availability of the patient’s
endogenous antithrombin. When the heparin – antithrombin complex is formed, two competing reactions take place.
First, factor Xa is neutralized by heparin-antithrombin complex and the residual factor Xa is quantified with a synthetic
chromogenic substrate. The paranitroaniline released is monitored kinetically at 405 nm and is inversely proportional to
the heparin level in the sample.
Plasma anti-Xa assay is a functional test that is used for monitoring patients on low molecular weight heparins (LMWHs),
unfractionated heparin (UFH) and fondaparinux. It can also be used to measure direct oral anticoagulants (DOACs) that
have anti-Xa activity.
Sample
Sample should be collected 4 hours after administration of subcutaneous LMWH, 6 hours after subcutaneous UFH
and 6 hours after dose change of UFH infusion.

The SOP is based on the test performed on ACL Elite Pro platform using liquid anti-Xa test kit.
1. Coagulometer
2. Centrifuge
3. Factor Xa reagent (Cat. No. 0020302610): 5 x 2.5 mL vial of a liquid preparation containing purified bovine factor Xa
(approximately 5.5 nkat/mL), tris-buffer, EDTA, dextran sulfate, sodium chloride and bovine serum albumin.
Unopened reagents are stable until the expiration date shown on the vial when stored at 2 - 8 0C. Opened reagent is
stable for 1 month at 2 - 8 °C or 3 days on-board the ACL Elite Pro Systems in the original vial.
4. Chromogenic substrate (Cat. No. 0020302620): 5 x 3 mL vial of liquid chromogenic substrate S-2732 (approximately
1.2 mg/mL) and bulking agent.
Opened reagent is stable for 1 month at 2 - 8 °C or 3 days on-board the ACL Elite Pro Systems in the original vial.
5. Heparin Calibrators Cat. No. 0020300600
6. LMW heparin Controls Cat. No. 0020300200
7. Cleaning Solution (Clean A) Cat. No. 0009831700
8. Cleaning agent (Clean B) Cat. No. 0009832700
Method
One-stage assay. Refer to the appropriate operator’s manual of the IL instrument for the complete assay procedure
instructions.
 Enable the test in the analyzer
 Go to Setup →Tests→ View/Define
 Click on the Show Enabled button to display all tests.
 Scroll down the test list and highlight the Anti-Xa test. Click on the Enable/Disable button to enable the test.
A check mark will appear in the enabled test column for this test.
 Press the Green Check to save and return to main database screen.
 Edit the test reference range/cutoff

 Go to Setup→ Tests→ View/Define
 Scroll down the list, highlight the Anti-Xa test, and press the Detail icon.
 Scroll down to the row for units in IU/mL and click on the Ranges button.
 Enter in the Min and Max values for the normal range.
 The reaction curve min and max may also be set. If the desired values are unknown, clear both the min and
the max to zoom curve display to full scale.
 Enter in the calibrator concentration

 Go to Setup→ Liquids
 Scroll down the Liquid ID column and highlight HepCal3.
 Scroll down the Used by column and highlight the Anti-Xa test.
 Click on the Assign Value button, and enter in the value 2.0
 HepCal 1 and HepCal 2 values are automatically calculated and do not need to be entered.
 Set up liquid positions
 The liquid positions for the Hep Cals(1,2,3), AnFXaRgt, and AnFXaSub are automatically assigned when the test
is calibrated, placed in a profile or run as a single assay.
 Refer to the materials map screen when running or calibrating the assay for placement.

 Setting up quality control
 Prior to setting up QC ranges, the QC material first needs to be defined.
 Verify that your system has the appropriate QC liquid defined. The anti-Xa test can utilize the following control
materials to verify assay performance: LMWH low control, LMWH high control
 Go to QC→ QC Review and Setup

 Scroll down the Liquid ID list and select the appropriate QC liquid. Press the Setup button to define.
 Configure the tests from the enabled tests list. Define the units, target mean, target SD and SD range for the test.
If the mean and SD range is not known, then initially leave it set to zero. Once the ranges are known, then you
can re-define and click on the QC Range check button for control result flagging.
 Calibrate the assay

 Heparin calibrators must be reconstituted according to directions and allowed to sit for 30 minutes prior to
calibration.
 Go to Calibration→ Calibrate
 From the calibrate screen, select the Anti-Xa test from the drop-down menu in the test to calibrate window.
 Confirm the necessary liquids are in place according to the material map.
 Press the Run key to begin the calibrate cycle.
 When the calibration is complete the graph and calibration curve data will be visible. If the curve data is
acceptable, click on the check to accept the use of the curve.
(One universal calibration curve for UFH and LMWH with a linear range of 0.04–2 IU/mL).
 Run test sample

 Invert and mix both chromogenic substrate and factor Xa reagent
 Place anti Xa reagent, substrate and factor diluent in assigned positions according to material map.
 Select analysis →single test pre analysis →current single test→ anti Xa
 Place the sample in sample tray and select sample position in the map.
 Select program sample
 Enter sample ID, patient ID, patient’s name, age, sex and ward number
 Select Tests → anti -Xa
 Press run to start analysis
 Enter the next available cuvette number for the test.
 After test is completed, anti -Xa value of the test sample appear automatically on screen.
 Results are transferred to LIS if available.
 Results are printed and issued with authorization signatures.
Reporting results
Results are reported as : Anti-factor Xa level ………. IU/mL

 Each laboratory should establish its own mean and standard deviation.
 Each lab should establish a quality control programme to monitor laboratory testing. Two levels of controls are
recommended for a complete quality control programme.
 For optimal stability remove reagents from the system and store them at 2-80C in the original vial.
 For optimum on-board stability, laboratory temperature and humidity should be controlled.

Sources of error
 Incorrect timing of sample collection in respect to the last anticoagulant dose
 Analyzer calibration errors
 Out dated reagents
 Low relative humidity is associated with increased evaporation of uncapped
reagents, which may decrease on-board stability.

 All clinical samples should be treated as possibly infectious and handled as per
guidelines on sample handling available in the laboratory.
 All parts and surfaces of the coagulometer should be considered as potentially
infectious since the instrument handles patient specimens.
 Appropriate personal protective gear should be worn when handling samples and
reagents.
 Disposal of waste should be done according to local, provincial or national
regulations.
 References
1. Operator manual ACL Elite Pro analyzer.
2. Liquid Anti-Xa Level on the ACL TOP, Stony Brook university medical centre.

SECTION 08
HIGHLY SPECIALIZED
HAEMATOLOGY TESTS

BONE MARROW EXAMINATION

IDENTIFICATION AND QUANTIFICATION OF HAEMOGLOBIN BY
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

IDENTIFICATION AND QUANTIFICATION OF HAEMOGLOBIN BY
CAPILLARY ELECTROPHORESIS

FLOW-CYTOMETRIC IMMUNOPHENOTYPING (FCM) FOR
LEUKAEMIA/LYMPHOMA/MYELOMA

FLOW-CYTOMETRIC IMMUNOPHENOTYPING FOR
PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA (PNH)
8.1
BONE MARROW EXAMINATION
Scope
The scope of this SOP is to guide medical officers on proper and safe performance of bone marrow examination (BME)
and the medical laboratory technologists on technical aspects of preparation of bone marrow (BM) smears.
Responsibility
Necessity of a BME on a patient should be determined by the consultant haematologist in collaboration with the relevant
clinicians, if necessary. Performance of BME as per the procedure stated in this SOP is the responsibility of consultant
haematologists or medical officers with special training in this procedure under the supervision of a consultant
haematologist. However direct supervision will not be necessary once competency is determined. Medical laboratory
technologists in haematology section of the laboratory are responsible for preparation of BM smears as per this SOP.
Principle
BME consists of two procedures namely bone marrow aspiration (BMA) and bone marrow trephine biopsy (BMT). BMA
is carried out principally to permit assessment of cellular morphology and enumeration of marrow cellular elements.
In BMT, a core of bone marrow is obtained to assess general BM architecture, cellularity, bones, vessels, stroma,
haemopoietic and any lymphoid or other tissues. If indicated, further specialized investigations can be carried out on
BMA and BMT. The aspirate and trephine biopsy specimens are complementary and when both are obtained, they
provide a comprehensive evaluation of the BM.
Indications for bone marrow examination

1. Investigation of unexplained cytopenias of the blood such as anaemia, leukopenia, thrombocytopenia presenting in
isolation or in combination as bicytopenia or pancytopenia.
2. Investigation of unexplained sustained increase of blood cells such as erythrocytosis, leukocytosis, thrombocytosis,
suspected myeloproliferative disorders and leukemias.
3. Investigation of a suspected haematological malignancy.
4. To monitor response to therapy in haematological malignancies.
STANDARD OPERATING PROCEDURES FOR HIGHLY SPECIALIZED HAEMATOLOGY TESTS 203

5. Investigation of abnormal morphologic changes observed in peripheral blood, i.e., tear drop poikilocytes, rouleaux
formation, presence of immature myeloid or lymphoid cells, leucoerythroblastosis and unexplained micro/
macrocytosis.
6. To evaluate BM involvement by suspected metastatic diseases.
7. Investigation of paraproteinemias / suspected plasma cell neoplasms.
8. For staging of lymphomas (if results of investigation will alter management)
9. To obtain cellular BM for cytochemistry, flow cytometry, cytogenetics, molecular genetics, immunohistochemistry,
microbial cultures, electron microscopy, and tissue culture.
10. As part of investigation for fever of unknown origin, to assess bone marrow involvement in infections, in
granulomatous disorders and in patients with unexplained lymphadenopathy and hepatosplenomegaly.
11. To evaluate BM involvement in suspected storage diseases and collagen vascular disorders.
12. To assess unexplained osteosclerosis and other abnormalities of trabecular bone detected by radiologic studies.
13. Investigation of suspected primary amyloidosis.
14. Investigation of suspected chromosomal disorders in neonates.
15. Confirmation of normal bone marrow in the donor, when bone marrow is being aspirated for allogeneic
transplantation (only in special situations if clinically relevant).
Contraindications/ precautions for bone marrow examination

1. Sternal aspirate is absolutely contraindicated in patients with diseases associated with marked osteoporosis, bone
resorption including multiple myeloma and in children.
2. Poor patient cooperation can be a contraindication, unless BME can be arranged under sedation or general
anaesthesia.
3. Isolated severe thrombocytopenia is not a contraindication to bone marrow examination if the procedure is
performed by a skilled clinician and technical difficulties are not encountered. Post procedure prolonged pressure
is indicated to achieve primary haemostasis. If technical difficulties are anticipated (e.g. obese patients with severe
thrombocytopenia) platelet transfusions to increase the platelet count above 15 x 109/ L is advisable.
4. Factor replacement therapy prior to the BME and hospital observation for 24 hours after the procedure may be
indicated in patients with acquired or congenital coagulation abnormalities.
5. If there is skin/ soft tissue infection or recent radiation therapy at the sampling site, a different site should be
chosen.
Equipment, reagents and consumables

1. Procedure bed/ table
2. Surgical spot light for better illumination
3. Sterile towels, gauze and gloves
4. Face mask
5. Sterile artery forceps
6. Pair of scissors
7. Pressure bandage /elastoplast adhesive
8 11G scalpel blade
9. Syringes (3 mL, 5 mL, 10 mL and 20 mL)
10. Needles (26G, 22G and 21G)
11. Bone marrow aspiration and bone marrow trephine biopsy needles; choose the appropriate sized needle for the
patient. (Table 8.1.1)

Table 8.1.1 Commonly used needle sizes for BME
Bone marrow aspiration needles Bone marrow trephine biopsy needles
For adults 15 gauge x 2.8” or 8 gauge x 4” or 6” or

15 gauge x 4” 11 gauge x 4” or 6”
For children 16 gauge x 2.8” 13 gauge x 2” or 3”
12. Antiseptic solutions (0.5% chlorhexidine/ 10% povidone iodine & 70% isopropyl alcohol)
13. 1% or 2% lignocaine
14. Sample collection tubes with ethylene diamine tetra-acetic acid (EDTA)
15. Appropriate sample collection tubes with relevant anticoagulants for additional investigations planned
16. Container with 10% formal saline for bone marrow clot biopsy
17. Container with appropriate fixative for BMT (Bouin’s fixative solution with picric acid + formaldehyde + acetic acid
is commonly used in many centres)
18. Microscope slides (preferably with a frosted end)
19. Pasteur pipette
20. Spreader
21. Marker pen/ pencil for labelling
Method
a) Patient assessment
 Evaluate the detailed medical history and clinical features.
 Examination of a blood film, assessment of results of a full blood count, other laboratory tests and radiological
investigations.
 Assess bleeding risk and need for platelet transfusion, coagulation factor replacement or discontinuation/dose
reduction of anticoagulants or antiplatelets.
 Do an assessment of comorbidities to decide on the site from which the biopsy is performed and the chosen mode
of analgesia/ sedation/ anaesthesia.
 The site of procedure should be chosen on the basis of the age, mobility of the patient and prior radiotherapy to a
proposed biopsy site.
 Inquire regarding allergy to latex, local anaesthetic or any antiseptics or sedatives that might be used.
b) Delivering information and obtaining informed consent

 The patient or parent/ guardian (in case of a child) must be provided with sufficient information (verbally and/ or
in writing) to allow him or her to understand the risks and likely benefits associated with a BME and must obtain
informed written consent for the procedure.
 When BME is to be performed under a general anaesthetic or deep sedation, consent should be taken separately.
 Provide information regarding care of the site after biopsy.
 Where appropriate, use an interpreter to ensure that informed consent and patient information is communicated in
a language in which he or she is fluent.
c) Plan for special investigations on bone marrow samples

If additional/ special investigations are indicated on BM samples, make appropriate sample collection containers
available and a method of transportation of samples to relevant laboratories arranged at the time of procedure.

d) Common anatomical sites for bone marrow examination
1. Posterior iliac crest (usually posterior superior iliac spine)
2. Anterior iliac crest 2 cm posterior and 2 cm inferior to the anterior superior iliac spine
3. Antero-medial surface of the tibia, just below the level of the tibial tubercle. ( For BMA of children up to the age of
18 months and for BM biopsy of neonates*)
4. First part of the body of sternum or manubrium
(Sternal aspiration should only be performed by an experienced operator of this procedure)
5. Other sites where focal bone marrow disease is identified by diagnostic imaging
* Please refer the reference No.9
e) Preliminary/ preparatory procedures

 No special preparation of the patient is needed, if BME is to be performed under local anaesthesia (LA) and patient
is allowed take usual medication on the day of BME except any drugs (antiplatelets or anticoagulants) which were
advised to be withheld prior to the procedure.
 If BME is planned to be done under general anaesthesia (GA)/ sedation, patient should be kept fasting for a minimum
period of 6 hours for solid food and milk and 2 hours for clear fluids prior to the procedure and it should be carried
out as an inpatient procedure.
 If the procedure is planned to be done under GA/ deep sedation with ketamine and propofol, procedure will be done
in an operatiing theatre.
 If sedation (usually with midazolam) is planned in conjunction with LA, procedure can be carried out either at the
ward or in the haematology unit.
 A medical laboratory technologist (MLT) and a trained assistant should be present to prepare the slides immediately
before the aspirated bone marrow sample gets clotted and to apply pressure to the wound to ensure adequate
haemostasis.
 Check pre-procedure pulse rate, respiratory rate and blood pressure.
f) Undertaking the procedure

 Either the aspirate or the trephine biopsy may be performed first depending on individual preference or local
protocols. However, the aspiration needle and biopsy needles should be introduced keeping approximately 0.5 – 1 cm
distance between two sites to obtain satisfactory samples.
 Depending on the selected site for BME, patient should be appropriately positioned.
 Identify the site of the biopsy using anatomical land marks and mark the puncture point.
 BME should be carried out under strict aseptic conditions.
 Sterilize the site with 10% povidone-iodine (Betadine) and 70% isopropyl alcohol. For individuals allergic to iodine,
use 4% chlorhexidine gluconate (surgical scrub).
 Administering local anaesthesia

 Use appropriate dose of lignocaine without adrenaline (3 mg/kg - maximum total dose of 200 mg) or lignocaine
with adrenaline (7 mg/kg -maximum total dose of 500 mg) to give adequate local anaesthesia
 Buffering of lignocaine with sodium bicarbonate is recommended (1 mL of 8.4% sodium bicarbonate per 10 mL
of local anaesthetic) to increase effectiveness of anaesthesia.
 The adequacy of anesthesia should be ensured before proceeding.
 Bone marrow aspiration

 When the identified site is numb, make a small incision in the skin with an 11G scalpel blade through which the
BMA needle with a stylet locked in place is inserted.
 Holding bone marrow needle with stylet in place, apply steady, controlled pressure carefully with a twisting
motion to advance the needle through subcutaneous tissue.

 Once the needle contacts the bone, advance it by slowly rotating clockwise and counterclockwise until the
cortical bone is penetrated and the marrow cavity is entered.
 When the needle is firmly in place with a decrease in resistance, it signals entry into the spongy marrow cavity.
 Penetrate a little further within the marrow cavity not extending beyond 1cm.
 Then remove the stylet and quickly attach a 10/ 20 mL plastic syringe to the needle hub.
 Plunger of the syringe is vigorously pulled back to aspirate approximately 0.5 – 1 mL marrow into the syringe.
If suction pain is distressing, slowing of the rate of aspiration is indicated.
 The minimum amount of bone marrow needed for the tests indicated should be aspirated because the greater
the volume of marrow aspirated the more dilution by peripheral blood occurs.
 Hand over the syringe to the assistant quickly to prepare smears and a finger is held over the needle opening to
prevent spillage of bone marrow.
 Use approximately 0.5 mL of the first draw of marrow, to prepare BM films.
 Place the additional aspirate in an appropriate concentration of EDTA to provide material for further smears if
required. These should be prepared within two hours.
 If needed, additional marrow samples should be collected with a second syringe attached to the aspiration
needle.
 In the event of a ‘dry tap’ or if no particles (‘fragments’) have been obtained, repeat the BM aspirate at a slightly
different angle or another site.
 When an adequate marrow aspirate has been obtained, remove the aspiration needle and apply pressure to the
aspiration site with gauze until any bleeding has stopped.
 Preparation of aspiration slides

 If smears cannot be prepared at the bedside, immediately place the aspirated material into a tube containing
EDTA anticoagulant, for smear preparation upon returning to the laboratory (within 2 hours).
 Prepare several wedge‐spread films containing small bone marrow particles and smears of crushed marrow
fragments. (At least six smears and two squash/crush slides are recommended by ICSH).
 To prepare smears it is better to use glass slides with a frosted end for labelling.
 Expel the BM aspirate into a small plastic or siliconized glass dish /slanted surface, and use a Pasteur pipette /
spreader to draw up particles, which are placed on glass slides and then smeared.
 Alternatively, place a drop of aspirate on each glass slide about 1 cm away from the end of the slide and the
excess blood drained off the slide by tipping the slide, or aspirated with a Pasteur pipette or plastic syringe,
before making the smear.
 Make the smears with a glass spreader with beveled edges so that the width of the spreader is narrower than the
width of the specimen slide to facilitate examination of the edges of the bone marrow film. Place the spreader in
front of the drop of aspirate at an angle of approximately 300 and pulled back until it touches the drop, to enable
the drop to spread along the line of contact with the slide. Then push the spreader forward at a 300 angle using
a rapid, even motion. The smear should end in a particle-rich feathered edge.
 Spreading of the smear should always be towards the frosted end to ensure the possibility of examination of
the thinnest part of the film, where cytological details are optimal, while the slide is firmly positioned on the
stage. (Figure 8.1.1) Spreading away from the frosted end towards the end of the slide sometimes make it very
difficult to examine an important part of the film by high power because the slide is not stable on the microscope
stage. (Figure 8.1.2)

Figure 8.1.1 Figure 8.1.2
Stained bone marrow films showing a film of A film that is too long and has been spread,
appropriate length, spread towards the frosted incorrectly, away from the frosted end where
end where the label is applied the label is applied
 To make a squash/crush preparation, place a drop of BM containing particles at one end of a clean microscope
slide and a second clean slide is placed directly over the drop of aspirate. With gentle pressure press the second
slide against the drop of aspirated marrow and pull across the full length of the long axis of first slide to crush
open and spread the marrow particles. (Figure 8.1.3)
Figure 8.1.3
Squash/crush preparation
 Label BM smears and squash preparations at the bedside with the surname and first name or initial, unique
patient identifier and date. If it is frosted glass at one end, details can be written with a pencil. Otherwise use a
permanent marker / diamond pencil for labeling.
 Label all aspirate tubes as bone marrow and enter patient identification details into the label.
 Collect excess aspirate using a needle or a slide into a small clot to make particle clot preparations (‘clot biopsy’),
otherwise draw a small amount of aspirate material into a plain syringe and allow it to clot on its own. Clot biopsy
should be placed into a container with 10% formal saline. The container must be labelled at the bedside with the
patient identifiers and date. (Clot biopsy is beneficial in the event of an inadequate aspirate, particularly if a trephine
biopsy is not taken. These do not require decalcification and can be used to assess marrow cellularity, megakaryocyte
morphology or tumour infiltrates, iron stores and can also be used for immunohistochemistry or FISH.)
 Bone marrow trephine biopsy

 The BM trephine biopsy may be performed either before or after the aspirate, but majority perform it after BMA.
 It should be performed through the same skin incision site used for the marrow aspiration but the needle is
angled slightly away from the aspirate cortical bone puncture site to avoid obtaining a damaged or haemorrhagic
trephine biopsy.
 Follow the steps described under performance of bone marrow aspiration to insert the biopsy needle through
the skin and subcutaneous tissue until it reaches the marrow cavity.
 Due to the larger caliber of the bone marrow biopsy needle, more force is usually required than with the aspirate
needle.
 Then remove the stylet from the needle. Rotation of the needle is continued until it penetrates deep enough in
to the bone marrow.

 To determine the length of the biopsy specimen in the needle, the stylet can be carefully reinserted into the
needle until resistance is encountered. Ideally, the core length from an adult should be 2 cm or greater.
 Vigorously rotate the biopsy needle 3600 in both directions several times while applying slight pressure to cut the
core biopsy well. Decreased resistance to rotation usually indicates detachment of the core from the surrounding
bone.
 Then apply the lock, if a core-securing device is available, to secure the core biopsy inside the needle.
 Slowly pull out the needle with biopsy in. Then unlock the biopsy needle.
 Insert a blunt stylet through the distal end of the biopsy needle to slowly expel the core from the proximal end
of the biopsy needle onto a clean glass slide. The specimen should be handled gently to avoid crush artefact and
distortion.
 Ideally the length of the core from an adult should be at least 2 cm after processing, particularly if there is
suspicion of focal infiltration. (Note - Biopsy specimens shrink by about 20 - 25% during processing.)
 Perform a second biopsy, preferably from the contralateral iliac crest, whenever the length of the initial
unprocessed specimen is less than 1.6 cm and the results of the procedure would be likely to alter the
management of the patient.
 Determine the adequacy of a BMT sample by considering the total length of the trephine biopsy core(s) obtained,
the location and number of anatomic sites sampled, and the gauge of the biopsy needle.
 Touch imprints should be prepared routinely from the trephine biopsy prior to placing it in fixative. (Imprints are
helpful to examine morphology, cell composition and cytological details, especially if there is a ‘dry tap’ on BMA).
 Place the fresh core biopsy specimen on a clean microscope slide. A second clean microscope slide is pressed
very gently against the biopsy core and rolled slightly from side to side. Prepare two or three microscope slides
with several imprints on each slide. (Figure 8.1.4)
Figure 8.1.4
Bone marrow touch imprint
 Place the core biopsy specimen into a container with appropriate fixative. The container must be labelled at the
bedside with the patient surname, first name or initials, unique patient identifier and date and time of collection,
so that the time when the biopsy specimen should be removed from the fixative can be calculated.
 Modified technique of bone marrow biopsy from neonates & preterm infants
 Use a 19 gauge, half-inch Osgood needle (Popper and Sons, New Hyde Park, N.Y.) – (Figure 8.1.5)
 After cleaning and giving local anaesthesia introduce the needle into the marrow space through the periosteum
of the flat surface of the tibia, approximately 2 cm below the tibial tuberosity.
 Once the firmness of the needle position is felt that the needle had penetrated into the tibia withdraw the trocar
completely.
 Advance the (hollow) needle by twisting, into the marrow space an additional 2-3 mm (advancing the hollow
needle trephinates marrow spicules into the needle).
 Then attach an empty sterile 3 mL syringe (without a lure-lock) to the needle hub and apply suction for a few
seconds, until the first sign of marrow (about 50 µL) entered the syringe hub.
 Withdraw the syringe and the attached needle from the tibia and then withdraw marrow specimen within the
needle into the syringe, and the small drop allowed to clot within the hub of the syringe.

 Gently dislodge and remove the clot from the hub using the tip of a needle, and place into formalin fixative
solution.
 Then process, section, and stain this specimen in a manner identical to a typical bone marrow trephine biopsy,
except that decalcification is not required.
Figure 8.1.5
Osgood needle
(This extremely fine & short gauge needle is commonly used
for procedures on babies & neonates)
 Collection of peripheral blood samples

 Collect a peripheral blood sample into an EDTA tube for full blood count and smear, if these have not been done
within previous 2 days, as the bone marrow biopsy should be interpreted together with them.
 Preparation of a finger prick blood film at the time of BME is beneficial for better morphology.
 Processing of bone marrow samples

 Follow the relevant SOPs for staining of bone marrow aspirate slides, crush /squash preparations and trephine
imprints and histological processing of trephine and clot biopsies.
g) Aftercare
 After obtaining all samples needed, remove the needle with syringe attached with slight twisting motion.
 No suturing is needed for the incision.
 Maintain pressure over the site for approximately 2 minutes until bleeding has stopped.
 Clean the biopsy site and apply a tight dressing.
 Proper disposal of the disposable needles or sterilization of the reusable bone marrow needles should be done.
 The patient must remain lying down in supine position with their weight concentrated over the wound for at least
30 – 60 minutes and is observed for bleeding.
 Monitor the patient's pulse, respiratory rate, blood pressure, and temperature for one hour or until they return to
normal.
 Advise the patient to wear the dressing and keep the biopsy site dry for 24 hours. Avoid shower, bathing or swimming.
 The patient should be able to resume most normal activities immediately. However patients who have received a
sedative often feel sleepy for the rest of the day; so driving, cooking, and other activities which require clear thinking
and quick reactions should be avoided.
 Prescribe a pain killer such as paracetamol to ease any discomfort felt at the biopsy site, and ice can be used to
reduce swelling.
 Instruct the patient and/ or parents to remove the dressing after 24 hours, observing for signs of infection, unusual
bleeding, or any other drainage on dressing. If either is noted, he/ she should seek medical attention. If biopsy site is
unremarkable, patient can get the site wet after 24 hours.
h) Documentation & discharge of the patient

 Enter a detailed record of the procedure in the bed head ticket (BHT) in in-patients or in the clinic books in
out-patients.
 Discharging of the patient should be planned depending on the anaesthesia used and findings of post- procedure
monitoring of the patient.

Complications of the procedure
BME is generally a safe procedure. Complications are rare, but can include;
1. Excessive bleeding
2. Short term or long-lasting discomfort at the biopsy site
3. Reactions to anesthetic agents, antiseptics used or latex
4. Infection
5. Haematoma formation at the site / retroperitoneal haematoma / bleeding into abdomen, buttock and thigh
6. Cardiac tamponade, pneumothorax, pneumopericardium (unique complications of sternal marrow aspiration)
7. Trauma to neighboring structures

 Reporting is done by the consultant haematologist.
 Report should include description of cellular morphology, interpretation of special stains if done, conclusion and
recommendations with clinical correlation.
Sources of error
 Complete failure of bone marrow aspiration (‘dry tap’) or aspiration of only blood (‘blood tap’) due to technical
difficulties/ poor technique/ anatomical variations
 Haemodiluted aspirate due to aspiration of large volume of sample
 Too thick aspirate smears
 Small clots on aspirate smears
 Excessive pressure for particle crush preparations
 Less cellular bone marrow aspirate due to sample aspirating from same site of core biopsy sampling
 Inadequate trephine specimen – too small, too thin
 Core biopsy consisting of hypocellular subcortical region
 Crushed core biopsy specimen
 Inclusion of disrupted tissues in the trephine biopsy specimen, if it is taken from the same site where a very large
aspirate is taken previously

 Ensure the competency of personnel performing the bone marrow biopsy by training and periodic assessment.
 Ensure the standards of the equipment and consumables.
 Ensure the competency of medical laboratory technologists in preparation and staining of BMA smears, crush
smears and BMT touch imprints and histological processing of trephine and clot biopsies, by training and periodic
assessment.

 All clinical samples should be treated as possibly infectious and handled as per guidelines available in the laboratory
on sample handling.
 Has to be extremely cautious when storing and handling chemicals and reagents;
 Picric acid  explosive
 Acetic acid  corrosive
 Proper disposal of waste and chemicals should be done as per guidelines available in the laboratory.

 References
1. Bain BJ. Bone marrow aspiration. Journal of Clinical Pathology, 2001; 54:657–663.
2. Bain BJ. Bone marrow trephine biopsy. Journal of Clinical Pathology, 2001; 54:737–742.
3. Riley RS, Hogan TF, Pavot DR, Forysthe R, Massey D, Smith E, Wright L, Ben-Ezra JM. A Pathologist’s
Perspective on Bone Marrow Aspiration and Biopsy: I. Performing a Bone Marrow Examination. Journal of
Clinical Laboratory Analysis, 2004; 18:70–90.
4. Wysokinski WE, McBane RD. Periprocedural Bridging Management of Anticoagulation. Circulation,
2012;126:486-490.
5. Oprea AD, Popescu WM. Perioperative management of antiplatelet therapy. British Journal of Anaesthesia,
2013;111(S1): i3–i17.
6. Bain BJ, Bates I, Laffan MA, Lewis SM. Dacie and Lewis Practical Haematology, 12th ed. 2017,Elsevier Limited,
2017.
7. Bone marrow specimen (aspirate and trephine biopsy) structured reporting protocol (1st Edition 2014) RCPA.
8. Bain BJ, Clark DM, Wilkins BS. Bone marrow pathology, 5th ed. Wiley-Blackwell Publishing, 2019.
9. Sola MC, Rimsza LM, Christensen RD. A bone marrow biopsy technique suitable for use in neonates. British
Journal of Haematology, 1999, 107, 458 – 460.
10. https://www.drugs.com /dosage/midazolam.html
11. https://www.medicines.org.uk/emc/product/6045/smpc
12. Lee SH, Erber WN, Porwit A, Tomonaga M, Peterson LC, For the International Council for Standardization in
Hematology. ICSH guidelines for the standardization of bone marrow specimens and reports. International
Journal of Laboratory Hematology, 2008 Oct; 30(5):349–364.
13. AnaesthesiaUK.comHomeResourcesPharmacologyLocal anaetheticsPharmacology regional
anaethesia (https://www.anaesthesiauk.com/SectionContents.aspx?sectionid=235).
14. Ruegg TA, Curran CR, Lamb T, MSN, RN, CNP, AOCNP. Use of Buffered Lidocaine in Bone Marrow Biopsies: A
Randomized, Controlled Trial. Oncology Nursing Forum, January 2009; Vol.36 (1): 52 – 60.
15. Mayo clinic Bone marrow biopsy and aspiration, https://www.mayoclinic.org/tests procedures/bone-
marrow-biopsy/about/pac-20393117.
16. https://www.surgeryencyclopedia.com/A-Ce/Bone-Marrow-Aspiration-and-Biopsy.html.
17. Raida I. Oudat RI. Bone Marrow: Indications and Procedure Set-up. Jordan Medical Journal, 2010; Vol. 44
(1):88-94.

8.2
IDENTIFICATION AND QUANTIFICATION OF
HAEMOGLOBIN BY
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
(HPLC)
Dr. Swarna Gunathilake
Scope
The scope of this SOP is to guide technical personnel on proper performance of thalassaemia testing by the HPLC method.
HPLC method provides a presumptive diagnosis of haemoglobinopathy.
Note- This SOP is prepared for the Bio-Rad VARIANT II Haemoglobin Testing System. While this can be used as a general
guide, users of other analyzers/ systems should follow manufacturer's instructions for the test procedure.
Responsibility
Performance of HPLC as per the procedure stated in this SOP is the responsibility of all medical laboratory technologists
who are assigned to perform this test in `Haemoglobinopathy diagnostic laboratories`. The overall supervision and
reporting are the responsibilities of the consultant haematologist.
Principle
The system utilizes the principle of ion-exchange high-performance liquid chromatography (HPLC). A mixture of Hb-
molecules (normal and variants) with a net positive charge is separated into its components by their adsorption onto a
negatively charged stationary phase in a chromatography column, followed by their elusion by a mobile phase. Mobile
phase is a liquid with an increasing concentration of cations, flowing through the column: the cations in the mobile phase
compete with the adsorbed proteins for the anionic binding sites. Thus the adsorbed positively charged haemoglobin
molecules are eluted from the column into the liquid phase at a rate related to their affinity for the stationary phase.
When separated in this way, they can be detected optically in the eluate and provisionally identified by their retention
time and quantified by computing the area under the corresponding peak in the elusion profile. For each haemoglobin
type, there is a characteristic retention time (RT) before they appear in the eluate.
In the currently used HPLC system the samples are automatically mixed and diluted on the sampling station and injected
to the analytical cartridge. A programmed buffer gradient of increasing ionic strength is delivered to the cartridge, where
the Hb A2 & Hb F are separated based on their ionic interactions with the cartridge material. The separated Hb A2 &
Hb F then pass through the flow cell of the filter photometer, where changes in the absorbance at 415 nm are measured.
An additional filter at 690 nm corrects the background absorbance.

A sample report and a chromatogram are generated for each sample. To aid in the interpretation of results, windows
(ranges) have been established for the most frequently occurring haemoglobins based on their characteristic RT.
Minor differences in the separation efficiency of individual analytical cartridges are corrected by the use of the
Haemoglobin A2/F (HbA2/F) calibrator.
Haemoglobinopathies are genetic disorders of the haemoglobin molecule giving rise to reduced globin chain production
and globin chain imbalance (thalassaemia) and structural abnormalities of globin chains (variant haemoglobins).
These disorders are inherited as autosomal recessive. The heterozygotes are usually asymptomatic and homozygotes/
compound heterozygotes show a spectrum of clinical features depending on the type of defect.
Sample
Whole blood collected in EDTA tubes.
Sample transport & storage- optimum temperature: 2 – 8 °C. Samples could be stored for 7 days.

1. Equipment- Bio-Rad VARIANT II analyzer
2. Reagents- Elusion buffer 1 and 2- Sodium phosphate buffer with <0.05% sodium azide as the preservative
3. Haemoglobin A2/F calibrator- Lyophilized human red blood cell haemolysate with gentamycin, tobramycin and EDTA
as preservatives.
4. Whole blood primer - Lyophilized human red blood cell haemolysate with gentamycin, tobramycin and EDTA as
preservatives.
5. Wash/ diluent solution - Deionized water with <0.05% sodium azide as the preservative.
Method
 Specimen preparation- no sample preparation is required.
Note: Low volume samples may require predilution before analysis.
 Preparation and storage of reagents/consumables;
a) Elution buffers and wash/diluent solution
 Allow the elution buffers and wash/diluent solution to reach room temperature before performing the assay.
Mix each bottle by gently inverting prior to installation.
 The elution buffers and wash/diluent solution will be stable until the expiration date when stored unopened
at 15 – 30 °C. After opening bottles, these reagents are stable for 60 days when stored at 15 – 30 °C.
b) Whole blood primer
 Use whole blood primer at the beginning of each run to condition the cartridge for analysis.
 The whole blood primer will be stable until the date of expiry when stored unopened at 2 – 8 °C.
 Prepare whole blood primer by adding 1.0 mL of deionized water to each vial.
 Allow to stand for 10 minutes at 15 – 30 °C.
 Swirl gently to dissolve and ensure complete mixing.
 Write reconstitution date on the label. The reconstituted whole blood primer is stable for 21 days when
stored at 2 – 8 °C.
c) Haemoglobin A2/F calibrator- reconstitute and store the Hb A2/F calibrator according to the manufacturer's
instructions.
d) Controls- reconstitute and store controls according to the manufacturer instructions.
e) Analytical cartridge- the analytical cartridge should be stored at 15 – 30 °C.

 Analysis - Note; Manufacture instructions should be strictly followed in steps 1 - 4.
i) Step 1 - Installing a New Reorder Pack Lot – should refer to manufacturer instructions.
ii) Step 2 - New cartridge installation, priming, and temperature adjustment-
 For all new cartridges (0 injections), a double-priming and cartridge temperature adjustment must be performed.
 Run set up for priming - Please refer instruction manual (Run two reconstituted whole blood primer (1 mL) in
PRIMER adaptors, two deionized water in BLANK adaptors, one reconstituted Hb A2/F calibrator (1 mL) in BLANK
adaptors, one reconstituted Hb A2/F calibrator (1 mL) in CALIBRATOR adaptor and STOP adaptor)
 After the run is complete, note the retention time of HbA2 in the second calibrator injection (tube number 6).
 Refer to manufacturer instructions for required temperature adjustments.
 If the retention time varies by more than 0.20 minutes, monitor the pump performance by measuring the flow
observing the pressure readings.
 For the remainder of the cartridge lifetime (250 injections), the cartridge should be single-primed with every run
setup. In most cases, the cartridge temperature will not require further adjustment; minor shifts in retention
time (<0.05 minute from the optimum retention time) reflect the random run-to-run variability of the assay
and does not require additional temperature adjustment. However, a retention time shift of >0.05 minute for
Hb A2 in the calibrator may occur due to the aging of the cartridge resin, and requires a second temperature
adjustment.
iii) Step 3 - Calibration- single-priming and calibration must be performed with every run.
iv) Step 4 - Sample run (Please refer instruction manual) (Run one reconstituted whole blood primer (1 mL) in
PRIMER adaptor, one deionized water in BLANK adaptor, one reconstituted Hb A2/F calibrator (1 mL)
in BLANK adaptors, one reconstituted Hb A2/F calibrator (1 mL) in CALIBRATOR adaptor, prediluted
control level 1 in CONTROL LEVEL 1 adaptor, prediluted control level 2 in CONTROL LEVEL 2 adaptor,
samples in primary tubes and STOP adaptor)
Note: After analysis of the calibrator, the calibration factors (CF) are automatically calculated and appears in the
calibration report. The calibration factors are used in the calculation of area percentage of Hb A2 and Hb F for
all subsequent analysis in the run.
 Calibration and IQC - criteria of acceptance,

i) The retention time for Hb A2 in the calibrator is 3.65 ± 0.10.
ii) The calibration factors for haemoglobins A2 and F must be >0.7 and <1.30.
iii) Retention times of A2 and F should be consistently in range.
iv) Total area of each analysis should range from 1.0 million to 3.0 million μvolt•second. Results should not be
reported if the area is outside this range. Samples with total areas < 1.0 million μvolt•second or > 3.0 million
μvolt•second should be manually diluted.
v) Quality control values should be in range.
vi) A2 and F results need to be within established linearity.
vii) Proper baseline construction of peaks should be present.
viii) Peak shape should be sharp and symmetrical.
i) Analyte identification windows
Analyte identification “windows” are intended to assist the laboratory in the interpretation of normal and
abnormal haemoglobins detected in patient samples. The “windows” are established time ranges in which
common variants have been observed to elute using the HPLC programme.
The retention time is the center of the window. Retention time is measured from the time of sample injection to
the maximum point of each peak. The band is the half-width of the window. (Table 8.2.1)

Table 8.2.1 Analyte identification windows
Analyte name Retention Time (minutes) Band (minutes) Window (minutes)
F 1.10 0.12 0.98 – 1.22
P2 1.39 0.11 1.28 – 1.50
P3 1.70 0.20 1.50 – 1.90
A0 2.50 0.60 1.90 – 3.10
A2 3.60 0.30 3.30 – 3.90
D-WINDOW 4.10 0.20 3.90 – 4.30
S-WINDOW 4.50 0.20 4.30 – 4.70
C-WINDOW 5.10 0.20 4.90 – 5.30
ii) Interpretation of the peaks
Unknown: Can be any abnormal haemoglobin which will elute in this window. Depending on the condition of
haemoglobin variant present in an individual, the area of the peak may vary.
F: Fetal haemoglobin – can be quantified.
P2: Specimens from diabetic patients typically exhibit an elevated P2 peak. The peak represents glycated Hb A. P2
peak will be present between 4 - 6 % in a non-diabetic individual. The peak will appear higher in diabetic patients
depending upon the degree of glycaemic control over last 2-3 months. Significantly high P2 peaks require further
evaluation. An additional peak, identified as “Unknown”, may also be present between the normal positions
of Hb F and P2. If this additional peak elutes within the F window when there is no Hb F present, it may be
misidentified as Hb F. Careful comparison of the sample’s F retention time with control can help to differentiate
this “Unknown” peak from Hb F.
P3: Known as residual peak/post A1c peak. Will not have clinical significance while reporting if <7%. P3 ≥7% could
indicate sample degeneration and the test should be repeated with a fresh sample.
A0: Adult haemoglobin (Hb A)
A2: HbA2 – can be quantified. Hb E, Hb Lepore and Hb D Iran elute within Hb A2 retention time.
D – Window: Haemoglobin D Punjab.
S – Window: Haemoglobin S.
C – Window: Haemoglobin C.
Final determination of specific variants eluting in the windows should be made by an experienced medical
specialist.
iii) Points to consider when interpreting the results;

 HPLC method provides an area percentage determination of Hb A2 and Hb F, as well as qualitative separation
of normal and commonly occurring abnormal haemoglobins. Other less frequently occurring variants may also
elute within the established analyte identification windows. For clinical purpose, an adequate presumptive
identification of a haemoglobinopathy requires a combination of two techniques and results being correlated
with clinical features.
 Severe iron deficiency anaemia (Hb < 8 g/dL) can reduce the Hb A2 level slightly (by up to 0.5%).
 Laboratory confirmation of a β-thalassaemia trait should be considered in conjunction with family history and
laboratory findings including haemoglobin level, red cell indices and S. Ferritin.
 With some instruments and programmes, Hb F may merge with the P2 peak when it is 0.6% or less. Conversely
elevated levels of glycosylated Hb A may lead to factitious elevation of Hb F.
 Haemoglobin S and other late eluting haemoglobin variants have minor component peaks that may coelute
with Hb A2. This may result in a falsely elevated area percentage value for Hb A2. Glycosylated Hb S may have
retention time similar to that of Hb A, so that patients with sickle cell anaemia may have small amount of Hb A.
 Haemoglobins E, D Iran and Lepore have been observed to coelute with Hb A2. Specimens determined
to have Hb A2 levels greater than 10% should be tested for the possible presence of haemoglobin variant
interference.

 Differentiation of haemoglobin A2 and other haemoglobin types can also be made by the area percentage
calculation on the sample report.
 Haemoglobins Bart’s and H elute prior to the start of integration ( within first minute). Increased bilirubin in
the plasma may lead to a sharp peak in Hb H area.
 Glycosylated haemoglobins have different elution times from non glycosylated forms.

 Interpretation and reporting should be done by a Consultant Haematologist.
 Report format -
The chromatogram that appears on the ANALYSIS MONITOR screen is automatically printed along with report data
for that analysis. The comments and conclusion following interpretation of the findings should be included in the
final report. The following information should be included in the report:
 Patient information: Name, Date of birth, Sex, Patient identification number
 Sample information: Sample identification number, receiving date, date of analysis, date of reporting
 Haemoglobin and red cell indices +/- blood picture findings & serum ferritin
 Chromatogram and table with summary of Hb quantities with reference ranges
 Interpretation and suggestions by Consultant Haematologist

 IQC - A set of normal (Hb F 1 – 2%, Hb A2 1.8 – 3.2%) and abnormal (Hb F 5 – 10%, Hb A2 4 – 6%) controls should be
run at the beginning and end of each group of patient specimens.
 Should participate in an EQA programme or inter-laboratory comparison.
Sources of error
 Patient related causes- post transfusion sample; should repeat with a fresh sample 120 days post transfusion.
 Sample related- due to sample degeneration; repeat with a fresh sample.
 Reagent/ calibrator related –
 reagents with discoloration, cloudiness, or precipitation
 reagents that show any signs of leakage
 calibrator or whole blood primer if the pellet is brown or the vial is broken
 lyophilized material containing insoluble matter
Precaution and hazards

patient specimens.
 Calibrators and whole blood primer should be handled with the same precautions as patient specimens.
 Some reagents contain sodium azide, which may react with copper or lead plumbing to form explosive metal azides.
Use caution in disposing of these reagents. If disposing to drain, flush with large volumes of water to prevent azide
buildup.
 References
1. Manufacture user guide for BIORAD VARIANT 2.
2. Bain B.J. (2006) Haemoglobinopathy Diagnosis, 2nd edition. ISBN: 978-1- 4051-3516-0.

8.3
Standard operating procedure
IDENTIFICATION AND QUANTIFICATION OF
HAEMOGLOBIN BY
CAPILLARY ELECTROPHORESIS
Scope
This SOP is to guide technical personnel on proper performance of haemoglobin quantification using the capillary
electrophoresis method.
Responsibility
Performance of haemoglobin quantification by capillary electrophoresis method as per the procedure stated in this SOP
is the responsibility of all medical laboratory technologists who are assigned to perform this test in the haematology
laboratory. The overall supervision and reporting are the responsibilities of the consultant haematologist.
Principle
This document is for CAPILLARYS 2 FLEXPIERCING system. While this can be used as a general guide, users of other
analyzers/systems should follow manufacturer instructions for the test procedure.
Samples are haemolyzed and injected into the anodic end of the capillary. Charged molecules are separated by their
electrophoretic mobility in an alkaline buffer (pH 9.4) with separation occurring according to the electrolyte pH and
endosmosis or electro-osmotic flow. The Electro-Osmotic Flow (EOF) is a stronger force than the Electrical Field. As a
result, particles are carried towards the cathodic end of the capillary. Haemoglobins migrate from the anodic end of the
capillary appearing in specific zones to the cathodic end where detection occurs at 415 nm. Results are assessed visually
for abnormalities with identification of normal and disease patterns.
Capillary zone electrophoresis allows clean separation of Hb E from Hb A2 and facilitates easier detection of Hb Bart’s
and Hb H. There is also improved detection of sickle cell disease due to separation of haemoglobin fractions which
enables differentiation of Hb S from other variants. (Figure 8.3.1)

Figure 8.3.1
Principle of haemoglobin electrophoresis using
capillary electrophoresis method
Haemoglobin quantification by Capillary Hb electrophoresis provides a presumptive diagnosis of haemoglobinopathy.
Haemoglobinopathies are genetically inherited and heterozygotes of the condition are usually asymptomatic while
homozygotes/compound heterozygotes show a spectrum of clinical features.
Sample
No special patient preparation is required.
The sample should be whole blood collected to an EDTA tube.
These samples can be stored at 2 - 8 °C for up to 7 days.

Equipment- CAPILLARYS 2 FLEXPIERCING system.
Reagents
1. CAPILLARYS HEMOGLOBIN(E) KIT- Buffer, hemolyzing solution (Keep at 2 - 8 0C and should bring to room
temperature before use)
2. Normal Hb A2 control
3. Pathological Hb A2 control
4. Hb AFSC control
5. Distilled/deionized water (Add 12 drops of clean protect solution for 1 L of distilled water)
6. CAPICLEAN solution (To clean capillaries)
7. Sodium hypochlorite solution (To clean the probe)
8. CAPILLARYS wash solution
9. Clean protect solution
Method
 Procedure
 Switch on analyzer and computer.
 Select "HEMOGLOBIN(E)" analysis programme and place the buffer and hemolyzing solution vials in the
instrument.
 Run QC samples.
NOTE: QC samples are in powder form and have to be dissolved before use. Add 1.6 mL of distilled water to the QC
sample vial and mix well for few minutes, then transfer the solution into a conical shaped tube. Every QC sample
has its own barcode, therefore paste the barcode on the conical shaped tube. Once prepared, a QC sample could
be used for about 10 runs, so that the QC samples should be handled carefully without contamination and should
be stored in the freezer soon after using.

 Place normal Hb A2 control samples on rack No. F0 intended for control blood sample with specific tubes for
controls, caps, and the wedge adapter for tubes. Insert the rack 0 and select ‘automatic dilution’ on the normal
Hb A2 control window. A segment of normal Hb A2 control is prepared automatically by Capillarys 2 flex piercing.
 After first analyses of normal Hb A2 control on all 8 capillaries using automatic dilution mode, reanalyze the
haemolyzed normal Hb A2 control segment on the rack 0 using ‘manual dilution’ if necessary.
 Place the patient sample tubes of whole blood (up to 8 on each sample rack positions 1 to 8); the bar code of
each tube must be visible in the openings of the sample rack.
NOTE: If the number of tubes to analyze is less than 8, complete the sample rack with capped tubes containing
distilled or deionized water.
 Position a new dilution segment on each sample rack. The sample rack will be ejected if the segment is missing.
 Slide the complete sample carrier(s) into the analyzer through the opening in the middle of the instrument.
Up to 13 sample racks can be introduced successively and continuously into the instrument.
 If the sample contain less than 1 mL of blood, the following procedure should be followed;
 Add 100 μL of whole blood in to a conical tube for ‘control’ and cap the tube. Place the tube with a wedge
adapter on a sample rack and slide the rack in to the analyzer at the beginning of an analysis series or add 50 μL
directly to the dilution segment and place an empty tube in the relevant position.
 Remove analyzed sample racks from the plate on the left side of the instrument.
 Remove the used dilution segments carefully from the sample rack and discard them.
 Haemoglobin identification and quantification

 At the end of the analysis, the haemoglobin fractions, Hb A, Hb F and Hb A2 are automatically identified; the Hb
A fraction is adjusted in the middle of the review window. The resulting electropherograms are evaluated visually
for pattern abnormalities.
 The potential positions of the different haemoglobin variants (identified in zones called Z1 to Z15) are shown on
the screen of the instrument.
 Hb variant fractions along with Hb A and Hb A2 fractions are automatically separated hence shown in the mosaic
screen to facilitate their interpretation.
 End of analysis sequence

 At the end of each analysis sequence, the operator must initiate the "shut down" procedure of the analyzer in
order to store the capillaries in optimal conditions.
 Filling of reagent containers

 Refer to the instructions for replacement of reagent containers according to the colour code for vials and
connectors.
 A message will be displayed when it is necessary to replace reagent containers. (E.g. Place a new buffer container/
empty waste container)
 Before filling the wash solution container, it is recommended to wash it with plenty of distilled or deionized
water.
 A new filter should be replaced when replacing the reagent container.

 The different migration zones of haemoglobin variants (Z1 to Z15) are shown on the screen of the instrument
and on the result worksheet (Figure 8.3.2). Passing the mouse cursor (on the top of the mosaic screen) over a
zone displays icon information containing possible haemoglobin variants that could be seen in this zone.
For each fraction, the maximum position (peak) defines the migration zone.
Figure 8.3.2 – Electropherogram
Facts to be considered during interpretation of results

 When an abnormal haemoglobin/variant is detected it should be confirmed by another method of
Hb-identification.
 Haemoglobin H presents a low isoelectric point.
Hb H disease which will usually present with an unstable and quickly disappearing Hb H (β4) fraction migrating
in Z15 in CE.
It migrates more anodic than haemoglobin A and may appear as one or several fractions.
 Weak haemoglobin fractions which migrate in zone Z12 are sometimes quantified with imprecision
(e.g:- too asymmetric Hb Bart’s). It is thus necessary to delete automatic quantification and then to quantify
them manually.
 When analyzing blood samples of newborn babies where Hb F is high, Hb A could be overestimated due to the
presence of deteriorated Hb F.
In addition, when some haemoglobin variants are present in more than 4% (such as Hb S, Hb C, Hb E or Hb
D-Punjab) with high Hb F (> 60 %) in a sample, it is necessary to perform a complementary analysis to confirm
the presence of Hb A.
 If a haemoglobin variant is clinically suspected despite a normal capillary electrophoresis in newborn babies
from age 6 to 9 months, it is recommended to analyze serial blood samples (collected monthly, for example) in
order to check the Hb F concentration. It will allow to verify the decrease of Hb F concentration and the potential
presence of a Hb variant. In case of uncertainty, it is advised to confirm by using complementary studies and to
analyze parents’ blood samples.
 When analyzing blood samples from transfused patients with sickle cell disease, with low Hb A level (< 10 %),
Hb S fraction may appear shifted from Z(S) zone to Z(D) zone.
In such instances, clinically correlate and perform complementary studies in order to confirm the presence of
Hb S.

 In a sample without any haemoglobin A or haemoglobin A2, the zones may not be identified. In such situations
the analysis has to be repeated with 50:50 mixture of normal Hb A2 control to identify Hb fractions. Following
method should be used,
 Vortex the patient blood sample for 5 seconds.
 Take a conical tube (tube used for controls) and mix 50 μL of the patient sample with 50 μL of normal Hb A2
control. Cap the tube and vortex for 5 seconds.
 Place the tube with a wedge adapter on a sample rack and perform the analysis.
 The result obtained with the mixed sample will enable presumptive variant identification due to the
positioning of the haemoglobin fractions in the appropriate identification zones. However, the relative
quantification of haemoglobins should be reported utilizing the initial, unmixed sample result.
 The presence of a Hb Constant Spring variant may be suspected when an additional haemoglobin fraction is
observed in Z(C) or Z(A2) migration zones. This fraction may also be due to plasma proteins from the sample.
To confirm/ exclude the presence of the variant, separate the red cells and re-analyze.
Method- Centrifuge the whole blood sample at 5,000 rpm for 5 minutes and discard plasma.
In a microtube, mix one volume (50 μL) of red blood cells from the test sample with 8 volumes (400 μL) of
haemolyzing solution. Vortex for 5 seconds. Apply 100 μL of prepared haemolysate in a well of a new dilution
segment. Place this dilution segment on the sample rack No. F0 and proceed to analyze by selecting "Sample"
with "manual dilution" in the window which appears on the screen.

 Interpretation and reporting should be done by a consultant haematologist.
 Report format -
The electropherogram that appears on the ANALYSIS MONITOR screen is automatically printed along with report
data for that analysis. The comments and conclusion following interpretation of the findings should be included
in the final report. The following information should be included in the report:
 Patient information: Name, Date of birth, Sex, Patient identification number
 Sample information: Sample identification number, receiving date, date of analysis, date of reporting
 Haemoglobin and red cell indices +/- blood picture findings & serum ferritin
 Electropherogram and table with summary of Hb quantities with reference ranges*
 Interpretation and suggestions by Consultant Haematologist
*NOTE: Reference values must be considered only when haemoglobin variants are absent.

 IQC should be run in the following instances;
 After changing the analysis buffer lot number or technique
 After a cleaning sequence with CAPICLEAN
 Before starting a new analysis sequence - run two analysis sequences with the normal Hb A2 control.
 In each run- include an assayed control blood (for example, a blood sample containing haemoglobin A, F, C and S,
such as Hb AFSC control) or a normal blood sample, the normal Hb A2 control, or the pathological Hb A2 control.
 EQA- the laboratory should participate in an EQA programme.
Sources of error
 Patient related causes- post transfusion sample; should repeat with a fresh sample 120 days post transfusion.
 Sample related- due to sample degeneration; repeat with a fresh sample.

patient specimens.
 References
1. Hemoglobinopathies: Current Practices for Screening, Confirmation and Follow-up. CDC.
2. Manufacturer’s guidelines. CAPILLARYS HEMOGLOBIN(E) - 2013/01.

8.4
FLOW-CYTOMETRIC
IMMUNOPHENOTYPING (FCM) FOR
LEUKAEMIA/LYMPHOMA/MYELOMA
Ms. A.M. Hemalie Kanchana Abeykoon
Dr. Dilini Jayaratne
Scope
The scope of this SOP is to guide technical personnel on proper performance of flow cytometric techniques and
requirements for its correct performance.
Responsibility
Performance of flow cytometry (FCM) as per the procedure stated in this SOP is the responsibility of all medical
laboratory technologists who are assigned to perform this test in a haematology laboratory. Interpretation, conclusions
and comments should be made by the consultant haematologist.
Principle
FCM measures optical and fluorescence characteristics of single cells (or any other particle, including nuclei,
microorganisms, chromosome preparations, and latex beads). Physical properties, such as size (represented by forward
angle light scatter) and internal complexity (represented by right-angle scatter) can resolve certain cell populations.
Fluorescent dyes may bind or intercalate with different cellular components such as DNA or RNA. Additionally, antibodies
conjugated to fluorescent dyes can bind specific proteins on cell membranes or inside cells. When labeled cells are
passed by a light source, the fluorescent molecules are excited to a higher energy state. Upon returning to their resting
states, the fluorochromes emit light energy at higher wavelengths. The use of multiple fluorochromes, each with similar
excitation wavelengths and different emission wavelengths (or “colors”), allows simultaneous multiparametric analysis of
physical and chemical characteristics of up to thousands of particles per second. Commonly used dyes include propidium
iodide, phycoerythrin, and fluorescein, although many other dyes are available. Tandem dyes with internal fluorescence
resonance energy transfer can create even longer wavelengths and more colors.
Inside a flow cytometer, cells in suspension are drawn into a stream created by a surrounding sheath of isotonic fluid
that creates a laminar flow, allowing cells to pass individually through an interrogation point. At the interrogation point,
a beam of monochromatic light, usually from a laser, intersects the cells. Emitted light is given off in all directions and is
collected via optics that direct the light to a series of filters and dichroic mirrors that isolate particular wavelength bands.
The light signals are detected by photomultiplier tubes and digitized for computer analysis. The resulting information
usually is displayed in histogram or two-dimensional dot-plot formats. (Figure 8.4.1)

Figure 8.4.1 Principle of FCM
Clinical applications of FCM immunophenotyping in haematological malignancies are;
1. distinction between neoplastic and benign conditions
2. diagnosis and characterization of lymphomas and leukaemias
3. quantification of the tumour burden
4. assessment of other neoplastic and preneoplastic disorders such as plasma cell dyscrasias and myelodysplastic
syndromes
5. detection of minimal residual disease in patients with acute leukaemia or chronic lymphoid malignancies
6. providing prognostic information
Sample
Peripheral blood and bone marrow aspirate in an EDTA tube (at least 1 mL and not exceeding the recommended volume
of the collection tube)
NOTE:
 sample should preferably be processed within 6 hours of collection.
 if delayed, sample must be kept at 2 – 8 0C and should be processed within 48 hours of collection.
 Bring samples to room temperature before analysis to optimize antibody binding.
Equipment, reagents and consumables
Equipment
1. Flow cytometer. (Refer to the appropriate instrument user’s guide for information of each type) – In this SOP we
focus on two types of machines – FACS brand (BD FACSCantoTM brand) and Navios brand
2. Falcon disposable 12 x 75 mm capped polystyrene test tubes
3. Micropipette with tips (1000 µL, 100 µL, 0 - 20 µL adjustable type)

4. 10 mL pipette rubber teat
5. Measuring cylinder - 100 mL and 50 mL
6. Centrifuge
7. Vortex mixer
8. Blotting papers
9. Tube rack
10. Beaker
Reagents
1. Sheath fluid (In Navios brand this is also called as Isotone)
2. Lysing solution
a. For FACSTM brand flow cytometer - FACS lysing solution (10X) (store at 4 0C )
Preparation of working solution (1X) - prepared by 1:9 dilution with distilled water - add 10 mL of FACS lysing
solution and 90 mL of distilled water and mix. Working FACS lysing solution (1X) is stable for 1 month when
stored in a glass or high-density polyethylene (HDPE) container at room temperature.
b. For Navios brand flow cytometer - Optilyse C lysing solution (store at room temperature)
3. Permeabilizing buffer
a. For FACSTM brand flow cytometer - Permeabilizing buffer/ Perm buffer (10X) (store at 40C)
Preparation of working solution (1X) - prepared by 1:9 dilution with distilled water - add 5 mL of Perm buffer
and 45 mL of distilled water and mix.
Working permeabilizing buffer (1X) is stable at room temperature for up to 1 month.
b For Navios brand flow cytometer – Intraprep permeabilization buffer (Perm Buffer – store at room temperature)
– This consists of 2 reagents; Reagent 1 – Fixative reagent, Reagent 2 – permeabilization reagent
4. Monoclonal antibodies (store at 4 0C and keep away from direct light)
5. BD Cytofix/CytopermTM Fixation/Permeabilization Kit for detection of Myeloma in FACSTM brand Flowcytometer.
BD Cytofix/CytopermTM Fixation/Permeabilization Kit components: -
 Fixation/Permeabilization solution
 BD Perm/WashTM Buffer- (dilute 1:10 in distilled water. Must be freshly prepared prior to use.)
6. BD Pharm LyseTM lysing solution (store at +40C) for detection of Myeloma in FACSTM brand flowcytometer.
Preparation of working Pharm Lyse solution (1X) - prepared by 1:9 dilution with distilled water. Add 5 mL of Pharm
Lyse (10X) and 45 mL distilled water and mix. Working Pharm Lyse solution (1X) is stable at +4 0C up to 1 month.
Consumables for quality control

In the FACSTM brand flow cytometer
1. Spherotech beads
2. Cytometer Setup and Tracking (CS & T) beads
3. Seven colour setup beads
4. Calibrite beads
5. Flow-Check™ Fluorospheres
6. IMMUNO-BRITE™ Standards Kit
7. Flow-Check™ Pro Fluorospheres
8. Cell counting beads
9. Antibody capture beads

In the Navios brand flow cytometer
1. Spherotech beads
2. Flow-Check™ Fluorospheres
3. IMMUNO-BRITE™ Standards Kit
4. Flow-Check™ Pro Fluorospheres
5. Flow count beads
6. Versa comp antibody capture beads
Method
 A well stained peripheral blood film or bone marrow should be reviewed by a consultant haematologist prior to
requesting and deciding upon an appropriate immunophenotype panel.
 Sample preparation
FACSTM brand flow cytometer
For leukaemia panels (Table 8.4.1)
Table 8.4.1 Acute leukaemia panels

FACS canto II 8 colour Acute leukaemia panel
PERCP-
V 450 V500 FITC PE Cy5.5 PE Cy7 APC APC H7
ALOT* cyCD3 CD45 cyMPO cyCD79a CD34 CD19 CD7 smCD3
BALL CD20 CD45 CD58 CD66c CD34 CD19 CD10 CD38
TALL CD4 CD45 nTDT CD99 CD5 CD2 CD1a CD8
AML 1 HLA DR CD45 CD64 CD13 CD34 CD117 CD33 CD14
AML 2-ext CD4 CD45 CD123 CD56 CD34 CD41a CD15 CD71
*Acute Leukemia orientation

Surface staining
1. Label the disposable Falcon tubes (12 x 75 mm) according to the panel provided in the lab.
2. Add appropriate volume of fluorochrome-conjugated monoclonal surface antibody (do not add cytoplasmic
antibodies) to appropriate Falcon disposable tubes.
3. Add 100 μL of well mixed whole blood or bone marrow aspirate to each tube.
(The WBC count of the sample must be 10,000 - 15,000/ μL. If WBC count is higher than 15,000/ μL, sample
must be diluted with sheath fluid.)
4. Vortex gently and incubate for 15 minutes in the dark at room temperature (20° to 25°C).
5. Add 2 mL of working (1X) FACS Lysing Solution.
6. Vortex gently and incubate for 15 minutes in the dark at room temperature (20° to 25°C).
7. Centrifuge at 500 g for 5 minutes. Remove the supernatant and blot on a filter paper.
8. Add 2 mL of sheath fluid and centrifuge at 500 g for 5 minutes. Remove the supernatant and blot on a filter
paper.
9. Add 0.5 mL of sheath fluid to all tubes and vortex.
10. Proceed with acquisition. Minimum number of events for acquisition is 20,000.
Surface and cytoplasmic staining

1. Label the disposable Falcon tubes (12 x 75 mm) according to the panel.
2. Add an appropriate volume of fluorochrome-conjugated monoclonal surface antibody to the appropriate Falcon
disposable tubes.

3. Add 100 μL of well mixed whole blood or bone marrow aspirate to each tube.
(The WBC count of the sample must be 10,000 - 15,000/ μL. If WBC count is higher than 15,000/ μL sample must
be diluted with sheath fluid.)
4. Vortex gently and incubate for 15 minutes in the dark at room temperature (20 - 25°C).
5. Add 2 mL of working (1X ) FACS Lysing solution.
6. Vortex gently and incubate for 15 minutes in the dark at room temperature.
paper.
9. Add 0.5 mL of permiabilizing buffer (1X Perm Buffer), mix by vortex, and incubate for 15 minutes in dark at room
temperature.
10. Add 1 mL sheath fluid and centrifuge at 500 g for 5 minutes. Remove the supernatant and blot on a filter paper.
11. Add cytoplasmic antibodies (e.g cyCD3, cyCD22, nTDT, MPO, CD79a etc.) in an appropriate volume to the
appropriate tube.
12. Incubate in dark for 15 minutes at room temperature.
paper.
For lymphoma panel (Table 8.4.2)

Table 8.4.2 Lymphoma panel
FACS canto II 8 colour Lymphoma panel
V 450 V500 FITC PE PERCP-CY5.5 PE CY7 APC APC H7
LST* CD20/CD4 CD45 CD8/smlg  CD56/smlgκ CD5 CD19/TCRүꝽ smCD3 CD38
BCLPD1 CD20 CD45 CD23 CD200 CD79b CD19 CD10 CD43
BCLPD2 (Hairy) CD45 CD123 CD103 CD11c CD19 CD25
T-CLPD CD4 CD45 CD30 CD26 CD7 CD2 CD25 smCD3
NK-CLPD CD16 CD45 CD57 CD56 HLADR CD19 CD94 smCD3
*LST - (Lymphoma screening tube - antibody pre coated tube
Surface staining
1. Label the disposable Falcon tubes (12 x 75 mm) according to the panel provided in the lab.
2. Add an appropriate volume of fluorochrome-conjugated monoclonal surface antibody to the appropriate Falcon
disposable tubes.
3. Add 100 μL of well mixed washed whole blood or bone marrow aspirate* to each tube (The WBC count of the
sample must be 10,000 - 15,000/ μL. If WBC count is higher than 15,000/ μL, sample must be diluted with
sheath fluid.)
*Preparation of washed whole blood or bone marrow aspirate sample
a. Add 500 µL of well mixed sample to a graduated centrifuge tube.
b. Add sheath fluid up to 10 mL.
c. Mix well by vortexing.
d. Centrifuge at 500 g for 5 minutes. Remove the supernatant and blot on a filter paper.
e. Repeat the washing cycle (b-d) two more times.

4. Vortex gently and incubate for 15 minutes in the dark, at room temperature (20 - 25°C).
5. Add 2 mL of working (1X) FACS lysing solution.
6. Vortex gently and incubate for 15 minutes in the dark at room temperature.
paper.
10. Proceed with acquisition. Minimum number of events for acquisition in LST is 100,000 and 50,000 in BCLPD1,
BCLPD2, TCLPD and NK CLPD tubes.
For myeloma panel (Table 8.4.3)

Table 8.4.3 Myeloma panel
FACS canto II 8 colour Plasma Cell Dyscrasia / Myeloma panel
V 450 V500 FITC PE PERCP-CY5.5 PE CY7 APC APC H7
CD138 CD45 CD38 CD56 CD27 CD19 cylgκ cylgλ
1. Label one disposable Falcon tube (12 × 75 mm).

2. Add 3 mL of working Pharm Lyse solution (1X) to Falcon tube.
3. Add 100 μL of well mixed washed whole blood or bone marrow aspirate* to the tube (The WBC count of the
sample must be 10,000 - 15,000 / μL. If WBC count is higher than 15,000/ μL sample must be diluted with
sheath fluid.) and mix well by vortexing.
*Preparation of washed whole blood or bone marrow aspirate sample
a. add 500 µL well mixed sample to a graduated centrifuge tube.
b. add sheath fluid up to 10 mL.
c. mix well by vortexing.
d. centrifuge at 500 g for 5 minutes. Remove the supernatant and mix by vortexting
4. Incubate for 15 minutes in the dark at room temperature.
6. Add 2 mL of sheath fluid, vortex and centrifuge at 500 g for 5 minutes. Remove the supernatant and blot on a
filter paper.
7. Add appropriate volume of fluorochrome-conjugated monoclonal surface antibodies (do not add Kappa and
Lambda antibodies ).
8. Vortex gently and incubate for 15 minutes in the dark at room temperature (20 - 25°C).
paper.
10. Add 250 µL of fixation/ permeabilization solution, vortex gently and incubate for 20 minutes in the dark at room
temperature (20 - 25°C).
11. Add 1 mL of BD Perm/ WashTM buffer working solution (1X).
12. Vortex gently and centrifuge at 500 g for 5 minutes. Remove the supernatant and blot on a filter paper.
13. Add appropriate volume of Kappa and Lambda antibodies, vortex gently and incubate for 20 minutes in the dark
at room temperature (20 - 25°C).
14. Add 2 mL of BD Perm/ WashTM buffer working solution (1X).
15. Vortex gently and centrifuge at 500 g for 5 minutes. Remove the supernatant and blot on a filter paper.
16. Add 500 µL of sheath fluid as final volume and vortex.

Navios brand flow cytometer
Table 8.4.4 Immunophenotyping panels for Navios brand flow cytometer
ACUTE
FITC PE ECD PC5.5 PC7 APC A700 A750 PB KO
LEUKEMIA
A LOT cyAMPO cyCD79a cyCD3 CD22 CD34 CD45

B-TUBE/BMRD CD58 CD73 CD19 CD86 CD34 CD10 CD20 CD38 CD81 CD45
MYELOID &
CD15 CD14 CD13 CD117 CD64 CD33 CD36 CD11b HLA-DR CD45
MONO
TALL TUBE CD1a CD34 CD3 CD5 CD56 CD2 CD4 CD8 CD7 CD45
Optional cyTDT cyCD61 cyCD41 CD45
CLPD FITC PE ECD PC5.5 PC7 APC A700 A750 PB KO
CLPD 1 Kappa Lambda CD19 CD5 CD200 CD10 CD20 CD38 CD23 CD45
CLPD 2 FMC7 CD11c CD19 CD5 CD25 CD103 CD123 CD49d CD45
CLPD 3 CD57 TCR g/d CD3 CD5 CD56+ CD16 CD2 CD4 CD8 CD7 CD45
Optional IgD CD180 CD19 IgM CD45
MM FITC PE ECD PC5.5 PC7 APC A700 A750 PB KO
cyKAPPA cyLAMBDA CD19 CD27 CD56 CD138 CD20 CD38 CD81 CD45
CD4/CD8

(Tetra cocktail) FITC PE ECD PC5.5 PC7 APC A700 A750 PB KO
CD45 CD4 CD8 CD3

TBNK Panel CD16 CD56 CD19 CD14 CD4 CD8 CD3 CD45
Surface staining
1. Label the tubes according to test panel. (Table 8.4.4)

2. Add 100 μL of peripheral blood or bone marrow aspirate (5,000 cells/ µL or 0.5 million/ tube)
3. Add 10 μL of each fluorochrome - tagged antibodies according to the panel.
4. Vortex and incubate for 15 - 20 minutes, in dark at room temperature.
5. After incubation, add 500 μL of Optilyse C and again incubate for 10 minutes in a dark room.
6. Add 4 mL of Isotone and centrifuge at 400 - 500 g for 5 minutes, and decant the supernatant.
7. Finally add 500 μL of Isotone solution and vortex.
Surface and cytoplasmic staining
1. Label the tubes according to test panel. (Table 8.4.4)

2. Add 100 μL of peripheral blood or bone marrow aspirate (5,000 cells/ µL or 0.5 million/ tube)
3. Add 10 μL of each fluorochrome - tagged antibodies (surface antibodies) according to the panel.
4. Vortex and incubate for 15 - 20 minutes in dark at room temperature.
5. Add 100 μL of Reagent 1 of Intraprep, vortex and incubate for 15 minutes at room temperature.
6. After incubation, add 4 mL of Isotone and centrifuge at 400 - 500 g for 5 minutes.
7. Remove the supernatant by aspiration.
8. Add 100 μL of Reagent 2 of Intraprep and wait for 5 minutes (without vortexing or shaking).
9. After 5 minutes, shake slowly 2 - 5 seconds by hand.

10. Add appropriate concentration of cytoplasmic antibodies and incubate for 15 minutes in the dark at room
temperature.
11. After incubation, add 4 mL of Isotone and centrifuge at 400 – 500 g for 5 minutes.
12. Discard the supernatant by aspiration, and add 0.5 mL of Isotone and vortex.
Kappa/Lambda staining
1. Take 300 μL of peripheral blood or bone marrow aspirate and add 3 mL of sheath fluid.
2. Centrifuge at 400 - 500 g for 5 minutes.
3. Discard supernatant and add 3 mL of sheath fluid.
5. Discard supernatant and add 3 mL of sheath fluid.
6. Take 100 μL from the pellet and add 10 μL of kappa/ lambda and CD19 antibodies each and vortex.
7. Incubate at room temperature for 15 minutes in dark.
8. Add 500 μL of Optilyse C, vortex and incubate for 15 minutes in dark.
10. Discard supernatant and add 500 μL of sheath fluid.
NOTE - For multiple myeloma, surface and cytoplasmic staining should be followed.
 Start up and shutdown procedure of flow cytometer

FACSTM brand flow cytometer
1. Check that sheath container is full and the waste tank is empty.
2. Turn on the flow cytometer main power switch.
3. Turn on the computer main power switch and start up BD FACSCANTO. Logon to the computer.
4. Run CS & T beads each time the instrument is turned on.
If CS & T failed - Check expiry date of the lot.
Prepare another tube and rerun.
Clean the tubing by running cleaning mode.
If not corrected, contact local agent.
5. Run samples.
6. Run the cleaning cycle according to the recommendation given by manufacturer.
7. Run the ‘fludic shutdown” procedure on “cytometer”, close the FACSDiva software, turn off the instrument and
shut down the computer.
8. Turn off computer.
9. All work surfaces must be decontaminated with appropriate disinfectant at the end of each day run.
Navios brand flow cytometer
1. Check that sheath container is full and the waste tank is empty.
2. Turn on the flow cytometer main power switch.
3. Turn on the computer main power switch and start up Navios. Logon to the computer.

4. Run cleanse panel.
5. Keep two tubes of cleaning solution and two tubes of water in the carousel.
6. Quality control
 Take 500 μL of Flowcheck fluorospheres and keep at location 1.
If QC failed – Check expiry date of the lot.
Prepare another tube and rerun.
Clean the tubing by running cleaning mode.
If not corrected contact local agent.
7. Run samples.
8. Shut down cycle - Repeat step No-4
9. Turn off computer.
10. Turn off the flow unit.
11. All work surfaces must be decontaminated with appropriate disinfectant at the end of each day run.
 A report should include the following

 Quantity of the abnormal cell clone
 Immunophenotypic characteristics of the abnormal cell clone
 Conclusion
 Any further suggestions to confirm the diagnosis
QC beads are very useful for instrument set up, daily monitoring process and evaluating instrument performance/
calibration.
In the FACSTM brand flow cytometer
Daily calibration:
 Run CS & T beads according to manufacturer’s recommendations.
 Run One flow setup beads according to manufacturer’s recommendations.
Monthly calibration : monthly calibration is done by the local agent of the vender.
 CS & T beads baseline
 One flow setup beads
 8 Colour compensation
In the Navios brand flow cytometer
 Electronic standardization, sensitivity and linearity, compensation, and instrument calibration needs to be
undertaken on daily basis.
 Because the instrument is a highly sensitive laboratory equipment, a routine sequence of quality control
procedures enables the end user to effectively monitor the instrument performances.
 The optical alignment generally does not need to be adjusted by the user and is best undertaken by a trained
engineer.
Sources of error
 Sampling issues
 Delayed sample (If delayed must be kept at 4 0C for < 48 hours)
 Suboptimal antibody binding in samples kept at temperatures lower than room temperature.

 Diluted bone marrow samples
 Degenerated samples
 Insufficient volume of sample
 Technical issues
 Missed antibody addition or wrong antibody addition.
 Inadequate RBC lysis leading to residual RBC.
 Inadequate cell washing leading to erroneous surface Ig results.
 Insufficient permeabilization leads to false negative cytoplasmic results.
 Misidentification of tubes when analysing leads to misdiagnosis.
 Autofluorescence, spectral overlap and non-specific antibody/ fluorochrome binding resulting in background
fluorescence causing errors in interpretation.
 Interpretation issues
 Use of wrong gate for the interested cell population.

 All parts and surfaces of the flow cytometer should be considered as potentially infectious since the instrument
 References
1. Practical Flow Cytometry in Haematology Diagnosis by Mike Leach, Mark Drummond, Allyson Doig (2013)
2. BD FACSCanto Clinical Software Reference Manual.
3. BD FACSCanto Flow Cytometer Reference Manual.
4. NAVIOS Flow Cytometer Reference Manual.

8.5
FLOW-CYTOMETRIC IMMUNOPHENOTYPING FOR
PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA
(PNH)
Dr. H.M.J. Priyanka Herath
Scope
This SOP is a technical guide for the medical laboratory technologists (MLTT) performing flow-cytometric
immunophenotyping for paroxysmal nocturnal haemoglobinuria (PNH). Method in this SOP is for `routine flow cytometric
analysis` for PNH which is capable of detecting a PNH clone as small as 1.0%.
Responsibility
The MLTT performing the test will be responsible for the technical procedures of the pre-analytical, analytical, and post
analytical phases mentioned in this SOP, while the consultant haematologist will be responsible for overall supervision,
data analysis and test reporting.
Principle
PNH is characterized by a somatic mutation in the X-linked phosphatidylinositol glycan class A (PIGA) gene, leading
to a deficiency of Glycosyl phosphatidylinositol (GPI) anchor protein on the cell surface (RBC and WBC). Therefore,
the antigens attached to these GPI anchors are also decreased or absent. Flow cytometry (FCM) relies on the use of
labeled antibodies to detect deficiencies of GPI linked proteins, CD59, CD24, CD14 on the cell surfaces of red blood cells,
neutrophils and monocytes respectively. FLAER (fluorescent labeled variant of the protein, Aerolysin) binds directly to
the GPI anchor, and used for WBC only.
If the GPI anchor is absent, using a combination of flurochrome labeled specific antibodies, FLAER and flow cytometric
gating strategies, the presence and the size of a PNH clone can be determined.
Flow cytometry (FCM) is the current ‘gold standard’ diagnostic test for PNH as it provides both quantitative and qualitative
information.
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired, life-threatening disease characterized by intravascular
haemolysis (by activation of the complement system) and high incidence of thrombosis. PNH could be associated with
other bone marrow disorders such as aplastic anaemia and myelodysplastic syndrome.

The `PNH routine diagnosis method` by FCM is able to detect a PNH clone in RBC/WBC with the sensitivity of detecting
a clone of 1% or more, and `high-sensitive method` is sensitive to detect a PNH clone as small as 0.01%. According
to several studies, a PNH granulocyte clone size more than 10% by FCM seems to be clinically significant in relevance
to PIGA gene mutation.
Sample
 Sample - 3 cc of freshly collected EDTA blood. No special preparation of the patient is needed. This test could be
performed in patients irrespective of red cell transfusion.
 Storage and transport of samples:
 it is strongly recommended to complete the test as soon as possible without refrigerating the sample.
 if samples are kept beyond 24 hours of collection, should be stored at 2 - 8 0C.
 neutrophil & monocyte are stable for 48 hours and RBC for 72 hours at 2 - 8 0C.
 temperatures below 2 0C and above room temperature (RT) should be avoided.
 Transport- at RT (18 - 22 0C).
Equipment, reagents and other consumables

Equipment
1. Flow cytometer (this SOP is for BD FACS Calibur Flow Cytometer)
2. Vortex mixer
3. Centrifuge
Reagents
1. BD FACS Lysing Solution
2. Sheath fluid
3. Distilled water
4. Calibrite beads
5. Monoclonal antibodies as per the panels* (Table 8.5.1)
Table 8.5.1 Monoclonal antibody panels for PNH detection

Monoclonal
Antibody Fluorochrome
Monoclonal antibodies for RBC CD59 FITC

FLAER
CD14 PE
Monoclonal antibodies for monocytes
CD45 PERCP
CD64 APC

FLAER
CD24 PE
Monoclonal antibodies for neutrophils
CD45 PERCP
CD15 APC
*Monoclonal antibodies
 should be stored at 2 - 8 0C.
 should not be frozen.
 should not be used after the expiry date.
 should not be exposed to direct light either during storage or during the test procedure as they are light sensitive.
 should be handled with care.
 take out of the refrigerator when ready to be used and should be returned to the refrigerator as soon as possible.

Other consumables
1. FACS tubes 5 mL (round bottom) – should not be reused
2. Micro pipettes – 1000 µL, 100 µL, 10 µL , 5 µL
3. Disposable pipette tips – should not be reused
4. Tube racks
5. Aluminum foil/ sheets to cover tubes
Method
 Calibration and calibration verification procedure- to ensure that the flow cytometer provides consistent results,
run BD FACSComp software with BD Calibrite beads. Daily calibration is preferred, however, should be calibrated at
least once a week.
If calibration fails;
First exclude any defect in the method followed (e.g. beads not added to the tubes/ method defect/ use of expired
calibrite beads). If this is excluded, run FACS clean solution and rerun the calibrite beads. If calibration is still failed,
contact the authorized service team of the local agent.
 Labeling of the tubes- label the tubes by the antibodies added. (Table 8.5.2)
Table 8.5.2 Labeling of the tubes

Tube Label
1 (RBC control) RBC (- / - / - / -)
2 RBC (CD 59 / - / - / -)
3 (WBC control) WBC (- / - / - / -)
4 Neutrophil (FLAER / CD24 / CD45 / CD15)
5 Monocyte (FLAER / CD14 / CD45 / CD64)
 Pipetting procedure
 Always use new pipette tips for every step of sample preparation.
 Reverse pipetting mode is recommended for optimal precision of the sample preparation.
 Before using 1000 µL and 100 µL pipettes, pre-rinse with the solution two to three times. SHOULD NOT PRE-
RINSE the pipettes used for probe dispensing (10 µL).
 Pipettes used for flow cytometry sample preparation should be checked for precision and accuracy
periodically.
 Testing procedure - testing should be done in a suitable area without direct light.
(i) Preparation of cells
 RBC preparation
a. Mix the EDTA blood sample with 8 - 10 complete inversions.
b. Take 5 µL of whole blood in to a FACS tube and add 2 mL of sheath fluid.
c. Gently mix well with the tip of the delivering pipette (use new pipette tips for each sample).
d. Label the FACS tubes (Table 8.5.2) and arrange the tubes (Table 8.5.3) as per the RBC panel.
Table 8.5.3 Arrangement of tubes for RBC panel
Tubes in RBC panel Monoclonal antibodies/probes

01 (-/-/-/-) RBC control - - - -
02 (CD59/-/-/-) CD59 FITC (10 µL) - - -

e. Pipette 50 µL of diluted EDTA blood to the FACS tubes of the RBC panel.
f. Add monoclonal antibodies (gently mix the antibody vial before taking the required volume, deliver to
the bottom of the tube and not to the side of the tube).
g. Vortex each tube gently for few seconds.
h. Cover tubes with aluminum foil, incubate in complete dark for 20 minutes at RT (18 - 22 0C)
i. After incubation, add 2 mL of sheath fluid to each tube.
j. Centrifuge the tubes at 500 g for 5 minutes.
k. Decant the supernatant and vortex the cell button.
l. Repeat another cell washing cycle - from (i) to (k)
m. Suspend the cell button in 500 µL of sheath fluid.
n. Run on pre-calibrated Flow-cytometer as soon as possible.
 WBC Preparation
a. Do FBC on the EDTA blood samples of the patient first and check the absolute neutrophil count (ANC).
Samples with low ANC have to be adjusted accordingly. (Table 8.5.4)
Table 8.5.4 Adjustment of neutrophil count in the sample
ANC Adjust the neutrophil count in the sample

≥ 5000 / µL No need of further concentration of the sample
1000-5000 / µL Take 1 mL whole blood, centrifugea and discard the supernatantb.
Resuspend the cell button in 200 µL of sheeth fluid.
500-1000 /µL Take 1 mL whole blood, centrifugea and discard the supernatantb.
100-500 /µL Take 3 mL whole blood, centrifugea and discard the supernatantb.
a centrifuge at 500 g for 5 minutes
b after centrifugation discard the supernatant by using a pipette.
b. Should use new FACS tubes for the test.

c. Label the tubes as per the WBC panel (Table 8.5.1).
d. Pipette 100 µL of the sample (whole/diluted blood) in to each tube.
e. Add monoclonal antibodies. (Table 8.5.5)
Table 8.5.5 Arrangement of tubes for WBC panel
Tubes in WBC panel Monoclonal antibody/probes

03 (-/-/-/-) WBC control - - - -
04 F/CD24/CD45/CD15 FLAER CD 24 PE CD 45 PERCP CD 15 APC
(Neutrophils) ( 5 µL ) ( 15 µL ) (10 µL) ( 5 µL)
05 F/CD24/CD45/CD64 FLAER CD14/ PE CD 45 PERCP CD 64 APC
(Monocytes) ( 5 µL ) ( 10 µL ) (10 µL) ( 5 µL)
f. Vortex each tube gently for few seconds.

g. Cover tubes with aluminum foil and incubate in dark for 20 minutes at RT (18 - 22 0C); after incubation
vortex tubes for few seconds.
h. Lyse by adding 2 mL of FACS lysing solution and vortex for few seconds.
i. Incubate in dark at RT for 12 minutes.

j. After incubation, centrifuge the tubes at 500 g for 5 minutes; decant the supernatant and break the pellet.
k. Wash by adding 2 mL of sheath fluid and centrifuge the tubes at 500 g for 5 minutes; decant and vortex
the pellet.
l. Repeat another cell washing cycle (k).
m. Suspend the cell button in 500 µL of sheath fluid.
n. Run on pre-calibrated flow-cytometer as soon as possible.
(ii) Acquisition
 Acquisition of RBC
a Start BD Cell Quest Pro Software.
b Select the PNH RBC acquisition template.
c Open Detectors/Amps window and change the mode of FSC and SSC to “Log/Log”.
d Run sample using this template and save the findings with patients details.
e Before running, mix the tubes well with racking. (rack the tube 10 times on the plastic tube rack, and until
no cell clumps are seen)
f Acquire 5,000 events to the RBC gate. (for routine purposes, analyzing 5,000 - 10,000 cells of interest is
generally sufficient to detect populations at a sensitivity of detecting a clone of 1%)
g Look for Type II (partial deficiency) and Type III (complete deficiency) cells in SSC/CD59 dot plot. If there
are no events in Type II or Type III areas of the dot plot, and in the statistic table, the acquisition can be
stopped. If there are any events in Type II/Type III area and in the statistic table, acquire up to 10,000
events.
 Acquisition of WBC
a Start BD Cell Quest Pro Software.
b Select the PNH WBC acquisition template.
c Mix the tubes well (may use the vortex) before acquisition.
d Run the sample using this template and rename the details of the patient.
e Acquisitions of events - acquire 10,000 events in neutrophils/monocyte gates. Acquisition of a smaller
number of monocytes is acceptable, if the result tallies with neutrophils.
 Data saving - Save information in the data saving folder.
Interpretation of results
1. PNH clone - RBC/Neutrophils/Monocytes population with absent GPI linked antigen expression. At least two GPI
linked antigens should be negative on one cell lineage.
2. To define PNH - PNH clones has to be present in at least two cell lineages, (RBC/neutrophils/monocytes).
3. Analysis RBC alone without WBC is not recommended.
4. There is no place for lymphocyte analysis.
5. The flow-cytometric findings should be correlated with the clinical and other laboratory information.
Reporting of results
1) If WBC or RBC cell populations with absent GPI linked antigens were not detected, it is reported as, “No evidence
of Paroxysmal nocturnal haemoglobinuria (PNH) clone”.
2) If WBC/ RBC cell populations with deficient/absent GPI linked antigens are detected, it is reported as “Paroxysmal
nocturnal haemoglobinuria (PNH) clone is present”
The size of the PNH clone (total of type II & type III cells) in each cell lineage has to be given as a percentage (%).

 Initially, the lab should run ten normal samples and develop the standard template for RBC and WBC.
 IQC -
a. Most of PNH positive samples will have at least a few normal cells and this could be used as an internal
control for both monocytes and granulocytic lineages.
b. Unstained RBC and unstained WBC tubes should be run as internal controls with each batch of samples.
c. If all cell populations are negative for all PNH-antibodies used, test should be repeated with a
normal control sample.
 EQA – there is no EQA programme available at present. Therefore, an alternative assessment policy is used.
Internal split samples – once in four months, one sample is chosen randomly and test done by two MLTT separately
and results are compared.
Acceptable criteria - 100% concordance in detecting PNH clones; If PNH clones detected the quantity (%) has to be
similar.
Sources of error
 Refrigeration of samples prior to testing can give rise to erroneous results.

 Loss of cells during sample processing.
 Failure to add antibodies / use of contaminated or expired antibodies.
 All parts and surfaces of the flow cytometer and other equipment should be considered as potentially infectious
since the instrument handles patient specimens.
 References
1. Rother RP et al. The Clinical Sequelae of Intravascular Hemolysis and Extracellular Plasma Hemoglobin. JAMA.
2005 Apr 6;293(13):1653-62.
2. Brodsky RA. In: R. Hoffman et al, eds. Hematology: Basic Principles and Practice. 4th edition (2005).
3. Rother RP et al. Discovery and development of the complement inhibitor eculizumab for the treatment of
paroxysmal nocturnal hemoglobinuria. Nature Biotechnology. 2007 Nov;25(11):1256-64.
4. Rosse WF et al. Immune-Mediated Hemolytic Anemia. Hematology Am Soc Hematol Educ Program 2004 (1):
48–62.
5. Wiedmer T et al. Complement-Induced Vesiculation and Exposure of Membrane Prothrombinase Sites in
Platelets of Paroxysmal Nocturnal Hemoglobinuria. Blood. Volume 82, Issue 4, 15 August 1993, Pages 1192-
1196.
6. Socié G et al. Paroxysmal nocturnal haemoglobinuria: long-term follow-up and prognostic factors. French Society
of Haematology. Lancet. 1996 Aug 31;348(9027):573-7.
7. Hillmen P et al. Natural History of Paroxysmal Nocturnal Hemoglobinuria. N Engl J Med 1995; 333: 1253-1258.
8. Hillmen P et al. Effect of the complement inhibitor eculizumab on thromboembolism in patients with paroxysmal
nocturnal hemoglobinuria. Blood (2007) 110 (12): 4123–4128.

9. Dacie JV, Lewis SM. SerHaematol 1972; 5: 3-23.
10. http://www.netflowconnect.com/
11. Parker C et al. Diagnosis and management of paroxysmal nocturnal hemoglobinuria. Blood 2005; 106: 3699-
3709.
12. Borowitz MJ et al. Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and
related disorders by flow cytometry. Cytometry B ClinCytom 2010; 78B: 211-230.
13. Sutherland DR et al. Practical guidelines for the high-sensitivity detection and monitoring of paroxysmal
nocturnal hemoglobinuria clones by flow cytometry. Cytometry B ClinCytom 2012; 82B: 195-208.
14. BD FACS CaliburTM Instructions for use - User manual.

SECTION 09
STAINS USED IN HAEMATOLOGY

LEISHMAN

MAY GRUNWALD - GIEMSA

NEW MODIFIED GIEMSA (NEW MGS)

PERLS (PRUSSIAN BLUE)

SUDAN BLACK B

PERIODIC ACID SCHIFF (PAS)

NON-SPECIFIC ESTERASE (NSE)

DOUBLE ESTERASE (DE)

TOLUIDINE BLUE

NEUTROPHIL ALKALINE PHOSPHATASE SCORE (NAP SCORE)
9.1
LEISHMAN
Mr. B.B. Isuru Namal Priyankara
Scope
The scope of this SOP is to guide technical personnel on correct performance of Leishman stain by providing the protocol
and requirements for the procedure.
Responsibility
Performance of staining with Leishman stain as per the procedure stated in this SOP is the responsibility of all medical
laboratory technologists, who are assigned to perform this staining in the haematology laboratory.
Principle
Leishman stain contains a methanolic mixture of an acidic dye called eosin Y, and a basic dye called polychromated
methylene blue. It colours the various cells in different ways allowing differentiation of each cell from the other. This
methanolic stock solution is stable and also serves the purpose of directly fixing the smear eliminating a prefixing step.
Leishman stain is a Romanowsky stain and is used in microscopy for staining blood and bone marrow smears. It is
generally used to differentiate and identify leucocytes, malaria parasites, and trypanosomes. When immunophenotyping
is unavailable, Leishman stain is important in identifying basic morphology of blood cell precursors and mature cells.
It colours nuclear and cytoplasmic details clearly. It is important for the diagnosis and helpful in determination of panel
for immunophenotyping by flowcytometry.
Sample
Air dried blood and bone marrow films. Prior fixation by methanol is not required.

1. Staining rack
2. Timer clock
3. Rack for drying slides
4. Wash bottle containing buffered distilled water
STANDARD OPERATING PROCEDURES FOR STAINS USED IN HAEMATOLOGY 245

5. Mortar and pestle (for grinding of stain powder in methanol)
6. Narrow range pH papers
7. Leishman stain powder
8. Absolute methanol
9. Distilled water
10. Disodium hydrogen phosphate (Na2HPO4 or Na2HPO4.2H2O)
11. Potassium dihydrogen phosphate (KH2PO4)
Method
1. Method of preparation of Leishman stain working solution
 Dissolve 0.2 g of powdered Leishman stain in 100 mL of absolute methanol and transfer the solution in to dark
colour bottle. (Grinding of stain powder with methanol may give more effective dissolving)
 Then warm it for 15 minutes at 50 0C with occasional shaking.
 Allow the bottle to cool and leave the stock solution for standing prior to use for improvement of the stain.
 Filter the required amount of stain for daily use into a dropping bottle. (Filtering of the stain before use is
mandatory to prevent deposition of stain particles on the film)
2. Preparation of buffered distilled water (Sörenson phosphate buffer – 66 mmol/ L). For general use buffered distilled
water with pH 6.8 is used. However, when looking for malaria parasites, a pH of 7.2 is recommended for better
observation of Schüffner dots.
 Prepare stock solutions of A & B as follows;
(A) 66 mmol/L KH2PO4 – 9.1 g of KH2PO4/ 1L distilled water
(B) 66 mmol/L Na2HPO4 – 9.5 g of Na2HPO4 or 11.9 g of Na2HPO4. 2H2O/ 1 L distilled water
 Mix A and B solutions at the proportion of 50.8: 49.2 to obtain the buffered water with the pH 6.8. To prepare
the buffer with pH of 7.2, need to mix A and B at the proportion of 28: 72.
 Check the pH of the solution using narrow range pH papers.
3. Method of staining a blood or bone marrow smear

 Freshly prepare a thin film and air dry rapidly.
 Prior fixation is not required as methanol in Leishman stain acts as the fixative.
 Filter the stain.
 Place the film on a staining rack and flood with Leishman stain.
 Leave for 30 seconds to 2 minutes to fix the smear.
 Add double the volume of buffered distilled water at pH 6.8 to the slide, preferably from a plastic wash bottle
to allow better mixing of the solution.
 Allow the diluted stain to act for 10 - 12 minutes.
 Then rinse the slide with tap water.
 Wipe clean the back of the slide and set it up right to drain and dry in air.

 Colour responses of blood cells to Romanowsky staining are as follows (Table 9.1.1)
Table 9.1.1 Colour responses of blood cells to Romanowsky stains
Cellular component Colour
Chromatin Purple
Nuclei
Nucleoli Light blue
Erythroblast Dark blue
Erythrocyte Dark pink
Reticulocyte Grey-blue
Lymphocyte Blue
Metamyelocyte Pink
Cytoplasm
Monocyte Grey-blue
Myelocyte Pink
Neutrophil Pink/orange
Promyelocyte Blue
Basophil Blue
Platelet Pale pink
Promyelocyte (primary granules) Red or purple
Basophil Purple-black
Granules Eosinophil Red-orange

Neutrophil Purple
Toxic granules Dark purple
Platelet Purple
Auer body Purple
Cabot ring Purple
Other Inclusions
Howell–Jolly body Purple
Döhle body Light blue
 Depending on the colour differences of cellular components, different cells and abnormalities on stained blood or
bone marrow films are identified and reported.

 Methanol used should be completely free of water, as it acts as the fixative of the smear. As little as 1% water may
affect the appearance of the films and a higher water content causes gross changes.
 Leishman stock solution should be stored in a tightly closed, dark colour container in a dry ventilated area protected
from bright light and sources of ignition.
 Leishman stock solution can be stored at room temperature for about 3 months. Therefore, the date of preparation
of the stain should be clearly mentioned in the label on the bottle.
 Leishman stain should be filtered correctly before use.
 To preserve the morphology of cells, films must be fixed without delay and the films should never be left unfixed for
more than a few hours.
 For quality control, the quality of stain should be compared with a well-made normal cover slipped slide on day-to-
day basis to detect the deterioration of the stain.

Sources of error
 Unfiltered solutions.
 Expired Leishman powder, methanol or Leishman stain stock solution
 Haemolyzed, partially air-dried or partially fixed blood or bone marrow smears
 Contamination of reagents
 Errors related to preparation of stain and staining method (Table 9.1.2)
Table 9.1.2 Factors giving rise to faulty staining
Appearance Causes
Too blue Incorrect preparation of stock, Eosin concentration

too low, Stock solution exposed to bright light,
Impure dyes, Staining time too short, Staining solution too acidic,
Smear is too thick, Inadequate time in buffer solution
Too pink Incorrect proportion of azure B: eosin Y in the stain powder, Impure dyes,
Buffer pH too low, Excessive washing in buffer solution
Pale staining Old staining solution, Incorrect preparation of stock, Impure dyes,
High ambient temperature.
Neutrophil granules not stained Insufficient azure B
Neutrophil granules
dark blue/ black (pseudotoxic) Excess azure B
Stain deposit on film Stain solution not filtered before use, Stain solution
left in uncovered jar, Stain solution is left for a prolonged
period on the slide without adding buffered water
Blue background Inadequate fixation or prolonged storage before fixation,

Blood collected into heparin as an anticoagulant

 As methanol is highly inflammable, this chemical and Leishman stock solution should be stored away from sources
of ignition.
 References
2. NCCLS guidelines (2003).
3. Hand book on I & J Giemsa and other Romanowsky staining methods for blood and bone marrow
smears. (ISBN - : 978-955-38758-0-8 ).
4. Gajendra S, Jha B, Goel S, Sahni T, Sharma R, Shariq M, et al. Leishman and Giemsa stain: a new reliable
staining technique for blood/bone marrow smears. Int J Lab Hematol. 2015;37(6):774-82.
5. Teerasaksilp S, Wiwanitkit V, Lekngam P. Comparative study of blood cell staining with Wright-Giemsa stain,
field stain, and a new modified stain. Lab Hematol. 2005;11(1):76-78.

9.2
MAY GRUNWALD - GIEMSA
Scope
The scope of this SOP is to guide technical personnel on correct performance of May – Grunwald - Giemsa Stain (MGG
stain) by providing the protocol and requirements for the procedure.
Responsibility
Performance of MGG staining as per the procedure stated in this SOP is the responsibility of all medical laboratory
technologists who are assigned to perform this staining in the haematology laboratory.
Principle
MGG stain is a Romanowsky stain which involves two steps that includes first staining with May – Grunwald stain followed
by Giemsa stain. It stains various cellular components in different colours allowing clear distinction of cells. This property
depends on its components; alkaline methylene blue and related azures (basic dyes) and Eosin Y, an acidic dye. The
cationic basic dyes stain negatively charged molecules (nuclei of blood cells, granules of basophils and RNA molecules
of the cytoplasm of granulocytes) while anionic acidic dyes stain positively charged molecules (red cells and granules of
eosinophils).
MGG stain is used for staining blood and bone marrow smears. It is generally used to differentiate and identify leucocytes,
malaria and other blood parasites such as trypanosomes. MGG stains can also be used to identify mast cells. When
immunophenotyping is unavailable, MGG staining is very important to identify basic morphology of blood cell precursors
and mature cells. It clearly colours nuclear and cytoplasmic details. It is important for the diagnosis and helpful in
determination of panel for immunophenotyping by flowcytometry.
Sample
Air dried blood and bone marrow films.

1. Staining rack
2. Timer clock
5 Pasteur pipette
6. Giemsa stain powder
7 Absolute methanol
8. Distilled water
9. Conical flasks
Method
1. Preparation of working stain solutions
May – Grunwald stain
 Weigh 0.3 g of powdered dye and transfer it into a conical flask of 200 mL – 250 mL capacity.
 Add 100 mL of methanol. (Grinding of stain powder with methanol may give more effective dissolving)
 Warm the mixture to 50 0C.
 Allow the flask to cool to 20 0C and shake several times during the day.
 After letting it stand for 24 hours, filter the solution.
Giemsa stain
 Weigh 1 g of powdered dye and transfer it into a conical flask of 200 mL – 250 mL capacity.
 Add 100 mL of methanol. (Grinding of stain powder with methanol may give more effective dissolving)
 Warm the mixture to 50 0C. Keep at this temperature for 15 minutes with occasional shaking.
 Filter the solution
Note: The stain will improve on standing for a few hours.
2. Method of staining blood or bone marrow smears

 Freshly prepare a thin film and air dry.
 Immediately after drying, fix the smear by immersing in a jar of methanol for 5 – 10 minutes.
(For bone marrow smears, allow a longer time for thorough drying and then fix by leaving them for
15 – 20 minutes in methanol).
 Transfer the fixed films into a jar containing May – Grunwald stain freshly diluted with an equal volume of
phosphate buffered water and keep for about 15 minutes.
 Transfer the film directly into a jar containing Giemsa stain freshly diluted with 9 volumes of phosphate buffered
water, pH 6.8.
 After 10 – 15 minutes, transfer the smears into a jar containing buffered water, pH 6.8.
 Rapidly wash in three or four changes of water and finally allow the films to stand undisturbed in water for
2 – 5 minutes.
(Naked eye colour of the film will be a good guide of proper differentiation)
 Stand the slides upright to dry.
Note: Individual laboratories may establish their own staining times based on the results of staining.

 Colour responses of blood cells to Romanowsky staining are as follows (Table 9.2.1)
Table 9.2.1 Colour responses of blood cells to Romanowsky stains

Chromatin Purple
Nuclei Nucleoli Light blue
Erythrocyte Dark pink
Lymphocyte Blue
Cytoplasm Metamyelocyte Pink
Monocyte Grey-blue
Myelocyte Pink
Neutrophil Pink/orange
Promyelocyte Blue
Basophil / Mast cell Blue
Promyelocyte (primary granules) Red or purple
Basophil / Mast cell Purple-black
Eosinophil Red-orange
Granules
Neutrophil Purple
Platelet Purple
Auer body Purple
Cabot ring Purple
Other Inclusions
Döhle body Light blue
Depending on the colour differences of cellular components, different cells and abnormalities on stained blood or
bone marrow films are identified and reported.

 Methanol used should be completely free of water, as it acts as the fixative of the smear. As little as 1% water may
 The stock stain solutions should be stored in a tightly closed, dark colour container in a dry ventilated area
protected from bright light and sources of ignition.
 It can be stored at room temperature for about 6 months. Therefore, the date of preparation of the stain should
be clearly mentioned in the label on the bottle.
 To preserve morphology of cells, films must be fixed without delay and the films should never be left unfixed for
 The slides should be transferred from one staining solution to the other without being allowed to dry.
 For quality control, the stain quality should be compared with a well-made normal slide with a cover slip on day-
to-day basis to detect deterioration of the stain.

Sources of error
 Expired stain powder, methanol or stain stock solutions.
 Haemolyzed, partially fixed or partially air-dried blood or bone marrow smears.
 Contamination of reagents.
 Changes of pH in solutions.

Appearance Causes
Too blue Incorrect preparation of stock, Eosin concentration

too low, Stock solution exposed to bright light,
Impure dyes, Staining time too short, Staining solution too acidic,
Smear is too thick, Inadequate time in buffer solution
Too pink Incorrect proportion of azure B: eosin Y in the stain powder, Impure dyes,
Buffer pH too low, Excessive washing in buffer solution
Pale staining Old staining solution, Incorrect preparation of stock, Impure dyes,
High ambient temperature.
Neutrophil granules not stained Insufficient azure B
Neutrophil granules
dark blue/black (pseudotoxic) Excess azure B
Stain deposit on film Stain solution not filtered before use, Stain solution
left in uncovered jar, Stain solution is left for a prolonged
period on the slide without adding buffered water

Blood collected into heparin as an anticoagulant

 As methanol is highly inflammable, this chemical and stock solutions should be stored away from sources of ignition.
 References
3. Gajendra S, Jha B, Goel S, Sahni T, Sharma R, Shariq M, et al. Leishman and Giemsa stain: a new reliable staining
technique for blood/ bone marrow smears. Int J Lab Hematol. 2015;37(6):774-82.

9.3
NEW MODIFIED GIEMSA
(NEW MGS)
Scope
The scope of this SOP is to guide technical personnel on correct performance of new Modified Giemsa Stain (MGS) by
providing the protocol and requirements for the procedure.
Responsibility
Performance of staining with new MGS as per the procedure stated in this SOP is the responsibility of all medical
laboratory technologists who are assigned to perform this staining in the haematology laboratory.
Principle
Giemsa stain contains a basic or cationic dye called methylene blue, and an acidic or anionic dye called azure B (Eosinated).
It colours various cells in different ways allowing differentiation of each cell from the other. This methanolic stock
solution is stable and also serves the purpose of directly fixing the smear eliminating a prefixing step. This New Modified
Giemsa Stain (MGS) is a modification of the Giemsa stain in which the staining time is shorter and provides improved
staining quality. The cost of staining is also low and user-friendly with lesser number of steps to follow, compared with
conventional staining methods.
Giemsa stain is used in microscopy for staining blood and bone marrow smears. It is generally used to differentiate and
identify leucocytes, malaria and other blood parasites such as trypanosomes. Giemsa stains can also be used to identify
mast cells. When immunophenotyping is unavailable, Giemsa staining is very important to identify basic morphology of
blood cell precursors and mature cells. It clearly colours nuclear and cytoplasmic details. It is important for the diagnosis
and helpful in determination of panel for immunophenotyping by flowcytometry.
Sample
Air dried blood and bone marrow films. Prior fixation by methanol is not needed.

1. Staining rack
2. Timer clock
5. Pasteur pipette
6. Giemsa stain powder
8. Distilled water
Method
1. Method of preparation of new MGS working solution
 Dissolve 0.15 g of powdered Giemsa stain dye in 100 mL of absolute methanol and filter into a dark bottle.
(Grinding of Geimsa powder with methanol may give more effective dissolving)
 Then warm it for 3 hours at 50 0C.
2. Method of staining a blood or bone marrow smear
 Prior fixation is not required, as methanol in the stain acts as the fixative.
 Place the film on a staining rack and flood with the stain.
 Leave for 1½ minutes to fix the smear.
 Add double the volume of distilled water to the slide, mix well by using a Pasteur pipette without touching the
smear.
 Allow the diluted stain to act for 10 minutes.
 Then rinse the slide with tap water.
 Wipe clean the back of the slide and set it up-right to drain and dry in air.

 Colour responses of blood cells to new MGS are as follows (Table 9.3.1)
Table 9.3.1 Colour responses of blood cells to new MGS

Chromatin Purple
Nuclei
Nucleoli Blue
Erythrocyte Pinkish Blue
Lymphocyte Blue
Metamyelocyte Pink
Cytoplasm Monocyte Light blue
Myelocyte Pink
Neutrophil Pink
Promyelocyte Pinkish Blue
Basophil Pinkish Blue
Platelet Pink
Promyelocyte (primary granules) Dark purple
Basophil Purple-black
Eosinophil Red-orange
Granules Neutrophil Purple
Platelet Purple
Auer body Purple/ magenta
Cabot ring Purple
Other Inclusions
Döhle body Blue
Depending on the colour differences of cellular components, different cells and abnormalities on stained blood or bone
marrow films are identified and reported.

 Methanol used should be completely water free, as it acts as the fixative of the smear. As little as 1% of water may
 The stock stain solution should be stored in a tightly closed, dark colour container in a dry ventilated area protected
from bright light and sources of ignition.
 It can be stored at room temperature for about 6 months. Therefore, the date of preparation of the stain should be
clearly mentioned in the label on the bottle.
 Stain should be filtered correctly before use.
 To preserve morphology of cells, films must be fixed without delay and the films should never be left unfixed for
 For quality control, the stain quality should be compared with a well-made normal slide with a cover slip on day-to-
day basis to detect deterioration of the stain.

Sources of error
 Expired Giemsa powder, methanol or Giemsa stain stock solution
 Haemolyzed, partially air-dried or partially fixed blood or bone marrow smears
Appearance Causes
Incorrect preparation of stock, Eosin concentration too low,
Stock solution exposed to bright light, Impure dyes, Staining time too short,
Too blue Staining solution too acidic, Smear is too thick,
Inadequate time in distilled water.
Incorrect proportion of azure B: eosin Y in the stain powder,

Too pink Impure dyes, Buffer pH too low, Excessive washing of the smear.
Pale staining Old staining solution, Incorrect preparation of stock,

Impure dyes, High ambient temperature.
Neutrophil granules not stained Insufficient methylene blue
Neutrophil granules
Excess methylene blue
dark blue/ black (pseudotoxic)
Stain solution not filtered before use, Stain solution left in uncovered jar,
Stain deposit on film Stain solution is left for a prolonged period on the
slide without adding distilled water

Blood collected into heparin as anticoagulant

 As methanol is highly inflammable, this chemical and Giemsa stock solution should be stored away from sources of
ignition.
 References
1. Gunawardena G, Priyankara I, Jayamanne H, Suresh S. Modified Giemsa Stain: A solution to improve the quality
of hypercellular bone marrow smears. Int J Lab Hematol. 2022 April 5. https://doi.org/10.1111/ijlh.13824.
4. Hand book on I & J Giemsa and other Romanowsky staining methods for blood and bone marrow smears.
(ISBN - : 978-955-38758-0-8 ).
5. Gajendra S, Jha B, Goel S, Sahni T, Sharma R, Shariq M, et al. Leishman and Giemsa stain: a new reliable staining
technique for blood/bone marrow smears. Int J Lab Hematol. 2015;37(6):774-82.

9.4
PERLS (PRUSSIAN BLUE)
Scope
The scope of this SOP is to guide technical personnel on correct performance of Perls (Prussian blue) iron staining by
Responsibility
Performance of Perls iron staining as per the procedure stated in this SOP is the responsibility of all medical laboratory
technologists, who are assigned to perform this staining in the haematology laboratory.
Principle
The test is based on the Perls reaction (Prussian blue reaction), where siderotic material (Haemosiderin) mainly in
macrophages or erythroid precursors react with potassium ferrocyanide to form a Prussian blue coloured compound,
ferriferrocyanide which is interpreted as positive Perls reaction, indicating presence of stored iron in the cells.
Perls stain allows assessment of both the amount of iron in macrophage stores and the availability of iron to developing
erythroblasts to be visualized. Absence of iron in bone marrow is diagnostic of iron deficiency or iron depletion (i.e. the
state where storage iron is absent but anaemia is not yet evident). Increased iron stores are seen in iron overloading
conditions such as thalassemia major /intermedia, dyserythropietic anaemias, sideroblastic anemia, haemochromatosis
and transfusional haemosiderosis, ect. Perls stain is also helpful in identifying characteristic ring sideroblasts. Ring
sideroblasts have been defined as erythroblasts with at least five siderotic granules surrounding at least one third of
the nucleus. The presence of >15% ring sideroblasts is a defining feature of Myelodysplastic syndrome (MDS) with ring
sideroblasts. Identification of this unique entity of MDS with a relatively favourable prognosis is important in deciding
the treatment options. Lesser number of ring sideroblasts are seen in several other conditions including megaloblastic
anaemia, other haematological neoplasms (e.g erythroleukaemia, myelofibrosis), alcoholism, lead poisoning, etc.

Sample
1. Freshly collected bone marrow sample into EDTA anticoagulant or directly prepared bone marrow smears without
using anticoagulant.
2. Freshly collected urine sample in case of testing for urine hemosiderin. (See SOP for demonstration of haemosiderin
in urine)
3. This method can be applied to films which have previously been stained by Romanowsky stains, even after years
of storage. However, such films should be kept in methanol overnight to remove the Romanowsky dyes as much as
possible. Then the films should be checked before carrying out Perls reaction, to ensure that there is no residual
blue staining that could obscure Prussian blue staining.

1. Staining jar
2. Staining rack
3. Timer clock
5. Immersion oil
6. 0.2 N HCl:
2 mL of concentrated HCl is poured carefully into 98 mL of cold distilled water with constant stirring.
(This solution is stable for a month at room temperature)
7. 2 g/dL potassium ferrocyanide:
Dissolve 2 g of potassium ferrocyanide in 100 mL of distilled water.
(This solution is stable for a month at room temperature if stored in a dark coloured bottle and kept away from light)
8. 10 g/L aqueous neutral red or Eosin (as counter stain):
Dissolve 1 g of neutral red or eosin in 100 mL of distilled water.
(This solution is stable for 2 months at room temperature)
10. Absolute ethanol
Method
 Freshly prepare a thin film and air dry rapidly. (Select a bone marrow smear with particles)
 Fix the blood or bone marrow film for 5 minutes in absolute methanol.
 Prepare the working solution by mixing equal volumes of 0.2 N HCl and 2 g/dL potassium ferrocyanide solution in a
staining jar, immediately before use.
 Place the slides in working solution at room temperature for 10 minutes. (Time can be extended up to 20 minutes)
 Wash thoroughly with running tap water for 20 minutes.
 Dip smears in eosin for 5 minutes for counterstaining.
 Rinse thoroughly in running tap water for 10 minutes.
 Then keep the slide on a staining rack and pour absolute ethanol on to the slide and keep for 1 minute or dip the
slide once, in a jar containing absolute ethanol.
 Again, wash with tap water for 10 minutes.
 Wipe clean the back of the slide and set it up-right to drain and dry in air.
 Assess iron stores under the light microscope at 5x, 10x and 40x objectives for stainable iron.
 Examine under oil immersion for the presence of ring sideroblasts.

 The reaction product is blue green or Prussian blue. The intensity of reaction product varies with the content of iron.
 Interpretation and conclusions on stained films should be made by the consultant haematologist.
 It is expected to review at least nine particles in one or more bone marrow films to make a valid judgment of
whether iron stores are reduced, normal, or increased.
 It is recommended to examine a minimum of seven particles to reliably establish the absence of stainable iron. If less
than seven particles are present and no iron is seen, the sample should be reported as lacking stainable iron, but the
assessment should be stated to be unreliable.
a) Reporting on iron stores -
In routine practice, assessment of iron stores is done semi-quantitatively and reported using the following two
methods.
Method 1 : Interpretation as absent / scanty / reduced / normal / increased iron stores.
Method 2 : Graded as 0 to 6+ (according to the method of Rath and Finch) as shown below (Table 9.4.1)
Table 9.4.1 Grading of iron stain
Grade Description
0 No stainable iron
1+ Small iron particles just visible in macrophages using an oil objective*
2+ Small, sparse iron particles in macrophages, visible at lower power *
3+ Numerous small particles in macrophages*
4+ Larger particles with a tendency to aggregate into clumps
5+ Dense, large clumps
6+ Very large clumps and extracellular iron
*Grades of 1+ to 3+ is considered normal.
b) Reporting ring sideroblasts –

In the absence of ring sideroblasts → No ring sideroblasts are noted.
When ring sideroblasts are seen → Ring sideroblasts are noted accounting for (….) % of bone marrow erythroblasts.

 A film made from a previously known iron positive bone marrow aspirate/ known tissue section positive for iron (e.g.
splenectomy specimen of a thalassaemia patient) should be used as a positive control to confirm the quality of the
stain. The control slide should always be stained together with the test slide/s.
 Iron free glassware should be used during the procedure. (Prepared by soaking them overnight in 3 mol/L HCl and
washing with distilled water before use)
 Working solution must be freshly prepared by mixing potassium ferrocyanide and HCl at each test.
 Potassium ferrocyanide solution should be stored in a dark coloured bottle and kept away from light to prevent
decomposition.
Sources of error
 Use of non-acid washed glassware.
 Use of already prepared working solution.
 Expired reagents.
 Use of haemolysed samples or partially air-dried/ partially fixed blood or bone marrow smears.
 Decomposition of potassium ferrocyanide solution due to exposure to light.

 Be extremely cautious when storing and handling concentrated HCl which is highly corrosive.
 When diluting acids, always note that concentrated HCl should be poured carefully into cold distilled water with
constant stirring. (Avoid pouring distilled water into concentrated HCl, as it can create a hazardous situations.)
 References
2. Bain BJ, Clark DM, Wilkins BS. Bone marrow pathology, 5th Edition, 2019.
3. Rath CE, Finch CA. Sternal marrow haemosiderin: a method for the determination of available iron stores in
man. J Lab Clin Med, 1948;33:81–6.

9.5
SUDAN BLACK B
Scope
The scope of this SOP is to guide technical personnel on correct performance of Sudan Black B (SBB) stain by providing
the protocol and requirements for the procedure.
Responsibility
Performance of Sudan Black B staining as per the procedure stated in this SOP is the responsibility of all medical laboratory
technologists who are assigned to perform this special staining in the haematology laboratory.
Principle
Sudan black B is a fat-soluble dye, which has a very high affinity for neutral fats and lipids. Therefore, it stains lipids such
as sterols, neutral fats and phospholipids which are present in azurophilic and secondary granules of myeloid lineage
cells (granulocytes and eosinophils) and lysosomal granules of monocytic cells. During staining, the dye leaves the solvent
because of its high solubility in lipids than the solvent and binds irreversibly to the cytoplasmic granule component of
these cells. On microscopic examination, varying degree of black colored pigments are seen in the positive reaction. It
gives comparable results to that of Myeloperoxidase staining.
SBB reactivity is essentially similar to Myeloperoxidase (MPO) in myeloblasts and monoblasts. However, its' specificity for
myeloid lineage is less than MPO. Positive cells usually stain more intensely with SBB than MPO. The staining becomes
more intense as cells mature from myeloblast to mature forms. Lymphoblasts are usually negative. However, in rare
cases, lymphoblasts contain granules which may stain light grey with SBB, contrasting to the black granules in neutrophils
and myeloblasts. Erythroblasts, basophils and megakaryoblasts are generally negative for SBB. Therefore, SBB staining
is useful for the differentiation of Acute myeloid leukemia (AML) from Acute lymphoid leukemia (ALL) and further
classification of AML subtypes especially when immunophenotyping is not available.
It has few advantages over MPO:
 As SBB reactivity is stable for months (practically for about 3 months) in unstained slides whereas MPO is stable
only for about 4 weeks.
 SBB stains both azurophilic and specific granules in neutrophils, whereas MPO stains azurophilic granules only.
 There is only a little fading of the SBB stain over time.

Sample
Air dried peripheral blood or bone marrow films. (Smears prepared with non- anticoagulated blood/ bone marrow are
preferred)

1. Staining rack
2. Coplin jar
3. Timer clock
5. Mortar and pestle (Optional)
6. Fixative – 40% formaldehyde solution (Formalin)
7. 70% ethanol (Prepared by diluting 70 mL of absolute ethanol to 100 mL with distilled water)
8. Stain (A) – Sudan Black B stock solution
Preparation of SBB stock solution;
 Put 0.3 g of Sudan Black B powder in to 100 mL of absolute ethanol into a volumetric flask and shake well
till the powder dissolves well. (It is better to keep the solution at 40 0C for 30 minutes) Otherwise, allow
the solution to stand at room temperature for one to three days and shake frequently until Sudan Black B
completely dissolves) Alternatively, the Sudan Black B may be ground in ethanol with a mortar and pestle and
the suspension heated.
 Filter the solution into a dark bottle.
 Stock solution is stable for 6 months at 25 0C. (Room temperature)
9. Phenol buffer (B)
Preparation of phenol buffer stock solution;
 Add 16 g of crystalline phenol into 30 mL of absolute ethanol and mix well. (Phenol – ethanol mixture).
(If available, liquid phenol can be used, calculating the required volume according to the density of phenol. e.g.
16.77 mL if 1g = 1.06 mL)
 Add 0.3 g of disodium hydrogen phosphate (Na2HPO4.12H2O) in to a 100 mL volumetric flask, then add distilled
water up to the mark and shake well. (Buffer solution)
 Add phenol – ethanol mixture into buffer solution and stir vigorously until all the phenol has dissolved well.
Then filter the solution into a dark bottle.
 Stock solution is stable for 6 months at 25 0C.
10. Counter stain – 1 % safranine
Preparation – Add 1 g of safranine powder in to 100 mL distilled water and filter it into a dark bottle.
(Giemsa or Leishman stain can also be used as an alternative counter stain)
Method
 Preparation of working SBB stain solution:
 Mix 30 mL of solution A with 20 mL of solution B and filter into a dark bottle.
 This solution may be reused repeatedly but should be replaced when lighter staining of control slides is noted.
 Working stain solution is stable for 2 - 3 months if kept at 4 0C.
 Fix air dried blood or bone marrow smears in formalin vapour for 10 minutes at room temperature.
 Prepare a formalin vapour bath by keeping a soaked filter paper or adsorbing material in the bottom of a Coplin
jar and closing with the lid. Leave for 15 minutes to allow vaporization.
 Then place the slides in the Coplin jar and replace the lid.
 After 10 minutes, keep slides outside for 1 - 2 minutes on air to evaporate the formalin vapour.
 Wash the smears gently in tap water for 5 - 10 minutes to remove formalin.

 Filter the working SBB stain solution into a Coplin jar and immerse the fixed smears in it.
 Close the jar and keep in an incubator at 37 0C for one hour.
 Transfer slides to a staining rack and immediately flood with 70% alcohol for 30 seconds. Tip the 70% alcohol off and
flood again for 30 seconds. Repeat this three times in total to remove excess stain, observing under the microscope.
 Wash gently under running tap water for 2 minutes and air dry.
 Then dip smears in a 1% safranine jar for 5 minutes for counterstaining.
 Rinse with tap water and allow to air dry.
 The prepared smears are stable for 3 months without fading.

 Interpretation and conclusions should be made by the consultant haematologist.
 Black and granular reaction product seen in the cytoplasm indicates positive reaction. Conventionally, ≥ 3% positivity
of total blasts is interpreted as ‘Sudan Black B – Positive’.
 Results are essentially similar to MPO staining both normal and leukemic cells.
 The only notable difference is in eosinophil granules, which have a clear core when stained with SBB.
 Rare cases (1 - 2%) of acute lymphoblastic leukaemia (ALL) show nongranular smudgy positivity not seen with MPO
staining.
 Basophils are generally negative but may show bright red/ purple metachromatic staining of the granules.
 Results are reaported as - Blasts are positive/ negative for Sudan Black B

 All reagents should be filtered correctly before use.
 Working stain solution should be filtered before use.
 Known positive AML smear or a peripheral blood film made from a sample with neutrophil leucocytosis can be used
as the positive control for the Sudan reaction and should be stained along with the test smear.
Sources of error
 Old working reagent and expired phenol buffer reagent.
 Haemolysed, partially air-dried or partially fixed blood or bone marrow smears

 References
2. WHO Classification of Tumours, Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues,
IARC Press, Lyon, 2001.
3. Giri D, Sudan Black B Stain: Purpose, Principle, Procedure and Interpretation, November 18, 2018,
Cytopathology, Hematology. (http://laboratorytests.org/sudan-black-b-stain/).
4. Crook L, Liu P I, Cannon A, Walker E M, Histochemistry of Bone Marrow Aspirations, Annals of Clinical and
Laboratory Science, Vol. 10, No. 4, Pg 290-304.
5. Cytochemical stainings – National Cancer Institute, Maharagama.

9.6
PERIODIC ACID SCHIFF (PAS)
Scope
The scope of this SOP is to guide technical personnel on correct performance of Periodic Acid - Schiff (PAS) stain by
Responsibility
Performance of Periodic Acid - Schiff (PAS) staining as per procedure stated in this SOP is the responsibility of all medical
laboratory technologists who are assigned to perform this special staining in the haematology laboratory.
Principle
Periodic acid oxidizes 1 - 2 glycol groups of glycogen to produce stable dialdehydes which gives a red reaction product
when exposed to Schiff’s reagent (leucobasic fuchsin). Although the reaction is not specific for glycogen, PAS stain is
principally used to detect glycogen in haemopoietic cells. PAS positivity is observed as a red color, the intensity of the
color being roughly proportional to the quantity of glycogen present in various types of cells.
In pre - immunophenotyping era, PAS reaction was frequently used to identify lymphoblasts. ‘Block positivity’ seen on a
clear background is most characteristic of lymphoblasts rather than myeloblasts. Although the PAS reaction is redundant
for the diagnosis of acute lymphoblastic leukaemia (ALL) with the availability of immunophenotyping, it can still be
useful to identify abnormal erythroblasts and dysplastic megakaryocytes in acute myeloid leukaemia and myelodysplastic
syndrome. The cytoplasmic blush demonstrated with PAS helps to confirm a diagnosis of acute promyelocytic leukaemia.
Sample
 Air dried blood and bone marrow films.
 Previously fixed, iron-stained or Romanowsky-stained blood/ bone marrow films after decolorizing them with
methanol.

1. Staining rack
2. Coplin jars
3. Timer clock
5. Conical flasks
6. Water bath
7. Whatman No.1 filter
8. Fixative - Methanol
9. 1% Periodic Acid solution (HIO4.2H2O)
Preparation;
 Dissolve 1 g of Periodic Acid Powder in 100 mL of distilled water

 Stable for one week in brown bottle at 4 0C.
10. Schiff’s reagent

Reagents for preparation of Schiff’s reagent;
 Basic Fuchsin – 1 g
 Potassium metabisulphite – 2 g or Sodium metabisulphite 2.2 g
 Distilled water – 200 mL
 2 mL of Conc. HCl
 3 g of activated charcoal
Preparation;
 Boil distilled water in conical flask.

 Add 1 g of basic fuchsin to the boiled distilled water (Take out the boiled water beaker from heating source
and add basic fuchsin)
 Mix well for 10 minutes.
 Allow the solution to cool down to 70 0C. Then filter the solution to a new conical flask.
 Then allow the filtrate to cool down to 50 0C.
Then add 2 g of potassium metabisulphite or 2.2 g of sodium metabisulphite and mix it well at least for
15 minutes.
 Filter the solution and allow to cool.
 Add 2 mL of Conc. HCl to the cooled solution. Mix well at least for 15 minutes. (while mixing you can observe
the change of colour from magenta to yellowish orange)
 Plug with cotton wool and keep overnight in a dark place for bleaching.
 Add 3 g of activated charcoal and mix for 1 minute and filter immediately through a large Whatman No.1 filter
into a dark bottle. (Solution should be clear after the filtration)
 Store the reagent in refrigerator.
11. Counterstain - Harris Haematoxylin

Reagents for preparation of Harris Haematoxylin;
 Haematoxylin 10 g
 Absolute ethanol 100 mL
 Ammonium/potassium alum 200 g

 Distilled water 2000 mL
 Mercuric oxide 5g
 Acetic acid 2 mL
Preparation;
Solution A ;
 Add alum 200 g in 2000 mL distilled water and boil it.
Solution B ;
 Dissolve 10 g of Haematoxylin in 100 mL of alcohol and keep in oven till it boils.
 After boiling, add solution B to solution A and add mercuric oxide. When dark purple colour appears, cool the
mixture in a cool water bath.
 Filter and add 2 mL of acetic acid.
Method
 Fix the air-dried smears in methanol in a Coplin jar for 15 minutes. Formalin vapour for 5 minutes,
formalin/ ethanol (10 mL 40 % formalin/ 90 mL ethanol) for 10 minutes or buffered formalin acetone for 45 seconds
are alternative fixatives.
 Wash the slides with distilled water and keep in gently running tap water for 10 minutes.
 Rinse the slide again with distilled water and then air dry.
 Flood 1% Periodic acid on to smears on slide rack and keep for 15 minutes.
 Rinse with distilled water and keep in running tap water for 10 minutes.
 Again, wash the smears with distilled water and air dry.
 Then, keep the slide/s on a slide rack and flood with Schiff’s reagent and keep for 15 minutes.
 Wash with distilled water and keep in running tap water for 10 minutes (If the stain is working well, you will observe
a magenta colour developing on smears) and air - dry.
 Observe under the microscope at x40 power to observe the colour development in neutrophils. (If not develop the
colors you can repeat from step 6)
 Rinse with distilled water and put smears in Harris Haematoxyline in Coplin jar for 2 minutes.
 Wash smears in running tap water for 5 minutes and dry in air.

 The reaction product is red (magenta) with intensity ranging from pink to bright red. Mainly, two types of PAS
staining patterns are reported, depending on which the different normal or abnormal cellular type/s are identified;
1. Cytoplasmic positivity – Could be diffuse or granular.

 Granulocyte precursors → show diffuse weak positivity.
 Neutrophils → show intense confluent granular positivity.
 Eosinophil → granules are negative, with diffuse cytoplasmic positivity.
 Basophils → may be negative but often show large irregular blocks of positive material not related to the
granules.
 Monocytes and their precursors → show variable diffuse positivity with superimposed fine granules, often
at the periphery of the cytoplasm.
 Normal erythroid precursors and red cells → negative.
 Abnormal erythroid precursors in acute erythroid leukaemia, an intense globular and diffuse reactivity.

 Megakaryocytes and platelets à variable, usually intense, diffuse positivity with superimposed fine
granules, coarse granules and large blocks.
 10 – 40% of peripheral lymphocytes → show granular positivity with negative background cytoplasm
(‘no blocks’), with no detectable differences between T and B cells.
2. Block positivity –
 Lymphoblasts → Magenta colour large cytoplasmic granules or blocks on a clear background.
 Block PAS positivity may also be observed in some lymphomas particularly in the T cell and Sezary cell
types.
 Results are reaported as - Blasts show block positivity for PAS/ Blasts are negative for PAS.

 All reagents should be filtered correctly before use.
 Schiff’s reagent should be prepared every two weeks.
 Periodic acid Schiff reagent should be stored at 4 0C.
 Known positive B ALL smear or a fresh peripheral blood smears containing adequate numbers of neutrophils can be
used as the positive control (Can have an idea about the reaction, by observing the colour of neutrophil cytoplasm
for red granules. If reaction happened well, it shows magenta coloured granules)
(Practical point – Put a drop of Schiff’s reagent into a beaker of water and observe for development of red colour within
1 – 2 minutes before use. If reagent is working well there should be a good colour development)
Sources of error
 Expired Schiff’s reagent and/ or sodium metabisulphite reagent.

 References
Laboratory Science, Vol. 10, No. 4, (1980) Pg 290-304.
3. Cytochemical stainings – National Cancer Institute – Maharagama.

9.7
NON-SPECIFIC ESTERASE (NSE)
Scope
The scope of this SOP is to guide technical personnel on proper performance of Non-Specific Esterase (NSE) stain by
Responsibility
Performance of NSE as per the procedure stated in this SOP is the responsibility of all medical laboratory technologists
who are assigned to perform this special staining in a haematology laboratory.
Principle
Non-specific esterase stain relies on an endogenous cellular esterase activity to hydrolyze an exogenous ester, α naphthyl
acetate substrate, to yield α -naphthyl which couples with hexazonium pararosaniline to give an insoluble brownish red
precipitate at the site of enzyme activity, which is visible under the light microscope.
When immunophenotyping is not available, NSE stain is used to confirm a diagnosis of acute myelogenous leukemia
with monocytic differentiation (FAB types M4 and M5). The strongly positive non-specific esterase staining is observed in
normal and leukaemic monocytes and megakaryocytes. However, weak, diffuse or focal positivity can also be observed
in some of the other haemopoietic cell types. The reaction with non-specific esterase is readily inhibited by sodium
fluoride.
Sample

1. Staining rack
2. Timer clock
5. Fixative – Buffered formal acetone

Reagents:
 20 mg Na2HPO4
 100 mg KH2PO4
 Acetone – 45 mL
 40% formalin – 25 mL
Preparation;
 Dissolve 20 mg Na2HPO4 and 100 mg KH2PO4, in 30 mL of distilled water. (Mixture A).
 Mix 45 mL of Acetone with 25 mL of 40% formalin in a beaker and add into mixture A and mix well.
 Measure the pH value. It should be 6.6. If required adjust the pH value.
 Store the fixative at 4 0C and use cold. Reagent is stable at 4 0C for 2 months.
6. Phosphate buffer 66 mmol/L, pH 7.6

Prepare stock solutions of A & B as follows;
 Solution A : 66 mmol/L KH2PO4 – Add 0.91 g of KH2PO4 in to 100 mL of distilled H2O.
 Solution B : 66 mmol/L Na2HPO4 – Add 4.75 g of Na2HPO4 or 5.95 g of Na2HPO4. 2H2O into 500 mL of
distilled water.
Preparation of working buffer solution;
 10 ml of working buffer solution should be freshly prepared by mixing 1.3 mL of Solution A and 8.7 mL of
Solution B. Adjust the pH value up to 7.6.
7. α naphthyl acetate substrate solution

Reagents:
 100 mg α naphthyl acetate
 5 mL ethylene monomethyl ether
Preparation;
 Dissolve 100 mg α naphthyl acetate in 5 mL ethylene monomethyl ether.
 Store at 4 - 10 0C
8. Coupling reagent
Reagent:
 1 g pararosaniline
 Distilled water
 5 mL of 2 mol/L HCl
 200 mg of NaNO2
Preparation;
 Pararosaniline stock solution –
 Add 1g of pararosaniline into 20 mL of distilled H2O. Then add 5 mL of concentrated HCl.
 Mix it well and warm gently.
 Filter when cool.
 Solution is stable for more than 3 months if stored at 4 0C.
 Stable only for 2 months if kept at room temperature in the dark.
 4% NaNO2 solution
 Dissolve 200 mg of NaNO2 in 5 mL of distilled water.
 Stable for 1 week at 4 -10 0C
 Hexazotised pararosaniline.
 Mix equal volumes of pararosaniline and 4% NaNO2 together for 1 minute before use.

9. Incubation mixture – Should be freshly prepared.
Preparation;
Add 0.5 mL of the α-naphthyl acetate substrate solution and 0.6 mL of Hexazotised pararosaniline to 9 mL of the
66 mmol/L phosphate buffer, pH 7.6, and mix well.
10. Counter stain – 2% Methyl Green is the preferable stain.

Add 2 g of methyl green into 100 mL of water and mix well.
Method
 Fix air dried smears by adding cold (4 - 10 0C) buffered formal acetone for 30 seconds.
 Rinse well in running tap water for 10 - 30 minutes and air dry completely.
 Filter the freshly prepared incubation mixture.
 Keep slides on a staining rack and flood with incubation mixture and keep for 45 minutes in a moist chamber.
 Wash with gently running tap water and air dry. (In double esterase staining. procedure starts from here).
 Counter stain the smears with 2% Methyl Green for 1 - 2 minutes.
 Wash with tap water and dry.

 The reaction product gives a diffuse reddish brown colour to the cytoplasm of the cells with esterase activity while
nucleus and background takes the counter stain colour. Eg – Green colour when methyl green is used. Variable
staining intensities are observed in different cell types as follows;
 Normal and leukaemic monocytes → Strongly positive.
 Megakaryocytes → Strongly positive.
 Normal granulocytes → Negative.
 Granulocytes in myelodysplasia or AML → Can give positive reactions of varying intensity.
 Leukaemic erythroblasts or megakaryoblasts → May show focal or diffuse positivity.
 Most T lymphocytes and some T lymphoblasts→ Show focal ‘dot-like’ positivity.
 Results are reaported as - Blasts are positive/ negative for NSE stain.

 Positive control films from a known AML M5a patient or from a patient with high monocyte count should be used.
 Incubation mixture must be freshly prepared.
 pH values must be adjusted to the expected values.
 Reagents should be stored at correct temperature.
Sources of error
 Blood smears prepared with delayed EDTA anticoagulated blood.
 Haemolysed, partially air-dried or partially fixed blood or bone marrow smears.

 References
2. Crook L, Liu P I, Cannon A, Walker E M, Histochemistry of Bone Marrow Aspirations. Annals of Clinical and
Laboratory Science, Vol. 10, No. 4, (1980) Pg 290-304.

9.8
DOUBLE ESTERASE (DE)
Scope
The scope of this SOP is to guide technical personnel on proper performance of Double Esterase (DE) stain by providing
the protocol and requirements for the procedure.
Responsibility
Performance of DE stain as per the procedure stated in this SOP is the responsibility of all medical laboratory technologists
who are assigned to perform this special staining in a haematology laboratory.
Principle
DE stain is a combination of chloroacetate esterase (specific esterase) and non-specific esterase stains performed on a
single slide. Esterase stains rely on endogenous cellular esterase activity to hydrolyze exogenous ester substrates, to yield
intermediate substances which combine with two separate coupling reagents to give insoluble coloured precipitates at
the sites of enzyme activity, which is visible under the light microscope. Chloroacetate esterase component demonstrates
myeloid differentiation in blue colour, whereas nonspecific esterase component demonstrates monocytic differentiation
in brown colour.
DE stain permits the simultaneous visualization of both specific and nonspecific esterase reactions on a single slide
providing an easy distinction between myeloid series and the monocytic series. This technique avoids the need to
compare results from separate slides and also reveals aberrant staining patterns. When immunophenotyping is not
available, double esterase stain is useful for identifying monocytic and granulocytic components in the samples, e.g acute
myeloid leukaemia FAB type M4 and chronic myelomonocytic leukaemia. In acute leukaemia, this abnormal simultaneous
cytochemical staining for both specific and non‐specific esterase seems to be specific for myeloid leukaemia and may
be used as supplementary evidence of myeloid differentiation of morphologically undifferentiated blasts in cases of
AML‐M0. Use of combination of esterase stains on a single slide also has the advantage of demonstrating pathological
double staining of individual cells, e.g in MDS and AML with dysplastic granulocytes. This may be helpful when a diagnosis
of MDS is not otherwise certain. However, the same abnormal pattern may be seen in some nonclonal dysplastic states
such as megaloblastic anaemia.

Sample

1. Staining rack
2. Timer clock
5. Fixative – Buffered formal acetone
Reagents:
 20 mg Na2HPO4
 100 mg KH2PO4
 Acetone – 45 mL
 40% formalin – 25 mL
Preparation;
 Dissolve 20 mg Na2HPO4 and 100 mg KH2PO4, in 30 mL of distilled water. (Mixture A).
 Mix 45 mL Acetone with 25 mL 40% formalin in a beaker and add in to mixture A and mix well.
 Measure the pH value. It should be 6.6. If required adjust the pH value.
 Store the fixative at 4 0C and use cold. Reagent is stable at 4 0C for 2 months.
6. Phosphate buffer 66 mmol/ L, pH 7.6

Prepare stock solutions of A & B as follows;
 Solution A : 66 mmol/ L KH2PO4 – Add 0.91 g of KH2PO4 into 100 mL of distilled H2O.
 Solution B : 66 mmol/ L Na2HPO4 – Add 4.75 g of Na2HPO4 or 5.95 g of Na2HPO4. 2H2O into 500 mL of
distilled water.
Preparation of working buffer solution;
 10 mL of working buffer solution should be freshly prepared by mixing 1.3 mL of Solution A and 8.7 mL of
Solution B. Adjust the pH value up to 7.6.
7. α naphthyl acetate substrate solution

Reagents:
 100 mg α naphthyl acetate
 5 mL ethylene monomethyl ether
Preparation;
 Dissolve 100 mg α naphthyl acetate in 5 mL ethylene monomethyl ether.
 Store at 4 - 10 0C
8. Coupling reagent
Reagent:
 1 g pararosaniline
 Distilled water
 5 mL of 2 mol/ L HCl
 200 mg of NaNO2

Preparation;
 Pararosaniline stock solution –
 Add 1g pararosaniline into 20 mL of distilled H2O. Then add 5 mL concentrated HCl.
 Mix it well and warm gently.
 Filter when cool.
 Solution is stable for more than 3 months if stored at 4 0C.
 Stable only for 2 months if kept at room temperature in the dark.
 4% NaNO2 solution
 Dissolve 200 mg NaNO2 in 5 mL distilled water.
 Stable for 1 week at 4 - 10 0C
 Hexazotised pararosaniline.
 Mix equal volumes of pararosaniline and 4% NaNO2 together 1 minute before use.
9. Incubation mixture – Should be freshly prepared.

Preparation;
Add 0.5 mL of the α-naphthyl acetate substrate solution and 0.6 mL of Hexazotised pararosaniline to 9 mL of
the 66 mmol/ L phosphate buffer, pH 7.6, and mix well.
10. Reagents for preparation of naphthol AS-D chloroacetate substrate solution (To be prepared freshly)
 0.5 mL N, N - dimethylformamide
 1 mg of naphthol AS-D- chloroacetate.
 10 mg fast blue BB salt
11. Counter stain – 2% methyl green is the preferable stain.

Add 2 g of methyl green into 100 mL of water and mix well.
Method
 Fix air dried smears by adding cold (4 - 10 0C) buffered formal acetone for 30 seconds.
 Rinse well in running tap water for 10 - 30 minutes and air dry completely.
 Filter the freshly prepared incubation mixture.
 Keep slides on a staining rack and flood with incubation mixture and keep for 45 minutes in a moist chamber.
 Wash with gently running tap water and keep slides in distilled water for 5 minutes.
 Freshly prepare the naphthol AS-D chloroacetate substrate solution:
 Take 0.5 mL N, N - dimethylformamide into a test tube and add 1 mg of naphthol AS-D chloroacetate.
 Add 10 mL phosphate buffer and mix
 Then add 10 mg of fast blue BB salt into it and mix thoroughly by using a vortex.
 Filter the naphthol AS-D chloroacetate substrate solution directly on to the slides and keep slides in a moist chamber.
 Incubate the slides at room temperature for 30 minutes.
 Wash the smears in running tap water slowly for 2 minutes and air dry.
 Then counter stain the smears with 2% methyl green for 1 minute.
 Wash with running tap water for 2 minutes and air dry.

 The reaction product of α naphthyl acetate esterase gives a diffuse reddish brown colour to the cytoplasm of the
cells of the monocytic series and chloroacetate esterase gives a granular blue black colour to cells of the myeloid
series. The nucleus and background take the colour of the counter stain. Eg – green colour when methyl green is
used.
 Results are reaported as - Blasts show dual positivity for DE stain.
 Should use positive control films from a known patient with AML M4 or from a patient with high monocyte and
myelocyte counts.
 Incubation mixture must be freshly prepared.
 pH values must be adjusted to the expected values.
 Reagents should be stored at correct temperature.
Sources of error
 Blood smears prepared by delayed EDTA anticoagulated blood.
 Haemolysed, partially air dried or partially fixed, blood or bone marrow smears

 References
3. Elghetany MT, Double esterase staining of the bone marrow contributes to lineage identification in a case of
minimally differentiated acute myeloid leukaemia (AML M0). International Journal of Laboratory Hematology,
1999 Aug;21(4):293-5.

9.9
TOLUIDINE BLUE
Scope
The scope of this SOP is to guide technical personnel on proper performance of Toluidine Blue stain by providing the
protocol and requirements for the procedure.
Responsibility
Performance of Toluidine Blue stain as per the procedure stated in this SOP is the responsibility of all medical laboratory
technologists who are assigned to perform this special staining in the haematology laboratory.
Principle
Toluidine Blue is a metachromatic stain which strongly binds to sulfonic acid groups on heparin in the granules of
basophils and mast cells and gives a bright red/purple colour. Nuclei stain blue and cells with abundant RNA may show
a blue tint to the cytoplasm.
When diagnosing AML, CML and other myeloproliferative disorders, the basophils may be dysplastic and poorly
granular and therefore may not be easily identifiable by Romanowsky stains, as may the mast cells in some acquired
mastocytosis. In such situations Toluidine Blue stain is useful in identification and enumeration of the basophils and
mast cells. Metachromatic positivity seen with toluidine blue is the most characteristic cytochemical reaction seen in
immature basophils and is helpful to differentiate between promyelocytes and the immature basophils that are observed
in acute basophilic leukaemia and CML in blast crisis. This stain may also be useful to differentiate between basophils and
neutrophils with extreme toxic granulations or Alder-Reilly anomaly.
Toluidine blue staining does not distinguish between basophils and mast cells. This differentiation can be achieved by
immunophenotyping. (e.g. by identifying expression of mast cell tryptase)
Sample

1. Staining rack
2. Timer clock
4. 1% w/v toluidine blue in methanol
Preparation;
 Add 1 g of toluidine blue to 100 mL absolute methanol and mix well.
 Keep the mixture on a roller mixture or a magnetic flea for 24 hours allowing to mix well.
 The stain can be used after incubation at 56 0C water bath for 4 hours.
 This stain is stable indefinitely at room temperature if kept tightly stoppered.
Method
 Prior fixation is not needed as methanol in toluidine blue stain acts as the fixative.
 Place the smears on staining rack and flood with the toluidine blue solution.
 Incubate at room temperature for 5 - 10 minutes or at 37 0C for 5 minutes.
 Rinse briefly in gently running tap water until clear.
 Then air dry the smears.

 Basophils and mast cells are identified by;
 bright red/ purple colour stained granules which appear discrete and distinct.
 blue colour stained nuclei
 blue tint in the cytoplasm of cells with abundant RNA
 The primary granules of promyelocytes can also stain red/ purple colour with prolonged incubation >10 minutes.
But they are easily distinguished as these granules are smaller and finer than the granules of mast cells or basophils.
 Results are reaported as - Blasts show metachromatic positivity for toluidine blue stain/ Blasts are negative for
toluidine blue stain.

 Working solution should be filtered each day before use.
 Known positive CML smear with a high basophil count can be used as a positive control.
Sources of error
 Unfiltered solutions
 Expired reagents


 As methanol is highly inflammable, this chemical and toluidene blue solution should be stored away from sources of
ignition.
 Toluidine blue is an animal mutagen. Therefore, it should not be handled by staff who are pregnant.
 Toluidine blue is an eye irritant. Therefore, avoid splashes or contact with eyes.
 References
2. WHO Classification of Tumours, Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues,
IARC Press, Lyon, 2001.
Laboratory Science, Vol. 10, No. 4, (1980), Pg 290-304.

9.10
NEUTROPHIL ALKALINE PHOSPHATASE SCORE
(NAP SCORE)
Scope
The scope of this SOP is to guide technical personnel on the performance of Neutrophil Alkaline Phosphatase (NAP) Score
by providing the protocol and requirements for its correct performance.
Responsibility
Performance of NAP Score as per the procedure stated in this SOP is the responsibility of all medical laboratory
technologists, who are assigned to perform this test in the haematology laboratory.
Principle
Azo – dye technique is used. This method uses naphthols as the substrate. At pH of 8.6, alkaline phosphatase
hydrolyzes naphthol AS phosphate to phosphate and the naphthol AS. The liberated naphthol immediately combines
with diazonium salt such as fast blue BB forming an insoluble blue azo- dye precipitate as the final reaction product at
the site of activity. This reaction gives different colour intensities according to the alkaline phosphatase content in the
cells. Although it is demonstrated as a granular reaction product in the cytoplasm, the enzyme activity is associated
with a poorly characterized intracytoplasmic membranous component distinct from primary or secondary granules.
Alkaline phosphatase activity is found predominantly in mature neutrophils with some activity in metamyelocytes. Other
leucocytes are generally negative, but rare cases of lymphoid malignancies show cytochemically demonstrable activity.
The cells are examined under oil and scoring is done according to the rating scale proposed by Kaplow.
In normal individuals, it is rare to find any neutrophils with a score of 3, and a score of 4 should not be present. Some
physiological variation in NAP scores is observed. Newborn babies, children and pregnant women have high scores and
premenopausal women have, on average, scores one-third higher than those of men.
In pathological states, the most significant diagnostic use of this test is in chronic myeloid leukaemias (CML). In chronic
phase of CML (CML- CP), the score is almost invariably low, usually zero and the score rises in myeloid blast transformation
or accelerated phase. Low scores are also found in paroxysmal nocturnal haemoglobinuria (PNH) and rarely hereditary
hypophosphatasia. Raised NAP score is noted in neutrophilia of pyogenic infections, polycythemia vera, primary
myelofibrosis, leukaemoid reactions, hairy cell leukaemia, Hodgkin’s disease, and aplastic anaemia.

A concurrent pyogenic infection in CML – CP with only a few neutrophils having a high score will result in a Kaplow score
within the normal range. In such cases, the alkaline phosphatase-stained smears must be assessed for the possibility of
a biclonal distribution of neutrophils and should correlate with clinical findings. A high NAP score is also not useful in
distinguishing between chronic neutrophilic leukaemia and a neutrophilic leukaemoid reaction since it is often high in
both conditions.
Sample
 Air dried blood smears prepared with non-anticoagulated blood from finger prick is preferred.
 If EDTA anticoagulated blood is used, smears should be made soon after blood collection, preferably within
30 minutes because neutrophil alkaline phosphatase (NAP) activity decreases rapidly in EDTA-anticoagulated blood.
 Once spread, the blood films should be stained within 6 hours. If any delay is expected, fix the smears, wrap in an
aluminum foil and store them in the freezer for 2 - 3 days until staining.

1. Staining rack
2. Timer clock
4. Wash bottle containing Tris buffer
6. Distilled water
7. Tris buffer (pH 9.0) 500 mL
Preparation;
 Weigh Tris - 18.165 g
 Add 250 mL of distilled water
 Adjust pH to 9.0 by adding 1N HCl in small quantities drop by drop.
 Add distilled water up to 500 mL
8. NAP substrate stock solution – 200 mL

Preparation;
 Dissolve 60 mg of naphthol AS phosphate in 1.0 mL of N,N dimethyl formamide
 Add Tris buffer (pH 9.0) up to 200 mL.
(This solution is stable for several months at 2 - 4 0C. But pH should be checked before use).
9. Diazonium salt of fast blue BB (Stored in freezer)
10. Fixative (4% formalin methanol)

Preparation;
 Add 1 volume of Formalin (40% formaldehyde) to 9 volume of absolute methanol
(Can be stored in freezing chamber [ - 20 0C] for 2 - 3 weeks)
11. 0.02% aqueous neutral red (Counter stain)
Method
 Fix freshly prepared air-dried blood film for 30 seconds in cold 4% formal methanol mixture.
 Rinse with tap water and air dry.
 Preparation of working substrate solution –
 Allow 10 mL of stock NAP substrate solution warm to room temperature.
 Then add 10 mg diazonium salt of fast blue BB and mix thoroughly until dissolved.
 Then filter the stain.

 Keep slides on a slide rack and pour the working substrate solution on to the slide.
 Incubate the slide for 15 minutes at room temperature.
 Then wash with tap water and air dry.
 Counter stain with 0.02% aqueous neutral red for 3 minutes.
 Rinse briefly and air dry.

 The reaction product is blue and granular. The intensity of reaction product in neutrophils varies from negative to
strong positive. Only mature neutrophils and bands should be scored by the Kaplow scoring method. An overall
score is obtained by assessing the stain intensity in 100 consecutive neutrophils, with each neutrophil scored on a
scale of 0 - 4 as follows:
Scoring
0 – Negative, no granules
1 – Occasional granules scattered in the cytoplasm
2 – Moderate numbers of granules
3 – Numerous granules
4 – Heavy positivity with numerous coarse granules crowding the cytoplasm, frequently overlying the nucleus.
The overall possible score will range between 0 - 400 per 100 cells.
Normal range : 30 – 120/ 100 cells
Ideally each laboratory should establish its normal range for NAP score.

 Perform procedure using positive controls. These can be obtained from patients with pyogenic leukocytosis, or
women in the third trimester of pregnancy or during the first few days postpartum. A negative control can be
prepared from a normal fixed smear by immersing it in boiling water for 1 minute to inactivate the enzyme.
Sources of errors
 Delayed preparation of blood smears with EDTA anticoagulated blood.
 Haemolysed, partially air-dried or partially fixed blood smears

 Glass tubes should be used to dissolve substrate as N,N dimethyl formamide may dissolve some plastics.
 References
Laboratory Science, Vol. 10, No. 4, (1980), Pg 290-304.

Manual of Laboratory Procedures in Haematology
Manual of Laboratory Procedures in Haematology
Manual of
Manual of
Haematolog
Haematolog yy
ISBN 978-624-5776-02-3
ISBN 978-624-5776-02-3
Published by
Published by
WijeramaWijerama
Mawatha,Mawatha,
ColomboColombo 07 Sri Lanka
07 Sri Lanka
282

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Manual of

Sri Lanka College of Haematologists 1 st

Sri Lanka College of Haematologists

First Edition - 2022

Page layout & Cover design

All rights reserved.

Dr. Asela Gunawardena

Dr. G. Sudath K. Dharmaratne

Dr. Thanuja Dissanayake

Dr. Indira Wijesiriwardena

5.6 ACTIVATED PARTIAL

4. 5 PACKED CELL VOLUME 6. 1 HAEMOGLOBIN H INCLUSIONS............................ 105

4. 8 ERYTHROCYTE SEDIMENTATION RATE (ESR) 6. 4 HAEMOGLOBIN S SOLUBILITY TEST..................... 113

6. 6 CRYOHAEMOLYSIS TEST........................................ 119

6. 10 KLEIHAUER TEST (ACID ELUTION TEST).............. 133 8. 2 IDENTIFICATION AND QUANTIFICATION OF

8. 3 IDENTIFICATION AND QUANTIFICATION OF

7. 3 COAGULATION FACTOR ASSAY........................ 153

7. 4 VON WILLEBRAND DISEASE TEST PROFILE........ 161 SECTION 09

9. 10 NEUTROPHIL ALKALINE PHOSPHATASE SCORE

Dr. Nipunika Senadheera

SAMPLE COLLECTION, TRANSPORT AND RECEPTION AT THE HAEMATOLOGY LABORATORY 13

Figure 1.1 Order of draw

14 MANUAL OF LABORATORY PROCEDURES IN HAEMATOLOGY

FBC, blood film, reticulocyte count and K2EDTA-preferred

Ratio of blood to anticoagulant

 Critical ratios between anticoagulant and blood should be maintained.

SAMPLE RECEPTION AT THE LABORATORY

SAMPLE ACCEPTANCE AND REJECTION CRITERIA

SAMPLE COLLECTION, TRANSPORT AND RECEPTION AT THE HAEMATOLOGY LABORATORY 15

Sample rejection criteria

 Samples collected into improper containers/ anticoagulant.

16 MANUAL OF LABORATORY PROCEDURES IN HAEMATOLOGY

Dr. Chandana Wickramaratne

GENERAL SAFETY IN THE HAEMATOLOGY LABORATORY 19

DISPOSAL OF SAMPLE TUBES

HANDLING OF SAMPLE SPILLS

PERSONAL PROTECTIVE EQUIPMENT AND GENERAL HYGIENE

20 MANUAL OF LABORATORY PROCEDURES IN HAEMATOLOGY

OTHER EMERGENCIES AND RISK ESTIMATION

TRAINING AND DOCUMENTATION

GENERAL SAFETY IN THE HAEMATOLOGY LABORATORY 21

Dr. Chandana Wickramaratne

GENERAL REQUIREMENTS FOR CALIBRATION

CALIBRATION AND MAINTENANCE OF EQUIPMENT, AND REAGENT MANAGEMENT IN HAEMATOLOGY 25

MAINTENANCE OF GLASSWARE/ GLASS PIPETTES

a. Mercury column thermometers

26 MANUAL OF LABORATORY PROCEDURES IN HAEMATOLOGY

MAINTENANCE OF WATER BATH

CALIBRATION AND MAINTENANCE OF EQUIPMENT, AND REAGENT MANAGEMENT IN HAEMATOLOGY 27

MAINTENANCE OF AUTOMATED FULL BLOOD COUNT ANALYZER

28 MANUAL OF LABORATORY PROCEDURES IN HAEMATOLOGY

MAINTENANCE OF AUTOMATED COAGULOMETER

More importantly, upon installation of a coagulometer, laboratory should verify following;

CALIBRATION AND MAINTENANCE OF EQUIPMENT, AND REAGENT MANAGEMENT IN HAEMATOLOGY 29

30 MANUAL OF LABORATORY PROCEDURES IN HAEMATOLOGY

4. 1 Standard Operating Procedure

4. 2 Standard Operating Procedure

4. 3 Standard Operating Procedure

4. 4 Standard Operating Procedure

4. 5 Standard Operating Procedure

4. 6 Standard Operating Procedure

4. 7 Standard Operating Procedure