Guideline N SOP For Government and Private Laboratories

GUIDELINE & SOP
FOR
GOVERNMENT AND PRIVATE
LABORATORIES

PREPARED BY
PROGRAMME MANAGER BAN BCT
&
LINE DIRECTOR - IHSM
DGHS. MOHAKHALI, DHAKA

CONTENT

Part- One : Guideline Page #
Chapter One : Guideline for Pathology laboratories at Primary level 01
Chapter Two : Guideline for Pathology laboratories at Secondary level 15
Chapter Three : Quality Assurance 29
Chapter Four : Bio-safety & Waste Disposal 43

Part- Two : Standard Operating Procedure (SOP)
Chapter Five : Biochemistry 55
Chapter Six : Microbiology 64
Chapter Seven : virology 101
Chapter Eight : Hematology 113
Chapter Nine : Histopathology 139

a. Advisory group members

1. Dr. Naima Moazzam, Prof of Microbiology, DMC
2. Dr. K.Z. Mamun, Prof. of Virology, DMC
3. Dr. A. Khaleque Akand, Prof of Pathology, SSMC
4. Dr. M.A Momen Talukdar, Prof. of Biochemistry, SSMC
5. Dr. Afzalunnessa Binte Lutfa, Prof. of Microbiology SSMC
6. Dr. Mohiuddin Ahmed Khan, Prof of Hematology, Hematology DMC
7. Dr. Kaniz Rasul, Associate Prof. Pathology, DMC
8. Dr. Nasima Sultana, Asst. Prof. Biochemistry, DMC
9. Dr. Be Nazir Ahmed, SSO, IEDCR, Dhaka.
10. Dr. A.S.M. Alamgir, MO, IEDCR. Dhaka.
11. Dr. S.A.J.M. Musa, DPM, DGHS. Dhaka
12. Dr. Md. Aminul Hasan, MO, DGHS. Dhaka
13. Dr. Mahbuba Zamil, Asst . Prof. IEDCR. Dhaka

b. Core working group members:

1. Dr. K.M Shahdul Islam, Associate prof, Microbiology, SSMC

2. Dr. Md. Nawful Islam. Associate prof. pathology, DMC

3. Dr. Lutfunnesa Siddique, Associate prof. Biochemistry, SSMC

4. Dr. Alamgir Kabir, Associate prof. Hematology, DMC

5. Dr. Enamul Kabir, Assistant prof. Pathology, SSMC

6. Dr. Nasima Sultana. Assistant. Prof. Biochemistry. DMC

7. Dr. Abdur Rahman. Assistant prof. Microbiology, DMC

8. Dr. Salma Afroz, Assistant Prof. Hematology. DMC

9. Dr. Mahbuba Zamil, SSO, IEDCR

10. Dr. Aktaruzzaman- Lecture, Microbiology, DMC

11. Dr. Shikha Paul, Lecture of Virology, DMC

Editing and compiled: Dr. ABM Tanjimul Hoque
Director (Hospitals and Clinics)
DGHS, Mohakhali, Dhaka.

Dr. S.A.J. Md. Musa
DPM (Training-Hospital)

Dr. Md. Aminul Hasan
MO (Hospital)

Final Reviewer: Prof. Dr. Md. Nazrul Islam
Chairman, Virology Department
BSMMU, Shahbag, Dhaka.

.
GUIDELINE & SOP FOR GOVERNMENT AND PRIVATE LABORATORIES
Programme Manager BAN BCT & Line Director IHSM, DGHS.
1

Part-One

GUIDELINE
FOR
PATHOLOGI CAL LABORATORI ES
AT
PRI MARY LEVEL

2

Guideline for a Pathological laboratory at
Primary Level

List of test for Primary level Laboratory:

S.L. Name of the Test Responsible
1. Stool R/E, M/E
Jr. Consultant, Lab Med.
2. Urine R/E, M/E
3. TC, DC, ESR, Hb, WBC, BT, CT, Platelet
4. Blood grouping
5. Blood screening, HbsAg, HIV, HCV
6. VDRL
7. Zehl Nelson stain for AFB
8. Gram staining
9. Aldehyde test
10. ASO Widal, RA
11. Blood sugar, Urea, Creatinine, Bilirubin,
Cholesterol, Pregnancy test, Alt, Ast.
12. Serum electrolyte
13. Lipid profile
14. Urine for total protein & C/S
15 PAP smear collection
16. ManLoux test
17. Pregnancy Test
Jr. Consultant, Lab Med

3

Manpower

Name of Post Quality Number
Junior Consultant
Laboratory Medicine
Postgraduate, Lab. Medicine 1
Medical Officer, Pathology MBBS 2
Senior Lab Technologist
Diploma in Health Technology (Lab)
with 3 yrs Experience.
1
Junior Lab Technologist Diploma in Health Technology (Lab)
2 (one must
be female)
Computer Operator cum
Office Assistant
H.S.C with Computer experience 1
Phlebotomist H.S.C with Science background 2
Cleaner Class VIII 1

Note:

(1) This setup will be under THFPO. The guard, Accountant and Office Assistant of
THC complex will be same.
(2) At Non-Govt. Level there will be a Lab Manager at least Masters in management
with experience in laboratory management.
(3) The recruitment of the laboratory manpower should be through a committee
where the consultant Lab. Medicine will be chairman.
(4) The consultant Lab. Medicine will be recruited by a panel of experts.
4

Proposed organogram for the laboratory of
Upazilla/Thana Health Complex

Thana Health & Family Welfare Officer
Indoor & Outdoor Service Lab Service Community Health
Service
Admin
Jr. Consultant Laboratory Medicine-01
Senior Lab Technologist-01
Computer Operator-01 Phlebotomist-02
Jr. Lab Technologist-02
Cleaner-01
MO, Pahology-01
MO, Pahology-01
5
Roles & responsibilities

Name of Post Responsibilities
Over all Supervision of the laboratory.
Reporting of Routine and referral specimen
Forwarding application of subordinate staffs
Budget preparation
Junior Consultant
Laboratory Medicine
Quality control
Reporting
Supervision of lab Technologist
Help in quality control
Medical Officer, Pathology
Store keeping
Supervision of table works
Assist the pathologist
Report checking
Sample distribution
Procedures for Biochemistry (Desk work)
Procedures Clinical Pathology (Desk work)
Procedures Routine Blood (Desk work)
Procedures Routine Urine (Desk work)
Procedures Routine Stool (Desk work)
Jr. Lab Technologist
Procedures Enzyme Study (Desk work)
Registration
Record Keeping
Report Typing
Report Delivery
Computer Operator
All other official works
Patient motivation
Sample collection
Sample labeling
Phlebotomist
Sample submission to the lab.
Cleaning of total laboratory premises
Cleaner
Disposal of waste products
6

Space distribution

Name Area Toilet Remarks
Pathologists Chamber 12 x 15
Attached bath with tiles
5x4
Within the
laboratory
separated by
glass.
Medical officer, Path 8 x 8
5x4
Within the
laboratory, bath
room common.
Patients waiting room 20 x 10
2 Bath rooms with tiles, 1
Male & 1 Female (5x 4)
Out side and in
front of the
laboratory.
Registration &
Sample collection
8 x 6 No bath room needed
Out side and in
front of the
laboratory.
Laboratory Proper 50 x 15
Both 5x 4, Wall plastic
paint, Floor mosaic.
Good quality
door and
window
Cleaning Room 8 x 8 Floor Tiles, Wall half tiles
Good quality
fittings
Store Room 12 x 15
Floor Tiles, Wall plastic
paint
Good quality
fittings
Waste disposal 12 x 15 Floor tiles, wall tiles
Common for the
hospital.

7

Furniture

Name of Area Description
Secretariat table 1 pc, Wooden
Wooden Armed Chair 1 pc, Wooden Armed less Chair 2 pcs.
Jr. Consultants
Room
Steel Cabinet 1 pc, Steel Almira 1 pc.
Medical Officer
Half Sec. Table 2 pcs, Wooden armed chair 2 pcs, wooden
armless chair 2 pcs, File cabinet (steel) 2 pcs
Patients waiting
room
Waiting Chair (half back), Steel, total 20 pcs, Sand full wooden
Dustbin 1 pc
Sample collection chair 1 pc, Sample collection pt. bed 1 pc
Lab. Table 4 x 2 x 2.5 , with three shelf & drawer
Stool 2 pcs
Registration &
Sample collection
Computer Table 1 pc, Computer chair 1 pc, File cabinet (medium)
Lab Table with Shelf 30 x 2 x 2.5 Preferably ceramic fixed to the
wall & fixed shelf (Good quality wood) to the upper part of the
wall.
Reading Table 1 pc, Armed less chair 3 pcs, Stool 3 pcs
Steel Almira 2 pcs, File cabinet 1 pc
Laboratory
Proper
Slides racks with drawer (4 x 1.5 x 5) x 3
Cleaning room Wooden rack fixed to the wall with three tier 1 pc, (3 x 3)
Steel almira 2 pcs, Wooden rack 8 x 1.5 x 8
Store room
Reading table, half (wooden), Armed less chair 1 pc,
Miscellaneous Table Cloth, Door Screen, Window Screen, Rexin (Good quality)

8
Instruments
Name Specification Unit Comments
Centrifuge Machine 8 holes, and good quality 2 pcs
Microscopes Olympus or equivalent. 3 pcs
Colorimeter Erma or equivalent digital 1 pc
Semi auto analyzer RA-50 or equivalent 1 pc
Electrolyte analyzer Biolyte or equivalent 1 pc
S.S. Drum for autoclave 12 4 pcs
Water bath Good quality 2 pcs
Refrigerator 10 cft 2 pcs For Lab
Refrigerator 12 cft 1 pc For Store
Refrigerator 10 cft 1 pc For Blood Bank
Balance (ordinary) Good quality 1 pc
Staining tray S.S 8 pcs
Emergency light (Charger light) Good quality 1 pc
Alarm Watch Good quality 1 pc
Stop watch Good quality 2 pcs
Micropepette (10 50 ) 2 pcs
Micropepette (100 1000 ) 1 pc
Micropepette (0.5 10 ) 1 pc
Measuring Pipette 5 ml 5 pcs
Measuring Scale 1 ft. Steel 2 pcs
Measuring Scale 4 plastic 2 pcs
Measuring Tape Good quality 1 pc
Cuscos Vaginal Speculum Medium size 1 pc
Cuscos Viginal Speculum Large size 1 pc
Cuscos Viginal Speculum Small size 1 pc
Microtomemacular Shandon 1 pc
Knife holder Shandon 1 pc
Parrafin bath Memmart 1 pc
Partic connter 18 parameter 1 pc
Ayers spatula Good quality 500 pcs
Endocervical brush For Cervical Cytology 500 pcs
Copline Jar Good quality, Glass 10 pcs
Spot light with stand Good quality 1 pc
Sponge holding forceps Good quality 2 pcs
Forceps holding cylinder S.S 2 pcs
Artery Forceps Medium size 4 pcs
Tissue forceps Medium size 4 pcs
Tooth forceps Medium size 4 pcs
Plane forceps Medium size 4 pcs
BP blade Medium size 12 pcs
BP handle Medium size 2 pcs
Kidney tray S.S. Good quality 4 pcs

9
Instruments

Petridish 3, Glass 12 pcs
Incubator Good quality 1 pc
Hot air oven Good quality 1 pc
Auto Clave machine Medium size 1 pc
Lab. Rotator Good quality 1 pc
Gauge cutting scissor Medium size, Good quality 1 pc
Dehumidifier Good quality 1 pc.

Chemicals
H
2
SO
4
Pure 5 ltr/year
HNO
3
Pure 2.5 ltr/year
Clorine Solution Pure 5 ltr/year
Acetic acid Pure 5 ltr/year
Eosin Pure 5 gm/year
Lugols iodine Pure 2.5 ltr/year
Chloroform Pure 2.5 ltr/year
Hydrogen per oxide Pure 2.5 ltr/year
New methylene blue Pure 10 gm/year
Normal Saline Pure 20 ltr/year
Methyl alcohol Pure 20 ltr/year
Formalin Pure 10 ltr/year
Savlon Pure 200 ltr/year
Finish Pure 200 ltr/year
Leishman powder Pure 500 gm/year
Carbal fuschin Pure 20 gm/year
Kit for Urea Good quality 2000 tests/year
Kit for Creatinine Good quality 2000 tests/year
Kit for Bilirubin Good quality 3000 tests/year
Kit for Cholesterol Good quality 1000 tests/year
Kit for Blood Sugar Good quality 10000 tests/year
Kit for Trighyceride Good quality 1000 tests/year
Kit for HDL Good quality 1000 tests/year
Kit for LDL Good quality 1000 tests/year
Kit for ASO titer Good quality 2000 tests/year
Kit for Widal Good quality 5000 tests/year
Kit for RA Good quality 500 tests/year
Kit for Blood group, Rh factor Good quality 5000 tests/year
Kit for HbsAg /ICT Good quality 2000 tests/year
VDRL Good quality 2000 test
Kit for HIV Good quality 200 tests/year
Kit for Urinary total protein Good quality 500 tests/year
Kit for Electrolyte. Good quality 500 tests/year
Kit for pregnancy test Good quality 3000 tests/year
Kit ALT
Kit AST
CPK
Uric Acid

10

Glass wear
Glass bottles 250ml 4 pcs/ year
Tongue depressor Good quality, S.S 24 pcs
Torch light Good quality 2 pcs
Cover slip Good quality 5000 pcs/year
Diamond pencil Good quality 2 pcs/ year
Surgical Gloves Sterile 1000 pcs/ year
Examination Gloves Unsterile 3000 pcs
Butterfly needle 23 G 1000 pcs/ year
Disposable Syringe 5 ml 5000 pcs/ year
Test tube 4 600 pcs/ year
Test tube holder Good quality 4 pcs / year
Urinometer with cylinder Good quality
Glass slide Good quality 3000 pcs/ year
Slide tray Wooden, Good quality 10 pcs
Surgical tray S.S Medium size 2 pcs
Adsorbent Cotton Good quality 30 lb
Leucoplast 3 roll 24 rolls
Micro pore 1 roll 250 rolls/year
Surgical Gauge (Bleached) Good quality 1000 pcs/ year
Beaker 250ml 4 pcs/ year
Beaker 1000ml 1 pc/ year
Kidney Tray S.S. Good quality 4 pcs/ year
Lancet Good quality 1200 pcs/ year
ESR tube Good quality 20 pcs/ year
Centrifuge tube Good quality 1000 pcs/ year
Wintrob tube (pvc) Good quality 2 pcs/ year
Pipette filler Good quality 2 pcs/ year
Plastic slides for VDRL etc. Good quality 24 pcs/ year
Test tube rack 36 holes 8 pcs/ year
Measuring cylinder (100 ml) 2 pcs
Bunsen burner Good quality 2 pcs/year
Spirit lamp Good quality 2 pcs
Wire loop Good quality 4 pcs
Measuring cylinder 1000 ml 1 pc
Flat Bottom Conical Flask 250 ml 2 pcs
Round Bottom Flask 500 ml 2 pcs
Stand for Flask Steel 2 pcs
Sputum container Plastic (Wide mouth) 5000 pcs
Stool container Plastic 20000 pcs
Neubers Counting
Chamber

11
Office Equipments

Name Specification Unit
Computer with
HP laser Printer
Pentium-4 Intel processor with 80 GB
capacity of Hard Disk and 256 MB
RAM, Key board, Mouse & Pad, Sound
system, CD writer (Rewritable), DVD
drive, Floppy disk drive, Built in
internet modem, USB port, 15 inch
color monitor,
1 unit
Scanner Cannon Laser Shot LBP-3000 1 unit
Register Books 16 Number, Good quality 4 pcs / year
Note Khata 320 Pages, Good quality 16 pcs/year
Reporting pad A4 size, Offset paper, Good quality 50000 pgs/year
Karnafully paper Good quality 24 pack/year
Offset paper Good quality 10 pack / year
Waste basket Good quality 10 pcs/ year
Paper basket Good quality 10 pcs/ year
Paper weight Good quality 10 pcs/ year
Stapler Good quality 4 pcs/year
Stapler Pin Good quality 24 boxes/ year
Punch machine Good quality 3 pcs/ year
Alpin Good quality 24 boxes/year,
Jems Clips Good quality 24 boxes/ year
Office files Good quality 24 pcs/year
Paper cutter Good quality 1 pc
Pen Stand Good quality 3 pcs
Toilet Soap Good quality 150 pcs/year
Laundry soap Good quality 150 kg/year
Bleaching powder Good quality 12 kg/year
Tissue paper Good quality 50 boxes/year
Duster Cotton 50 pcs/year

12
Electric Devices

Ceiling Fan 56 7 pcs
Air Conditioner (Store room) 1.5 ton 1 pcs
Air conditioner (Consultant) 1.5 ton 1 pcs
Air Conditioner (Lab) 2 ton 1 pcs
Table lamp Good quality 2 pcs
UPS 600 VA 6 pcs
Exhaust fan for toilet Good quality 4 pcs
Tube light with blast and holder Good quality 20 pcs
Multi-plug Good quality 2 pcs

Sanitary fittings

Basin Medium size, BISF 4 pcs

Sink Medium size, BISF 4 pcs

Elbo Tap S.S. 8 pcs

Commode BISF 2 pcs

Mirror with glass shelf Good quality 2 pcs

Ceramic Soap case Medium size, BISF 2 pcs

Dust bin with lead Plastic, Medium size 5 pcs

Bucket plastic Medium size 4 pcs

Bucket steel Large size 2 pcs

Chopping Knife Medium size, S.S. 1 pc

Chopping board Medium size 1 pcs

Bodna Plastic, Medium size 12 pcs

Mug Medium size, Plastic 12 pcs

Gas Burner Single, Auto spark 1 pc

Plastic Drum 200 ltr. Good quality 2 pcs

Cloth Hanger S.S. 4 pcs

13

Disposal policy

An incinerator (small size) will be kept in the waste disposal room

Specimen
Name
Duration of preservation
Disposal
procedure
Stool 24 hrs. in tight container with 0.5 % chlorine sol. Incineration daily
Urine 24 hrs. in tight container with 0.5 % chlorine sol. Incineration daily
Blood sample 24 hrs. in tight container with 0.5 % chlorine sol. Incineration daily
Sputum 24 hrs. in tight container with 0.5 % chlorine sol. Incineration daily
Body fluid 24 hrs. in tight container with 0.5 % chlorine sol. Incineration daily
Blood slides 7 days in slide rack. Routine wash
AFB slides According NTP policy in slide box -
MP slides According to malaria control policy in slide box -
Requisitions 3 months Burned out
Left over Reports 3 months Burned out
Register books 2 years Burned out
Access papers 3 months Burned out
Papers and
documents
Note books 2 years Burned out
Broken
instruments
1 months Burned out
Broken
furniture
1 months Burned out

14

Chapter-Two

Guideline for laboratory at secondary level

15

Guideline for a laboratory at Secondary level

List of test for Secondary level Laboratory:

S.L. Name of the Test
1. Stool R/E, M/E
2. Urine R/E, M/E
3.
TC, DC, ESR, Hb, WBC, BT, CT, Reticulocyte count, Peripheral blood film,
M.P. Platelet
4. Blood grouping
5. Blood screening, HBsAg, HIV, ELISA for HBV, HCV, Dengue
6. VDRL, TPHA, C/S
7. Nelson stain for AFB
8. Gram staining
9. Aldehyde test
10. ASO Widal, RA
11. Blood sugar, Urea, Creatinine, Bilirubin, Cholesterol, Pregnancy test,
SGPT, SGOT, Alk.Phos, CKMB, CPK, Uric acid, STP, CSF for sugar, Protein,
Electrolyte
12. Serum electrolyte, ICT for different test.
13. Lipid profile
14. Urine for total protein & C/S
15 PAP smear collection, FNAC, Cell cytology
16. Histopathology and Cytopathology

16

Manpower

Name of Post Quality Number
Senior Consultant
Laboratory Medicine
Postgraduate, Lab. Medicine with 3
years experience in respective
discipline.
1
Junior Consultant
Laboratory Medicine
Postgraduate, Lab. Medicine 2
Medical Officer, Pathology MBBS 4
Diploma in Health Technology (Lab)
with 3 yrs Experience.
2
Junior Lab Technologist Diploma in Health Technology (Lab)
4 ( one must
be female)
Computer Operator H.S.C with Computer experience 2
Office Assistant H.S.C 1
Phlebotomist H.S.C with Science background 3
Cleaner S.S.C 3

Note: This setup will be under Superintendent of district level hospitals. The
ancillary man power will be provided by the hospital.
17

Proposed Organogram for the laboratory of district
level hospital

Superintendent of district level hospitals
Indoor & Outdoor
S i
Lab Service
Sr. Consultant Laboratory Medicine-01
Senior Lab Technologist-02
Computer Operator-02 Phlebotomist-02 Jr. Lab Technologist-04 Cleaner-02
Jr. Consultant Lab. Med-01
Jr. Consultant Lab. Med-01
MO, Pathology-02
MO, Pathology-02
Administration
18

Roles and Responsibilities

Name of Post Responsibilities
Forwarding application of subordinate staffs
Budget preparation
Quality control
Senior Consultant
Laboratory Medicine
Maintenance of store.
Assist senior consultant according to need
Junior Consultant
Laboratory Medicine
Reporting
Supervision of lab Technologist
Medical Officer, Pathology
Store keeping
Supervision of table works
Assist the pathologist
Report checking
Quality control
Sample distribution
Procedures for Biochemistry (Desk work)
Procedures Clinical Pathology (Desk work)
Procedures Routine Blood (Desk work)
Procedures Routine Urine (Desk work)
Procedures Routine Stool (Desk work)
Procedures Enzyme Study (Desk work)
Jr. Lab Technologist
Histo and cyto processing
Registration
Record Keeping
Report Typing
Report Delivery
Computer Operator
All other official works
Patient motivation
Sample collection
Sample labeling
Phlebotomist
Sample submission to the lab.
Cleaning of total laboratory premises
Cleaner
Disposal of waste products
19

Space distribution

Name Area Toilet Remarks
Senior consultant Lab
Medicine
12 x 15
5x4
Good quality door and
window
Junior consultant Lab
Medicine
(10 x 12) x 2
5x4
window
Medical officer, Path (8 x 8) x 2
5x4
Within the laboratory,
bath room common.
Patients waiting room 30 x 20
2 Bath rooms with tiles, 1
Male & 1 Female (5x 4)
Out side and in front of
the laboratory.
Registration &
Sample collection
12 x 10 No bath room needed
window
Laboratory Proper 75 x 15
Both 5x 4, Wall plastic
paint, Floor mosaic
window
Cleaning Room 12 x 10
Floor Tiles, Wall half
tiles
Good quality fittings
Store Room 20 x 15
Floor Tiles, Wall plastic
paint
Good quality fittings
Waste disposal 30 x 15 Floor tiles, wall tiles Good quality fittings

20

Furniture

Name of Area Description
Secretariat table 1 pc, Wooden
Wooden Armed Chair 1 pc, Wooden Armed less Chair 2 pcs.
Senior
Consultants Room
Steel Cabinet 1 pc, Steel Almira 1 pc.
Secretariat table 2 pcs, Wooden
Wooden Armed Chair 2 pcs, Wooden Armed less Chair 4 pcs.
Junior
Consultants Room
Steel Cabinet 2 pcs, Steel Almira 2 pcs.
MO, Pathology
Half Sec. Table 4 pcs, Wooden armed chair 4 pcs, wooden
armless chair 4 pcs, File cabinet (steel) 4 pcs
Patients waiting
room
Waiting Chair (half back), Steel, total 50 pcs, Sand full wooden
Dustbin 2 pcs
Sample collection chair 2 pcs, Sample collection pt. bed 2 pcs
Lab. Table (4 x 2 x 2.5 ) x 2, with three shelf & drawer
Stool 4 pcs
Registration &
Sample collection
Computer Table 1 pc, Computer chair 1 pc, File cabinet
(medium)
Lab Table with Shelf 50 x 2 x 2.5 Preferably ceramic fixed to
the wall & fixed shelf (Good quality wood) to the upper part of
the wall.
Reading Table 2 pcs, Armless chair 6 pcs, Stool 6 pcs
Steel Almira 4 pcs, File cabinet 2 pcs
Laboratory Proper
Slides racks with drawer (4 x 1.5 x 5) x 3
Cleaning room Wooden rack fixed to the wall with three tier 1 pc, (3 x 3)
Steel almira 2 pcs, Wooden rack 8 x 1.5 x 8
Store room
Reading table, half (wooden), Armed less chair 1 pc,
Miscellaneous
Table Cloth, Door Screen, Window Screen, Rexin (Good
quality),

21

Instruments

Centrifuge Machine 12 holes, and good quality 4 pcs
Microscopes Olympus or equivalent. 4 pcs
Colorimeter Erma or equivalent digital 2 pcs
Semi auto analyzer RA-50 or equivalent 2 pcs
ELISA with reader & washer Good quality 1 set
Electrolyte analyzer Biolytic or equivalent 2 pcs
S.S. Drum for autoclave 12 8 pcs
Water bath good quality 2 pcs
Refrigerator 12 cft 2 pcs For Store
Refrigerator 10 cft 2 pc For Lab
Refrigerator 8 cft 1 pc For Blood Bank
Balance (ordinary) good quality 1 pc
Staining tray S.S 8 pcs
Emergency light (Charger light) Good quality 2 pcs
Alarm Watch Good quality 2 pcs
Stop watch Good quality 4 pcs
Micropipette (10 50 ) 4 pcs
Micropipette (100 1000 ) 2 pcs
Micropipette (0.5 10 ) 2 pcs
Measuring Scale 1 ft. Steel 4 pcs
Measuring Scale 4 plastic 4 pcs
Measuring Tape Good quality 2 pcs
Cuscus Vaginal Speculum Medium size 2 pcs
Cuscus Vaginal Speculum Large size 2 pc
Cuscus Vaginal Speculum Small size 2 pcs
Microtome machine Shandon 1 pc
Knife holder Shandon 1 pc
Paraffin bath Memmart 1 pc
Partic connter 18 parameter 1 pc
Ayers spatula Good quality 3000 pcs
Endocervical brush For Cervical Cytology 3000 pcs

22
Instruments
Copline Jar Good quality, Glass 40 pcs
Spot light with stand Good quality 2 pcs
Sponge holding forceps Good quality 4 pcs
Forceps holding cylinder S.S 4 pcs
Artery Forceps Medium size 8 pcs
Tissue forceps Medium size 8 pcs
Tooth forceps Medium size 8 pcs
Plane forceps Medium size 8 pcs
BP blade Medium size 24 pcs
BP handle Medium size 4 pcs
Kidney tray S.S. Good quality 8 pcs
Petri dish 3, Glass 24 pcs
Incubator Good quality 2 pcs
Hot air oven Good quality 2 pcs
Autoclave machine Large size 2 pcs
Lab. Rotator Good quality 2 pcs
Gauge cutting scissor Medium size, Good quality 2 pcs
Dehumidifier Good quality 2 pcs
Incinerator Capacity 20 kg/day 1 unit
Dissecting set Good quality 1 set
Miscellaneous (Small Tools
and devices)

23

Chemicals

H2SO4 Pure 20 ltr/year
HNO3 Pure 10 ltr/year
Acetic acid Pure 20 ltr/year
Eosin Pure 20 gm/year
Lugols iodine Pure 10 ltr/year
Chloroform Pure 10 ltr/year
Hydrogen per oxide Pure 10 ltr/year
New methylene blue Pure 40 gm/year
Normal Saline Pure 80 ltr/year
Methyl alcohol Pure 80 ltr/year
Formalin Pure 40 ltr/year
Savlon Pure 800 ltr/year
Finish Pure 800 ltr/year
Leishman powder Pure 1000 gm/year
Curbal fuschin Pure 500 gm/year
Kit for Urea Good quality 8000 tests/year
Kit for Creatinine Good quality 8000 tests/year
Kit for Bilirubin Good quality 12000 tests/year
Kit for Cholesterol Good quality 4000 tests/year
Kit for Blood Sugar Good quality 40000 tests/year
Kit for Trighyceride Good quality 4000 tests/year
Kit for HDL Good quality 4000 tests/year
Kit for LDL Good quality 4000 tests/year
Kit for ASO titer Good quality 4000 tests/year
Kit for Widal Good quality 20000 tests/year
Kit for RA Good quality 2000 tests/year
Kit for Blood group Good quality 20000 tests/year
Kit for HbsAg ICT Good quality 8000 tests/year
VDRL Good quality 8000 test
Kit for HIV ICT Good quality 800 tests/year
Kit for Urinary total
protein
Good quality 2000 tests/year
Kit for Electrolyte. Good quality 2000 tests/year
Kit for pregnancy test Good quality 12000 tests/year
Kit for Particle counter 18 parameter 10000 test/year For TC, DC,
Pt-Count

24

Glass wear

Bunsen burner Good quality 4 pcs/year
Tongue depressor Good quality, S.S 48 pcs
Torch light Good quality 4 pcs
Cover slip Good quality 20000 pcs/year
Diamond pencil Good quality 4 pcs/ year
Surgical Gloves Sterile 4000 pcs/ year
Examination Gloves Unsterile 12000 pcs
Butterfly needle 23 G 4000 pcs/ year
Test tube holder Good quality 8 pcs / year
Urinometer with cylinder Good quality
Glass slide Good quality 6000 pcs/ year
Slide tray Wooden, Good quality 20 pcs
Surgical tray S.S Medium size 4 pcs
Adsorbent Cotton Good quality 120 lb
Leucoplast 3 roll 100 rolls
Micro pore 1 roll 1000 rolls/year
Surgical Gauge (Bleached) Good quality 4000 pcs/ year
Kidney Tray S.S. Good quality 8 pcs/ year
Lancet Good quality 2400 pcs/ year
ESR tube Good quality 40 pcs/ year
Centrifuge tube Good quality 2000 pcs/ year
Wintrob tube (pvc) Good quality 4 pcs/ year
Pipette filler Good quality 4 pcs/ year
Plastic slides for VDRL etc. Good quality 48 pcs/ year
Test tube rack 36 holes 16 pcs/ year

25
Glass wear
Spirit lamp Good quality 4 pcs
Wire loop Good quality 8 pcs
Measuring cylinder 1000 ml 2 pcs
Round Bottom Flask 500 ml 4 pcs
Stand for Flask Steel 4 pcs
Sputum container Plastic (Wide mouth) 20000 pcs
Stool container Plastic 1000000 pcs
EDTA vial Plastic 10000/year

Office Equipments

Name Specification Unit Remarks
Computer with
HP laser Printer
Pentium-4 Intel processor with 80 GB
capacity of Hard Disk and 256 MB RAM,
Key board, Mouse & Pad, Sound system,
CD writer (Rewritable), DVD drive, Floppy
disk drive, Built in internet modem, USB
port, 15 inch color monitor,
2 units
Scanner Cannon Laser Shot LBP-3000 2 units
Register Books 16 Number, Good quality 8 pcs / year
Note Khata 320 Pages, Good quality 80 pcs/year
Reporting pad A4 size, Offset paper, Good quality 200000 pgs/year
Karnafully paper Good quality 100 pack/year
Offset paper Good quality 40 pack / year
Waste basket Good quality 20 pcs/ year
Paper basket Good quality 20 pcs/ year
Paper weight Good quality 20 pcs/ year
Stapler Good quality 8 pcs/year
Stapler Pin Good quality 100 boxes/ year
Punch machine Good quality 6 pcs/ year
Alpin Good quality 100 boxes/year,
Jams Clips Good quality 100 boxes/ year
Office files Good quality 50 pcs/year
Paper cutter Good quality 2 pcs
Pen Stand Good quality 6 pcs
Toilet Soap Good quality 600 pcs/year
Laundry soap Good quality 6000 kg/year
Bleaching powder Good quality 50 kg/year
Tissue paper Good quality 200 boxes/year
Duster Cotton 100 pcs/year

26
Electric Devices

Ceiling Fan 56 7 pcs
Air Conditioner (Store room) 1.5 ton 1 pcs
Air conditioner (Consultant) 1.5 ton 1 pcs
Air Conditioner (Lab) 2 ton 1 pcs
Table lamp Good quality 2 pcs
UPS 600 VA 6 pcs
Exhaust fan for toilet Good quality 4 pcs
Tube light with blast and holder Good quality 20 pcs
Multi-plug Good quality 2 pcs

Sanitary fittings

Basin Medium size, BISF 4 pcs

Sink Medium size, BISF 4 pcs

Albo Tap S.S. 8 pcs

Commode BISF 2 pcs

Mirror with glass shelf Good quality 2 pcs

Ceramic Soap case Medium size, BISF 2 pcs

Dust bin with lead Plastic, Medium size 5 pcs

Bucket plastic Medium size 4 pcs

Bucket steel Large size 2 pcs

Chopping Knife Medium size, S.S. 1 pc

Chopping board Medium size 1 pcs

Bodna Plastic, Medium size 12 pcs

Mug Medium size, Plastic 12 pcs

Gas Burner Single, Auto spark 1 pc

Plastic Drum 200 ltr. Good quality 2 pcs

Cloth Hanger S.S. 4 pcs

27

Disposal policy

An incinerator (small size) will be kept in the waste disposal room

Specimen Name Duration of preservation
Disposal
procedure
Stool 24 hrs. in tight container with 0.5 % chlorine sol. Incineration daily
Urine 24 hrs. in tight container with 0.5 % chlorine sol. Incineration daily
Blood sample 24 hrs. in tight container with 0.5 % chlorine sol. Incineration daily
Sputum 24 hrs. in tight container with 0.5 % chlorine sol. Incineration daily
Body fluid 24 hrs. in tight container with 0.5 % chlorine sol. Incineration daily
Blood slides 7 days in slide rack. Routine wash
AFB slides According NTP policy in slide box -
MP slides According to malaria control policy in slide box -
Requisitions 3 months Burned out
Left over Reports 3 months Burned out
Register books 2 years Burned out
Access papers 3 months Burned out
Papers and
documents
Note books 2 years Burned out
Broken
instruments
1 months Burned out
Broken furniture 1 months Burned out

28

Chapter-Three

Quality Assurance

Quality: Quality means meeting the predetermined requirements of the users for a
particular substance (e.g. : a chemical reagent , stain etc ) or service ( eg : a
laboratory test to diagnose a disease).

Users perception Of quality in laboratory practice:

1. Courteous personnel.
2. Test results are timely.
3. Test results are consistent.
4. Sample mix-ups are extremely rare.
5. Test reports reach the right persons.
6. Needs of the users is understood and met.
7. Complaints and problems are swiftly addressed and not repeated.

Factors affecting the quality:

Some important factors that influence quality are:

Sample (Clinical Specimen): The most important factor. Selecting the right
specimen, collecting in a right manner, adequate quantity, proper identification of
specimen, proper transportation to the lab, sample processing etc are crucial.

Personnel: The qualities of lab results are directly proportional to the training,
commitment and motivation of lab personnel.

Environmental factors: Adequate lighting, workplace, ventilation, safe working
conditions produce quality lab results.

Pre analytic factors: Pre analytic factors include selection and collection of right
of specimen, identification of specimen i.e. labeling of specimen, transport of
specimen in proper condition to the appropriate laboratory.

Analytic factors: The quality of reagents, chemicals, stains, culture media,
equipment and efficiency of lab personnel etc influence lab results.

Post analytic factors: Incomplete reports, improper interpretation influence lab
results.

29

Good Laboratory Practices:

All activities of the Lab can be classified under Good Laboratory Practices which
indicates performance of all the activities of the laboratory in the best possible way so
that the results are of highest possible accuracy . The various components of Good
Laboratory Practices are:

1. Proper collection of the appropriate samples
2. Appropriate identification of specimens with special labels on hazardous
samples.
3. Prompt transportation to the laboratory at appropriate temperature.
4. Accurate performance of the test.
5. Release of reports after proper scrutiny.
6. Delivery of the report to the correct destination in the shortest time.
7. Cordial relationship with the users.

Quality Assurance: Quality assurance is a wide ranging concept covering all
matters that alone or altogether influence the quality of a product .

Components of a quality Assurance program:

1. Personnel with adequate training and experience.
2. Proper collection of the appropriate specimen.
3. Reagents and equipments of good quality.
4. Using techniques with precision and accuracy.
5. Proper performance of tests.
6. Efficient processing of results.
7. Methods to detect errors.
8. Corrective steps when analyses go out of control.
9. Proper maintenance of equipments.
10. Continuous training of staff.
11. Documentation.
12. Coordination.
13. Timely feedback.

The objectives of quality assurance:
To improve the quality of health care
To generate results (e.g.: Laboratory diagnostic test reports) that are
reliable and reproducible.
To establish inter laboratory comparability in lab tests
To establish credibility of the laboratory among the clinicians and the public.
To motivate the staff for further improvement.
Prevention of legal complications (e.g.: law suits)

30
Benefits of a quality assurance programme:

1. Production of a quality product.
2. Generation of a reliable service ( a diagnostic test).
3. Helps physicians to establish proper diagnosis.
4. Creation of a good reputation for the laboratory.
5. Motivation for staff to work better.
6. Prevention of legal suites.

Quality Control :

a) Internal quality control (IQC) : It is a set of procedures undertaken by the
staff of a health facility ( e.g.: a diagnostic laboratory ) to continuously and
concurrently assure quality of lab work so that quality results are produced .

b) External quality assurance: It is a system of objective assessment of a lab
work done by an outside agency.

Internal Quality Control (IQC):

Requirements of IQC:
Comprehensive: covers all steps from sample collection to reporting results
Regular continuous monitoring
Rational: focus on critical factors
Practical
Economical

Each laboratory should have Standard Operating Procedure manuals (SOPMs) which
should include the following information about the infrastructure of the lab .

Bio-safety precautions Described in chapter- 4 (four)
Disposal of infectious waste Described in chapter- 4 (four)
Collection, Transportation and storage of specimens
Criteria to reject any sample
Sample processing
Sample testing;
Equipment maintenance
Recording results
Quality control procedure
Referral
31

Sample Collection:
1. Apply all possible strict aseptic precaution throughout the procedure .
2. Hand-wash before and after the procedure.
3. Sample is to be collected at the appropriate phase of the disease.
4. It is to be ensured that the specimen is the representative of the disease
process (sputum and not saliva in pulmonary tuberculosis).
5. Adequate amount of specimen to be collected.
6. Specimen is to be collected sterile/appropriate clean leak proof container.
7. Container containing the sample is to be appropriately labeled.
8. The sample container is to be sent to the lab without any delay.
9. In case of any delay in transportation of sample to the lab, for some sample
(urine and stool), transport media can be used. For stool sample, Cary-Blaire
transport media can be used. For urine, 1% w/v (0.1 gm in 10 ml) of Boric
acid powder may be used. Remember that, after using this preservative, the
urine sample cannot be used for culture and sensitivity test but only for
microscopy.
Rejection of sample: 1. When the identification is missing /Inadequate.
2. Insufficient quantity.
3. Inappropriate container.
4. Contaminated specimen.
5. Inappropriate transport /storage.
6. Unknown duration of delay.
7. Degraded specimen.

Maintenance of equipment: Good quality equipment is absolutely essential to
generate good quality results. The quality control steps for certain commonly used
equipment is given below.

Autoclave: 1. Clean and change water regularly.
2. Adjust water level before each use.
3. For each run, record time, temperature and pressure .
4. Inspect gasket in the lid regularly.
5. Technical inspection every 6 months.

Incubator: 1. Clean inside walls once a month.
2. Record temperature at the start of each working day.
3. Technical maintenance every 6 months.

Hot Air Oven: 1. Clean inside walls once a month.
2. Record temperature at the start of each run.
3. Technical maintenance every 6 months.
32

Microscope: 1. Wipe lenses with lens paper at the end of each days work .
2. Protect the microscope from dust, dirt, vibrations & moisture
3. Technical maintenance every 12months.

Balance : 1. Keep the balance and weights clean and dry.
2. Do not put material directly on the pan. Always use a paper or a
container .
3. Protect the balance from drafts of air.

Refrigerator: 1. Place at least 10 inches away from the wall.
2. Clean and defrost every 2 months.
3. Record temperature daily.
4. Technical maintenance every 12months.

Water Bath: 1. Check water level daily.
2. Check temperature before and during use.
3. Clean monthly
4. Technical maintenance every 6 months

Centrifuge: 1. Wipe the inner walls once a week with antiseptic solution.
2. Check brushes and bearings every 6 months

Glassware: 1. Discard chipped glassware.
2. Keep them free of detergents
3. Do not store sterile glassware for more than 3 weeks before it is
used

Culture Media:
1. Do not over stock culture media. Store which will be used. up over
the next 6 12 months .
2. When not in use, secure caps tightly when not in use.
3. Store in a cool, dark, ventilated place.
4. Keep a record of the receipt and opening of media container.
5. Rotate the media stock in the manner first in, first out.
6. Discard all dehydrated media that have darkened or caked.
7. For media preparation, strictly follow manufacturers instruction
8. Prepared media should be protected from sunlight and heat.
9. After preparation of the media, its sterility, pH and performance
testing should be done.
10. Do not use prepared culture media if they become dehydrated or
contaminated before they are used .
33

Quality control procedure:

Quality control of laboratory: The quality of clinical laboratory depends a great
deal on the quality of the materials used. In the vast majority of cases, the reason for
bad analyses is poor quality of the materials used, contaminated or deteriorated
standards, reagents glassware not cleaned properly, use of uncalibrated instruments,
improperly collected or preserved specimens.

Pipettes:
1. Every lab should have a written procedure for pipetting.
2. There should not be any personal variation in the standard
procedure.
3. The pipette should be of the correct size, free from chips, water spots,
broken jagged edges.
4. Do not dip the pipette more than 2 inches into the liquid.
5. Do not directly introduce the pipette into the reagent /standard.
Use a separate beaker.
6. Hold the pipette in a vertical position.
7. After taking up the measured amount of liquid, wipe its lower outside with
a gauze before dispensing.
8. Do not touch the bottom of the container after dipping the pipette.

Glassware:
1. Shall be of the right size, free of spots chips, cracks .
2. Do not dip the pipette more than 2 inches into the liquid.
3. Use a separate beaker and do not introduce pipette directly into the
reagent under preparation.
4. Always hold the pipette vertically when in use.
5. Deliver the liquid in suitable glassware.
6. Do not place the tip of the pipette on the bottom of the container. It will
obstruct the flow.
7. At the end, do not leave the pipette in the liquid. The liquid will move back
into the pipette.
34

Cleaning glassware:
1. Always use the appropriate liquid to clean glassware.
2. After cleaning with the appropriate liquid, clean finally using distilled
water .

Reagents, Standards, Chemicals :

1. Should be properly selected, used and stored .
2. Should be stored in an appropriate container.
3. Keep the containers tightly closed when not used.
4. Never store organic solvents in plastic container because they dissolve
plastic.
5. Store them in amber coloured glass bottles.
6. Store photosensitive substances in amber coloured glass bottles .

Culture media:
1. Discard media showing presence of turbidity, precipitate, altered colour,
signs of dehydration ( eg : cracks ) .
2. Use the appropriate method of sterilization. After preparation, select one
plate from the batch , incubate it aerobically at 37 deg Celsius and then
look for growth of contaminants . If present , discard the whole batch .
3. Determine the ability of the media to support the growth of bacteria that it
is supposed to by inoculating the media by a diluted preparation of a
standard strain of the bacteria .

Quality System: Quality is assured through a well defined quality system . Each
procedure is undertaken in such a manner that it delivers the desired result through
a well defined system approach.

Input Processing Output
( Raw material ) ( Product )

In the health laboratories, clinical samples from the patient constitutes the input and
the output is the report of the tests done in the lab. The health laboratory will strive
to assure the quality of the product through systematic efforts of organizational
structures and efficient utilization of resources. Quality system is an overall quality
management that aims to ensure consistency, reproducibility, traceability and
efficiousness of the health laboratory services .

The key elements of the quality system in a health laboratory may be :

1. Organizational management and structure
2. Reference standards
3. Documentation
4. Assessment ( Monitoring and Evaluation )
5. Training

35
The key elements of the quality system in a health laboratory-

1. Organizational Management and structure: In a health laboratory, the
responsibility of the design , implementation, management and improvement
lies with the laboratory management. The laboratory management will
delegate responsibility and authority to appropriate individuals who are
directly responsible for quality implementation .Depending on the status of
the laboratory, (Referral, Tertiary, Intermediate or peripheral) all labs must
follow the structure and compostion with regards to staff, space, other
facilities (e.g.: water supply, electricity, waste disposal, equipments and
supplies, tests to be performed etc).

2. Reference standards: These need to be followed to meet the regulatory
requirements and monitoring the functioning of the laboratory.

3. Documentation: These are records of any information or instructions eg :
policy statement, quality manuals, procedures, specifications, calibration
tables, reports, job descriptions, regulations, standards, examination
procedures . The quality system of the laboratory will define, document and
maintain procedures to control all documents. Examples of documents
include:

(a) Accession list - a list of all specimens that the lab can receive.
(b) Requisition form - a record on which the test of the clinical specimen is
requested by the doctor which includes name, age, sex, registration no of
the patient, presumed diagnosis, treatment received, name of the
specimen, tests requested, date of specimen collection etc.
(c) Test report - This record convey the laboratory data ( i.e. result of the test
done in the laboratory ) to the doctor.

4. Monitoring and evaluation: The laboratory management shall develop
and implement quality indicators to systematically monitor and evaluate the
laboratorys efforts in health care. They shall also identify opportunities for
improvement. This is done by Internal Quality Assessment (IQA) and External
Quality Assessment (EQA).

5. Training: Training should be competency based and must follow a post
training support to provide a continuous support.

Quality Assurance in Health Laboratory: Quality assurance in a health
laboratory should be comprehensive and must cover all aspects from the decision to
collect clinical specimen (sample) to the interpretation of the report.
A simple step by step details of quality assurance in health laboratory.
Step 1: Identify the right patient
Step 2: Order relevant test (s).
Step 3: Collect appropriate specimen of adequate quality and quantity
Step 4 : Label the specimen appropriately .
Step 5 : Rapidly Transport the specimen to the laboratory .
Step 6 : Proper and accurate and precise analysis .
Step 7 : Proper and timely documentation and reporting .
Step 8 : Proper interpretation .
Step 9 : Timely action on right patient .
36
Guidelines for quality assurance parameters:
Parameter Guidelines
Specimen collection and
transport
1. Provide clear cut instructions for sample collection and
transport.
2. Establish clear cut criteria for sample acceptance and
rejection.
Procedure manual 1. Define test procedure, limits, specimen acceptance,
reagent preparation, reporting, and quality assurance.
2. Review annually
3. Make available in work area.
Personnel 1. Use sufficient personnel depending on workload and
complexity.
2. Provide continuous technical education.
3. Evaluate annually.
Quality Control records 1. Record all quality control records on prescribed Forms.
2. Report all out of control observations
3. Note all corrective actions on quality control form.
4. Review quality control records.
Patient reports 1. Report only to authorized personnel
2. Notify the test requesting doctor about important values.
3. Provide normal ranges wherever available.
4. Correct errors in patient reports
5. Retain records for at least 2 years.
Referral specimens 1. Use authorized referral laboratory.
2. Use the name of the referral laboratory on patients reports.
Quality assurance
schemes
1. Adopt internal quality assessment program
2. Participate in External Quality assessment scheme.
Equipment performance 1. Document function checks of equipments
2. Perform as frequently as manufacturer suggests.
3. Retain maintenance records for the life of the equipment.
Culture media 1. Inspect each shipment for cracked media or Petri-dish,
hemolysis, unequal filling, too much bubbles, color,
consistency, depth, smoothness and contamination.
2. Test media for pH.
3. Note the date of preparation.
4. Do not use beyond expiry date
5. Document deficiencies, take corrective action and inform
the manufacturer.
Stains reagents and sera 1. Label containers appropriately and completely regarding
contents, preparation date, date received, shelf life.
2. Discard expired reagents, reagents that fail to perform.
3. Always test along with positive and negative control.
4. Store as per manufacturers recommendations.
Commercial diagnostic
kits
Test each batch as per manufacturers recommendations

37

Internal Quality Assessment:

Internal quality assessment is similar to external quality assessment except that the
material is prepared, distributed, evaluated and the results assessed internally.

The sample (eg: clinical specimen, smear on a slide etc) is split in three. One is tested
by the technician as routine (the identity of the sample will be unknown to him).
Another specimen labeled as QA (Quality Assessment) will be tested by the
technician. The third sample will be tested by a senior consultant or similar authority
(he may know the identity of the sample before testing). The results of all three tests
will be observed, analyzed and reviewed and any errors will be corrected and any
problem solved any shortcoming attended .All will be documented. The test sample
will differ from time to time.

External Quality Assessment Scheme (EQAS):

The external quality assessment scheme (EQAS) is a schematic way of quality
assessment by way of an external agency.

Objectives and benefits of EQAS:
1. To monitor laboratory performance.
2. To evaluate quality control measures.
3. To influence reliability of testing
4. To ensure credibility of the laboratory.
5. To Stimulate performance improvements.
6. Identify errors and correct them
7. Encourage use of standard methods, reagents and trained personnel .

Process of External Quality Assessment Scheme (EQAS) :

EQAS requires two things. A well equipped, experienced lab at the center to act as
the organizing laboratory and a fairly reasonable number of laboratories as the
participating labs. These participating labs are the customer of the organizing lab.

The participants will aim to:

1. Compare performance and results.
2. Minimize errors.
3. Self appraisal
4. Identification of training needs
38

The organizing laboratory, in their efforts, should integrate the following:
1. Clinical relevance and match with the mandate of the participating
laboratory.
2. Should cover a large number of tests to satisfy the needs of diverse
laboratories.
3. May provide a combination of tests that the participants can pick up.
4. Frequency of distribution should be adequate to allow the participants to
regularly assess their functions.
5. Materials distributed from the organizer should be realistic and relevant.
6. Availability of repeat.
7. Timeliness of feedback from the organizer to the participants.
8. Should not be influenced by commercial interest.

Requirement of EQAS:

1. The material supplied: should be homogenous, all participants should
receive the same sample; the stability of the material should be stated. It
should also be stable enough to remain intact during its transportation from
the organizer to the participants.

2. Documents accompanying the material: Should be unequivocal, should
state the return address of the results. The last date of return should also be
state.

3. The manner of performing the test: The manner of testing the specimen
should be the same as that employed during testing similar routine specimens.

4. Number of participating labs: The higher, the better for its usefulness.

5. Statistical analysis of the results: The Statistical analysis of the results
and the methods of displaying them should be easily understood by the
participants. Results should also show whether the performance of the
participating lab is improving or deteriorating.

6. Turn around time and frequency: The turn around time is the period
between sending out the material and reception of results by the participating
labs. It should be as short as possible. The frequency is the number of times
EQAS is carried out. It may be quarterly.

7. Anonymity of the Participating Labs: This is a matter of choice .

39

Recommendations to set up a system of EQAS in Bangladesh:

1. First, we recommend that, in order to carry out systematic EQAS in
Bangladesh, a permanent Central National Reference Laboratory (CNRL) be
set up which will act as the organizer. It should receive adequate, appropriate
and continuous financial, logistic and staff support. It should comprise a
central committee and working staff comprised of experts from all specialties
of Laboratory services. The experts will come from government (both civil and
military), semi government, autonomous and private establishments.

2. As long as the CNRL is established built, the work of such may be carried out
by a temporary reference lab set up in similar fashion. This may be BSMMU,
BIRDEM, DMC, AFIP etc.

3. Several regional referral centers may be set up with the objective to act as
organizer for the peripheral labs who will be their participants .The different
departments of different medical colleges can work.

4. The members of the CNRL will visit the regional referral centers and
peripheral labs to monitor, evaluate, aid to improve the services provided by
them.

40

QUALITY CONTROL

It is the system to reduce the variance in laboratory results by improving
accuracy and precision.
Or
Everything done to produce the correct laboratory data.

The main emphasis of Quality control is to monitor the precision and
accuracy of the performance of analytical methods.

Reasons:
1. Ethical and legal responsibility to supply correct analysis.
2. Fast and reliable support for treatment.
3. Obligation to keep costs low.

Importance:
1. Immediate information whether the result is correct and acceptable. If errors
are detected, it must be repeated.
2. Quality of work improves step by step.
3. Number of repeated test can be reduced, saving time and money.
4. Poor methods are identified and can be substituted by reliable method.
5. Early recognition of reagent or instrument damage.
6. Manager of the laboratory gets objective information on quality of staff
performances.
7. Technicians have simple and reliable tool to control and improve their
performance and gain more self confidence.
8. Reputation and acceptance of the laboratory increase.

QUALITY CONTROL PROGRAM

It is divided in to two phases.

A. Internal Quality Control program:

1. Prospective or Preventive phase:
Precaution taken before starting of actual lab work. (i.e. in planning stage)

2. Retrospective or corrective phase:
Usually practiced and important
(i) Control of precision: Every test series should include a precision control.
(ii) Control of accuracy
(iii) Individual or Clinical control

41

B. External Quality Control program:

Before a laboratory begins to participate in an external quality survey it should
be assured that it works on a high level of quality.

There are several essential elements of a quality control program.

Commitment:
Dedication to quality service must be assured, otherwise quality goals are
not likely to be achieved.

Facilities and resources:
Laboratories must have the administrative supports necessary to provide
the quality of service that is desired.

Technical competence:
High quality personnel are essential for high quality services.

Technical procedures:
Good technical procedures are necessary to provide quality laboratory
services.

Problem solving mechanism:
Approach to problem solving is the use of quality team that meets to
analyze problem and identify solutions.

NETWORK DEVELOPMENT

Meetings between heads of laboratories at the central laboratory may be helpful
in exchanging technical knowledge and discussing meaningful and operational
problem. To the same end, visits from the central and or district laboratory to the
peripheral laboratory should also be organized in order to provide technical guidance
and necessary supervision.

42

Chapter-Four

BIOSAFETY AND WASTE
DISPOSAL

43

I. Laboratory Safety Programme

It is essential that every laboratory should have safety program to establish safe work
practices. It should be designed to prevent injury and illness in all laboratory
personnel medical, technical and ancillary and to protect other people. All
laboratory personnel have a responsibility to adhere to and enforce the programme
at all times and should avoid complacency. Overcrowding, heavy workloads,
incorrectly installed and poorly-maintained equipment and badly-designed premises
are frequent contributing factors for occupational injuries.

Occupati onal i nj uri es and i llness may result from:
Bad practices
Ignorance
Inexperience
Failure to follow safety instruction and procedures

i. Essential Objectives of the Safety Program
Appropriate design of premises
Safe management of the laboratory environment
Effective training of staff

ii. Common Sources of Hazards in The Laboratory
People
Environment
Fabric and fittings
Equipment and furniture
Reagents
Specimens and cultures
Waste

iii. Zones for Laboratory Safety Precaution
Safer Zone: Reception, sample collection, entrance-exit area, washing
area, canteen, place of safe equipment etc. Every staff can move this area
freely.
Low Hazard Zone: Space between safe and high hazard zone. Limited
movement of the laboratory staff.
High Hazar d Zone: Space where dangerous reagent, electric supply
center, deep x-ray room etc. Restricted movement of the laboratory staff.
Only skilled and trained concerned personnel can move these areas of the
laboratory.

44
IV. Safety Measures:
i. Common Safety Measures (For patient, attendant, visitor etc.)
Position and place of the laboratory: Easy wide accessible place.
Wide upstairs, lift and also emergency exit like ladder.
Keep effective fire protection instrument in every floor of the laboratory.
Keep first-aid box in every floor.

ii. Safety Measures for Laboratory Staff
Laboratory personnel should practice following safety measures:
Mouth pipeting is prohibited.
Provide mechanical pipeting devices.
Eating, drinking, smoking, food storing and applying cosmetics are not
permitted in the laboratory work area.
The laboratory must be kept neat, clean and free of materials not
pertinent to the work. Provide special disposal containers for needles and
lancets.
Work surface are decontaminated at least once a day and after each spill
of viable material.
Persons should wash their hands after handling infectious materials and
animals, when they leave the laboratory.
All the procedures are conducted carefully to minimize the creation of
aerosols.
All contaminated liquid or solid wastes are decontaminated before being
disposed of or otherwise handled.
Laboratory coats, gowns, or uniforms must be worn in the laboratory.
Safety glasses face shields or other protective devices must be worn to
protect the eyes and face from splashes and impacting objectives.
Laboratory doors are kept closed when work is in progress.
Hypodermic needles and syringes are not used as a substitute for
automatic pipeting devices in the manipulation of infectious fluids.
Gloves must be worn for all procedures necessitating direct contact with
infectious materials.
All spills, accidents, and overt or potential exposures to infectious
materials must be reported immediately to the laboratory supervisor.
If possible provide safety hood.

45

iii. Decontamination of Work Surface
Flood the spillage area and the broken container with adequate
disinfectant (usually double the volume).
Leave undisturbed for 10 minutes and mop with cotton wool or
absorbent paper.
Wear disposable gloves, apron and goggles while cleaning the
spillage.
Disinfect the used dustpan, brush or forceps.
Use hypochlorite (10 gm/L) for blood or viruses.
Do not use hypochlorite solution in centrifuges.
For viral decontamination use activated gluteral-dehyde (20 gm/L) on
surfaces.
Restrict the entry to accident area until decontamination process is
complete.

iv. Emergency and other Safety Provisions:

All laboratories should have contingency plans for dealing with accidents and
natural disasters-fire, flood, storm, earthquake, etc.

46

II. Management of Laboratory Waste

Healthy laboratory wastes and contaminated materials can produce a hazard
both to laboratory workers and to the community. The uncontrolled dumping of solid
and liquid, chemical and biological laboratory wastes can also threaten the
environment. The presence of contaminated items in public sites, such as used
syringes and hypodermic needles may pose an infection risk. Similar fears may arise
if wastes bearing the radioactive label are found on public access sites.
The safe disposal of laboratory waste is therefore of prime importance. Laboratory
waste should be decontaminated or autoclaved before disposal.

Type of Waste

Laboratory wastes may be of the following types:
A. Sharps
B. Chemicals (other than radioactive substances)
C. Radioactive substances
D. Infectious materials
E. Pressurized containers
F. General, non-hazardous waste (Municipal solid waste)

A. Sharps
These are items that can cause a cut or puncture, such as needles, syringe,
scalpels, saws, blades and broken glass. Contaminated sharps offer the
greatest infection hazard. Sharp items may be of two types disposable and
reusable

i. Management of Disposable Items
These should be placed in sharps containers at the work station. To
avoid needle stick injury, needles should not be removed from syringes
or resheathed; the complete syringe and needle assembly should be
placed in the sharps container. Sharps containers should be
incinerated.

ii. Management of Re-usable Items
These should be placed in a puncture-resistant metal or plastic
container at the work station. The contents should be chemically
disinfected or autoclaved before cleaning and further processing.
47

B. Chemical Waste
Chemical waste and redundant chemicals and residues have the same
hazardous properties as that of pure substances. Chemical waste includes
chemicals, e.g. from diagnostic and experimental work and cleaning,
housekeeping and disinfection procedures. Some of these may be hazardous
like:
Toxic or highly toxic (including carcinogens and dermatogens).
Corrosive.
Flammable.
Reactive ( explosive, water reactive).

i. Management of Chemical Waste
It is necessary to store chemicals for some time before disposal. Some
chemicals may be disposed directly while others need prior treatment before
disposal. Choice of containers is important. Some chemicals, when stored in
metal containers for long periods, can cause corrosion. Several disposal
methods are available for chemical wastes which can be directly disposed -
disposal to the public sewer, disposal to the atmosphere and disposal to
landfill sites.

ii. Direct Dispose of Chemical
a. Disposal to Public Sewer
Dilution with water and dispersal into the sewerage system is
often convenient for small amounts.
Strong acids and bases should first be diluted to near-neutral pH
and not poured into sewers with mineral salts, such as azides,
cyanides, hypochlorite and sulphides.
Disposal to the sewer is not suitable for highly toxic, malodorous
or water-non immiscible chemicals, nor for chemicals that can
react with metal drainage piping to produce dangerously
reactive substances; e.g. solutions of sodium azide and picric
acid.

b. Disposal to the Atmosphere
Dilution by air and disposal to the atmosphere may be
acceptable for small volumes of gases and volatile liquids.
The gas or vapour should be released in such a way that it cannot
re-enter the laboratory or affect adjacent buildings and near by
public areas.

c. Disposal to Landfill Sites
Subject to local and national regulations or codes many non-or
low- hazardous chemicals may be dispersed in small amounts.
Toxic wastes, such as the salts of arsenic antimony, barium,
cadmium and mercury, may be buried in deep landfill.
48

iii. Disposal of Chemical Waste after Treatment
Dangerous chemical wastes and residues are to be treated before disposal that
can be done in two methods chemical treatment and thermal treatment. In
areas where incinerators are not available, it may be acceptable to burn such
wastes in shallow, open trays or pits in open field. This should be well away
from residential areas and be done by experienced staff.

C. Radioactive Waste
Radioactive wastes may be liquid, solid or gaseous. All action related to
disposal of radioactive wastes to the environment must be carried out in full
compliance with national regulations on radiation protection. Laboratories
handling radioactive materials they must have their own system for radio
active waste disposal.

D. Infectious Waste
Infectious waste includes any material which contains potential pathogens. In
view of the potential risks to health it is essential that no infected or
potentially infected material shall leave the laboratory unless it has been
sterilized (e.g. autoclaved) or disinfected by a proven effective process.

i. Management of Infectious Waste
a. Segregati on
Infectious waste should be carefully segregated from other kinds of
waste by placing it in colour coded bags. These should be sealed when
three parts are filled. These bags should be supported in metal or
autoclavable plastic boxes to minimize damage and retain spillages.

b. Tr eatment and Di sposal of I nfecti ous Waste
Infectious wastes may be treated in four ways - autoclaving,
disinfection, incineration and landfill.

i . Autoclaving
Autoclaving is one of the safest and satisfactory method of treating
infectious waste.

i i. Di si nfecti on
Some of the infectious waste, and surfaces or materials contaminated
with infectious wastes may be treated with chemical disinfectants.

i ii . I nci ner ati on
If facilities for autoclaving of infectious waste are not available,
incineration should be done. Waste which is awaiting incineration
should be securely stored under cover and steps should be taken to
prevent access by people, animal and birds.
49

i v. Landfill
Burial of decontaminated waste in a special landfill site is an acceptable
option only when incineration is technically impossible or is not
permitted for practical or legal reasons. Waste disposed of in this
manner should first have been autoclaved. The waste should be
deposited in trenches, covered with earth and compacted daily. The
controlled fill should be fenced and scavenging strictly prohibited.

E. Pressurized Containers
Aerosol cans and other vessels containing residual gas under pressure should
never be placed in containers with other waste but carefully punctured in the
open air and left there for sufficient time to allow any residual gas to escape.
They may then be placed in non-hazardous waste containers.

F. General, Non-hazardous Waste (Municipal Solid Waste)
General, non-hazardous waste includes packaging and other substances that
do not pose a special handling problem or hazard to human health or the
environment. This is known as municipal solid waste. It is usually collected by
the local authority and either incinerated or land-filled, where this service is
not available it may be burned on site or buried.
50

III. Safety and First Aid

Safely training of the staff should be carried out before starting work in the
laboratory. It should be a continuous process. First aid training should also be given
to all staff for the management of most likely accidents occurring in the laboratory.
The minimum first-aid facilities consists of
A first aid box
First aid equipment
Eye irrigation equipment
Antibodies to poisonous chemicals used in the laboratory and instructions for
their use.
Protective clothing and safety equipment for the person rendering first-aid.

Bio-Safety Guidelines

Many people get illness following infection associated with laboratory investigation
of microorganisms or contact with infectious material. Victims are laboratory
workers, laboratory associated workers, visitors and general public. To prevent this
laboratory associated infections laboratory workers should know the following
principles:
o The route by which infections are acquired in the laboratory.
o To identify the hazardous organisms to take early precautions.
o To identify hazardous techniques which may be replaced by safer
ones.
o To protect himself or herself against infection.

Routes and sources of Laboratory associated infections:
In the laboratory:
o Skin and Eyes
o Through the mouth
o Through the respiratory tract.

Outside the laboratory:
Routes are same but the sources are different. General public may be infected as a
result of escape of microorganisms during transport of infectious specimens to
or between the laboratories and if public comes into contact with infectious waste
material. Therefore, biosafety guidelines are set up focusing on safe guarding
human and animal health and the environment.
51
Biosafety is used to describe the policies and procedures adopted to ensure the
environmentally safe application of modern biotechnology. It is a term that is gaining
wider currency as more countries get benefit from the application of modern science
without endangering public health or environmental safety. Biosafety guidelines are
applicable for all research work.
Objectives for bio-safety guidelines

To ensure safe transfer, handling and use of living organisms.

Application of bio-safety guidelines depend on risk assessment. Such as

The character of the organisms
The manner in which organisms or their products are to be used.
For the safe use or transfer of organisms or their product.

Risk management measures

Appropriate information and training
Monitoring procedures in such a way that appropriate measures can be
taken in case of unexpected effects.
Following the principles of good laboratory practice.
Insurance of qualified personnel, appropriate facilities, equipments and
materials.
Maintenance of records of the qualifications, training, and job
descriptions for each professional and technical individual.
Availability of safety precautions.
Work according to the risk group for the hazardous organisms.
Consideration of containment procedures.

World Health Organization has formulated hazardous microorganisms into four
groups :

1. Risk Group I-Unlikely to cause human diseases. They are-
Spoilage bacteria,
Common moulds
Yeasts etc.
52
2. Risk Group II-Offer moderate risk to the laboratory worker and limited risk to
the community. They are-
Staphylococci
Streptococci
Enterobacteria except Salmonella typhi
Polio virus
Hepatitis virus
Leishmania etc.

3. Risk Group III- Provides high risk for the laboratory worker but low risk for the
community if they escape from the laboratory. They are-
Mycobacterium tuberculosis
Salmonella typhi
Human immunodeficiency virus etc.

4.Risk Group IV- Offers high risk for the laboratory worker and for the community.
They are- Marburg virus
Lassa virus
Ebola virus
Encephalitis viruses
Certain arboviruses etc.

Work with organisms in different Risk Group needs different conditions of
containment. The World Health Organization describes three kinds of laboratory-
Basic laboratory
Containment laboratory
Maximum containment laboratory

Basic laboratory:
o It is for work with organisms in Risk Group I and II.
o Have adequate hand washing facilities
o Be equipped with autoclave.
o Work is done on open bench top.
53

Containment laboratory:

o Is used for work with organisms in Risk Group III.
o Separate room with controlled access by authorized staff only.
o Should be fitted with biological safety cabinet ventilation of
which should be arranged so that air flows into it from other
rooms and corridors and flows out to the atmosphere through
the filters of the safety cabinet.
o Need highly skilled and experienced staff.

Maximum containment laboratory:

o Is for work with Risk Group IV organisms.
o These laboratories are separate buildings.
o Strictly controlled access through air locks and exit through
decontaminant shower.
o Have pressure gradients between their various rooms and all air
from rooms and safety cabinets is filtered twice before discharge
to the atmosphere.

Containment: means to protect the personnel in the laboratory, personnel outside
the laboratory and the environment.

Biological safety cabinets: are intended to protect a laboratory worker from
aerosols and airborne particles. They will not protect the person from spillages and
the consequences of mishandling and poor technique. They are classified as class I,
Class II, Class III type.

Class I cabinet: The operator sits at the cabinet, looks through the
glass and works with hand inside. Any aerosols released, are
retained because current of air passes in at the front of the cabinet
and sweeps the aerosols up through the filters, which remove all, or
most of the organisms.

54
Class II cabinet: About 70% air is recirculated through filters so
that working area is bathed in clean air. The air flow carries any
aerosols are removed by the filters. About 30% air is exhausted to
atmosphere and is replaced by room air which enters at the working
face.

Class III cabinet: is totally enclosed and is tested under pressure
to ensure that no particles can leak from it into the room. The
operator works with gloves, which form part of the cabinet.

Good Laboratory Practices:

o Avoiding infection from spillages and breakages
o Avoiding infection from centrifuge accidents
o Safe pipetting
o Safe use of syringes and needles
o Careful uses of inoculating loops and looping out.
o Careful Shaking and homogenization etc.

55

Part-Two
Standard Operating Procedure

56

Chapter-Five

Biochemistry

SAMPLE

Sample or specimens analyzed in primary and secondary Biochemistry laboratories
include whole blood, serum, urine and CSF .

BLOOD:
For Biochemistry laboratory venous blood is usually the specimen of choice, and
venipuncture is the method used to obtain this specimen. Venipuncture is the
process by which a blood specimen is obtained from a patients vein.

Serum: The clear liquid that separate from on clotting.

Preliminary steps for Blood collection
Before a specimen is collected, the phlebotomist (who collects the blood) should ask
patients to verify their identities by asking them to state their names. If appropriate,
the phlebotomist should verify that patients are fasting. Patients should be
comfortably seated or in supine position and should have been in the position for 20
minutes before the blood drawn. This standardization minimizes minimizes
differences in concentrations of blood constituents due to variation in blood
volume(hemoconcentration or hemodilution).

Location
The median cubital vein in the antecubital fossa is the preferred site for collection of
venous blood in adults.
In severely ill patients or those requiring many I/V injections, an alternative blood
drawing site should be selected. Selection of a vein for puncture is facilitated by
palpation.
An arm containing a cannula or arteriovenous fistula should not be used without
consent of the patients physician.
If fluid is being infused intravenously into a limb, the fluid should be shut off for 3
minutes before a sample is obtained.

Preparation of the site
Cleaning of the puncture site with a prepackaged alcohol swab and it is performed in
a circular motion and outward from the site. the skin should air dry.

Venous occlusion
After the skin is cleaned tourniquet is applied 4-6 inches (10-15cm) above the
intended puncture site to obstruct the return of venous blood to the heart and
distend the vein. A tourniquet usually needs to be left in place no longer than 1
minute, otherwise the composition of blood changes. Pumping of the fist before
venipuncture should be avoided because it causes an increase in the plasma
potassium, phosphate and lactate concentration.
57

Blood collection with a sterile disposable syringe.

The syringe and needle should be aligned with the intended vein and the needle
pushed into the vein at an angle approximately 15 degree to the skin.
When blood collection is complete, the needle is withdraw and the patient asked to
hold a dry gauze pad over the puncture site. After removal of the needle from the
syringe drawn blood should be transferred quickly by gentle ejection into prepared
tubes. The tubes then should be capped, if they contain additives or anticoagulant
they should be mixed gently.
Venipuncture in children- The techniques for venipuncture in children and adults are
similar.

Factors affecting blood collection

Factors affecting the collection of a blood sample include the use of anticoagulants
and preservatives, site of collection and hemolysis of a collected sample.

Anticoagulants and preservatives for blood

To prepare a whole blood or plasma sample, an anticoagulant must be added to the
specimen during the collection procedure. A number of anticoagulants are used,
including heparin, EDTA
( ethylene diamine tetra acetic acid), sodium fluoride, citrate, oxalate and sodium
iodoacetate.

Hemolysis
Hemolysis is the disruption of the red cell membrane, which cause the release of
hemoglobin. Serum shows visual evidence of hemolysis when the hemoglobin
concentration exceed 20mg/dl. Sight hemolysis has little effect on most test values,
severe hemolysis of blood is not suitable for any test, because it may affect test
results.

UNIRE:

Various types of urine specimens are collected and a variety of techniques are used to
preserved them.

Types of urine specimen:
Random specimen
Fast- morning specimen
Timed specimen
24-hour specimen
Clean- catch specimen
Catheter specimen
Suprapubic specimen

In primary and secondary biochemistry laboratory urine specimen is required for
determination of sugar in urine, protein in urine and 24-hour urine specimen for
determination of total protein of urine.

58

Method of collection of 24-hour urine
The first morning urine is discarded, not put into the container then subsequent
voiding are collected and put into container(with preservative) up to the first
morning urine of the following day. the urine specimen will complete the 24-hour
collection. Specimens are preserved by refrigerator in between collection.

Cerebrospinal fluid (CSF)

CSF sugar, protein and electrolytes are estimated in secondary biochemistry
laboratory. Specimen is collected by physician and sent to the laboratory for test.

Maintenance of specimen for identification/Registration and labeling.

Proper identification of the patients with the correct specimen begins whenever the
specimen is collected. This identifying link must be maintained throughout the
collection process, transport of the specimen to laboratory, subsequent analysis and
preparation of a report.
Labeling includes- the patients name, hospital identification number, ward and bed
number, date and time of collection are commonly found on the label. For some tests
label must include the time of collection of the specimen and the type of specimen.
A properly completed request slip should accompany all specimen sent to the
laboratory.

Handling of specimens

a. Preservation of specimens
The specimen must be treated properly from the time of its collection, during its
transport to the laboratory, to the point at which it is analyzed. For some tests,
specimens must be preserved at 4
0
C from the time the blood is drawn until the
specimen is analyzed or the serum or plasma is separeted from the cell. For all the
test constituents that are thermolabile, serum and plasma should be separated
from cells in a refrigerated centrifuge, specimen for bilirubin must be protected
from both day light and fluroscent light to avoid photodegradation.

b. Separation and storage of specimens
Plasma or serum should be separated from cell as soon as possible and certainly
within 2-hours after collection. Premature separation of serum however may
permit continued formation of fibrin and lead to the obstruction of sample probes
in testing equipment. If centrifugation of a blood specimen within 2-hour is not
possible, the specimen should be stored at room temperature rather than at 4
0
C
to decrease hemolysis and the risk thereof. If the specimen can not be analyzed at
once, the separated serum usually should be stored in a capped tube at 4
0
C until
the time of analysis. If a specimen for a particular test is unstable at 40C, the
serum specimen should be maintained at -20
0
C.
Specimen tubes should be centrifuge with stopper in place.

59

c. Specimen Transport

The time required to transport a specimen from collection until it reaches the
laboratory varies from a few minutes to as long as 72-hour. The container or tube
used to hold a specimen( primary container) should be constructed so that the
contents do not escape if the container is exposed to extreme of heat, cold, or
sunlight.
The request form and full identification with labeling must be placed on the
outside of the container.

Rejection

A specimen is rejected if the specimen container identification does not match
exactly the identification on the request form for that specimen.
Severely hemolysed blood specimen is rejected. A specimen is also rejected if the
specimen is not properly labeled, preserved, storage and transported.

_________________________

60

MANAGEMENT OF INSTUMENTS

Temperature influenced instruments

Many determinations in the clinical laboratory are based on measurements of
radiant energy emitted, transmitted, absorbed, or reflected under controlled
conditions. The optical principles involved in Photometry, Spectrophotometry,
Flame photometry and Atomic absorption spectrophotometry and various types
of Autoanalyzers.
These instruments are very sophisticated and they have very fine operating
procedure. These instruments must be handle by expert operating personnel.
Some times these sophisticated instruments are heat and dust sensitive and may
affect the tests results if suitable environment are not available for these
instruments.
The environment for the proper functions of these instruments are must be as
follows
a. Well spacious laboratory room
b. The laboratory room must be well electrified and must have stabilizer for each
instruments
c. The laboratory room must be air conditioned and dust free
d. Laboratory should have fire extinguisher

Maintenance of instruments should be as follows
a. Handle by expert personnel
b. Control should be run in every weak or whenever required
c. Must be cleaned after use
d. Must be covered after finishing the work
e. The electric switch must be off at the end of work
f. It is better if the personnel wears sterile cloths
g. The laboratory room must be a restricted area.

MANAGEMENT OF OTHER INSTRUMENTS

CENTRIFUGES:

The horizontal-head, or swinging-bucket, centrifuge allows the tube placed in the
cups of the rotor to assume a horizontal position when the rotor is in motion and
a vertical position when it is at rest. During centrifugation, particles travel
constantly along the tube while the tube is positioned at right angle to the shaft of
the centrifuge; thus the sediment is distributed uniformly against the bottom of
the tube.
Fixed- angle, or angle-head centrifuge, in the rotor tube are held in fixed positions
at angles from 25to 40 degrees to the vertical axis of rotation, on the start of
centrifugation, particles are driven outward horizontally but strike the side and
bottom of the tube, with the surface of the sediment parallel to the shaft of the
centrifuge.

61
ULTRACENTRIFUGE:

An ultracentrifuge is a high-speed centrifuge that typically uses a fixed-head
rotor. The most common application of an ultracentrifuge in the clinical
laboratory is the separation of lipoproteins. Because the separation may require
hours or days and generate considerable heat as a result of high speed friction,
ultracentrifugation require a refrigerated chamber.

BALANCE:

Double and single pan and electronic balances frequently are used in the clinical
laboratory. Balance always should be placed with in a cage.

LABORATORYWARE:

Glassware: Various types of glass are used in the manufacture of laboratory
glassware, and various processes are used to clean the glassware.
Important glassware in biochemistry laboratories are as follows
a. Pipette- i. Transfer pipette, ii. Measuring pipette, iii. Micropipette, iv.
Semiautomatic and automatic pipette.
b. Burette
c. Measuring cylinder
d. Volumetric flask
e. Beside these reagent and dropping bottle, beaker, funnel, evaporating basin,
watch glass and test tube are also used.

Cleaning of glassware : For general washing without an automatic washer, most
laboratories prefer detergents that are nonionic, metal free, and not highly
alkaline. Again special care must be taken to ensure adequate rinsing. All clean
laboratory ware should drain with a continuous thin film of water. Imperfect
wetting, or the presence of discrete droplets of water, indicates that the vessels is
not sufficiently clean.
Air-drying or oven drying at temperature below 100
0
C with the laboratory ware
placed bottom up, is the preferred drying method.

Plastic ware:
Many types of laboratory ware are manufacture from plastic and possess unique
qualities that make them ideal for use in situation that require high corrosion
resistance and unusual impact and tensile strength.

Cleaning of plastic ware: Linear polyethylene, polypropylene, teflon, polymethyl
pentene, and polycarbonate plastics are cleaned in ordinary glass ware washing
machines.
some plastic ware may be autoclaved repeatedly under normal condition.

TEST PROCEDURE

The Biochemical tests for primary and secondary level are:
Blood sugar
Blood urea
Serum creatinine
Serum bilirubin
62
Serum cholesterol
Lipid profile
Serum electrolyte(Na
+
, K
+
, Hco3
-
)
SGPT
SGOT
Serum alkaline phosphatase(ALP)
Serum creatine kinase(CKMB)
Serum creatine phosphokinase(CPK)
Serum uric acid
CSF for sugar and protein
Urine for total protein

Now a days all these tests are done by kit method according to the manufacturer
instruction. The procedure of principle are different from one to other
manufacturer company. And the reference value also vary according to the
procedure of principle. So all the tests are done according to the manufacturer
instruction.

UNIT OF MEASUREMENTS OF DIFFERENTS VALUE
Values are according to the Tietz Text Book Of Clinical Chemistry, 3rd Edition.
Test are given according to alphabetically
Test specimen Age Reference interval
Conversion
factor
(multiply)
Reference
interval
(International
unit)
ALT/SGPT
(37
0
C)
Serum Adult
Male
Female
Newborn/
Infant

10-40U/L
7-35U/L
13-45U/L

AST/SGOT
(30
0
C)
Serum Adult
Newborn
Infant
8-20U/L
25-75U/L
15-60U/L

Albumin Serum

CSF

Urine
0-4day
4day-14yrs
Adult
>60yrs

3mon-4yrs
>4yrs

Adult
2.8-4.4gm/dl
3.8-5.4gm/dl
3.5-5.2gm/dl
3.2-4.6gm/dl

0-45mg/dl
10-30mg/dl

1-14mg/dl
10

10

10
28-44gm/L
38-54gm/L
35-52gm/L
32-46gm/L

0-450mg/L
100-300mg/L

10-140mg/L
Bicarbonate
(Hco3
-
)
Serum 21-28mmol/L
(artery)
22-29mmol/L
(venous)
1 same
Bilirubin (Total)
Direct
Serum 0.3-1.2mg/dl
0-0.2mg/dl
17.1 5-21mol/L
0-3.4mol/L
Calcium Serum
CSF
Adult

8.6-10mg/dl
4.2-5.4mg/dl
0.25
0.25
2.115-
2.5mmol/L
1.05-
1.35mmol/L
63
Conversion
factor
(multiply)
Reference
interval
(International
unit)
Chloride Serum or
Plasma

CSF
Adult
0-30days
Adult
Infant
98-107mmol/L
98-113mmol/L
118-132mmol/L
110-130mmol/L
Same

Same
Cholesterol
(Total)

HDL-C

LDL-C
Serum
Plasma
(EDTA)

Serum
Plasma
(EDTA)
Serum
Plasma
(EDTA)
Greatly vary
with age
Desirable

Desirable

Desirable

<200mg/dl

>37mg/dl

<130mg/dl

0.0259

<5.18mmol/L

0.96mmol/L

<3.37mmol/L

Creatine
kinase(Total)
30
0
C

CKMB (immuno-
enzymatic)
Serum

Serum
Adult
Male
Female
Newborn

15-105U/L
10-80U/L
10-200U/L
0-9g/L
CKMB
relative
index(%)=
CKMB
g/LTotal
CK X100

Creatinine Serum or
plasma
Adult
Male
Female
Child
0.7-1.3mg/dl
0.6-1.1mg/dl
0.3-0.7mg/dl
88.4
62-115mol/L
53-97mol/L
27-62mol/L
Glucose Serum
(Fasting)
Whole
blood

CSF

Urine
Adult

Adult
Child

74-106mg/dl

65-95mg/dl

40-70mg/dl
60-80mg/dl

1-15mg/dl
0.0555 4.1-5.9mmol/L

3.5-5.3

2.2-3.9mmol/L
3.3-4.5mmol

0.1-0.8mmol/L
GTT
Oral-
75g glu.-non
pg.,100g glu-
pg.,1.75g/kg
upto75g-child
serum Non pg. adult
Fasting

after 30 min

after 60 min

after 90 min

after120 min
Unit (mg/dl)
70-105,Diabetic
>140
110-170,
Diabetic 200
120-170,
Diabetic200
100-140
Diabetic 200
70-120,
Diabetic140
0.0555

Unit(mmol/L)
3.9-5.8,
Diabetic >7.8
6.1-9.4,
Diabetic 11.1
6.7-9.4,
Diabetic 11.1
5.6-7.8,
Diabetic 11.1
3.9-6.7,
Diabetic 7.8
Phosphatase
(Alk) 37
0
C
Serum Adult
Male
Female

53-128U/L
42-98U/L

Phospholipid
Serum Plasma
(EDTA)
Adult 125-275mg/dl 0.01 1.25-2.75g/L
Potassium(K
+
) Serum 3.5-4.5mmol/L 1 same
64
Conversion
factor
(multiply)
Reference
interval
(International
unit)
Plasma
CSF

70% of plasma
level
2.5-3.2mmol/L
Protein(Total) Serum

Urine
(24-hr)
Adult
child3yrs

6.4-8.3g/dl
6-8g/dl

1-14mg/dl
50-80mg/day
10 64-83g/L
60-80g/L

10-140g/L
Sodium(Na
+
) Serum or
Plasma
Adult 136-145mmol/L 1 same
triglyceride(TG) serum
12hr fast
Desirable <250mg/dl 0.013 <2.83mmol/L
Urea nitrogen Serum Adult
child
High after
protein intake
6-20mg/dl
5-18mg/dl
0.357 2.1-7.1mmol/L
1.8-6.4mmol/L

Uric acid serum Adult
Male
Female
Child

4.4-7.6mg/dl
2.3-6.6mg/dl
2.0-5.5md/dl
0.059 0.26-
0.45mmol/L
0.13-
0.39mmol/L
0.12-
0.32mmol/L

65

Chapter-Six

MICROBIOLOGY

66

An Overview of Microbiological Tests from collection of
specimen to report delivery.

Laboratory reporting of microbiological tests is a chain of works, which include

a) Collection of specimens
b) Processing of specimens
c) Microscopy Wet Film & Staining (when indicated)
d) Culture & sensitivity
e) Serological tests
f) Record keeping of the test results
g) Report delivery

Mistakes in any of the above mentioned steps would produce incorrect result even if
the test procedure were correctly done. It is to be noted here that different lab
personnel are involved in different steps of the chain. So all concerned lab personnel
performing different steps of the chain should have adequate knowledge to carry out
their task correctly. An expert consultant microbiologist even by using high tech-
equipments may fail to produce quality report if pre-analytic, analytic and post
analytic processes are not carried out properly.

In Upazilla & District hospitals, the scope of microbiological tests is limited. Culture
& sensitivity test are not done at present but Gram staining, AFB staining & some
serological tests are done almost in every Upazilla & District hospitals. Blood also
collected from the patients for demonstration of malarial parasites and in some
centers for microfilaria. Proper collection of specimens, processing and staining for
these tests deserve special attention for good laboratory results.

A) Collection of Specimens:

Putting ID no. correctly in the sample collecting container is the prerequisite of
correct laboratory test. One must be very careful in doing that.
Before collecting the specimen you must explain the whole mechanism to the
patient, specially the part which has to be done with the patient.
Throat swab, wound swab and sputum are usually taken for Gram staining at
Upazila & District hospitals.
Throat swab preferably be taken in the morning before breakfast. Patient may
wash his/her mouth with water but should not use toothpaste. Breakfast or
use of toothpaste may interfere the bacterial load at least a couple of hours gap
should be allowed for collection of swab after tooth paste mouth wash or food.
Wound swab should be taken from the area where pus is present. Do not
clean the wound with spirit or any other antiseptics before taking the swab (it
will reduce the quantity of bacteria and will lead to an incorrect report of
Gram staining).
67

In all Upazila Health Complexes & District hospitals, sputum is collected for
AFB staining.
1st morning cough is preferable, as this sample of sputum likely to contain
maximum number of AFB.
For demonstration of malarial parasite in blood film the ideal time of
collection of blood is during febrile stage i.e. during height of temperature. In
this stage of fever, higher numbers of malarial parasites are present in
peripheral blood.
On the other hand, for demonstration of microfilaria in blood film for
diagnosis of filariasis the ideal time of collection of blood is during night.
During night, microfilaria come in the peripheral blood. However, blood may
be collected during daytime by provocative test. Give tablet Diethyl
Carbamazine 100mg to your patient and then collect blood after half an hour
to one hour.

B) Processing of Specimens:

Processing of specimens for microbiological tests is very important. Throat
swab or wound swab may get dry if kept for longer time. You will not get
the correct result if you make a smear by a dry swab for staining. If you can
not make immediate time to do Gram staining you should at least prepare a
smear and complete the step of heat fixation. You can keep the heat fixed
smear for longer time for Gram staining.
If you keep sputum for a longer time before Gram staining there will be over
growth of bacteria, which will give incorrect result in reporting Gram staining.
So a smear for Gram staining should be prepared as soon as possible and may
be kept longer time after heat fixation if required.
Sputum collected for AFB staining should be treated with 5% Sodium
hypochlorite solution. Add equal volume of 5% Sodium hypochlorite solution
to sputum collected in a container. Keep it for 24 hours and then centrifuge it
to collect the deposit for AFB staining. Hypochlorite causes liquefaction of the
sputum and also kills AFB and other bacteria present in the sputum. The
chance of finding AFB under microscope increases when a smear is prepared
from centrifuged deposit.

A thick film of blood prepared for demonstration of malarial parasites and
microfilaria may take half an hour or more to become dry at room
temperature. So the slide should be kept in a petri dish to prevent mixing of
the dusts with the blood smear.
The test tube containing blood should be centrifuged after clotting for
separation of serum for serological tests. This will prevent haemolysis of blood
and you will get clear serum for the tests.
One must be very careful in writing ID no. during sample processing.
68

C) Microscopy Wet Film & Staining:
In Upazilla & District hospitals, microscopic examination of urine, stool, Gram
staining & Ziehl-Neelsen (AFB stain) staining are commonly done. The
examination of urine and procedures of the staining techniques are described in
the next text of this manual.

D) Culture & Sensitivity Test / Serological Tests:
The pathological laboratories of Upazilla & District Hospitals are not equipped for
culture & sensitivity test. However, some serological tests are performed
depending on the supply of reagents. The serological tests, which can be
performed at Upazilla & District hospitals, are described in this manual.
In near future, Upazilla & District hospitals will be equipped for culture and
sensitivity test. So techniques of culture & sensitivity are also described in this
manual.

E) Record Keeping:
Laboratory must keep the records of the patients ID number, age, sex, test
name, test result and date of examination.

F) Report Delivery:
Laboratory personnel should not interpret the test result while delivering the
report to the patient. There is every possibility of misinterpretation, so in
answer to patients query lab personnel should advice the patient to discuss
with the clinician who has advised the test.
Gr am stai ni ng

The microscopic examination of Gram Stained preparation of the different samples
reveal the morphology, relative size and arrangement of microorganisms. It also
assists in the detection of cells, especially pus cells. Bacteria can also be differentiated
by their staining reaction- Gram positive or Gram negative organisms.

Importance: A presumptive diagnosis can be done by examining a Gram stained
smear.

Procedure:
A) Labeling of slides:
Every slide should be labeled clearly with patients ID number. Care should be
taken so that the label is not washed off during the staining process.

B) Smear preparation:
Smears should be spread evenly covering an area of about 15-20 mm
diameter. The purulent specimens or culture colonies require emulsification in
distilled water before being spread thinly.
Non-purulent fluids (like CSF, urine) should be centrifuged and smear should be
prepared with a drop of well- mixed sediment. In case of a swab, the swab should be
rolled gently on a slide. This is particularly important if looking for intracellular
69
bacteria such as N. gonorrhoeae (in urethral or cervical swabs). Rolling the swab
avoids damaging the pus cells.

C) Drying of smears: After making smears, the slides should be left in a safe
place to air-dry.

D) Fixation of smears: The purpose of fixation is to fix the microorganisms
with the slide so that they are not washed away from the slide during washing in the
staining procedure. Fixation help the stain to penetrate the stain into organizes
Smears may be fixed by heat or chemicals:
Heat fixation: This is widely used method but can damage organisms if excessive
heat is used. Heat fixation also damages leucocytes and is therefore unsuitable for
fixing smears, which contain intracellular organisms such as N. gonorrhoeae and N.
meningitides.

Pr ocedur e of Heat Fi xati on:
1. Rapidly pass the completely air-dried smear 3 times through the flame of a
spirit lamp or a Bunsen burner.
2. Allow the smear to cool before staining.

Chemi cal Fi xati on:
Chemical fixation is commonly done with alcohol. This from of fixation is far less
damaging to microorganisms than heat. Cells, especially pus cells, are also well
preserved. Alcohol fixation is therefore recommended for fixing smears when
looking for Gram-negative intracellular diplococci ( in urethral discharge, cervical
swab).

Procedur e of Alcohol fixation:
1. For the detection of intracellular Gram negative diplococci, one or two drops
of absolute methanol or ethanol are poured over the completely air-dried
smear.
2. Leave the alcohol on the smear for a minimum of 2 minutes or until the
alcohol dries on the smear.

E) Primary staining:
Cover the smear with crystal violet and keep for 1 minute. Then rapidly wash off
the stain with clear water.

F) Mordanting:
Cover the smear with Lugols iodine and keep for 2 minute. Then wash off the iodine
with clear water.

G) Decolourization:
Decolourization is done by washing the smear with alcohol or acetone alternately
until the violet colour stops to come out from the smear.

H) Counterstaining:
Cover the smear with dilute carbol fuchsion of Ziehl-Neelsens carbol fuchsin and
keep for 30 seconds. Then wash off the stain with clean water.

I) Clean the back of the slide and place in a draining rack for the smear to air-
dry.

70
Microscopic Examination of Gram smears:
Examine the smear microscopically first with the 40X objective to check the staining
and to see the distribution of the material, and then with the oil immersion objective
(100X) to look for bacteria and cells.

Findings:
Gram positive bacteria and yeast cells look violet or dark purple. Gram negative
bacteria, pus cells and epithelial cells look red.

Observation :

Gram positive cocci Gram positive cocci Gram negative diplococci
(arranged in clusters) ( arranged in chains ) eg : Neisseria
eg : Staphylococcus eg : Streptococcus (Neutrophils are also seen)

Gram negative bacilli Gram stained Candida
( eg : E.coli , Klebsiella , Proteus , pseudomonas etc )

Variations in Gram reactions:
Occasionally Gram-positive organisms may stain as Gram negative. The followings
are among the reasons why this may occur:
Damaged cell wall due to antibiotic therapy or excessive heat fixation of the smear.
Over decolourization of the smear.
Use of an old iodine solution.
Smear prepared from an old culture.
Occasionally Gram-negative organism may not be fully decolourized and appear as
Gram positive.
Reporting of Gram smears:
The report should include the following information:
a. The number of bacteria present, whether many, moderate, few, or scanty.
b. The gram reaction of the bacteria, whether Gram positive or Gram negative.
c. The morphology of the bacteria, whether cocci, rods, or coccobacilli. Also the
arrangement intracellular, diplococci, in clusters, in chains, etc.
d. The presence and number of pus cells.
e. Presence of yeast and epithelial cells.

71
Reagents required:
Crystal violet stain.
Lugols iodine
Acetone/alcohol decolourizer
1/10 Carbol Fuschin

Solutions :
Cr ystal vi olet stai n

Crystal violet 5 g
Distilled water 1 litre

The solution should be made up in bulk and filtered.

Lugols i odine soluti on

Iodine 10 g
Potassium iodine 20 g
Distilled water 1 litre

Dissolve 20 g potassium iodine in 250 ml water, and then add 10 g iodine; when
dissolved make up to 1000 ml with water.

Counterstai n- 1/ 10 Carbol
Fuschi n

Absolute Carbol Fuschin 10 ml
Distilled water - 90 ml

Ziehl-Neelsen Stain :

Flow chart for sputum examination procedures

The flow chart for sputum examination procedures is as follows:

Flow chart for sputum examination procedures

Sputum collection

Registration

Smearing
_____________________

Non- microscopy center Microscopy center
Transportation of smear to
Microscopy center Staining
Drying
Examination
Recording
Reporting

72
Sputum Examination Procedures

A. Sputum collection:

i) Number of sputa and timing of collection

ii) For diagnosis
For tuberculosis suspects three sputum specimens should be collected, but
if the first two are positive the third one can be omitted. For outpatients,
the first sputum will be collected at the time of presentation (spot
specimen). A second container will then be handed to the suspect, who will
be instructed to collect an early morning sample in that container and
bring that to the laboratory. A third container will then be given to collect
another spot specimen. For inpatients, early morning sputum should as
much as possible be examined, because this yields the best chance to find
Acid Fast Bacilli (AFBs).

iii) During treatment
During treatment smear examinations should be done at the following
intervals:
Cat. 1 treatment: after 2, 5 and 6 months or, if the initial phase was
extended with one month: at 2, 3, 5 and 6 months
Cat. 2 treatment: after 3, 5 and 8 months or, if the initial phase was
extended with one month: at 3, 4, 5 and 8 months
Cat. 3 treatment: after 2 and 6 months
At each of above occasions only one specimen should be examined,
preferably an early morning one. This implies that the patient should be
given a sputum container during the last visit before the examination is
due.

iv) Method of sputum collection
Sputum collection is the most dangerous procedure in tuberculosis
laboratory work, and must be done in the open air and at a distance of
other people.
NEVER COLLECT SPUTUM I N THE LABORATORY.
The laboratory technologist should give a new sputum container to each patient
for whom sputum examination for TB is requested, And demonstrate the patient how
to use the container. The patient should be explained:
the importance of sputum examination for diagnosis of TB
how to open and close the container
that real sputum is needed and not saliva
how to produce good sputum, by repeated deep inhalation and
exhalation of breath followed by cough from as deep inside the chest as
possible
how to avoid contamination of the exterior of the container (by careful
spitting and tight closure)
how to proceed for early morning sputum, including advise on careful
handling (wrapping the container in waste paper or cloth and keeping it
out of reach of children sputum collection)
73

v) Checking the quality of sputum samples
Sputum should be checked immediately after it is presented at the laboratory,
without opening the container. Suitable TB sputum can have various colors and
aspects. Only if the sample is clear and liquid as water, without particles or
streaks of mucous material, the sample should be rejected and the patient be
encouraged to try to produce a better sample. The exception is follow-up sputum
that should be accepted and examined even if its looks like saliva, because these
patients can often not produce a better sample. If the patient has difficulty to
produce sputum and the aspect looks right, very small sample can be accepted.
The smear then should be made immediately, before drying up of the sputum.
Blood-streaken sputum is suitable, but pure blood should not be examined. These
patients should never be encouraged to cough more, but be referred to a Medical
Officer immediately.
Samples of more than one, even two weeks old are still suitable for examination,
but, as a general principle, sputum should be examined as soon as possible.

vi) Identification of sputum samples
A sputum identification number is given at the time the patient brings the first
sputum. The first number due to be issued is taken from the TB Laboratory
Register.
The number should be written on the side of the container and not on the lid,
using a grease pencil, marker pen or a normal pen and label.

vii) Transport of sputum samples to the laboratory
Transport of sputum samples to the laboratory should not be done. It is safer to
transport fixed smears, prepared by trained non-laboratory staff. Chances of
reliable and quick results are better if the patients is referred to the diagnostic
center, or if a relative brings the samples and waits for the results.

B. Smear preparation and Ziehl-Neelsen staining procedure:

i) Smear preparation
There are a number of important points to be covered carefully in order to get the
best results in AFB smears:
Choice of the part fo the sputum to be smeared: choose purulent parts,
if there are. Otherwise the thicker mucous part. Avoid watery or bloody
parts whenever possible.
Smears should be of correct thickness. When too thin, false negative
may occur. Too thick smears or parts of smears cannot be decolorized
sufficiently and may also cause errors.
Timing for the different staining steps is not very strict, but a few rules
should be respected.
Carbolfuchsin must be left on the smear long enough, at least 10
minutes, while a bit longer will not harm as long as it does not dry up.
Good heating, but not boiling, will also result in better staining.
74
Watery acids such as sulphuric acid 25% will not easily decolorize AFB
that were well stained easily, so that they can be left a bit longer or
repeated if necessary.
Methylene blue should be used just enough to provide a background for
focusing and for hiding traces of red that are usually still there. One
half to one minute should be enough, if longer times are needed it
means that the smear or de-staining was bad and there will be a risk of
hiding AFB under the strong blue.
Fuchsin stain fades rapidly in direct sunlight and more slowly under
condition to high temperature/ humidity, especially in thin smears or
after poor staining. So stained smears are better examined without
delay, and should always be kept out of sunlight.

ii) Slide identification
First mark the necessary slides with there respective TB laboratory register
numbers that should already be present on the sputum containers, and place each
slide on its corresponding container. Markings should be indelible, using
diamond pencil or lead pencil in case of frosted slides. If there is no alternative,
grease pencil can be used or a label (paper or leucoplast) attached, but these will
be spoiled if no care is taken during staining (avoid covering this writing with
stains or water).

iii) Procedure of Smear preparation
Verify if the number corresponds with that on the sputum container, open the
container carefully, and using bamboo stick or other tool pick up a little bit from a
good part of the specimen. Close the container, then apply the sputum in the
middle of the slide, and smear it out as evenly as possible over an area of 3 cm by
2 cm, using slow circular and spiral movements. If too much sputum is taken,
remove some using the other part of the stick. The thickness is correct if the
newspaper print under it can only just be read. Place the slide on 9 slide rack to
dry.
Drying should be complete and may take at least half an hour; if humidity is very
high, drying can be assisted by heating the slide over the spirit lamp just a little
bit, and repeating this may be one or two times. Excess heat will spoil the AFB
when it is still wet.

iv) Fixation
Fixation is necessary to kill the TB bacilli, and to make the smear stick to the
slide. Fixation is usually done by passing the completely dry smears three times
through the blue part of the flame of a spirit lamp, smears uppermost, and
holding it with a slide forceps. Avoid overheating; it will damage the AFB and
even the slide. If needed, fixation can be done by covering the smear with ethanol
or methanol and letting it dry up.

v) Staining procedure
Cover the slide with freshly filtered carbol fucshin and heat until steam rises. The
slide may be heated with a toarch by twisting a small piece of cotton wool on the
tip of a wire and soaking it in methylated spirit before lighting. Heat is applied
under the slide till vopour starts coming up from the stain. Allow the slide to stain
for 5 to 10 minutes, heat being applied at intervals to keep the stain hot. Stain
must not be allowed to evaporate and dry. So care should be taken to add
75
adequate carbol fuchsine on the smear to start with. If necessary, pour on more
carbol fuchsine to keep the smear covered.
Use a forcep to drain off the stain, and gently rinse with pure water till no visible
stain comes off anymore. For rinsing, do not use tubing attached to a tap that may
become colonized by mycobacteria but a jug that is easy to clean. Let drain off
excess water and replace the slides on the rack. Cover them with sulphuric acid
25% for about 3 minutes, and rinse again. Repeat this process. Decourisation is
complete when after washing the film is very faintly pink. Let the water drain off
and replace the slides on the rack, then cover the slide with methylene blue 0.1%
for maximum one minute. Let the stain drain off and rinse with pure water. Take
slides from the rack, clean the underside by wiping with toilet paper soaked in
alcohol if necessary, and place them on drying rack out of sunlight. Microscopy
should be done on well dried smears only.

C. Examination of smears:

i) Acid Fast Bacilli (AFBs)
AFBs has special cell-wall that makes them resistant to destining by acids or
alcohol. Other types of bacilli and cells loose the red stain and take up the blue
color of methylene blue. AFBs are slender, slightly curved, lying single, in small
groups or sometimes in clumps.

ii) Technique of AFB-microscopy
This requires a good binocular microscope, with 10 x eyepieces and 100 x oil
immersion objectives. The condenser should be brought up almost fully and it is
better not to use the blue filter so as to obtain maximum brightness.
Apply immersion oil by letting a drop fall on the smears without touching yhe
smear to avoid possible contamination of the oil dropper.
Examination should be from one end to the other, selecting a line with many
blood cells and mucous materials, but not so thick that it will be difficult to see
details. Check each field for red slender rods (bacillus), while moving slowly to
the next field. If you observe red shapes examine these more closely for AFB.
Examine 100 fields, before declearing the smear negative. If few bacilli are seen,
count the number and examine 200 fields to obtain better quantification. If
numerous AFBs are seen, examination of 20 fields will be sufficient, while
estimating whether there are less than 10 per field or 10 or more. Report the
result as follows:

Number of AFBs seen Result and grading Numbers of field to be examined
None in 100 fields Negative 100
1-9 in 100 fields Scanty, report exact
number of AFB
200
10-99 in 100 fields 1+ 100
1-10 per field 2+ 20
>10 per field 3+ 20
76

iii) Storage of examined smears
All examined smears, irrespective the result, should be preserved until a
supervisor can make a selection for external quality assessment (EQA). Cleaning
the xylene is not needed, it is sufficient to remove most of the oil by one careful
sweep using toilet paper, or to let oil drain on toilet paper. Slides should be stored
according to number. After the first slide of a suspect, two empty slots must be
left to accommodate the other slides smears later on.
Supervisors, and not the laboratory technologist of the microscopy center, should
select the sample for EQA from the TB laboratory Register and request the
technologist to retrieve these slides from the boxes. Thereafter all slides that have
not bee selected for EQA must be discard. Stained smears are not infectious. They
can be reused, but not for AFB examination. If you run out of slides completely,
only negative slides may be reused. In that case clean off the smears and then
soak then in 5% household bleach for several hours to destroy eventually present
AFB.

D. Preparation of stains for Ziehl-Neelsen staining:
Good stains, especially good carbolfuchsin, are essential to detect high numbers
of AFB-positives. Only a good stain will be able to show the AFB, also when they
are rare. AFB damaged by treatment is difficult to stain, so all follow-up smears
will often be negative when the quality of carbolfuchsin is substandard.
Stain preparation requires some equipment for weighing and measuring, and also
pure water without mycobacteria from the environment. Therefore preparation of
stains should be done at one laboratory at districts level, with checks for quality
before they are distributed to the peripheral laboratories.

i) Equipment needed
The following equipment is needed for preparation of the stains;
A balance or weighing scale, sensitivity 0.1 gram
Measuring cylinders, one of 100 ml and one of 500 or 1000 ml
Conical flasks or flat bottomed balloon flasks
A spirit lamp for heating
Containers for newly prepared stains. These can be well closing plastic
bottles of 1 liter
Labels for the bottles
Brushes for clean the bottles before re-use
Big funnel for each solution to fill up the bottles
77

ii) Chemicals needed for AFB microscopy
For carbolfuchsin; basic fuchsin powder; phenol crystals of good quality;
alcohol; denatured 95% ethanol or pure methanol; water, distilled or
purified
For acid solution: concentrated sulphuric acid, which can be of industrial
grade if it is not too dirty; water, distilled or purified
For methylene blue solution: methylene blue powder of good quality;
water, distilled or purified.
Water for preparation of stains does not have to be distilled, but it should not
contain AFB. This can be obtained by regular cleaning of water containers and by
using a household candle type filter to prepare water if distilled is not available.
The filter should be maintained regularly.

E. Preparation of stains

i) One liter of carbolfuchsin (1% fuchsin, 5% phenol)
Weigh 10 grams of basic fuchsin and 50 grams of phenol crystals. Measure 100 ml
of alcohol and pour it in a conical flask. Add the phenol, swirl the flask till it is
dissolved. Add the basic fuchsin powder and continue to swirl until complete
dissolution of the fuchsin powder. Heat slightly over the spirit lamp if dissolution
is difficult. Only after fuchsin is completely in solution, 900 ml of water should be
added and mixed by further swirling.

ii) One liter of sulphuric acid 25%
Use the 5 litre flask of good quality glass; fill it with 750 ml of pure cold water.
Measure 250 ml of concentrated sulphuric acid in a cylinder, and pour it slowly
into the flask of water, letting it drain along its side. This will generate a lot of
heat; usually it is necessary to stop a few times, swirling the flask to let it cool off a
bit, or even holding it under the tap with cold water. NEVER DO THE REVERSE;
ADDING WATER TO ACID WILL MAKE IT BOIL IMMEDIATELY AND IT MAY
EVEN JUMP INTO YOUR FACE. In case of accident with acid, rinse the part of
body immediately with plenty of water.

iii) One liter of methylene blue 0.1%
Weigh 1 gram of methylene blue powder and add it to 0.5 liter of pure water into a
conical flask.
Make it dissolve by swirling, then add another 0.5 liter of water and mix again.
Let the flasks with freshly prepared stains stand till quality control has been done;
then fill the solutions into clean bottles and label them.
Especially carbolfuchsin bottles must be cleaned carefully before reuse, the
crystals sticking to the bottom are hard to remove. Use acid alcohol and a bottle
brush. Label should mention the name of the stain and the date it was prepared.
Carbolfuchsin should be filtered during the process of staining by the users. The
other stains do not need to be filtered.

iv) Quality control
Quality control is necessary to ensure that the stains work well, and that they do
not contain contaminating AFB. For the first purpose, two low positive smears
will be uses, for the second two negative or fake smears. The stain-preparing
laboratory should always do this for each batch prepared. Thus it may be
78
advantageous to prepare bigger batches if big flasks are available. And this
laboratory should keep careful records, to defend itself against possible
complaints of bad stains. The batches should be identified by name of reagent and
preparation date.
Quality control is done using one or more freshly prepared stains and the staining
procedure is as described. EXCEPT THAT NEGATI VE CONTROLS WI LL
BE STAI NED THREE TI MES BEFORE EXAMI NATI ON. This is to make
the contaminants in acid or methylene blue visible by putting again carbolfuchsin
after the first staining cycle, and to give possibly present contaminants more
chance to stick to the smears. Positive controls must be stained only once.
Examine all controls carefully, and record results in a QC register.

v) Keeping of stains
Well-prepared stains will keep for several months, or even a year, also at
temperatures over 35C. However, they have to be stored in clean and well-closed
bottles, OUT OF DI RECT SUNLI GHT. Stains should preferably be kept in a
closed cabinet.

F. Recording and reporting:

i) Sputum examination request form
Name and address of the patient; necessary to trace cases found to be
positive but not returning for their result. The address must be detailed
enough to make this possible.
Reason for examination. Can be found out by questioning the patient
concerning ongoing treatment. The registration number must be added
for follow-up patients
Specimen identification number: only for specimens sent from outside
the laboratory. Specimens collected directly by the technologist should
immediately receive the laboratory serial number that is the first
unused number in the laboratory register.

ii) Maintenance of Laboratory register

G. Laboratory Safety:

i) To destroy microorganism present in the specimen or container use of
chemical sterilization (given in waste management) or autoclave or
boiling method can be used.
ii) To prevent environmental microorganism (normally present in hair,
hand, cloth or bench) to enter the specimen- use of
disinfectant,detergent, antiseptics for contacts equipment working
space advised.
iii) Safety of laboratory personnel-
Use of musk, gloves and provision of vaccination should be encouraged
79

H. Waste Management:

Use of disinfectant before disposal or reuse*
Target Disinfectant
Strength to use
(Disinfectant/
Material v/v)
Application
Time of
exposure
Skin Ethanol
Iodine
Povidone iodine
Quaternary
Ammonium comp
70%
1%
1%
Direct
Direct
Direct
Direct
2 minutes
2 minutes
2 minutes
2 minutes
Urine Cresol (pH 9) 5% 1:1 4 hours
Sputum Cresol (pH 9) 5% 1:1 4 hours
Work
benches
Lysol
Cresol
Hypochlorite
Chloramine- T
5%
1%
5%
Direct
Direct
Direct
Direct
4 hours
4 hours
4 hours
4 hours
Glassware Hypochlorite 1% Direct 4 hours
Laboratory
instrument
Hypochlorite
Isopropanol
0.1%
70%
Direct
Direct
4 hours
4 hours

* Based upon: Basics of quality assurance: WHO/EMRO, 1992, page 162

Serological Tests

Many diseases can be diagnosed by detecting antigen or antibody produced against
the microorganisms (e.g. bacteria, viruses, parasites) causing those diseases.

Detection of antigen or antibody in the serum is called serological test. Here
antigen is the component of the causative microorganism and antibody is the
immunoglobulin produced in the patients serum against that antigen of the
causative microorganism.

COMMON METHODS of detection of antigens or antibodies in the laboratory:
1) Agglutination
2) Precipitation

AGGLUTINATION:
The interaction between antibody and a particulate antigen results in a visible
clumping called agglutination.

Direct Agglutination:
Antibody directly binds with particulate antigen to form visible clumps.
Example: Blood Grouping

Indirect Agglutination:
The soluble antigens are coated on particulate substances (ie., on latex particles,
carbon particles, erythrocytes, etc.) to make the Ag-Ab reaction visible.
Example of common Agglutination tests:
Widal test, Pregnancy test, ASO titre, RA test, Rose Waaler test, RPR, HBsAg by
latex, CRP
80

PRECIPITATION:
Soluble antigens bind with specific antibodies at or near equivalent concentration to
form an insoluble precipitate.
Example of common precipitation test: Immunochromatography (ICT)

ICT can be performed at the Upazila and District hospitals. So the method is
described in this manual.

WIDAL TEST

Indication:
The test is done for laboratory diagnosis of Enteric fever (Typhoid & Paratyphoid).

Principle:
The Widal test is a serological (agglutination) test that detects antibodies against
Salmonella typhi and Salmonella paratyphi A, B, and C. Patients serum is tested for
O and H antibodies. The stained bacterial antigens are used. The presence of a
visible agglutination is related with the presence of the corresponding antibody
concentration in the samples tested.

Specimen:
Serum is stable for 7 days at 2
0
C- 8
0
C. Avoid haemolysed or lipaemic samples. For
longer preservation, keep the sample at 20
0
C.

Storage of the reagents:
Reagents must be storage upright, well closed at 2C-8C.
Do not freeze any of the reagents.

Methods:
Two methods are there
1) Rapid Slide Titration method.
2) Tube Agglutination test method this method is not done now a days due to
1) requirement of longer time to perform and 2) unavailability of the reagents.

Rapid Slide Titration method:

Procedure:
1) using a suitable pipette, deliver 80, 40, 20, 10 or 5 l of undiluted serum on to
a row of 3 cm diameter circles on a white tile.
2) Shake the bottles containing O and H antigens. Add one drop of the
appropriate suspension (S. typhi O/H, S.paratyphi A, B & C O/H) to each
serum aliquot. S. paratyphi C is very rare in Bangladesh.
3) Mix and spread the mixture over the entire area of the circle.
4) Slowly rotate (at 100 r.p.m.) the tile or card for 1 minute and observe for any
agglutination (clumping).
81

Result:
Examine macroscopically the pattern of the agglutination. The reactions seen in
the circles will be approximately equivalent to those, which would occur in a tube
agglutination test with serum dilutions of 1/80, 1/160 and 1/320.
20 l = 1/80
10 l = 1/160
05 l = 1/320
The rapid slide titration therefore, provides an approximate titre for the test
serum. For higher titres, serum should be further diluted.

Interpretation:

Although Widal test has some limitations, it can be a useful aid for the diagnosis of
enteric fever, especially in the endemic areas provided interpretations are made
carefully.
The titre is defined as the highest dilution showing a positive result (agglutination).

1. Titres 1/160 is significant titre for enteric fever.

2. O (TO/AO/BO/CO) 160 with corresponding H 160 indicates active
infection is present.

3. O (TO/AO/BO/CO) 160 with corresponding H < 160 indicates early stage of
active infection.

4. H (TH/AH/BH/CH) 160 with corresponding O < 160 indicates anamnestic
reaction or past infection.

ASO (Latex Agglutination Test)

Indication:
ASO latex agglutination test is done in -haemolytic (Group A) streptococcal
infection and rheumatic fever.

Specimen:
Fresh serum is obtained by centrifuging clotted blood. The sample may be stored at
2 C to 8C for 48 hours before performing the test. For longer preservation, keep the
sample at 20
0
C.
Haemolytic, lipaemic or contaminated serum should be discarded.

Storage of the reagents:
Reagents must be storage upright, well closed at 2C-8C.
Do not freeze any of the reagents.

Qualitative method (Slide screening method):

Procedure:
1. Allow the reagents and serum to reach room temperature.
2. Gently shake the latex reagent to disperse the particles.
82
3. Take a slide. Place a drop of undiluted serum into the circle of the test slide
using the disposable pipettes provided.
4. Add one drop of the latex reagent next to the drop of serum.
5. Spread the reagent and serum sample over the entire area of the test circle
with a clean wooden stick.
6. Gently tilt the test slide backwards and forwards approximately once every
two second for two minutes. Observe for the presence of agglutination
(clumping).

Results:

Presence of agglutination indicates ASO antibody 200 IU/ml.
Positive and negative controls should be included at regular intervals.
At the end of the test, rinse the test slide with distilled water and dry. Normal
laboratory precautions should be maintained while handling patients samples.

Semi-quantitative determination

The semiquantitative test can be performed in the same way as the qualitative test
using dilutions of the serum in saline, phosphate buffered saline or glycine Saline as
follows:

Dilutions 1/2 1/4 1/8
Sample serum 50l - -
Saline 50l 50l 50l
Transfer 50l from the mixer to the next and
discard 50l from last dilution
50l

50l

Final volume of sample 50l 50l 50l
IU/ml 400 800 1600

Normal levels: Adults <200 IU/ml.

83
Results:

The titre is expressed as the reciprocal of the highest dilution showing macroscopic
agglutination e.g. if this occurs in dilution 3, the titre is 1600.

Interpretation of results:

Positive results may indicate an acute streptococcal infection. In which case the test
should be repeated at weekly intervals to determine the progression of infection.
Elevated levels of ASO have also been found in patients suffering from scarlet fever,
acute rheumatic fever, tonsillitis other streptococcal infections as well as healthy
carriers.

PREGNANCY TEST (HCG LATEX TEST)

Human chronic gonadotropin (HCG) excreted in the urine during pregnancy, is
determined qualitatively using a latex agglutination reaction.

Principle
The HCG latex reagent is a suspension of polystyrene latex particles coated with
monoclonal antibodies to HCG. The presence of the hormone in the urine induces
the particle agglutination. When HCG is absent in the urine no agglutination occurs.

Sample collection
Any fresh urine sample can be used. But the first morning urine is the most suitable
as it contains greatest concentration of HCG hormone. It must be collected in a clean
plastic or glass container. The samples can be stored between 2 C - 8 C for 48
hours. For longer periods the sample must be frozen.

Reagents
Latex reagents :Latex particles with monoclonal anti-HCG.
Positive control
Negative control
Test slide
Mixing sticks.

Storage of reagents
The reagent is stable until the expiry date printed on the label, if stored between
2 C - 8 C. Do not freeze. Shake the latex before use to have a uniform suspension of
latex particles.

Procedure
1.Allow the reagent and controls to reach room temperature.
2.Gently shake the reagent to disperse and suspend the latex particles uniformly.
3.Place 50l of urine sample in a circle of the slide.
4.Add one drop of HCG latex reagent next to the urine drop.
5.Mix both with a stick, covering the full surface of the circle.
6. Rotate the slide manually or with a rotatory shaker for 2 minutes.
7. After 2 minutes read the presence or absence of agglutination (clumping).

84
Both controls should be run with each batch of test to distinguish possible
granularity from agglutination.

Sensitivity of the test
The test is standardized to detect 200 IU of HCG in urine or higher.
A positive reaction is therefore possible 5 days after a missed period. If the test is
proved negative, it is recommended to repeat the test after 7 days.

Interpretation
The test is positive in pregnancy. It is also positive in hydatid mole.
False negative results may be obtained in case of fetal death, abortion and toxemias.
In these cases the excretion of HCG in urine is decreased below the detection level.
RPR Test

(RPR is mistakenly referred as VDRL test; no reagent is available for now VDRL test.
RPR is an agglutination test while VDRL is a flocculation test.)

Principle

The RPR carbon reagent is a suspension of cholesterol crystals coated by cardiolipin,
lecithin added to adjust the sensitivity and charcoal particles to improve the reading
(visualization) of the reaction. The reagent acts as antigen against antibodies present
in persons suffering from syphilis.

Sample

Use fresh serum obtained by centrifugation of clotted blood. The sample may be
stored at 2
0
C - 8
0
C for 48 hours before performing the test. For longer periods of
time the serum must be frozen. Haemolytic, lipaemic or contaminated serum must
be discarded.

Reagents

RPR carbon reagents
Positive control
Negative control
Test slide
Mixing sticks

Shake the reagent (RPR carbon) before use to have a uniform suspension without
visible clumping. The reagent has to be dispensed through the needle and dispensing
bottle supplied or with an automatic pipette adjusted to 20l. Sensitivity of the test
depends on the drop volume. Do not use any other droppers but those provided and
place the dropper perpendicular to the slide surface. Any variation of this way will
modify the result of the reaction.
The reagent and controls have to be stored at 2
0
C - 8
0
C. Do not freeze.

Procedure

A) Qualitative determination (slide screening method)

85
1. Before using the reagents allow the components to reach room
temperature.
2. Gently shake the reagent to disperse the particles.
3. Place a drop of undiluted serum (50l) on to a a circle of the slide.
4. Add a drop of RPR carbon (20l) through the dispensing bottle and
needle, or pipette measuring 20l, next to the drop of serum.
5. Mix both drops spreading them over the full surface of the circle.
6. Rotate the slide manually or with a mechanical rotator at 80 100 rpm
for 8 minutes.
7. Read the presence or absence of visible agglutination in this period of
time.
8. Non-specific agglutination could appear if the test is read later.

Interpretation of reactions

Read the results immediately after 8 minutes rotation. Read with naked eye
preferably in good daylight.
A negative reaction will appear as a concentration of smooth carbon in the center of
the circle or as a smooth, even gray suspension throughout the test circle.
A weak positive reaction is characterized by a concentration of fine black aggregates
surrounded by a diffuse area of fine black aggregates.
A positive reaction is the production of black aggregates most commonly observed
throughout the test circle.

Semi-Quantitative Test

The semi-quantitative test is performed in the same way like the quantitative test by
preparing a serial double dilution of the serum sample in normal saline (NaCl 9 g/L).
The last circle in the dilution series that contains any black aggregates gives the titre
of the sample.

Significance

Syphilis is a sexually transmitted disease caused by Treponema pallidum. The test is
positive in primary and secondary syphilis. It may be negative in late latent stage of
the disease.
Biological false positive results occur in acute viral infections (measles. Infectious
mononucleosis), mycoplasma pneumonia, malaria, after vaccinations, leprosy,
autoimmune disease, etc.
86
TPHA (Treponema Pallidum haemagglutination Test )

Principle

The TPHA for syphilis is an indirect haemaglutination test for the detection and
titration of specific antibodies against Treponema pallidum. Avian erythrocytes are
sensitized with antigens of the Nichols strain of Treponema pallidum. In the
presence of positive syphilitic serum, the red cells aggregate to form characteristic
patterns on the bottom of the microplate wells.

Sample

Use fresh serum obtained by centrifuging clotted blood. The sample may be stored at
2
0
C - 8
0
C for one week before performing the test. For longer period of time, the
serum must be frozen. Haemolytic, lipemic or contaminated serum must be
discarded.

Reagents

1. Test cells
2. Control cells
3. Sample diluent buffer
4. Positive control
5. Negative control
All reagents are ready for use, however both test and control cells should be
thoroughly resuspended prior to use. The reagents should be stored at 2
0
C - 8
0
C.

Additional equipment
U-well microtitration plates

Procedure (of Qualitative test)

1. Bring all reagents and samples to room temperature before use.
2. Dilute serum sample 1/20 with diluent buffer. Use the dilute serum the same
day (serum10ml +190ml of diluent buffer)
3. Dispense into adjacent wells of microtitration plate.

Control well Test well
Sample (1/20) or control 25 l 25l
Control cells 75 l -
Test cells - 75 l

4. include a positive and negative control for each batch of test.
5.Gently tap all four sides of the plate to ensure the contents of each well are
thoroughly mixed.
6. Cover the plate and place on a flat, white surface away from vibration and
direct sunlight. Leave for 45-60 minutes at room temperature or overnight
before reading results.

Interpretation of results

87
Results are read visually by comparing with positive and negative control sera.
Readings are scored by the degree of haemagglutination and reported as
4+,3+,2+,1+ or negative. The final diagnosis should be based on a correlation of
the test results with the patients clinical history.

Interpretation of the results
Degree of haemagglutination Reading Report
Smooth mat of cells covering entire
bottom of well, sometimes with
folded edges
4+ Reactive
Smooth mat of cells covering part of
well bottom.
3+ Reactive
Smooth mat of cells surrounded by
red circle
2+ Reactive
Smooth mat of cells covering less area
and surrounded by a smaller red
circle
1+ Reactive
Button of cells having a small hole in
center
+/- Borderline
Definite compact button of cells,
sometimes with a very small hole in
the center.
- Nonreactive

The negative control must show a non-agglutinated pattern with both test and
control cells.
The positive control must show agglutination with test cells but not with control
cells.
Sera showing agglutination with control cells indicate the presence of non-specific
agglutinins and should be retested after absorption.
Borderline sera should be retested and reported as non-reactive if the same pattern is
reproduced.

RA Test (Rheumatoid Arthritis Latex Test)

Principle

An abnormal protein occurs in the serum of many patients suffering from
rheumatoid arthritis. This protein behaves as if it is an IgM antibody directed against
determinants of IgG globulins. Detection of the rheumatoid factor protein is of value
in the diagnosis of rheumatoid arthritis. Rheumatoid factor in serum sample can be
detected using a suspension of fine plastic granules coated with human gamma
globulins, which were agglutinated in the presence of rheumatoid factor. The RA
latex reagent is a sensitive, standardized preparation of purified human IgG fraction
coated polystyrene latex particles.

Sample preparation

Use fresh serum obtained by centrifuging clotted blood. The sample may be stored at
2
0
C - 8
0
C for 48 hours before performing the test. For longer periods of time the
serum must be frozen. Haemolytic, lipaemic or contaminated serum must be
discarded.

88

Test reagents

Latex test reagents
Positivecontrol
Negative control
Test slide
Mixing sticks

Procedure of Qualitative Method

1. Allow each component to reach room temperature.
2. Gently shake the latex reagent to disperse the particles.
3. Place a drop of undiluted serum onto the circle of the test slide using the
disposable pipettes provided. Positive and negative controls should be
included.
4. Add one drop of the latex reagent next to the drop of serum, positive and
negative controls.
5. Using the other end of the pipette (broad end) mix the reagent and serum
sample and spread over the entire area of the test circle.
6. Gently tilt the slide backwards and forwards approximately once every two
seconds for two minutes. Observe for agglutination.

Results
Presence of agglutination indicates RF concentration in the sample more than 8
IU/ml.
The lack of agglutination (a smooth homogenous milky suspension) indicates a level
of RF in the sample less than 8 IU/ml.

Normal levels: Adults < 8 IU/ml
Immunochromatography Test (ICT)

Objective:
At the end of the session the trainees
1) will know the principle of the test.
2) will know the procedure of the test.
3) will be able to perform & maintain the quality of the test.
4) will be able to report the test.

Principle

Immunochromatographic device or strip contains a membrane embedded with
antigen (Ag) or antibody (Ab) and colloidal conjugate to detect the counterpart (e.g.
Ag or Ab).

Specimen

Whole blood or serum or plasma may be used according to the instruction of the
manufacturer of ICT device or strip.

89
Explanation of the test

The ICT device or strip contains a Test Line and a Control Line. Both the Test
Line & Control Line are not visible before applying any samples.
The Control Line is used for procedural control. Control line should always appear if
test procedure is performed properly and test reagents of control line are working.
The Test line becomes visible when the sample contains specific antigen or antibody.
The appearance of the Test Line after the specified time (mentioned by the
manufacturer) is not considered valid.

Interpretation
Positive Result:
The appearance of both Control Line & Test Line within the time is considered as
positive.
Negative Result:
The appearance of only Control Line is considered as negative.

Invalid Result:
If the Control line C is not visible, the result is considered invalid. The specimen
must be retested using a new test device.

N.B. One must follow the instruction of the manufacturer (provide

Aldehyde Test (Formol Gel Test)

The Aldehyde test is widely used for the diagnosis of kala-azar. The test is non-
specific. It detects large amounts of non-specific polyclonal globulin in the patients
serum. It becomes positive about 3 months after infection and negative about 6
months after successful treatment.

Procedure
1. Collect about 5 ml of venous blood in a dry glass tube and leave to clot. Separate
the serum.

2. Transfer about 1 ml of serum to a small tube. Add 2 drops of concentrated formalin
solution (40% formaldehyde) and mix. Allow to stand for up to 2 hours.

3. Positive test: Serum whitens and gels like the white of a hard boiled egg within 20
minutes (often within 5 minutes). A milky appearance without the solidification of
serum may occur in early cases of kaka-azar.

90
4. Negative test: Serum remains unchanged or gelling and whitening only occurs
after 20 minutes.

Aldehyde test may be positive in multiple myeloma, chronic liver disease, and
leprosy. Variable result may be found in kala-azar patient infected with HIV.

Skin Fungus- lab Diagnosis.

Objective:
At the end of the session the trainees
1) will know the principle of the test.
2) will know the procedure of the test.
3) will be able to perform & maintain the quality of the test.
4) will be able to report the test.

Fungal infections are usually divided in to four groups

1) Superficial
2) Cutaneous
3) Subcutaneous
4) Systemic or deep

Here we are concerned with superficial and cutaneous fungal infections. Malassezia
furfur is the most common fungal agent causing superficial fungal infection in the
skin. The disease is known as Tinea versicolor, in Bangladesh, it is commonly
known as Chuli.
Dermatophytes cause cutaneous infection which is known as Dermatophytosis or
Ring worm. In Bangladesh, it is commonly known as Dad or Daud.
Cadida spp. (mainly Candida albicans) cause infection in the skin and mucus
membrane. Infection of oral cavity by Candida albicans is called oral thrush and that
of vagina is called vaginal thrush.

Dermatophytosis or Ring Worm

Collection of samples

Skin scraping
1. Select the most suitable lesion. Suitable areas are: raised
and/or reddened margin.
2. Clean the lesion with 70% alcohol and dry.
3. Scrap the area with the back of the surgical blade.
4. Avoid bleeding.
5. Collect the skin fragments in black paper and keep in
Petri-dish.
Nail scraping
1. Clean the affected nail as described for skin.
2. Scrap off dead tissues and scales until pain is felt.
3. Then scrap the nail with backside of the scalpel and collect
the specimen for examination.

91

Hair
1. Suitable hair samples are:
Twisted hair
Broken hair
Bifurcated hair
Twisted bundle hair
2. Pluck hair with a forcep.
3. Collect in a petridish.
4. Cut hair samples into small pieces (5 mm length)

Sample processing

1. Put 1 2 drops of 20% KOH/NaOH on a glass slide.

2. Mix the sample with 20% KOH/NaOH.

3. Put a cover slip and place it in a covered petridish for one
hour.
In case of nail, keep it for longer time for better dissolve of the
keratin present in the nail, that will help better visualization of
fungal hyphae.
4. Before examination under microscope, give gentle pressure
on the cover slip to spread the sample uniformly.
5. see under microscope at 10X and with 40X with condenser
closed.
6. Look for
- branching hyphae or
- chains of angular or rounded arthrospores or
- mixture of hyphae and arthrospores.

Interpretation

1. Septate fungal hyphae with arthrospores are definitive findings of
dermatophytes.

2. Septate fungal hyphae without arthrospores are suggestive of
dermatophytes.

3. Short curved hyphae with clusters of round cells are definitive of
Malassezia furfur.

4. Aseptate fungal hyphae are suggestive of non-dermatophytes.

92

Microscopic view of Malassezia furfur
( long hyphae and clusters of spores seen )

Microscopic View of Dermatophytes
( long branched hyphae with chains of spores seen )

Candidiasis or Thrush

Specimen Collection

Wet the cotton swab with normal saline. Then rub the moist swab over the lesion in
the mucus membrane.

Sample processing

1. Put 1 2 drops of normal saline on a glass slide.

2. Mix the sample with normal saline.

3. Put a cover slip on it and examine under microscope.

4. See under microscope at 10X and with 40X with condenser
closed.
5. Look for
- Yeast like cells with buddings & pseudohyphae.

Interpretation

Yeast like cells with buddings & or pseudohyphae are suggestive of Candida spp.

Microscopic view of Candida

93

(spherical yeast like cells with long pseudohyphae) ( Yeast like cells of Candida
showing budding )

Examination of Urine

1. Collection

a. Urine should be collected in a wide mouthed clean and dry container. For
routine analysis first morning sample should be collected since this urine is
most concentrated. At least 15-30 ml of urine should be collected. Casts tend
to dissolve and are generally less in number in dilute urine. If the sample
cannot be examined immediately, it should be placed in a refrigerator in 4C -
8C.

b. Label and number the sample immediately.

2. Preservation

Urine should be properly preserved if it is not immediately processed in the
laboratory.

a. Refrigeration

Store all urine specimens in refrigerator at 4C - 8C whether any preservative
has been added or not. Refrigeration prevents the growth of bacteria and helps to
preserve casts, red and white blood cells, and an acid pH for short period of time
(up to about 8- 12 hr.)

b. Boric acid preservative

Boric acid is a good preservative of urine. At a concentration of 10 g/l (1% w/v),
bacteria remain viable without multiplying. White cells, red cells, and casts are
also well preserved and there is no interference in the measurement of urinary
protein and glucose.

Deterioration of urine

The following changes occur when unpreserved urine is left at room temperature:

Bacteria in the urine will multiply so that the bacterial count will be
unreliable.
94

If the urine contains urease-producing organism, the ammonia released
will increase the pH of the specimen. The increased pH will destroy cells
and casts.

Bacteria will also break down any glucose, which may be present.

If white cells, red cells and casts are present these will begin to lyse.

The concentration of protein in the urine will be altered.

Bilirubin and urobilinogen will not be detected as these are oxidized.

3. Physical examination

a. Colour and appearance: Normal urine colour is usually straw to amber. If the
colour is different from normal then it should be noted and reported.

b. Reaction: Twenty four hour urine is normally acidic. The normal pH range is
5.0- 7.5. It is measured by pH paper. With Esch. coli infections, the urine is
markedly acidic. Alkaline urine is found with Proteus infections.

c. Specific gravity: The normal specific gravity of urine is 1.010- 1.020 or greater.
It is measured by urinometer.

4. Chemical examination

Detection of protein or albumin

Urinary protein (albumin) can be detected by various methods namely heat
coagulation test or sulphosalicylic acid tests.

Procedure of heat coagulation test

This test is a quqlitative method.

a. Take a clean tube and fill 2/3
rd
of the tube with urine.
b. Hold the tube in its lower part and heat the upper 1/3
rd
with burner.
c. Add few drops of 5% acetic acid.
d. Heat again. If the turbidity disappears then it is due to the phosphates present
in the urine. But if the turbidity remains or increases then it is due to albumin.

Report as follows:

No turbidity Negative
Slight turbidity Protein +
Moderate turbidity Protein ++
Marked turbidity Protein +++
Marked turbidity with precipitate Protein ++++
95

Procedure for sulphosalicylic acid tests

1. Pour 2 ml of urine in a clean test tube. If the urine is cloudy, centrifuge it to obtain
clear urine.

2. Test the pH of the urine with a pH paper. If the urine is neutral or alkaline, add a
drop of glacial acetic acid.

3. Add 2- 3 drops of 20% sulphosalicylic acid reagent.

4. Hold tube against a dark background, examine for any cloudiness and report as
follows:

No cloudiness Negative --
Slight cloudiness Protein +
Moderate cloudiness Protein ++
Marked cloudiness Protein +++
Cloudiness with
precipitate
Protein ++++

False positive results may be obtained if the patient is receiving tolbutamide,
penicillin, para-amino salicylic acid, sulphonamides.

Test for reducing substances (sugar)

Reducing substances in urine is detected by Benedicts qualitative test. This test
is not specific for sugar. False positive results may be obtained if urine contains
reducing substances such as other sugars (galactose, lactose, fructose, pentose),
salicylates, penicillin, streptomycin, INH, PAS, vitamin C etc.

PROCEDURE
l. In a test tube, take 5.0 ml of Benedict's solution and heat to boil.

2. If no change occurs then add 8 drops (0.5 ml) of urine.

3. Mix and heat again. Allow the solution to cool to room temperature and read
as follows:

Interpretation of Benedict's test
Colour Result Sugar
concentration
Blue
Green, no ppt
Green, ppt
Yellow, with ppt
Orange, with ppt
Brick red, with ppt
Negative
Trace
+
++
+++
++++
-
Trace
0.5 g%
1.0 g%
1.5 g%
2.0g%

96
Microscopic Examination

1. Mix the urine thoroughly and take about 10 ml of urine in a conical
centrifuge tube.
2. Centrifuge for 7-10 minutes at 1500-2000 rpm.
3. Pour off the supernatant carefully.
4. Then place a drop on a glass slide and cover with a cover slip.
5. Examine under microscope with 10 X objective to obtain an overall
picture
of the deposit.
6. Use the 40 X objective to examine objects more closely. Look for cells,
casts, crystals, parasites, etc. as mentioned below.

Epithelial Cells

These are easily seen with the 10x objective. They are nucleated and vary in
size and shape. They are usually reported as number of cells per low or
high power (10 x or 40 x objective) field.
It is normal to find a few epithelial cells in urine. When seen in large
numbers. They usually indicate inflammation of the urinary tract or vaginal
contamination of the specimen

White blood cells (pus cells)

White blood cells (WBC) are round, 10-15 m in diameter that contain
granules. In urinary tract infections they are often found in clump.

WBCs are usually reported as the number per high power field (HPF),
e.g. 10-15 WBC/HPF. Normal urine may contain a few white cells (less than
5/HPF).

Bacteriuria without pyuria (pus cells in urine) may occur in early urinary
infections, in diabetes, enteric fever, and in bacterial endocarditis.

Pyuria with a sterile routine culture may he found with renal tuberculosis,
gonococcal urethritis, and leptospirosis, or when a patient with UTI has
been treated with antimicrobials.

Red Cells
These are smaller (6-8 m) and more refractile than white blood cells. They
have a definite outline and contain no granules and nucleus. When the
urine is isotonic, they have a ringed appearance. If the urine is hypertonic,
the red cells will appear smaller than normal and often crenated or spiky. If
the urine is hypotonic, the cells will appear larger than normal

RBCs are reported as number of RBC/HPF.

97
Yeast Cells

Yeast cells are oval shape and usually show budding. If it is confused with RBC,
put a drop of dilute acetic acid under the cover slip. Red cell will be haemolysed
by the acid and disappear, but not yeast cells.

Yeast cells are usually reported as few, moderate, or many per HPF. They can be seen
in the urine of women with vaginal candidiasis, and in specimens from diabetic
patients.

Casts

These can usually be seen with the 10 X objective. They are cylindrical in shape. There
are several types of casts:

Hyaline casts are colourless and empty. They are associated with damage to the
glomerular filter membranes. A few may be seen following strenuous exercise or
during fever.

Waxy casts are thicker and denser than hyaline casts, often appear indented or
twisted, and may be yellow in colour. They usually indicate tubular damage and can
sometimes be seen in renal failure.

Cellular casts contain white cells or red cells. Red cell casts appear orange red. They
indicate hemorrhages into the renal tubules or glomerular bleeding. White cell casts
are found when there is inflammation of the kidney pelvis o r tubules. Yellow-brown
pigmented cast, may be seen in the urine of jaundiced patients.

Granular casts contain irregular sized granules originating from degenerated
cells and protein. They are also associated with renal damage.

Crystals
Normal urine contains many chemicals from which crystals can form. These
have a characteristic refractile appearance, and therefore the finding of most
crystals has little importance.

Common crystals found in urine
Triple phosphate crystals are occasionally found in alkaline urine. They have
little or no clinical significance.

Calcium oxalate crystals are frequently seen. When found in freshly passed
urine ,they may indicate calculi in the urinary tract.

Uric acid crystals are yellow or pink-brown. They can sometimes be found
with calculi.

Parasites found in urine

Sometimes ova and adult worms of E. vermicularis (usually found in female
children) are found in urine. Sometimes urine may contain Trichomonas,
microfilaria of W. bancrofti and ova of Schistosoma.

98
Microscopic view of different cells and casts in urine :

Red blood cells White blood cells WBC cast in urine

Microscopic view of different crystals in urine :

Phosphate crystals Calcium Oxalate crystals Cystine crystals
99
Examination of Stool

Stool from patient is examined to detect :

1. Adult worms
2. Segments of tapeworms
3. Ova and Cysts of Parasites
4. Larvae
5. Trophozoites
6. WBC , RBC , Pus Cells , Macrophages etc

Collection of stool sample :

1. Stool sample is to be collected in a wide mouthed leak proof container with a tight
fitting lid.

2. Amount: 20 40 gram of solid stool or 5 6 tablespoonful of liquid stool .Care
is to be taken to prevent contamination with urine , dirt etc .

3. Patient is to be warned not to take any medicine or medicinal substance before
collection of the sample.

4. Stool sample must not be collected from bedpan containing disinfectants.

5. The container should be properly labeled with patients ID no., the name of the
test that is desired by the clinician.

6. The stool sample is to be kept in a cool shady place but not to be frozen.

7. Stool sample is to be transported to the laboratory without any delay.
Examination of stool is to be done within 30 minutes of stool sample collection
(not within 30 minutes after reception of stool in the lab).

Examination of stool is divided into

a) Naked eye examination (Macroscopic Examination)

b) Chemical examination

c) Microscopic examination

a) Naked eye examination (Macroscopic Examination):

Consistency: whether the stool sample is formed, soft, loose or watery.
Presence of blood and mucus
Presence of round worm, thread worm or tapeworm proglottides
Colour and smell of stool
100

b) Chemical examination

Stool is usually acidic but it will be alkaline in bacillary dysentery (shigellosis).
The reaction is tested by litmus paper.

c) Microscopic examination
Two different types of slide preparations can be made for microscopic
examination of stool. The saline wet mount and the Iodine wet mount. Both can
be made directly from fecal specimens or from the concentrated specimens.

The saline wet mount detects worm eggs, trophozoites and cysts of protozoa,
larvae of intestinal nematodes, RBCs, WBCs, etc .

The Iodine wet mount stains the nuclei and glycogen of cysts of protozoa and thus
differentiates between the cysts of Entamoeba histolytica and Entamoeba coli .

Procedure

Step1: On one end of a clean glass slide, one drop of normal saline is placed and
on the other end, one drop of iodine is placed.

Step 2: Using an application stick, a small portion of stool (the size of a match
stick head) is placed in the saline drop and another portion is placed in the iodine
drop.

Step 3: Both the drops are covered with cover slips.

Step 4: The slide is observed under microscope using 10 and 40 magnifications.

The cysts, trophozoites, ova, cells etc are identified based on their structural
features .

101

Different Common Eggs Seen In Stool Microscopy :

Egg of Ascaris lumbricoides Egg of Ascaris lumbricoides
( Fertilized ) ( Unfertilized )

Egg of Trichuris trichiura Egg of Hook worm

Larva of strongyloides stercorales Cyst of Giaridia intestinalis

Cyst of Entamoeba histolytica

102

Chapter-Seven

VIROLOGY

103

A. Guide lines for laboratory testing and diagnosis of viral diseases at
health care settings of District and sub district (Upazila)Level,
Bangladesh

COMMON FOR ALL THE TESTS

Specimen collection and preparation:
With aseptic precaution collect 3 ml blood by venipuncture.
Allow the blood to clot at room temperature (20 to 25
o
C) for 30 min.
Centrifuge at 1200-1500 rpm for 15 min.
Separate the serum as soon as possible, and keep in another container.

Processing Blood Collected by Venipuncture
The following steps are recommended for processing blood collected by venipuncture

1. Collect blood from the patients vein into a sterile tube. For serum, blood is
collected in a plain test tube (without anticoagulants). For plasma, blood is collected
in a tube with anticoagulants, e.g., EDTA. For safety reasons, the use of an evacuated
blood collection system (e.g., Vacationer tube) is recommended. Let the blood stand
for at least 20-30 minutes, and then remove plasma carefully with a pipette so as not
to get too many red blood cells.

2. Centrifuge the specimen to separate the serum (without EDTA) or plasma (with
EDTA). If blood is collected for serum, allow the blood to stand for at least 20-30
minutes so that a clot will form before centrifuging. In general, the specimen should
be centrifuged at 300-400 g or 1,200 to 1,500 RPM for 10 minutes.

3. After the specimen is centrifuged or has had time to separate, use a clean pipette
(do not pour) to remove an aliquot of 0.5 to 2.0 ml off the top layer. Transfer it to
another sterile labeled tube (plastic, not glass) or cryovial (1.5-2.0 ml with screw cap)
and tighten the cap. The specimen is ready for storage and testing.

Storing Serum and Plasma Collected by Venipuncture

To store serum and plasma, consider the following:
Make sure the cap is tight on the labeled cryovial or plastic tube. (Do not use
glass tubes for storing specimens). Place the cryovials in a cardboard freezer
box with a partitioned insert.
If the specimens are to be transported to the testing laboratory, immediately
pack the box upright in a cooler containing cold packs to produce an ambient
temperature of 4 C within the cooler.
If specimens are held at the collection site for longer than 3 days prior to
shipment to the laboratory where testing will be performed, freeze specimens
at -20 C in a non frost- free freezer. For long-term storage, specimens should
ideally be frozen at -70 C in a non-frost-free freezer.
Limit the number of freeze/thaw cycles to five because multiple thaws will
affect antibody levels and therefore test results.

Labeling Specimens
104
The plastic tube, cryovial, or filter paper containing the specimen must be labeled
with a code with a permanent markers at the time of collection and processing. Make
sure the label is placed on the side of the tube, not on the cap.
For unlinked anonymous testing, label the tube only with a new code unlinked to
personal identifying information.

Registration, labeling and handling:
Each specimen must be accompanied by a request form, which will
contain
Patients name, age and sex
Identification number
Ward, Bed or name of out-patient department
Date and Time of collection
Type of investigation requested
Clinical note about patients illness.

Rejection:
Missing or inadequate identification
Insufficient volume
Unknown time delay
Contamination
Grossly hemolysed or lipaemic samples.

I nternal Quali ty Control:
Always validate quality control.
Determine mean absorbance of negative control/anti-HCV non-reactive control.
Determine mean absorbance of positive control/anti-HCV strongly reactive
control.
Calculate cut-off value.

External Quality Control:
The results of the tests should be reproducible by same lot of reagents in different
standard laboratory.

Data collection and Record keeping:
Personal data of the patient
Proper registration and control in the laboratory
Appropriate evaluation of the laboratory results
Record keeping to
i. calculate requirements for laboratory managements
ii. meet the demands of the patient

Laboratory safety:
i. Always clean the bench after working.
ii. Use protective articles such as lab coats, disposable gloves and protective
glasses.
iii. Do not eat, drink, smoke or apply cosmetics in the assay lab.
iv. Do not pipette solutions by mouth.
v. Handle assay specimens and positive controls carefully.
vi. Carefully handle chromogen, substrate, and blocking or stop solution.
105

Waste management:
i. Needles of disposable syringes should be crushed or cut and kept into a box
before final disposal.
ii. Sharp instruments should be kept immersed in savlon water.
iii. Used disposable syringes and tips should be kept into a separate container.
iv. Autoclave all the glass wares, like pipette etc.
v. Dispose all specimens and materials used to perform the tests. Preferred
method-autoclaving.
vi. Send disposable materials for final disposal.
vii. Liquid wastes, not containing acid may be decontaminated with 1-3% sodium
hypochlorite solution.
viii. Liquid wastes, containing acid may be neutralized with proportional amount
of base first. Then apply 1-3% sodium hypochlorite solution.

Store management:
A. Sample:
No need for refrigeration of serum if tested within 2 hours
Serum should be refrigerated at 2 to 8
o
C if tested within 48 hours.
Serum should be stored at () 20
o
C or below this temperature if not tested within
two days.

B. Reagents:
Store all reagents at 2-8 C
Keep all reagents away from intense light and heat.
Note expiry date of each component and use before expiry.
Work with one lot.
Do not mix specific reagents from different lots.
Avoid repeated freeze-thaw of the reagents
Follow instruction of the manufacturer for storage.

Required Instruments:
Photometer
Shaker/Incubator 37c 2 c
Manual / Automatic equipment for washing well of ELISA plate
Micropipettes with tips (various sizes),
Multichannel pipettes(6-12 channel)
Distilled water
Glass wares
Timer (0-1 hour).
Disposable gloves (various sizes)
Biohazardous waste container
Absorbent tissue
ELISA Reader
Hot air oven
Auto Clave
Water bath
Test tube rack.

106

Principle of ELISA:

Enzyme-linked immunosorbent assay, commonly known as ELISA depends on an
enzyme. An enzyme conjugated with an antibody reacts with a colourless substrate
and generates a coloured reaction product. This substrate is called a chromogenic
substrate. The product can be detected with a photometer.

It is solid state sandwich assay to detect antigen.

1. Plastic well coated with antibody.
2. Antigen to be detected in the serum sample which is added to the well.
-- Incubated for usually 30 minutes to 60 minutes at 37
0
C in a shaker incubator.
3. After incubation the wells are washed for 5 times by wash buffer.
4. Add antibody conjugated with engyme.
-- Incubated for usually 30 minutes to 60 minutes at 37
0
C in a shaker incubator.
5. After incubation wash the well for 5 times by wash buffer.
6. Add substrate (Chromogen) to the well.
-- Incubate for 30 minutes at room temperature away from light.
7. After incubation add blocking reagent / stop solution (acid or alkali) in to the well
to stop the reaction between the enzyme and substrate (Chromogen).
8. Measure the absorbance (optical density or OD value) of the coloured fluid in the
well produced by reaction with enzyme and substrate in the ELISA reader with
appropriate filter.
9. Calculate the cut off value from the absorbance of negative control wells.
10. There should be positive control wells for reference.
11. The samples with the OD value above the cut off value are considered as positive.

To detect antibody the plastic well is coated with antigen and conjugate used in this
test is prepared by enzyme conjugated with antigen. All other steps are similar.

107

ELISA FOR HEPATITIS B VIRUS (HBV) Surface antigen (HBsAg)

Sample: Either serum or plasma. Usually serum is used.
Reagent components for HBsAg detection:
Coated strips
Conjugate/Enzyme tracer
Negative control
Positive control
Tracer diluent
Wash buffer
Chromogen
Substrate
Blocking agent/Stop solution

Preparation
Follow instruction to prepare working solution of concentrated reagents.
Test Procedure for detecting HBsAg:
Bring all reagents to room temperature

Dispense reagents into strip well according to mentioned scheme
Reagents No. of replicates Volume
Negative control 3 100l
Positive control 3 100l
Samples 1 100l

Leave an empty well for the blank.

Incubate at 37 c (with or without shakers) for one hour.

Aspirate the liquid. Fill up the well with wash buffer and aspirate, repeat this for 5
times. Dispense the conjugate into all wells except blank well. Incubate at 37
0
C for
one hour.

Aspirate the liquid. Wash with wash buffer for 5 times.
Dispense chromogens/substrate into all wells.

Incubation in dark at room temperature for 30 minutes.

Dispense blocking/ stop reagents into all well.

Read absorbance values with a photometer ELISA Reader within one hour of the stop
solution earliest the better.
108

I nterpretati on
Presence or absence of HBsAg is determined by comparing absorbance values of
unknown samples to that of the cut-off value. Determination of cutoff value is to be
done according to instructionn.

B) Rapid Tests

Interest in the development of HIV antibody, HCV-antibody, HBsAg tests that
provide same-day results and do not require reagents or equipment, contained in the
kit led to the currently available rapid tests. Most HIV rapid tests contain antigens to
HIV-1 and HIV-2 and detect antibodies to both .

Performing a Rapid Test ( Particle agglutination test)

Rapid tests can be performed with serum, plasma, whole blood, and oral fluids.
Confirmed test results can be obtained in less than 45 minutes after the specimen is
applied to the device or strip. Manufacturers instructions provided with the test kit
should be followed. Most results are easy to read and are indicated by a spot, dot, or
line, depending on the test format.
The following are critical to the success of conducting a rapid test:
Use of test kits that have not expired
Training with the technology being used
Adherence to manufacturers instructions
Correct interpretation of results by person reading results
The person taking the reading should have normal eye sight with or without glass
and must not be colour blind.

109

Chapter- Eight

Hematology

110

Collection of blood

Blood must be collected with care and adequate safety precautions to ensure test
results are reliable, contamination of the sample is avoided and infection from blood
transmissible pathogens are prevented. Protective gloves should be worn during
collection and handling blood samples. Lancets, needles, and syringes must be
sterile, and dry, and blood collecting materials must be discarded safely to avoid
injury from needles and lancets.

Capillary blood
Capillary blood is mainly used when the patient is an infant or young child and the
volume of blood required if small, e.g. to measure haemoglobin, perform a WBC
count, and to make thick and thin blood films. In district laboratories, capillary blood
is also used to monitor anaemia during pregnancy and post-operatively. Thick blood
films for malaria parasites are best made from capillary blood (anticoagulated blood
is more easily washed from slided furing staining).

Disadvantages in using capillary blood for blood tests include:
Capillary blood can be used for only a few tests.
Greater possibility of sampling errors particularly when the blood is not free-
flowing, e.g. dilution of the sample with tissue juice can occur if the puncture
area is squeezed excessively.
Difficulty in obtaining sufficient blood particularly when it is required for
more than one test. Rapid clotting of blood in a pipette is common,
particularly in tropical temperatures.
Tests cannot be repeated immediately of further tests performed when results
are unexpected of seriously abnormal. A patient may have also received
treatment following collection of the capillary sample, e.g. an antimalarial
drug which will make it difficult to find parasites at a late stage.

Capillary blood can be collected from:
- The ring finger of a child or adult as shown in Fig.1. Do not stick the thumb
of index finger as these are the most sensitive.

- The heel of an infant up to one year old as shown in Fig.2. Care must be taken
not to damage the heel by sticking it too near the edge or by holding if too
forcibly.

111
Techni que for collecti ng capillary blood
Make sure the puncture area is warm to allow the blood to flow freely. On cold days
soak the hand or foot of an infant in warm water prior to collecting a sample.

1 Clean the puncture area with 70% ethanol. Allow the area to dry.
2 Using a sterile pricker or lancet, make a rapid puncture, sufficiently deep to
allow the free flow of blood.
3 Wipe away the first drop of blood with a dry piece of cotton wool and use the
next drops for the tests. Do not squeeze too hard because this will result in an
unreliable test result.
4 When sufficient blood has been collected, press a piece of dry cotton wool over
the puncture area until bleeding stops.

Techniques for collecti ng venous blood
Laboratory staff must not collect venous blood unless they have been adequately
trained in the procedure. Newly qualified staff must be supervised until they have
gained sufficient experience. When venous blood is required from infants, this
should be collected by a medical officer. Do not collect blood for haematological tests
from intravenous lines.

1. Select a sterile, dry, preferably plastic syringe of the capacity required, e.g. 2.5
ml, or 10 ml. Attached to it 19 or 20 SWG needle ( disposable one). If the
patient is a child or adult with small veins, use a 23 GWG needle.
Note: When not using a disposable syringe or needle for any blockage, directing the
syringe and needle safely away from the patient. Ensure all air is expelled from the
syringe and always use a disposable needle and syringe.

Evacuated tube collection systems: These disposable blood collecting
containers are available from several manufacturers but they are more expensive to
use for collecting venous blood that a syringe or needle. The containers has a vacuum
which is used to draw the blood into the container. One end of the needle is situated
in the patients vein and the other end through the cap of the container. Evacuated
collection systems minimize contact with blood, help to ensure the correct amount of
blood is added to anticoagulant, and simplify multiple sample collection.

2. Apply a soft tubing tourniquet or
velcro fastening arm band to the
upper arm of the patient (see
Fig.3) to enable the veins to be
seen and felt. Do not apply the
tourniquet too tightly for longer
than 2 minutes. Ask the patient to
make a tight fist which will make
the veins more prominent.

3. Using the index finger, feel for a
suitable vein, selecting a
sufficiently large straight vein
that does not roll and with a
direction that can be felt.

If a vein cannot be felt, apply a
112
pressure cuff above the elbow and raise the pressure to 80 mm (deflate the
cuff once the needle is in the vein).

4. Clean the puncture site with 70% ethanol and allow to dry. Do not re-touch
the cleansed area.
5. With the thumb of the left hand holding down the skin below the puncture
site, make the venepuncture with the bevel of the needle directed upward in
the line of the vein. Steadily withdrawn the plunger of the syringe at the speed
it is taking the vein to fill*. Avoid moving the needle in the vein.

*If the plunger is withdrawn too quickly this can cause haemolysis of the
blood and the collapse of a small vein.

6. When sufficient blood has been collected, release the tourniquet and instruct
the patient to open his or her fist. Remove the needle and immediately press
on the puncture site with a piece of dry cotton wool. Remove the tourniquet
completely. Instruct the patient to continue pressing on the puncture site until
the bleeding has stopped.
7. Remove the needle from the syringe and carefully fill the container(s) with the
required volume of blood. Discard the needle safely. Do not attempt to re-
sheath it because this can result in a needle-stick injury.

Important: Do not fill a container with the needle attached to the syringe.
Forcing the blood through the needle can cause haemolysis.

8. Mix immediately the blood in an EDTA or citrate anticoagulated container.
When required, make a thick blood film from the blood samples at once,
before collecting blood from another patient.
9. Check that bleeding from the venepuncture site has stopped. Cover the area
with a small dressing.

Avoiding haematoma when collecting venous blood

Bleeding from a vein into the surrounding tissue (haematoma) can cause
unnecessary distress to a patient and result in subsequent bruising. Haematoma can
be avoided by ensuring an appropriate vein is selected and the needle is well
positioned in it and not accidentally pulled out of the vein when withdrawing the
plunger of the syringe. When removing the needle, always release the tourniquet first
and apply pressure immediately to the puncture site, maintaining it until the
bleeding has stopped completely (always check).

White cell count:
Principal: The red cells to be lysed and a coloring agent can be added to aid
recognition of leukocyte.
Blood sample: EDTA anticoagulated blood or capillary blood can be used for
counting white cell.

Equipment:
Counting chamber: Improved Neubauer counting chamber.
Counting chamber cover glasses: Special optically cover glasses of defined
thickness (designated for use with hemocytometer) are required.
Pipettes
113
Reagent:
W.B.C. diluting fluid: 2% (20 ml/l)
Acetic acid colored pale violet
with gentian violet.

Composition of reagents:
White cell diluting fluid
Glacial acetic acid 2 ml
1% Methylene Blue 1-2 drops
Distilled water 98 ml

Test method:
1. Measure 0.38 ml of diluting fluid and dispense it into a small container or
tube.
2. Add 20 l (0.02) ml, 20cmm) of well-mixed EDTA anticoagulated venous
blood or free-flowing capillary blood and mix.
3. Assemble the counting chamber:
Make sure the central grid areas of the chamber and the special
haemocytometer cover glass are completely clean and dry.
Slide the cover glass into position over the grid areas and press down
on each side until rainbow colors (Newtons rings) are seen. Prior
moistening of the chamber surface on each side of the grid areas will
help the cover glass to adhere to the chamber.
4. Re-mix the diluted blood sample. Using a capillary, Pasteur pipette, or plastic
bulb pastette held at an angle of about 45
0
, fill one of the grids of the chamber
with the sample, taking care not to overfill the area.
Important: The chamber must be refilled if the sample overfills into the
channel beyond the grid or an air bubble forms in the grid area.
5. Leave the chamber undisturbed for 2 minutes to allow time for the white cells
to settle.
Note: To prevent drying of the fluid, place the chamber in a petri dish or
plastic container on dampened tissue or blotting paper and cover with a lid.
6. Dry the underside of the chamber and place it on the microscope stage.
Using the 10 X objective with the condenser iris closed sufficiently to give
good contrast, focus the rulings of the chamber and white cells. Focus the cells
until they appear as small black dots.
7. Count the cells in the four large corner squares of the chamber marked W1,
W2, W3, W4 in Fig.4 (total area of 4 mm
2
). Include in the count the cells
lying on the lines of two sides of each large square as shown in Fig. 5.
114

8. Report the number of white cells per litre of blood using the following simple
calculation:
No. of cells counted
Count (/l) = ----------------------------- X dilution X 10
6

Volume counted ( l)
Thus, if N cells are counted in 0. l,
then the leucocyte count per litre
= N x 10 x (dilution) x 10
6

= N x 200 x 10
6
/1
(= N x 200 per l).
9. After performing the count, before the sample dries, dismantle the chamber,
wash and dry it. Store it with the cover glass in a safe place.

Interpretation
Normal range
Adults 4-11 X 10
9
/l
Infants at birth 10-24 X 10
9
/l
Infants 1 year 6-18 X 10
9
/l
Children < 10 yrs 5-14 X 10
9
/l

Quality control of WBC counts
Whenever possible perform WBC count in duplicate. The difference
between the two counts (as a percentage of the mean) should not be more
than 20%.

How to calculate the % difference between two counts
1. Record the number of cells counted in Count 1 and Count 2.
2. Calculate:
- the difference in the number of cells counted between the two counts.
- The mean of the two counts.
3. Calculate the difference of the two counts as a percentage of the mean.
Example
Cells counted in Count 1 = 88, Count 2 = 76
- Difference in number of cells between the two counts 88-76 = 12

- Mean of Count 1 and Count 2 = = 82

- difference of the two counts as a % of the mean = 14.6%

Note: When the difference between the two counts is more than 20%, repeat the
counts.
Check that the diluting fluid is free from particles which could be mistaken
for WBCs. To do this, fill a counting chamber with a sample of the diluting
fluid and examine the grid areas microscopically using the 10X objective
with greatly reduced condenser iris. If the fluid contains particles
resembling WBCs, filter it and recheck of discard the fluid and prepare
fresh diluting fluid.
When examining the blood film, check that there is no major discrepancy
between the total white cell count and white cells seen in the blood film.
88+76
2
12X100
82
115

External quality assessment
Whenever possible the Regional or Central Haematology Laboratory should send
control blood samples to district laboratories for abalysis.

Sources of error in manual WBC counts
Incorrect measurement of blood due to poor techniques (see Text Method) or
using a wet of chipped pipette.
When using anticoagulated blood, not mixing the blood sufficiently of not
checking the sample for clots.
Inadequate mixing of blood with diluting fluid.
Not checking whenever the chamber and cover gloves are completely clean.
Not using a haemocytometer cover glass.
Over-filling a counting chamber of counting cells when the sample contains
air-bubbles.
Not allowing sufficient time (2 minutes) for the cell to settle in the chamber.
Using too intense a light source of not reducing the iris diaphragm sufficiently
to give good contrast (poor focusing and difficulty in seeing clearly the cells
and rulings are common when using non-metallized haemocytometers, see
previous text).
Not completing counting of the cells before the sample begins to dry in the
chamber.
Counting too few cells (see previous text). Precision with the number of cells
counted.
Not correcting a count when the sample contains many nucleated RBCs (see
previous text).

Platelet count

Value of test: A platelet count may be requested to investigate abnormal skin and
mucosal bleeding which can occur when the platelet count is very low (usually below
20 X 10
9
/1). Platelet counts are also performed when patients are being treated with
cytotoxic drugs or other drugs which may cause thrombocytopenia.

Principal of test
Blood is diluted 1 in 20in a filtered solution of ammonium oxalate reagent which
lyses the red ells. Platelets are counted microscopically using an Improvement
Neubauer ruled counting chamber and the number of platelets per litre of blood
calculated.

Blood sample
Whenever possible use EDTA anticoagulated venous blood. The collection of venous
blood is described in subunit fig.3. Capillary blood should not be used because there
is a tendency for the platelets to clump as the blood is being collected. When the
patient is a young child and venous blood is difficult to obtain, it is possible to use
capillary blood providing it is free-flowing and the skin is first smeared thinly with
vaseline which helps to prevent the platelets from clumping.

Equipment
An Improved Neubauer ruled Bright-line counting chamber and other equipment as
described previously for WBC counting are required for counting platelets.
116

Platelet haemocytometers

Thin glass chambers for counting platelets by phase contrast microscopy are
available. Such chambers are expensive and break easily. They are not essential for
counting platelets.

Reagent
Ammonium oxalate 10g/1
(1% w/v) diluting fluid.

Composition of reagents:
Platelet fluid
Ammonium oxalate 1 gm
Glass distilled or deionized water 100 ml
Filter and centrifuge at 1200 1500 g for 15 minutes prior to use.

Test method
Perform a platelet count within 2 hours of collecting the blood (venous sample).
1. Measure 0.38 ml of filtered ammonium oxalate diluting fluid and dispense it
into a small container or tube.
2. Add 20 l (0.02 ml, 20 cmm) of well-mixed anticoagulated venous blood and
mix.
3. Assemble the counting chamber and fill it with well-mixed sample as
described previously in steps 3 and 4 of the method for counting white cells.
4. Leave the chamber undisturbed for 20 minutes. To prevent drying of the fluid,
place the chamber in a petri dish or plastic container on dampened tissue of
blotting paper and cover with a lid.
5. Dry the underside of the chamber and place it on the microscope stage. Using
the 10 X objective, focus the rulings of the grid and bring the central square of
the chamber into view. Change to the 40 X objective and focus the small
platelets. They will be seen as small bright fragments (refractile).

Note: If available, use phase contrast microscopes.

6. Count the platelets in the small squares marked P as shown in Fig. 4,5
7. Report the number of platelets in 1 liter of blood. This is the actual number of
platelets counted X 10
9
.

Example
If 150 platelets are counted, the platelet count is 150 X 10
9
/1.

Platelet calculation details

Platelet count (per liter) =

where * = 1 in 20 dilution of blood
1
= 1.2 mm
1

Cells counted X 20* X 10*
0.2
1
X 0.1
1

117
Quality control
In district laboratories the most feasible quality control of platelet counts is to follow
the test procedure exactly, perform duplicate counts, and examine the platelet fluid
microscopically (at the time of performing the count) to ensure it does not contain
refractile particles resembling platelets. The mean of the two counts should be
calculated a described previously for WBC counts.

Sources of error in counting platelets
Sources of errors when counting platelets are similar to those mentioned previously
for WBC counts. Special care must be taken when counting platelets:
To check there are no clots in the blood sample.
To ensure the blood is well mixed with the diluting fluid.
Not to mistake debris from haemolyzed red cells or particles in the diluting
fluid for platelets.
To ensure the platelets are evenly distributed and not in small clumps (if
clumps are present, obtain a new blood sample).
Not to use too intense an illumination.

Note: In some disorders, large platelets may be present.

Interpretation of platelets counts
In health there are about 150-400 X 10
9
platelets/litre of blood. Platelet counts
from capillary blood are usually lower than from venous blood and are not as
reproducible. Platelet counts are lower in Africans.

Blood films

Value of blood films: Examination of thin blood films is important in the
investigation and management of anaemia, infections, and other conditions
which produce changes in the appearance of blood cells and differential white cell
count. A blood film report can provide rapidly and at low cost, useful information
about a patients condition.

Technique of making a thin blood film
1. Make a blood spreader from a slide which has ground glass polished sides as
follows:
*Only good quality slides will have ground glass polished sides. Such sides are
not necessary for most routine work but they are essential to make blood
spreaders.
Examine each end of the slide and select the end which is completely
smooth, i.e. no chips in the glass. If one end of the slide is frosted, use
the non-frosted end (ensure it is smooth).
Using a glass marker,
etch across a corner of
the slide as shown in
Fig. 6.
Holding the slide
between a piece of
cloth, break off the
corner and discard
safely the broken off
piece of glass.

118

2. Place a drop of blood on the end of a clean
dry slide as shown in Fig. 7. Avoid
making the drop too large (if too large,
use a drop from the excess blood to make
the film).
Slides on which to make blood films
Whenever possible use double frosted end
slides because these provide an area for
writing clearly the patients name and
number. It is essential to ensure slides are
washed free from traces of detergent and
the surface of the slide is completely clean
and not greasy.

3. Using a clean smooth edged spreader,
draw the spreader back to touch the drop of blood and allow the blood to
extend along the edge of the spreader. Holding the spreader at an angle of
about 30
0
, spread the drop of blood to make a film about 40-50 mm in length
(two thirds of the slide) as shown in Fig. 7.
Note: When the blood is from an anaemic patient, increase the angle of
spreading and spread the blood more quickly. When the blood is thick and
viscous, reduce the angle of spreading and spread the blood more slowly.
4. Wipe clean the end of the spreader.
5. Immediately air dry the film by waving the slide back and forth. Protect the
dried film from dust and insects.
Note: When not using a frosted ended slide, write the patients name and
number on the dried blood at the top or along the side of the film using a lead
pencil.
6. When completely dry and within a few minutes of making the blood film, fix it
in absolute methanol.
7.

Feature of a well made film
Not too thick, nor too long
Free from lines and holes
Has a smooth tail

Staining

Leishmans method
1. Support film in a horizontal position on two parallel glass or metal rods 5 cm
apart, over a sink.
2. Flood the slide with Leishmans stain and leave for two minutes to fix the
film. In hot weather, evaporation will be rapid and the stain will precipitate
on the film; this can be avoided by adding more stain.
3. Add double the volume of buffered distilled water, making sure that none of
the diluted stain runs off. The stain and water can be mixed by blowing
gently over the surface, or by using a Pasteur pipette with a rubber teat,
making sure that the surface of the film is not touched. A metallic scum will
appear on the surface.
4. Leave for eight minutes.
119
5. Wash the stain off with buffered water until no blue washes off; give a final
rinse with running tap water. Immediately clean the back of the slide with a
tissue and stand upright to dry; if it is very dirty, adding a drop of methanol
to the tissue will quickly remove any stain deposit. DO NOT BLOT THEM
DRY.
6. When completely dry, the thinner end of the film should be covered with a
22 X 22 mm coverglass, using a mountant.

Tap water: If the tap water is highly acidic, resulting in too pink a blood film
or highly alka;ine, resulting in too blue a blood film, try using boiled cooled
water or filtered rain water. If neither of these are suitable, wash the film
with pH 6.8 buffered water.

Quality control

When a new batch of stain is prepared, decide the best staining time to use,
e.g. stain films made from the same blood at different times, e.g. 5, 7, 10, 12, 15
minutes. Compare the results with a stained control blood film (protect the
control film from light).

Check the pH of newly prepared buffered water and re-check it at weekly
intervals. The pH of the buffered water used to dilute the stain must be
correct. It is mainly responsible for the staining reactions

Maintain consistency in the staining procedure by following exactly a standard
operating procedure (SOP). If the quality of staining changes, always report
this to the person in charge of the laboratory and ensure the fault is rectified.
The staining procedure should be checked at the beginning of each week.

External quality assessment: Whenever possible the Regional or Central
Haematology Reference Laboratory should send fixed blood films to district
laboratories fro staining and reporting (films and report to be returned to the
Reference laboratory), and control stained blood films for reporting and
comparing with films being stained in the district laboratory (these control
stained films to be retained by the district laboratory).

Staining results
Red cells..Pink-red
Nucleus of cellsPurple-violet

Cytoplasm
Neutrophils, eosinophilsPale pink
Large lymphocytes.. Clear blue
Small lymphocytesDarker clear blue
MonocytesGrey-blue

Granules
Eosinophils.Orange-red
Neutrophils.Mauve-purple
Toxic granules.Dark violet
Basophils.Dark blue-violet

120
Platelets..Purple-blue

Inclusions
Malaria pigment (in monocytes).Brown-black
Howell-Jolly bodyPurple-violet
Auer body (in myeloblast)...Purple-red

Differential white blood cell count
A differential white cell count provides information on the different white cells
present in the circulating blood, i.e. neutrophils, lymphocytes, monocytes,
eosinophils, basophils (rarely seen). Providing the total WBC count is known, the
absolute number of each white cell type, i.e. number of each cell per litre of blood,
can be calculated and an assessment made of whether the number of a particular
cell type is increased or decreased (compared with the accepted reference range).

Method
As previously discussed, it is only possible to report blood films reliably providing
the thin blood film is well made and correctly stained. Allow the stained film to dry
completely before examining it.
1. Place a drop of immersion oil on the lower third of the blood film and
cover with a clean cover glass.
2. Examine the film microscopically. Focus the cells using the 10X objective
with the condenser iris closed
sufficiently to see the cells clearly.
Check the staining and distribution
of cells.
3. Move to a part of the film where the
red cells are just beginning to
overlap & bring the 40X objective
into place. Open the iris diaphragm
more.
4. Systematically examine the blood
film & count the different white
cells seen in each field, preferably
using an automatic differential cell
counter, or if this is not available,
record the count in chart form as
shown in fig 8
5. Calculate the absolute number of
each white cell type by multiplying
the number of each cell counted
(expressed as decimal fraction) by
the total WBC count.

Example
If: Percentage of neutrophils counted = 80% i.e. 0.80 when expressed as a
decimal fraction.
If total WBC count = 8.6 X 10
9
/l
Absolute neutrophil count is 0.80 X 8.6 = 6.88 X 10
9
/L

121
Reporting blood films
Reporting Romanowosky stained thin blood films includes:

White cell morphology
Left shift of neutrophils and toxic granulation
Hypersegmented neutrophils
Reactive (atypical) lymphocytes
Malaria pigment in white cells.

Red cell morphology
Variation in red cell staining, e.g. hypochromic cells, polychromatic cells,
dimorphic blood picture. If normal, refer to the red cells as normochromic.
Variation in red cell size, e.g. microcytes, macrocytes, spherocytes,
significant anisocytosis. If normal in size, refer to the red cells as
normocytic.
Cells of abnormal shape or form, including pencil cells, sickle cells, target
cells, fragmented cells (schistocytes), tear drop cells, bite cells, cells
showing rouleaux, significant poikilocytosis.
Cells with inclusions, e.g. nucleated red cells, megaloblasts, cells containing
Howell-Jolly bodies, cells showing basophilic stippling, cells containing
malaria parasites, cells containing Bartonella organisms.
Nucleated red cells, target cells, sickle cells, megaloblasts, malaria parasites,
Howell-Jolly bodies, pencil cells & other abnormal red cells can be reported
as many, moderate numbers, few, occasional.

Platelet morphology
Platelets size, variation & large platelet.

Making and staining thick blood films
Thick blood films are widely used in the diagnosis of malaria. A relatively large
volume of blood may be scrutinized in a short time and parasites seen even if present
in very small numbers. The Giemsa method is more suitable for staining large
number of films.

Making thick blood films
Make a thick film by placing a small drop of blood in the centre of a slide and spread
it out with a corner of another slide to cover an area about four times its original
area. The correct thickness for a satisfactory film will have been achieved if, with the
slide placed on a piece of newspaper, small print is just visible.
Allow the film to dry thoroughly for at least 30 min at 37
0
C before attempting to stain
it. Alternatively, leave the slide on the top of a microscope lamp, where the
temperature is 50
0
-60
0
C, for c 7 min. Absolutely fresh films, although apparently dry,
often wash off in the stain.

Giemsas stain
Dry the films thoroughly as above, immerse the slides for 20-30 min in a staining jar
containing Giemsas stain freshly diluted with 15-20 volumes of buffered water.
Gently wash the films with buffered water. Stand the slides upright to dry. Do not
blot.
Sometimes the films are overlaid by a residue of stain or spoilt by the envelops of the
lysed cells being visible. These defects can be minimized either by soaking the stained
and dried films for a few minutes in 9 g/l NaCl or by staining the films in the first
122
instance in Giemsas stain which has been diluted in buffered 9 g/l NaCl, pH 6.8,
instead of water.

ABO and Rh-D Grouping

ABO and Rh-D grouping must be performed by an approved technique with
appropriate controls. Before use, all new batches of grouping reagents should be
checked for reliability by the techniques used in the laboratory. Grouping reagents
should be stored according to the manufacturers instructions.

ABO Grouping:

Correct interpretation of the patients ABO grouping requires confirmation,
whenever possible, by tests on the patients serum or plasma(except for newborn
infants upto 4 months of age in whom naturally occurring ant-A and ant-B are
normally absent). Ideally both cell and serum(or plasma) grouping should be carried
out.

Reagents:
Commercially available anti-A, anti-B, anti-A,B reagents are traditionally used for
cell grouping tests. The anti-A,B reagent (group O serum) acted as an additional
check on red cells which agglutinated by anti-A or anti-B and detected weaker A or B
antigens.

Methods:
ABO grouping may be carried out by tube or slide methods. In an emergency ABO
grouping may be carried out rapidly on slides or tiles and the method is satisfactory if
potent grouping are used.

( I ). Cell grouping:
To perform the cell grouping, patients red cells(one volume e.g one drop of 50% red
cell suspension in normal saline ) are added to one volume (one drop) of specific
anti-sera: anti-A, anti-B, anti-A,B at room temperature. Mix the content in the slide
with a cleanpiece of applicator stick for each (separate stick use for each). Tilting
gently the slide or tile from side to side and look for agglutination. Careful
examination for any agglutination. In case of confusion, microscopic examination to
confirm agglutination is required. Agglutination appears as clumped RBCS
Agglutination indicate specific antigen present on the red cells and no agglutination
means specific antigen is absent on the RBCs.

Note: Do not delay reading the results beyond 2 minutes because false agglutination
may result from drying of mixture.

Red cells plus
ABO group Anti-A Anti-B Anti-A,B
A ++++ - ++++
B - ++++ ++++
AB ++++ ++++ ++++
O - - -

123
( II ). Serum grouping(Reverse grouping):

Patients serum is added to groupA, groupB red cells. The presence of antiA and or
antiB in the patients serum will give a specific pattern of agglutination. Mixed and
rotate the test solution for 3 to 5 minutes. Examine carefully for agglutination.
Agglutination indicate specific antibody is present in the patients serum.no
agglutination means absent of specific anti-body in the patients serum.

Patients serum plus
ABO group A - RBC B- RBC
A - ++++
B ++++ -
AB - -
O ++++ ++++

Control:
Positive and negative controls must be included in every test. Anti-A reagent should
be tested against group A(positive control) and B(negative control) cells. The anti-A
reagent against group-B(positive control) and group-A(negative control) cells.

Preparation of red cell suspension:
In tube method 3 to 5 % and in slide method 50% cell suspension are used.
1. 0.5 ml EDTA blood or red cells from clotted blood are taken into 5ml
physiological saline (0.9% normal saline).
2. Centrifuge at about 1000g for 2 to 3 minutes
3. Discard the supernatant fluid and repeat the procedure with adding 5 ml
normal saline.
4. Make 3 to 5% red cells suspension by mixing 1 drop of red cells(sedimented
cells) in 20 to 25 drops of normal saline.(hold the plastic pipette vertically).

Rh-D Grouping
This is usually performed at the same time as ABO grouping, each sample should be
tested in duplicate, at least for first time patients, as there is no counterpart of
reverse grouping as in ABO grouping.

Reagents: the availability of high titre monoclonal IgM anti-D reagents has made it
possible to use the same techniques as for ABO grouping. This reagents work well
equally at room temperature and at 37C. Non-potentiated monoclonal reagents
should be used in preference to potentiated reagents which do not cause
agglutination in saline unless potentiators( e.g;albumin, polyethylene glycol) have
been added to the diluent.
All anti-D grouping reagents should be checked by the method used in the laboratory
for specificity with positive (OR'r or OR
1
R
2
) and negative(Orr or Or'r) controls.

124

H HA AE EM MO OG GL LO OB BI IN N D DE ET TE ER RM MI IN NA AT TI IO ON N B BY Y T TH HE E C CY YA AN NM ME ET TH HA AE EM MO OG GL LO OB BI IN N
M ME ET TH HO OD D

Measurement of haemoglobin concentration in a whole blood sample is a basic
screen for anaemia or for polycythaemia.

P Pr ri in nc ci ip pl le e

Blood is diluted is a solution containing potassium cyanide and potassium
ferricyanide (Darbkins solution). Haemoglobin, methaemoglobin and carboxy-
haemoglobin are all converted to cyanmethaemoglobin. The absorbance of the
solution is measured against a reference standard solution of cyanmethaemoglobin.
T Te es st t S Sa am mp pl le e

Venous or capillary blood is collected into dry EDTA. Alternatively, venous blood or
free flowing capillary blood may be measured and added to the diluting fluid without
anticoagulation.

S St ta an nd da ar rd d: :

Standard solutions of cyanmethaemoglobin are widely available commercially. They
have a shelf life of 5 years and contain 700 150 mg of cyanmethaemoglobin per
litre. Each ampoule is labeled to the nearest mg of cyanmethaemoglobin. Accurate
calibration of a blood sample which can serve as a standard can be prepared by
making a dilution of blood in Darbkins solution and measuring the extinction at 540
nm. One millimole of cyanmethaemoglobin (M.Wt.64,458) has an extinction
coefficient of 44.0.
R Re ea ag ge en nt ts s: :

Darbkins solution

It is usual to prepare this solution by diluting an ampoule as directed by the supplier.
Alternatively the solution may be prepared as below:
Composition of the Darbkins Solution (P
H
7.0 7.4)

Potassium ferricyanide 200 mg.

Potassium Cyanide 50 mg.

Potassium dihydrogen orthophosphate 140 mg.

Nonidet p40 (Shell Chemical Co) 1 ml.

Distilled water to 1 Littre

125
Check P
H
and store solution in a dark bottle at room temperature. It must not be
allowed to freeze. Its absorbance should be zero at 540 nm against a water blank.

M Me et t h ho od d: :

1. Add 20 l of blood to 4 ml of cyanmathaemoglobin reagent.

2. Mix well and leave to stand at room temperature for at least three minutes.

3. Read the absorbance on a photoelectric colorimeter at 540 nm against a reagent
blank.

4. Read the absorbance of the contents of an ample of cyanmathaemoglobin
standard at 540 nm against a reagent blank.

C Ca al l c cu ul l a at t i i o on n: :

Absorbance of test sample Concentration standard X dilution factor
X
Absorbance of standard 100

= Haemoglobin (g/l)
N No ot t e es s o on n t t e ec ch hn ni i q qu ue e: :

The blood sample must be properly mixed before sampling and allowed to warm.

The standard solution of cyanmathaemoglobin should be at room temperature
when its absorbance is measured.

When the haemoglobin level of a number of blood samples is to be measured it is
convenient to prepare a table relating colorimeter readings to haemoglobin
concentration.

When blood samples contain more than 40 x 10
9
white cells/litre the diluted
samples should be centrifuged at 3000 rpm for 10 minutes before reading to
avoid a false reading due to turbidity imparted by the white cells.

Care should be exercised when handling Potassium cyanide.

Prolonged pressure on the arm during vene-puncture must be avoided as this
can increase the haemoglobin concentration. The haemoglobin concentration is
lower when the specimen is collected from the patient in the supine as compared
to the erect posture. The variation may be as great as 15 gm/L.

C Co om mm me en nt t : :

The cyanmathaemoglobin method is the reference method for haemoglobin
estimation because all haemoglobin compounds except sulphaemoglobin are
estimated. Highly reliable and stable standards are available.

Interpretation:

126
Normal range (Sea level)

Adult males 155 25 g/l

Adult females 140 25 g/l

New born infants 165 30 g/l

Children at 3 months 110 15 g/l

Children at 1yr 120 10 g/l

Children at 3-6 yrs 130 10 g/l.

E Er ry yt th hr ro oc cy yt te e s se ed di im me en nt ta at ti io on n r ra at te e ( (W We es st te er rg gr re en n t te ec ch hn ni iq qu ue e) )

V Va al l u ue e o of f t t e es st t : :
The erythrocyte sedimentation rate (ESR) is a non-specific test. It is
raised in a wide range of infectious, inflammatory, degenerative, and
malignant conditions associated with changes in plasma proteins,
particularly increases in fibrinogen, immunoglobulins, and C-reactive
protein. The ESR is also affected by many other factors including
anaemia, pregnancy, haemoglobinopathies, haemoconcentration and
treatment with anti-inflammatory drugs.

Moderately raised sedimentation rates can sometimes be found
in healthy people, particularly those living in tropical countries and a
normal ESR cannot exclude disease. In many tropical countries ESR
measurements have been discontinued because they add little to
diagnosing disease, assessing its progress and monitoring response
to treatment. When performed, test results must be interpreted in
conjunction with clinical findings and the results of other laboratory
tests.

P Pr ri i n nc ci i p pl l e e o of f t t e es st t : :

When citrated blood in a vertically positioned Westergren pipette is left
undisturbed, red cells aggregate, stack together to form rouleaux, and
sediment through the plasma. The ESR is the rate at which this
sedimentation occurs in 1 hour as indicated by the length of the
column of clear plasma above the red cells, measured in mm.
Fibrinogen, immunoglobulins, and C-reactive protein (CRP) increase
red cell aggregation. Sedimentation is increased when the ratio of red
cells to plasma is altered, e.g. in anaemia. Sedimentation is reduced
when the red cells are abnormally shaped, e.g. sickle cells. High
temperatures (over 25C) increase sedimentation.

E Eq qu ui i p pm me en nt t

Westergren ESR pipette

127
Glass Westergren pipettes or when available, disposable plastic
Westergren pipettes can be used. Westergren pipettes measures
300mm in length (plastic pipettes are often shorter) and are graduated
from 0-200mm. The diameter should not be less than 2.55mm. When
reusing pipettes, care must be taken to ensure the pipettes are
completely clean and dry.

Westergren ESR stand with levelling device*

*Most Westergren stands are designed for use with disposable closed tube
ESR systems to minimize biohazard risks associated with handling blood
Unfortunately such systems are expensive.

R Re eu us sa ab bl l e e E ES SR R s sy ys st t e em ms s

When using a reusable ESR system, an ESR stand must be used
which enables blood to be aspirated safely, not mouth-pipetted. A
simple reusable system suitable for use in a district laboratory is that
blood is aspirated using 2 ml. plastic syringes. A plastic connector
attaches the syringe to the pipette.

Timer capable of timing accurately 1 hour, e.g. mechanical 60
minute timer.

R Re ea ag ge en nt t : :
Tri-Sodium citrate, 32 g/l (3.2 %
w
/
v
) anticoagulant. Store the reagent at
4-8C. Renew if it becomes cloudy.

S Sp pe ec ci i m me en n: :
Either venous blood collected directly into sodium citrate and tested
within 2 hours, or EDTA anticoagulated blood diluted in sodium citrate
can be used. If EDTA blood is used and kept refrigerated at 4-8C,
citrate dilution of the blood and testing can be delayed for up to 6
hours.

T Te es st t M Me et t h ho od d: :

1 Pipette 0.4 ml of sodium citrate anticoagulant into a small container.
Note: If using previously prepared sodium citrate containers, check that there
has been no leakage of the anticoagulant.

2 Add 1.6 ml of venous blood or EDTA anticoagulated blood and mix
well. Remove the cap of the container and place the sample in the ESR
stand (make a note of the number in the patients notes). Insert a
Westergren pipette and ensure it is positioned vertically.

3 Using a safe suction method, draw the blood to the 0 mark of the
Westergren pipette, avoiding air bubbles.

128
4 Check that the ESR stand is level by making sure that the bubble in the
spirit level is central. If required, adjust the screws on the bottom of the
stand. Re-check that the pipette is vertical.

5 Set the timer for 1 hour. Ensure the ESR stand and pipette will not be
exposed to direct sunlight.

6 After exactly 1 hour, read the level at which the plasma meets the red
cells in mm*.

*Some Westergren pipettes are marked 0. 10. 20. etc. and some are
marked 0.1 (equivalent to 10 mm). 2 (equivalent to 20 mm.) etc.
therefore read the graduations carefully.

7 After reading the ESR, return the blood to its container, remove
carefully the pipette and soak it in sodium hypochlorite 2,500 ppm av Cl
(0.25%) disinfectant.

Dispose of the blood safely and decontaminate the container before
washing it and also the ESR stand as described in subunit 3.4 in Part 1
of the book.

Q Qu ua al li it ty y c co on nt tr ro ol l: :

The most practical way of controlling ESR tests is to follow the test method exactly.
S So ou ur rc ce es s o of f e er rr ro or r: :

Using the wrong volume of blood to anticoagulant.

Blood not sufficiently mixed with anticoagulant.

Clots in the blood. Even the smallest fibrin clot in the sample will invalidate the
test result.

Air bubbles at the top of the column.

Testing blood samples at the hottest time of the day, or leaving tests in direct
sunlight. Temperatures over 25C increase sedimentation.

Using a pipette, which is, not clean or not dry.

Pipette not positioned vertically. Even slight variations from the upright increase
sedimentation.

Not checking whether the ESR stand is level on the bench.

Placing an ESR stand on the same bench as a centrifuge where vibration will
interfere with sedimentation.

Measuring the ESR when a patient is dehydrated.

129

I In nt te er rp pr re et ta at ti io on n o of f E ES SR R t te es st t r re es su ul lt ts s: :

Reference range:

MenUp to 10 mm/hour*
WomenUp to 15 mm/hour*
ElderlyUp to 20 mm/hour*

*These figures should be checked locally. Higher values may need to be applied in tropical
countries.

Note: Higher values are obtained during menstruation, pregnancy, puerperium.

Because of the many factors that affect red cell sedimentation, slightly and
moderately raised values may not be significant, particularly in tropical countries.
Markedly raised values should be investigated.

C Ca au us se es s o of f a a s si i g gn ni i f f i i c ca an nt t l l y y r ra ai i s se ed d E ES SR R: :

Anaemia due to any cause.

Acute and chronic inflammatory conditions and infections including:

q HIV disease
q Tuberculosis*
q Acute viral hepatitis
q Arthritis
q Bacterial endocarditis
q Pelvic inflammatory disease
q Ruptured ectopic pregnancy
q Systemic lupus erythematosus

*When the ESR is used to monitor progress during treatment. ESR values do not fall when a
patient with tuberculosis is also co-infected with HIV.

African trypanosomiasis (rises rapidly)
Visceral leishmaniasis
Myeloma, lymphoma, Hodgkins disease, some tumours
Drugs, including oral contraceptives

Note: The ESR is not usually significantly raised in typhoid, brucellosis, malaria, infectious
mononucleosis, uncomplicated viral diseases, renal failure with heart failure, acute
allergy, peptic ulcer.
130

R Re ed du uc ce ed d E ES SR R: :

Sedimentation is falsely low in Polycythaemia, dehydration, dengue haemorrhagic
fever, and other conditions associated with haemoconcentration.

Abnormally shaped red cells as in sickle cell disease also lower sedimentation rate.

Other situations in which sedimentation is reduced include DIC (fibrinogen is
consumed) and treatment with anti-inflammatory drugs.

H HA AE EM MA AT TO OC CR RI IT T O OR R P PA AC CK KE ED D C CE EL LL L V VO OL LU UM ME E ( (P PC CV V) )
D DE ET TE ER RM MI IN NA AT TI IO ON N

P Pu ur r p po os se e: :

The haematocrit (packed cell volume PCV) is the percentage of a volume of blood
occupied by red cells. It is a screening test for anaemia, or polycythaemia, and when
accurate measurements of the haemoglobin level and red cell count are available the
absolute values for red cells can be calculated.

P Pr ri i n nc ci i p pl l e e: :

A volume of anticoagulated (or capillary) blood is placed in a glass tube which is
centrifuged so that the blood is separated into its three main components red cells,
white cells and platelets (buffy coat) and plasma. Ideally there should be complete
separation of cells and plasma, but this is not achieved. The haematocrit is the ratio
of the height of the red cell column to that of the whole blood sample in the tube.

T Te es st t s sa am mp pl l e es s: :

1. Venous blood, anticoagulated with dry K
2
EDTA.

2. Capillary blood, freely flowing, drawn up directly into a microhaematocrit
capillary tube which has been coated inside with heparin.

E Eq qu ui i p pm me en nt t : :

M Mi i c cr ro oh ha ae em ma at t o oc cr ri i t t m me et t h ho od d: :

Microhaematocrit centrifuge, with a pre-set speed providing a centrifugal force
of 12,000 g.

Capillary tubes, 75 mm long and internal diameter of 1 mm.

131
a. Plain for anticoagulated blood.
b. heparinized for capillary blood.

Plasticine sealer (e.g. Cristaseal) or

Bunsen burner.

M Ma ac cr ro o- -m me et t h ho od d o of f W Wi i n nt t r ro ob be e: :

Wintrobe graduated haematocrit tubes.

Long-stemmed capillary pipette (the delivery tip must reach the bottom of the
haematocrit tube).

Bench centrifuge capable of generating at least 2500 g at the bottom of the
cup.

M Me et t h ho od d: :

With both methods, the blood sample should be as fresh as possible and well mixed.

M Mi i c cr r o o m me et t h ho od d: :

1. Using capillary action, allow blood to enter micro-haematocrit capillary tube,
stopping at 10 15 mm from the end. Wipe the outside of the tube.

2. a. Seal the dry end by pushing into the Plasticine two or three times.

b. If heat sealing is used rotate the dry end of the capillary tube in a fine
Bunsen burner flame.

3. Place the tube into one of the centrifuge plate slots, with the sealed end
against the rubber gasket of the centrifuge plate. Keep a record of patients
name against centrifuge plate numbers.

4. If using a Hawksley microhaematocrit centrifuge, screw down solid plate then
close lid. If using a Crist microhaematocrit centrifuge, close lid.

5. Centrifuge for five minutes.

6. Read the PCV in the Microhaematocrit Reader by placing the base of the red
cell column on the 0 line, the meniscus of the plasma on the 100 line, then
moving the silver line on the adjuster until it is touching the red cell/white cell-
platelet interface. Avoiding parallax error read the PCV from the scale.

U Un ni i t t s s: :

The haematocrit is the ratio of the packed red cells to the volume of blood
(1/1), e.g. 0.45.
132

C Co om mm me en nt t s s: :

It is preferable to perform tests in duplicate, as the fragile microhaematocrit
capillary tubes may shatter, but if large numbers are being tested, it is
impracticable.

- Shattering of the capillary tubes is due to the very rapid acceleration of
high speed microhaematocrit centrifuges. This can be prevented to
some extent by first spinning the centrifuge head by hand so that the
bottom of the capillary tube is in firm contact with the shoulder of the
centrifuge plate.

- It is preferable, although not essential, to balance the microhaematocrit
capillary tubes in the centrifuge.

- The amount of blood added to the EDTA- tube should be as indicated
on the label. An excess of EDTA leads to red cells shrinkage.

- Oxygenation of the blood produces a 2 % decrease in the PCV and,
therefore, the sooner the PCV is measured the better. Prolonged
mixing will usually aerate the blood satisfactorily.

- With heat sealing, a rubber gasket is not required, with Plasticine-
sealed tubes a gasket is necessary.

- With a raised haematocrit, as in polycythaemia, there may be reduced
red cell packing even with a high speed centrifuge; centrifugation for a
further five minutes may be required. A further 30 minutes is required in
the Wintrobe method.

- Red cell size and shape may influence the PCV reading; abnormally
shaped red cells such as spherocytes, sickled cells, microcytic
hypochromic cells and macrocytes, etc. may lead to increased plasma
trapping.

- Inspection of the plasma and buffy coat will sometimes provide
valuable diagnostic information, e.g. jaundice, thrombocytosis,
leukaemia.
133

I In nt t e er rp pr r e et t a at t i i o on n: :

N No or rm ma al l r ra an ng ge e

Males : 0.40 - 0.52
Females : 0.37 - 0.47

M Me et th ha ae em mo og gl lo ob bi in n r re ed du uc ct ti io on n t te es st t ( (R RC CT T) )

Reticulocyte count

Value of test: Reticulocytes are immature red cells normally present in small
numbers in the blood (up to 2 %). Reticulocyte numbers increase when there is an
increase in erythropoietic activity. A reticulocyte count assesses bone marrow
activity, e.g. whether there is an effective erythropoietic response when there is a
reduction in the number of red cells due to haemolysis or haemorrhage. A
reticulocyte count is also of value in monitoring the erythropoietic response of an
anaemic patient following treatment.

P Pr ri in nc ci ip pl le e o of f t te es st t: :

An isotonic solution of a supravital stain (i.e. one that stains living material) such as
New methylene blue or brilliant cresyl blue is incubated with a few drops of blood. To
detecet ribosomal RNA in reticulocytes, the red cells must be stained while they are
still living (not fixed). A thin preparation is made and the reticulocytes counted
microscopically. Reticulocytes are recognized by the violet-blue stained granules of
ribosomal RNA (reticulin) they contain. The reticulocyte count is expressed as a
percentage, as an index (RI), or preferably in absolute numbers when an RBC count
is available.

R Re ea ag ge en nt t: :

10 g/l (1%
w
/v) New methylene
blue or brilliant cresyl blue.

*New methylene blue gi ves better stai ni ng of r eti culi n i n r eti culocytes than
br i lli ant cr esyl blue. I t i s chemi cally di ffer ent fr om other for ms of methylene
blue

Keep the stain refrigerated and always filter it before use.

Specimen: Use well-mixed EDTA anticoagulated blood or if the patient is a young
child, use free flowing capillary blood.
134

T Te es st t m me et th ho od d: :

1. Filter 2-3 drops of the stain into a small tube or vial.

2. Add about 4 drops* of EDTA anticoagulated blood or capillary blood and mix
well.

*The amount of blood used is not critical. Use at least twice the volume of blood
to stain if the patient is severely anaemic.

3. Incubate at room temperature for 20 minutes or 10-15 minutes at 35-37C.

4. Mix gently to resuspend the red cells and using a capillary or plastic bulb pipette,
transfer a drop of the stained blood to each of two slides. Spread to make two
evenly spread thin films. Wave the slides back and forth to air-dry the films.
Protect the films from dust and insects until the count can be performed.

5. Count the reticulocytes microscopically. Use the 10X objective (with reduced
condenser iris diaphragm) to check the distribution of the red cells. Select an area
where the red cells can be seen individually, add a drop of immersion oil, and
examine using the oil immersion objective (open more the condenser iris
diaphragm).

6. Count systematically (i.e. consecutive fields), 500 red cells (1000 if there are very
few reticulocytes), noting the number that are reticulocytes. Calculate the
percentage of reticulocytes.

A Ap pp pe ea ar ra an nc ce e o of f r re et ti ic cu ul lo oc cy yt te es s: :

Reticulocytes appear as pale green-blue stained cells containing dark blue-violet
inclusions in the form of small granules, distributed irregularly. Mature red cells
stain pale green-blue.

Counting reticulocytes: A convenient method of counting reticulocytes is to
reduce the size of the microscope field by inserting in each eyepiece a circular
piece of black (opaque) paper which has a punched out hole of about 5 mm.

T To o c ca al lc cu ul la at te e % % o of f r re et ti ic cu ul lo oc cy yt te es s: :

q Using a hand tally counter, count a total of 500 red cells, noting on paper the
number of cells that are reticulocytes (alternatively use two hand tally
counters or a white cell differential counter).

q Multiply the number of reticulocytes counted by 2.

q Divide the figure by 10 to obtain the percentage figure.
7. Report the reticulocyte count as the average of the two counts performed (from
the two separate preparations).
135

Q Qu ua al li it ty y c co on nt tr ro ol l: :

Quality control of reticulocyte counts should include filtering the stain before use and
checking the best staining time to use when a new batch of stain is made. Always
perform duplicate counts (preferably using a different person for each count). The
two counts should agree within 20 % of each other.

Sources of error

Not mixing adequately the stained blood prior to making the blood films.

Not counting the cells accurately or counting too few cells.

Using stain which has become contaminated and has not been filtered before use.

Confusing Heinz bodies or precipitated stain for the reticulin of reticulocytes.

I In nt te er rp pr re et ta at ti io on n o of f r re et ti ic cu ul lo oc cy yt te e c co ou un nt ts s: :

Reference range*

*Figures should be checked locally

Infants at birth... 2 5%
Children and adults... 0.5 2.5 %

Note: Whenever an RBC count is available, express the reticulocyte in absolute numbers.

R Ra ai is se ed d r re et ti ic cu ul lo oc cy yt te e c co ou un nt ts s: :

Found when there is an increase in red blood cell production as occurs in:

q Haemolytic anaemias (with effective erythropoiesis).

q Following acute blood loss.

q After iron therapy for iron deficiency anaemia or specific therapy for
megaloblastic anaemia.

Reticulocyte responses are higher with haemolysis than haemorrhage.
Decreased reticulocyte count : Associated with in effective erythropoiesis or
decreased production of red cells.

Reticulocyte index: When a RBC count is not available, it is recommended that the
reticulocyte count be expressed as a reticulocyte index (RI)

RI = Reticulocyte % x PCV
45

136

Chapter-Nine

Histopathology

137

SOP of Histopathology Specimens for Thana hospital
(Primary level Hospitals)

Objectives:

1. To know the nature of Histopathology specimens.
2. Procedure of collection and preservation of Histopathology specimens.
3. Procedure of labeling and transport of Histopathology specimens

1. Nature of Histopathology specimens
a. Surgical and post mortem specimens
b. Surgical specimens includes Resection specimens and biopsies
Resection specimens: are the whole or part of an organ removed for a
previously diagnosed condition.
Biopsies are sample of tissue removed from a patient for diagnostic
purposes.

2. Specimen collection and fixation:

Receive the specimen in 10% formalin and keep at room temperature.

If the specimen is not in 10% formalin, Take a clean and dry jar made of
glass or plastic with wide mouth with sufficient space to contain the
specimen

Do not hold tissue with forcep, it may injure tissue. Use a stiff paper to
drop the tissue into the jar.

Pour sufficient formalin so that the specimen remains always under the
formalin solution.

If the specimen is small or tiny, it should have 50 times more formalin
than the volume of the specimen. If large add 10 times more formalin
than the volume of the specimen. If the biopsy material is 0.50.5cm then
amount of fixative required is 6-10 ml. If the biopsy material is 22 cm
then amount of fixative required is 90 ml.

If the specimen is very large, it should be cut into pieces after taking
measurement, so that formalin penetrates easily into tissue.

Make 1 cm slice in such a way that the specimen received can be
subsequently reconstituted during gross examination.
138

Fig: 1. Amount of fixative and tissue size

A request form with an authorized signature must be submitted with the specimen
containing

Name--------------------------------------------- Age------------- Sex----------
----------
Address of patient--------------------------------------------------------------------------
-----------
Referring consultant----------------------------- Ward ------------ Unit--------
Bed-------
Name of specimen--------------------------------- Registration No.----------------
-----
Clinical diagnosis----------------------------------- Ocupation------------------------
-----------
Date of operation------------------------------------- Clinical note---------------------
------------
Investigation required----------------------------------------------------------------------
-----------
Laboratory Registration No.------------------------- Hospital/OPD--------------------
------------

Write a registration number for the specimens/specimens on the jar
containing specimen/specimens.
Enter the particulars of the patient with the registration number into a
register provided for that purpose.

139
Checklist for specimen collection and request form:
Correct specimen has been sent or not.
Correct fixative has been used or not.
Name of tissue has been mentioned or not.
Clinical diagnosis/differential diagnosis/short clinical note/x-ray in case
of bone has been included or not.
Registration number has been provided or not on the jar and also into a
register.

2.1. Preservation of histopathology specimens:

Fixatives- The most simple to prepare fixatives is 10% formalin.

Q. What is formaldehyde?
Ans: This saturated solution is available commercially as 40 % (v/v)
formaldehyde or formalin.

A. Preparations of 10% formalin
.
*Add 40% formaldehyde-100ml
*Distilled water or tap water-900 ml to prepare 10% formalin

Caution: Concentrated formaldehyde solution is a toxic chemical and its vapor
is irritating to eyes and mucous membrane. So, it should be handled with great
care in a well-ventilated room.

3. Procedure of labeling and transport of histopathology specimens

Fig: 2. Steps of transport of specimen.

Cut out a small rectangle of stiff paper (about 31). Use a lead pencil to
write on the stiff paper to be dropped into the bottle or jar. Write on it the
name of the patient, the nature of specimen and date of collection and
registration number of the patient
Drop the stiff paper into the bottle
24 to 48 hour may pass before the specimen is cut and stained.
The specimen can be preserved for at least a week
Screw the cap or stopper of the bottle with adhesive plastic.
140
Place the bottle in a box, together with requisition form containing age,
sex, name of specimen with clinical and differential diagnosis and wrap it
on the body of container.
Pack well with nonabsorbent cotton wool.
Dispatch the fixed material to the nearest referral hospital without delay.

SOP of Cytopathology
Specimens for Thana hospital
(Primary level Hospitals)

*Value of cytopathology
A cytology specimen is of high diagnostic
value if it is a
Representative sample.
Preserved and fixed by wet alcoholic
fixation or air-dried.
Processed and stained optimally.
Properly mounted for microscopic
study and storage.

*Objective: To learn about

a. Nature of Cytopathology specimens.
b. Procedure of collection and labeling and
preservation of cytopathology specimens.
c. Procedure of transport of Cytopathology
specimens
d. Waste disposal and laboratory safety for
Cytopathology specimens

a. Nature of Cytotopathology specimens
Cytopathology involves the examination and
interpretation of cells rather than solid
tissue. It is usually done for diagnosis of
cancer and precancerous and inflammatory
lesion.

Exfoliative cytology -cells sheded from, scraped or brushed off an
epithelial surface, e.g. sputum, cervix, bronchus,
Fluid cytology -cells removed with the fluid in which they are suspended,
e.g. pleural fluid, peritoneal, pericardial, CSF, Urine, synovial fluid,
141
washing cells from flushing out a organ with an irrigating fluid such as
bronchial washing from the lung,
Fine needle aspiration cytology-FNAC -cells sucked out from a solid
tissue by a needle fitted to a syringe, e.g. lymph node, breast lump,
thyroid, salivary gland.

b. Procedure of collection, labeling, and preservation of cytopathology
Specimen

All specimens must be accompanied by a laboratory request form duly filled by an authorized
person containing informations of
Name------------------------------------ Age------------- Sex----------------
Referring consultant--------------------------- Ward ---------- Unit------ Bed-------
Name of specimen----------------------------- Registration No.--------------------

If any important and relevant information is missing in the request form,
effort must be made to obtain it
The attending medical technologist should write a registration number on
the jar containing specimen.
The attending medical technologist should enter the particulars of the
patient with the registration number into a register provided for that
purpose.
The specimen received must be entered into the register and a registration
number should be given on the specimen container as well as on
laboratory request form.

Name of organ from where it has been obtained.
Clinical diagnosis/differential diagnosis/short clinical note/x-ray in
case of bone has been included or not.
142
Registration number has been provided or not on the jar and also into
a register and the request form.

Specimen collection:
1.Fine needle Aspiration Cytology (FNAC)
FNAC: Superficial body masses can be collected by FNAC, Smear prepared by
surgeon or Pathologist. Staining is done by medical technologist.

2. SOP for Urine cytology
143

It is a form of exfoliative cytology where it is used primarily for diagnosis of
diseases that involves the collecting system of kidney.
It may contain cell exfoliated from the tumors of pelvis, kidney, bladder
and ureter.
It is simple, safe and inexpensive method to detect most aggressive
urothelial neoplasms and carcinoma in situ.
It is essential for proper evaluation and follow up of patients with
urothelial cancer

Urine specimen:
1. Freshly voided urine
2. Catheterized urine from bladder or ureter or pelvis
3. Brushing and washing specimen such as bladder washing, urethra or
ureter washing or brushing

Check unsatisfactory specimen:
Patient is not well hydrated
Urine is alkaline (it be acidified to an optimum pH-5 by taking I
gm Vit-C).
Collected not from a collecting bag
First morning specimen

Ideal specimen
Three freshly voided urine specimen
Specimen obtained by simple catheterization
Bladder wash
Washing urethra by saline
Direct swabbing with a saline moistened cotton applicator
Retrograde catheterization with brushing or lavage

Collection and preservation of specimen:
Fresh voided urine should be collected-three specimen is optimum
Avoid fresh voided technique if there is residual urine and severe
vaginitis or urethritis
Let the patient drink one glass of water for every 15 minutes for 2 hours-3
hours
At the end of 2 hour, have patient void urine and discard it.
One hour later, have patient void in the container.
Cup or jar may be used for collection
Bring it immediately to the lab
Repeat the procedure for successive 5 days
Urine should be collected with equal volume of 50% alcohol as a fixative
in a jar
144
Clean catch voided urine should be processed immediately
Can be refrigerated for a short time (hours).
If delay is inevitable immediate fixation with 50% ethanol may preserve
specimen for several days Urine should be collected with equal volume of
50% alcohol as a fixative in a jar

Method of staining: Paps stain

3. SOP for other Body fluid specimens

*Pleural and peritoneal fluid is common specimen in cytology lab.
*Pericardial, joint fluid and CSF are less common.

Collection and preservation:
Should be submitted fresh and unfixed or heparinized (three units of
heparin per ml of fluid).
Heparin preparation: powdered heparin with lithium salt-4gm and
distilled water 1 litre.
Cells degenerate in effusion so process it immediately
Degenerate rapidly in presence of blood
Collected by paracentesis in a flask or suitable container
Place heparin into the receiving flask
Gently agitate the flask for proper mixing
Alternatively, if heparin is not available add equal volume of 95%
ethanol and processed immediately.
May be preserved for 24 hours with refrigeration.
Cytological specimen with high protein content can be preserved for a
maximum of 48 hours in a refrigerator. If the Pleural, peritoneal,
pericardial, and joint fluid contain blood, add 0.2 ml of anticoagulant
(25.5 gm of sodium citrate and 8.0 gm of citric acid dissolved in 100
ml of distilled water) immediately for 10 ml of specimen.

Cytopreparation and staining

Materials required
1.Four numbered albuminized slides
2.15 ml capacity conical centrifuge tubes numbered
3. Pasteur pipette with dropper bulb
4. Cotton swab
5. Paper towels or absorbent paper
145
Preparation:
60 ml of fluid is collected
Distribute in four conical tubes
Balance tubes in centrifuge
Centrifuge at the rate of 3000 rpm for ten minutes
Decant supernatant
Place one or two drops of sediment on the slide.
Place a second slide over the sediment and allow it to spread
evenly
Gently pull the slides apart
Fix immediately into 95% ethanol.

Staining method: Papinacolou stain.

4. SOP for CSF cytology

Value of CSF cytology
It is useful for diagnosis of tumors, infections, vascular disorders, trauma
and demyelinating diseases.
Useful for monitoring CNS chemotherapy
It is a part of complete neurological examination
A single abnormal or foreign cell can be a significant findings

Prerequisites
A complete and accurate clinical history is important.
Sign and symptom
Pertinent lab report
Radiological findings

Procedure of collection, preparation and preservation of CSF
CSF is collected by lumber puncture
Specimen is collected in two or more plastic tubes
Small amount of CSF in first tube
5 ml specimen is necessary for better cytologic evaluation of the
submitted specimen
Third tube is recommended for cytological diagnosis
Repeated specimen may be necessary
Cells should be processed as soon as possible as cell deterioration is
rapid
Should be processed within 2 hour even if refrigerated
Cells can be preserved for one day in a solution of equal parts
balanced salt solution and 20% human serum albumin
146
50% ethanol allow longer preservation
For maximum cells yield few preparation are helpful such as
membrane filter (Gelman, milliporenuclepore)
Cytocentrifugation-method of choice
For maximum cells yield sedimentation may be done
Coated slides
Cytospin preparation is easier and faster then filters
Method of staining:
The CSF is processed in cytospin and the slides are stained by
Papinicolou method.

5. SOP for Sputum collection and processing

Value: It increases the detection rate of primary bronchogenic carcinoma from
45% to 95%

Materials required:
One wide mouthed bottle, jar, or cup or Petri dish for each day.
It should be clean and dry and no fixative is required.

Collection of specimen:
Fresh unfixed morning specimen for each day for 5 days.
Sputum may be collected by patient himself or herself. In case of sputum,
and bronchial aspirates, if there is delay in sending it should be preserved
in a refrigerator for 24 hours.
Do not submit 24 hours specimen
First morning specimen to be collected
Instruct the patient to cough deeply upon awakening and expectorate all
sputum in to the cup (Saliva should not be collected),
Send it to the lab
Repeat the procedure for 5 days

Processing of sputum

Materials required:
Take four sliders numbered with diamond pencil
Petri dish identified with case number
Two wooden applicator
Paper towel
Jar with fixative

Method:
Note the description of specimen
147
Transfer specimen into a Petri dish
Transfer multiple white streak portions, blood mixed area to slides by wooden
applicator
Gently mesh specimen between two slides until large lumps disappear
Dip immediately in 95% ethanol containing jar (Coplin jar) for 30 minutes.

c. Transport of cytopathology specimens
A Coplin Jar with fixative or separate bottle of fixative for each patients
smear is preferable. A big sized wide mouthed bottle can be used. Cells
dry rapidly so obtained specimen should be immediately dropped into the
fixative.
Mailing of unstained smears can be done by coating fixative like 10%
carbowax in 95% ethanol and spray coating fixatives.
Wet smears are immediately dipped in 10% carbowax solution, removed,
placed on paper towel to dry smear. Alcohol evaporates leaving
protecting wax coating on the slide and protects from drying effect.
Just before staining, the carbowax must be removed by placing into 95%
ethanol or 95% rectified sprit or 80% propanol for 30 minutes.
Hair spray containing high concentration of alcohol without lanolin or
other oily substances can be used. Immediately after collection, the smear
is placed on table and sprayed for 5-10 min from a distance of 5-10
inches and it become ready for transport after 5 minutes.
Glycerine method of mailing can be done by fixing the smear in 95%
ethanol for 15 minutes and removed. Two drops of glycerine are placed
on smears and covered with a clean glass slide. They may be wrapped in
wax paper and mailed to the referellaboratory in a suitable container.

d. Procedure for waste disposal and laboratory safety
In case of leaking or spilling, put on the gloves and put the specimen
in another container. The old container and laboratory request form
are placed in plastic bag for disposal (autoclaved or flooded with 1%
phenol). The tray or table is wiped up with paper towel, which are
disposed off in the waxed paper bag.

Specimen containers are to be carefully opened when received and a
portion of the specimen is smeared on a slide with the help of forceps,
spatulas or wooden sticks. Glass tubes or plastic tubes can be used for
centrifugation of specimen. All the articles used for cytopreparation
must be sterilized by autoclaving or socked in 1% phenol.
Preferably, all articles should be sent to the incinerator.
Unused portion of specimens are poured into a metal container
containing 1% phenolic disinfectant. When it is full, this mixture is
poured down the sink drain.
148
The work area should be wiped with 1% phenolic disinfectant several
times a day. Drains, centrifuge machine and floor area are also to be
cleaned with 1% phenolic disinfectant.
All specimens are to be handled as if infectious and laboratory safety
procedure is to be practiced. Infection may be acquired through direct
contamination of hands with subsequent invasion through cuts,
abrasions, mucous membranes and through air borne mechanisms.
Food is prohibited from storage in the work area and in refrigerator
and smoking and eating and drinking is not permitted. Mouth pipe ting
is forbidden.

SOP of Histopathology Specimens for District Hospital (Secondary level)

Objectives:

1. To know the nature of Histopathology specimens.
2. Procedure of collection and preservation of Histopathology specimens.
3. Procedure of labeling and transport of Histopathology specimens
4. Tissue processing and staining technique

1. Nature of Histopathology specimens
c. Surgical and post mortem specimens
d. Surgical specimens includes Resection specimens and biopsies
Resection specimens: are the whole or part of an organ removed for a
previously diagnosed condition.
Biopsies are sample of tissue removed from a patient for diagnostic
purposes.

2. Specimen collection and fixation:

Receive the specimen in 10% formalin and keep at room temperature.

If the specimen is not in 10% formalin, Take a clean and dry jar made of
glass or plastic with wide mouth with sufficient space to contain the
specimen

Do not hold tissue with forcep, it may injure tissue. Use a stiff paper to
drop the tissue into the jar.

Pour sufficient formalin so that the specimen remains always under the
formalin solution.
149

If the specimen is small or tiny, it should have 50 times more formalin
than the volume of the specimen. If large add 10 times more formalin
than the volume of the specimen. If the biopsy material is 0.50.5cm then
amount of fixative required is 6-10 ml. If the biopsy material is 22 cm
then amount of fixative required is 90 ml.

If the specimen is very large, it should be cut into pieces after taking
measurement, so that formalin penetrates easily into tissue.

Make 1 cm slice in such a way that the specimen received can be
subsequently reconstituted during gross examination.

Fig: 1. Amount of fixative and tissue size

A request form with an authorized signature must be submitted with the specimen
containing

Name--------------------------------------------- Age------------- Sex----------
----------
-----------
Referring consultant----------------------------- Ward ------------ Unit--------
Bed-------
-----
-----------
150
------------
-----------
------------

Write a registration number for the specimens/specimens on the jar
containing specimen/specimens.
Enter the particulars of the patient with the registration number into a
register provided for that purpose.

Correct fixative has been used or not.
Name of tissue has been mentioned or not.
Clinical diagnosis/differential diagnosis/short clinical note/x-ray in case
of bone has been included or not.
Registration number has been provided or not on the jar and also into a
register.

2.1. Preservation of histopathology specimens:

Fixatives- The most simple to prepare fixatives is 10% formalin.

Q. What is formaldehyde?
Ans: This saturated solution is available commercially as 40 % (v/v)
formaldehyde or formalin.

A. Preparations of 10% formalin
.
*Add 40% formaldehyde-100 ml
*Distilled water or tap water-900 ml to prepare 10% formalin

Caution: Concentrated formaldehyde solution is a toxic chemical and its vapor
is irritating to eyes and mucous membrane. So, it should be handled with great
care in a well-ventilated room.
151

3. Procedure of labeling and transport of histopathology specimens

Fig: 2. Steps of transport of specimen.

Cut out a small rectangle of stiff
paper (about 31). Use a lead
pencil to write on the stiff paper
to be dropped into the bottle or
jar. Write on it the name of the
patient, the nature of specimen
and date of collection and
registration number of the
patient
Drop the stiff paper into the
bottle
24 to 48 hour may pass before
the specimen is cut and stained.
The specimen can be preserved
for at least a week

4. Tissue section preparation
Q. What is fixation?
Fixation is a complex series of
chemical events, and differs for the
different groups of chemical
substances found in tissue. It is the
foundation of subsequent steps of
tissue processing. The most commonly
used fixative is 10% formalin.
Q. What are the aims of fixation?
1. Prevention of autolysis and
152
bacterial attack.
2. Help to prevent change of shape and volume in any subsequent steps of tissue
processing
3. Helps in clear staining of tissue
5. Maintain the tissue close to their living state as far as possible without loss or
rearrangement of their component.
Steps of tissue processing.
1. Check the label of tissue with registration No.
2. Gross examination of tissue by the Histopathologist.
The submitted specimen must be measured in cm and weight should be taken in
gram. Representative blocks should be taken, preferably measuring approx.
2.52.50.4 cm each. All measurement and pertinent informations obtained
from the submitted specimen/specimens must be noted in the histopathology
register. Number of blocks and their source must be mentioned in the register.
To achieve sections 5-10 micron thick, the tissue must have consistency suitable
for cutting. For this they are processed through a series of alcohols and liquid
paraffin. Liquid paraffin penetrates the finest tissue space and produces a good
cutting consistency. Then the tissue after cutting with microtome, are mounted
on a microscope slide and stained after deparrafinized by xylene.

Principles of tissue processing

The necessary steps involved in tissue processing include

153
a. Dehydration-tissue is immersed first in 70% ethanol, and then progressed
through 95% to 100% ethanol. Other aalternatives include acetone and
methanol.
b. Clearing-xylene, alternative-Tolune
c. Impregnation-replacing clearing agent with the embedding medium,
mostly used is paraffin wax.
d. Embedding-

Flow chart of steps involved in processing of tissue and staining by
haematoxylin and eosin.

1. Fixation-10% formalin-24 hours

2. Dehydration-
i) 50% alcohol-90 minutes
ii) 70% alcohol-90 minutes
iii) 80% alcohol-90 minutes
iv) 95% alcohol-90 minutes
v) 100% alcohol-90 minutes
vi) 100% alcohol-24 hours

3. Clearing-i) xylene-I-1 hour
ii) Xylene-II-1 hour

4. Paraffin impregnation- a paraffin bath with temperature 50-60
o
C is used.
The melting point of paraffin is 58
o
C. The tissue ware treated in

Melted paraffin-I-30 minutes
Melted paraffin-II-30 minutes

5. Paraffin embedding-For embedding the tissue in melted paraffin, metallic
moulds are used. At first lubrication of the metallic moulds are done by
liquid paraffin. After that, melted paraffin is poured into that. The tissue is
carefully embedded in proper plane at the bottom of the metallic moulds.
The melted paraffin is allowed to harden at room temperature. After
hardening, the metallic moulds were removed and the blocks were properly
trimmed. Then the blocks are kept in the ice chamber of a refrigerator for
some time before cutting the sections.

154
6. Section cutting-After sharpening the microtome knife in knife sharpener,
each block of tissue is fitted in the microtome machine. A water bath with a
regulated temperature of 45-50 oC is used for floatation of the sections.
Sections are cut at 4-5 micron thickness. Ribbons of good sections are
selected and floated on lukewarm water in water bath. The sections are then
taken on albuminized glass slides. The slides are then kept in inclined
position to drain off water and allowed to dry at room temperature.

Steps of haematoxylin and eosin staining

1. Deparrafinization- slides are kept in paraffin bath - 15 minutes
Xylene-I-5 min
Xylene-II-5 min

2. Hydration of the tissue-
a) absolute alcohol-3 min
b) absolute alcohol-3 min
c) 95% alcohol-2 min
d) 70% alcohol-2 min
e) 50% alcohol-2 min
f) Running tap water-2 min

3. Staining
a) Harriss haematoxylin-10 min
b) 1% acid alcohol; for differentiation-1 min
c) Tap water for bluing till the section become pale blue- 8-10min
d) Counterstaining with aqueous solution of Eosin-1 min

4. Dehydration
a) 70% alcohol-5 min
b) 95% alcohol-2 min
c) absolute alcohol-5 min
d) absolute alcohol-5 min

5. Clearing

a) Xylene-I-5 min

b) Xylene-II-5 min

c) Xylene-III-2min.

6. Mounting
155

The slides are mounted with DPX using coverslips.

7. Results Nuclei-Blue
Cytoplasm-Shades of pink to red.

SOP of Cytopathology Specimens for District Hospital (Secondary level)

*Value of cytopathology
A cytology specimen is of high diagnostic value if it is a
Representative sample.
Preserved and fixed by wet alcoholic fixation or air-dried.
Processed and stained optimally.
Properly mounted for microscopic study and storage.

*Objective: To learn about

1. Nature of Cytopathology specimens.
2. Procedure of collection, labeling, and preservation of Cytopathology
specimens.
3. Procedure of transport of Cytopathology specimens
4. Paps staining
5. Waste disposal and laboratory safety for Cytopathology specimens

1. Nature of Cytotopathology specimens
Cytopathology involves the examination and interpretation of cells rather than
solid tissue. It is usually done for diagnosis of cancer and precancerous and
inflammatory lesion.

Exfoliative cytology -cells sheded from, scraped or brushed off an
epithelial surface, e.g. sputum, cervix, bronchus,
Fluid cytology -cells removed with the fluid in which they are suspended,
e.g. pleural fluid, peritoneal, pericardial, CSF, Urine, synovial fluid,
washing cells from flushing out a organ with an irrigating fluid such as
bronchial washing from the lung,
Fine needle aspiration cytology-FNAC -cells sucked out from a solid
tissue by a needle fitted to a syringe, e.g. lymph node, breast lump,
thyroid, salivary gland.

156
2. Procedure of collection, labeling, and preservation of cytopathology
Specimen

All specimens must be accompanied by a laboratory request form duly filled by
an authorized person containing in formations of

Name--------------------------------------------- Age------------- Sex----------
Referring consultant----------------------------- Ward ------- Unit------ Bed------

If any important and relevant information is missing in the request form,
effort must be made to obtain it
The attending medical technologist should write a registration number on
the jar containing specimen.
The attending medical technologist should enter the particulars of the
patient with the registration number into a register provided for that
purpose.
The specimen received must be entered into the register and a registration
number should be given on the specimen container as well as on
laboratory request form.

Name of organ from where it has been obtained.
Clinical diagnosis/differential diagnosis/short clinical note/x-ray in
case of bone has been included or not.
Registration number has been provided or not on the jar and also into
a register and the request form.

Specimen collection:
1. FNAC: Superficial body masses can be collected by FNAC, Smear prepared
by surgeon or Pathologist. Staining is done by medical technologist.

157

2. SOP for Urine cytology: It is a form of exfoliative cytology where it is used
primarily for diagnosis of diseases that involves the collecting system of kidney.
It may contain cell exfoliated from the tumors of pelvis, kidney, bladder
and ureter.
It is simple, safe and inexpensive method to detect most aggressive
urothelial neoplasms and carcinoma in situ.
It is essential for proper evaluation and follow up of patients with
urothelial cancer

Urine specimen:
4. Freshly voided urine
5. Catheterized urine from bladder or ureter or pelvis
6. Brushing and washing specimen such as bladder washing, urethra or
ureter washing or brushing

Check unsatisfactory specimen:
Patient is not well hydrated
Urine is alkaline (it be acidified to an optimum pH-5 by taking I
gm Vit-C).
Collected not from a collecting bag
First morning specimen

Ideal specimen
Three freshly voided urine specimen
Specimen obtained by simple catheterization
Bladder wash
Washing urethra by saline
Direct swabbing with a saline moistened cotton applicator
Fig:3
158
Retrograde catheterization with brushing or lavage

Collection and preservation of specimen:
Fresh voided urine should be collected-three specimen is optimum
Avoid fresh voided technique if there is residual urine and severe
vaginitis or urethritis
Let the patient drink one glass of water for every 15 minutes for 2 hours-3
hours
At the end of 2 hour, have patient void urine and discard it.
One hour later, have patient void in the container.
Cup or jar may be used for collection
Bring it immediately to the lab
Repeat the procedure for successive 5 days
in a jar
Clean catch voided urine should be processed immediately
Can be refrigerated for a short time (hours).
If delay is inevitable immediate fixation with 50% ethanol may preserve
specimen for several days Urine should be collected with equal volume of
50% alcohol as a fixative in a jar

Method of staining: Paps stain

3. SOP for other Body fluid specimens

*Pleural and peritoneal fluid is common specimen in cytology lab.
*Pericardial, joint fluid and CSF are less common.

Collection and preservation:
Should be submitted fresh and unfixed or heparinized (three units of
heparin per ml of fluid).
Heparin preparation: powdered heparin with lithium salt-4gm and
distilled water 1 litre.
Cells degenerate in effusion so process it immediately
Degenerate rapidly in presence of blood
Collected by paracentesis in a flask or suitable container
Place heparin into the receiving flask
Gently agitate the flask for proper mixing
Alternatively, if heparin is not available add equal volume of 95%
ethanol and processed immediately.
159
May be preserved for 24 hours with refrigeration.
Cytological specimen with high protein content can be preserved for a
maximum of 48 hours in a refrigerator. If the Pleural, peritoneal,
pericardial, and joint fluid contain blood, add 0.2 ml of anticoagulant
(25.5 gm of sodium citrate and 8.0 gm of citric acid dissolved in 100
ml of distilled water) immediately for 10 ml of specimen.

Cytopreparation and staining
Materials required
1. Four numbered albuminized slides
2.15 ml capacity conical centrifuge tubes numbered
3. Pasteur pipette with dropper bulb
4. Cotton swab
5. Paper towels or absorbent paper
Preparation:
60 ml of fluid is collected
Distribute in four conical tubes
Balance tubes in centrifuge
Centrifuge at the rate of 3000 rpm for ten minutes
Decant supernatant
Place one or two drops of sediment on the slide.
Place a second slide over the sediment and allow it to spread
evenly
Gently pull the slides apart
Fix immediately into 95% ethanol.

Staining method: Papinacolou stain.

4. SOP for CSF cytology

Value of CSF cytology
It is useful for diagnosis of tumors, infections, vascular disorders, trauma
and demyelinating diseases.
Useful for monitoring CNS chemotherapy
It is a part of complete neurological examination
A single abnormal or foreign cell can be a significant findings

Prerequisites
A complete and accurate clinical history is important.
Sign and symptom
Pertinent lab report
Radiological findings

Procedure of collection, preparation and preservation of CSF
160
CSF is collected by lumber puncture
Specimen is collected in two or more plastic tubes
Small amount of CSF in first tube
5 ml specimen is necessary for better cytologic evaluation of the
submitted specimen
Third tube is recommended for cytological diagnosis
Repeated specimen may be necessary
Cells should be processed as soon as possible as cell deterioration is
rapid
Should be processed within 2 hour even if refrigerated
Cells can be preserved for one day in a solution of equal parts
balanced salt solution and 20% human serum albumin
50% ethanol allow longer preservation
For maximum cells yield few preparation are helpful such as
membrane filter (Gelman, milliporenuclepore)
Cytocentrifugation-method of choice
For maximum cells yield sedimentation may be done
Coated slides
Cytospin preparation is easier and faster then filters

Method of staining:
The CSF is processed in cytospin and the slides are stained by
Papinicolou method

5. SOP for Sputum collection and processing

It increases the detection rate of primary bronchogenic carcinoma from 45% to
95%

Material:
One wide mouthed bottle, jar, or cup or Petri dish for each day.
It should be clean and dry and no fixative is required.

Collection of specimen:
Fresh unfixed morning specimen for each day for 5 days.
Sputum may be collected by patient himself or herself. In case of sputum,
and bronchial aspirates, if there is delay in sending it should be preserved
in a refrigerator for 24 hours.
Do not submit 24 hours specimen
First morning specimen to be collected
Instruct the patient to cough deeply upon awakening and expectorate all
sputum in to the cup (Saliva should not be collected),
Send it to the lab
Repeat the procedure for 5 days
161

Processing of sputum

Materials required:
Take four sliders numbered with diamond pencil
Petri dish identified with case number
Two wooden applicator
Paper towel
Jar with fixative

Method:
Note the description of specimen
Transfer specimen into a petridish
Transfer multiple white streak portions, blood mixed area to slides by wooden
applicator
Gently mesh specimen between two slides until large lumps disappear
Dip immediately in 95% ethanol containing jar (Coplin jar) for 30 minutes

Staining method: Paps stain

3. Transport of cytopathology specimens
A Coplin Jar with fixative or separate bottle of fixative for each patients
smear is preferable. A big sized wide mouthed bottle can be used. Cells
dry rapidly so obtained specimen should be immediately dipped into the
fixative.
Mailing of unstained smears can be done by coating fixative like 10%
carbowax in 95% ethanol and spray coating fixatives.
Wet smears are immediately dipped in 10% carbowax solution, removed,
placed on paper towel to dry smear. Alcohol evaporates leaving
protecting wax coating on the slide and protects from drying effect.
Just before staining, the carbowax must be removed by placing into 95%
ethanol or 95% rectified sprit or 80% propanol for 30 minutes.
Glycerine method of mailing can be done by fixing the smear in 95%
ethanol for 15 minutes and removed. Two drops of glycerine are placed
on smears and covered with a clean glass slide. They may be wrapped in
wax paper and mailed to the referral laboratory in a suitable container.

Note:
Alcohol is a coagulative fixative and Ethyl alcohol mixture is the best
preservative. 95% ethanol is the fixative widely used.
Alternatively, 95% rectified sprit or 100% methanol or 80% isopropanol
can be used also.

162
4. Procedure of Paps stain technique
After fixing in 95% ethyl alcohol for 30 minutes
95% ethyl alcohol-10 dips
Three changes to water
Haematoxylin-1 min
Ammonium alcohol-45 seconds
Orange G-2 min
EA-36 2 min 30 seconds
100% absolute ethyl alcohol-10 dips
100% absolute ethyl alcohol-10 dips
Xylol-15 minutes-overnight

Mount in DPX mountant

Result:
1. Nucleus-Bluish purple
2. Nucleoli-Red
3. Cytoplasm-Superficial cells-pink
Intermediate cells-Green or greenish blue
Endocervical, endometrial cells- Bluish, green
or purple

5. Procedure for waste disposal and laboratory safety
In case of leaking or spilling, put on the gloves and put the specimen
in another container. The old container and laboratory request form
are placed in plastic bag for disposal (autoclaved or flooded with 1%
phenol). The tray or table is wiped up with paper towel, which are
disposed off in the waxed paper bag.

Specimen containers are to be carefully opened when received and a
portion of the specimen is smeared on a slide with the help of forceps,
spatulas or wooden sticks. Glass tubes or plastic tubes can be used for
centrifugation of specimen. All the articles used for cytopreparation
must be sterilized by autoclaving or socked in 1% phenol.
163

Preferably, all articles should be sent to the incinerator.

Unused portion of specimens are poured into a metal container
containing 1% phenolic disinfectant. When it is full, this mixture is
poured down the sink drain.
The work area should be wiped with 1% phenolic disinfectant several
times a day. Drains, centrifuge machine and floor area are also to be
cleaned with 1% phenolic disinfectant.
All specimens are to be handled as if infectious and laboratory safety
procedure is to be practiced. Infection may be acquired through direct
contamination of hands with subsequent invasion through cuts,
abrasions, mucous membranes and through air borne mechanisms.
Food is prohibited from storage in the work area and in refrigerator
and smoking and eating and drinking is not permitted. Mouth pipe ting
is forbidden.

T H E P A P T E S T

Title of SOP: Pap test

Authorized signature Number
Date

Staff able to perform test
Unsupervised: List names
Under supervision: List names

Cost of test

VALUE OF THE TEST

Early detection of cervical cancer when appropriate therapeutic measures can be
undertaken and complete cure of this deadly disease is possible.

The test also detects certain other non-neoplastic and infectious disorders affecting
the genital tract.

PRINCIPLE OF TEST

Pap test is a form of abrasive cytology in which cytological samples are obtained from
the cervix by scraping with a spatula. The sample thus obtained are then quickly
smeared on glass slides, wet fixed in 95% ethanol and then stained, usually, by
Papanicolaous method. Microscopic examination of these slides may reveal certain
abnormalities of cells which help in detecting cervical cancer.
164

The cervical cancer, a relatively common form of cancer among females of
Bangladesh, starts as a pre-invasive lesion and remains so for several years before it
actually becomes invasive. Pap test can detect abnormality of the cells at this pre-
invasive stage when therapy may be effected to completely cure this disease.

SPECIMEN

Specimen is collected from the cervix by a wooden spatula (Ayres spatula) and
smeared on glass slides. This is then immediately dropped into a Coplin jar
containing 95% ethanol (ethyl alcohol).

EQUIPMENT

1. Ayres spatula or scraper (Figure 2).
2. Cuscos bivalve vaginal speculum
3. Sims vaginal speculum
4. Gynecological examination table
5. Glass slides
6. Diamond glass marker.
7. Coplin jars
7. Staining jars
8. Microscope- binocular with good quality optics.
9. Trolley

REAGENTS / STAINS

1. Ethyl alcohol- 100%
2. Haematoxylin
3. Eosin-Y
4. Glycerol
5. Potassium alum (Aluminium potassium hydroxide)
6. Potassium iodate
7. Orange-G crystals
8. Light green SF Yellow
9. Bismarck brown
10. Phosphotungstic acid
11. Lithium carbonate
12. Xylene
13. DPX

METHOD OF TEST

1. Patients clinical data: Patients name, registration no, accession number, age
and relevant clinical data with a minimum, the date of last menstrual period should
be entered into the request form and must be accompanied by each of the patients
sample.

2. Identification marks on glass slides: Glass slides should be marked with the
accession number (Patient identification number) by diamond glass marker.

165
3. Preparation of patient:

Prerequisites

1. The presence of a third party, preferably a female relative or nurse.
2. The consent of the patient
3. The patients bladder must be empty.
4. A good light, well suited. An electric lamp with a long stand may prove to be
sufficient for the purpose.
5. The Patient should not douche for 24 hours before the smears are obtained.

Procedure
o The Patient should lie on her back in lithotomy position on the examination
table.

o A trolley should be ready with all the necessary instruments including glass
slides, diamond marker, Coplin jars with fluid fixative (95% ethyl alcohol) for
specimen collection.

o A speculum (Cuscos bivalve) is introduced to visualize the cervix under direct
vision. Under no circumstances, the speculum should be lubricated with
medical jellies, since foreign material may readily contaminate the smear. If
there are difficulties in introducing the speculum, a few drops of normal saline
solution may be used to moisten it.

FIGURE 1. Collection of sample using Cuscos bivalve vaginal speculum to visualize
the cervix.

4. Collection of sample (Figure 1.): Sample is collected by scraping the cells at
ectocervix including the junction between ectocervix and endocerix (transformation
166
zone). Ayres spatula is used to scrape out cervix. One end of the scraper is somewhat
longer than the other so that it fits the external os and reaches endocervical canal.

o Introduce the Ayres scraper through the Cuscos bivalve vaginal speculum to
reach the cervix.
o Place the longer end of scraper into the external os.
o Rotate the scraper under pressure using the longer end as a pivot within the
external os. (see Figure 3.).

FIGURE 2. Ayres scraper.

5. Preparation of smears:

o Place the material obtained by scraping with spatula on a clean glass slid and
make a thin, uniform smear. Considerable skill and practice are required to
prepare excellent smears by a single, swift motion without loss of material or
air drying. Excessive crushing of material must be avoided.
o Put the smeared slides immediately into the fixative.

FIGURE 3. Method of obtaining sample from the uterine cervix by means of Ayres
scraper.
6. Fixation: Two types of fixatives are commonly used: Fluid fixatives and spray
fixatives.

Fluid fixative: 95% ethanol is most suitable.
o Take the fixative in a Coplin jar with cover.
167
o Place the smears in the container with fluid fixative while still wet, making
sure that there is sufficient fixative to cover the smeared area.
o Close the container immediately with cap and send to the laboratory together
with laboratory form.
o Alternatively, in case where the laboratory is at a distant place, the smears can
be removed from the fixative after 15 to 30 minutes and placed in an
appropriate container e.g. a cardboard box and sent to the laboratory.

Spray fixatives (Figure 4). Spray fixatives protect the smears from drying by forming
an invisible film on the surface of the slides. Spray fixatives may be used in lieu of
fluid fixatives, that is, immediately after the process of smear preparation has been
completed. The distance between the nozzle of the spray and the smear to be sprayed
must be about 10 to 12 inches. If the nozzle is held too close to the smear, the cells
may be dislodged by the force of the spray. Smears coated with spray fixative are air
dried and placed in cardboard slide containers and forwarded to the laboratory in
this fashion.

FIGURE 4. Use of coating fixative for smears of uterine cervix obtaining with a
wooden spatula (Ayes spatula). Immediate application of fixative is essential.

7. Staining. Once the slides reaches the laboratory, the next step is staining of the
fixed smeared slides. Papanicolaous staining method is commonly employed.

THE PAPANICOLAOU STAIN

The use of Papanicolaou stain results in well-stained nuclear chromatin, differential
cytoplasmic counterstaining, and cytoplasmic transparency. The following method is
168
a quick progressive staining using Carazzis haematoxylin. Preparation of stains and
solutions will be found in the appendix-A.

Rapid Papanicolaous Stain

Stain the fixed smeared slides by undertaking the following steps sequentially. The
staining and other solutions should be taken in staining jars with covers.
Alternatively, Coplin jars with cover can also be used. It is important to cover the
containers air tight to prevent evaporation of the alcohol containing stains and
solutions.

1. Tap water 10 dips
2. Carazzis haematoxylin 45 seconds
4. Lithium carbonate (bluing solution) 10 dips
6. 95% alcohol 10 dips
8. Orange G 6 45 seconds
11. Eosin-azure-65 45 seconds
16. Xylene 10 dips
17. Xylene 10 dips
18. Mount with mounting medium (DPX) and coverslip.

8. Mounting: Mounting medium creates a permanent bond between the slide and
the coverslip. This permanent bond protects the cell film from mechanical damage,
air-drying effect, and stain fading. DPX is a commonly employed mounting medium
with satisfactory results.

It is important to maintain the pH of the mounting medium as close to neutral as
possible to prevent the fading of stains. One gram of 2,6-di-tert-butyl-p-cresol (J.T.
Baker Chemical Company, Phillipsburg, N.J. 08865) can be added to 100 ml of any
mounting medium to inhibit the fading of stains. Mounting medium will thicken as
the solvent evaporates. For consistency to be maintained, each new bottle of
mounting medium should be checked by counting the number of drops that fall from
a pipette filled with the fresh medium for 5 seconds. The number of drops should be
noted on the label for future reference. Each week the medium should be checked in
a similar manner to determine if the addition of a solvent (e.g. xylene) is necessary.

9. Coverslips: No. 1 glass coverslips in size 24 x 50 to 60 mm are recommended.
However, coverslips with smaller surface area (e.g. 22 x 22 mm) can also be used.

Practice is necessary to achieve well-mounted slides, free of air bubbles and artifacts.
A minimum of mounting medium should be used. Too much mounting medium
interferes with microscopic detail, making the cell film appear hazy or milky when
169
examined with the high dry (x40) objective. If the mounting medium and coverslip
are applied too slowly, a common artifacts appears as a brown, retractile pigment-
like substance on the surface of the cells.

FIGURE 4. Method of coverslipping.

This artifact is caused by air trapped on the surface of the cell when xylene is allowed
to evaporate. If this artifact occurs, the slide may be soaked in xylene, absolute
alcohol, and 95% alcohol, rinsed in running tap water, and restained in OG and EA.
In stubborn cases, after the running water rinse, the slide may be placed in glycerine
for hour and rinsed well in tap water prior to reapplication of the counterstains.

Method of coverslipping (Fig.- 4)

1. Remove slide from xylene.
2. Place one or two drops of mounting medium on the glass slide or coverslip.
3. Lower the coverslip over the glass slide to which mounting medium has been
applied. Bubbles should be avoided at this point. Alternatively , if the
mounting medium is applied to the coverslip, lower the glass slide over the
coverslip and then turn slide right side up.
4. Gently tease bubbles from under the coverslip with an applicator stick and
wipe excess xylene and mounting medium from slide.

REPORTING RESULTS / INTERPRETATION

A good communication between the laboratory and clinician is necessary in the
interpretation of results. The Bethesda System (TBS) for reporting cervical/vaginal
cytologic diagnoses was developed at a National Cancer Institute (NCI)-sponsored
170
workshop in December 1988 and modified in 1991 to provide uniform diagnostic
terminology that would facilitate communication between the laboratory and the
clinician. The format of TBS report includes an evaluation of specimen adequacy and
a descriptive diagnosis.

It is recommended that laboratory report includes each of the following elements:

a) A statement of adequacy of the specimen for diagnostic evaluation.
b) A general categorization of the diagnosis (within normal limits or other)
c) The descriptive diagnosis, using the following terminology and classification

Defi ni ti on and Cri teri a for Speci men Adequacy

The specimen is Satisfactory for evaluation indicates that the specimen has all
of the following:

1. Appropriate labeling and identifying information
2. Relevant clinical information
3. Adequate numbers of well-preserved and well-visualized squamous epithelial
cells. The epithelial cells should cover more than 10% of the slide surface.
4. An adequate endocervical/transformation zone component (form a patient
with a cervix). It means, at a minimum, of two clusters of well-preserved
endocervical glandular and/or squamous metaplastic cells, with each cluster
composed of at least five cells. In cases of marked atrophic changes in post-
menopausal women, absence of transformation zone component, however,
does not affect specimen adequacy.

A specimen is Satisfactory for evaluation but limited by... if any of the
following apply:

1. Lack of pertinent clinical information (age, date of last menstrual period as a
minimum; additional information as appropriate)
2. Partially obscuring blood, inflammation, thick areas, poor fixation, air-drying
artifact, contaminant, etc. that precludes interpretation of approximately 50%
to 75% of the epithelial cells.
3. Absence of an endocervical/transformation zone component as defined above.

Satisfactory for evaluation but limited by... indicates that the specimen provides
useful information but interpretation may be compromised. Absence of
transformation zone component does not necessarily require a repeat smear. Patient
factors such as location of transformation zone, age, pregnancy, and previous therapy
may limit the clinicians ability of obtain an endocervical sample. The ultimate
determination of specimen adequacy rests with the clinician, who must correlate
cytopathology report with clinical knowledge of the individual patient.

A specimen is Unsatisfactory for evaluation... if any of the following apply:
1. Lack of patient identification on the specimen and/or requisition form.
2. A slide that is broken and cannot be repaired.
3. Scant squamous epithelial component (well-preserved and well-visualized
squamous epithelial cells covering less than 10% of the slide surface).
171
4. Obscuring blood, inflammation, thick areas, poor fixation, air-drying artifact,
contaminant, etc. that precludes interpretation of approximately 75% or more
of the epithelial cells.

The Unsatisfactory... designation indicates that the specimen is unreliable for the
detection of cervical epithelial abnormalities.

However, if abnormal cells are detected, the specimen is never categorized as
Unsatisfactory. Such cases may be regarded as Satisfactory or Satisfactory but
limited by... based on the above criteria.

Descriptive Diagnoses

Benign Cellular Changes

I nfecti on

Trichomonas vaginalis
Fungal infection e.g. Candida spp.
Predominance of coccobacilli consistent with shift in vaginal flora.
Bacteria morphologically consistent with Actinomyces spp.
Cellular changes associated with Herpes simplex virus
Other

Reacti ve Changes
Reactive cellular changes associated with:

Inflammation (including typical repair)
Atrophy with inflammation (atrophic vaginitis)
Radiation
Intrauterine contraceptive device (IUD)
Other

Epithelial Cell Abnormalities

Squamous Cell

Atypical squamous cells of undetermined significance (ASCUS):
Low-grade squamous intraepithelial lesion (LSIL)
Encompassing: Human papillomavirus (HPV)/mild dysplasia/cervical
intraepithelial
neoplasia (CIN) 1
High-grade squamous intraepithelial lesion (HSIL)
Encompassing: Moderate dysplasia, severe dysplasia, and carcinoma in situ/CIN
2
and CIN 3
Squamous cell carcinoma

Glandular Cell

Endometrial cells, cytologically benign, in a postmenopausal woman
172
Atypical glandular cells of undetermined significance (AGUS): Qualify
Endocervical adenocarcinoma
Endometrial adenocarcinoma
Extrauterine adenocarcinoma
Adenocarcinoma, NOS

APPENDICES

Appendix-A

Preparation of Stains and Chemicals used in Papanicolaous Stain

Graded Alcohols. The formulas are listed in the Table 2.

Table 2
Preparation of Graded Alcohols (1,000 ml)

Desired Volume of Volume of
concentration Water 95% Alcohol*
(%) (ml) (ml)

50 474 526

70 263 737

80 160 840

* Prepare 95% ethyl alcohol by mixing 50 ml of water with 950 ml of absolute
alcohol.

Bluing Solutions. There are several formulas for bluing solutions of
approximately equal value.

a) Ammonium hydroxide in 70% alcohol
Add 15 ml of NH4OH (28% to 30% weight/volume concentration) to 985 ml of 70%
ethanol

b) Lithium carbonate
Stock solution: 1.5 g of LiCO3 in 100 ml of water.
Working solution: Add 30 drops of stock solution to 1,000ml of water.

c) Tap water can be used as a bluing agent if pH is higher than 8.

Carazzis Haematoxylin. This double-strength modification of Carazzis
original formula can be used as a progressive stain.

Ingredients:
173

Haematoxylin 2 g
Glycerol 200 ml
Potassium alum (Potassium aluminium hydroxide) 50 g
Potassium iodate 0.4 g

Procedure:
1. Dissolve the haematoxylin in the glycerol.
2. Dissolve the alum in 700 ml of water.
3. Allow to stand overnight.
4. Add the alum solution to the haematoxylin solution, mixing thoroughly.
5. Dissolve the iodate in the remaining 100 ml of water. Avoid heating if
possible.
6. Add the iodate solution to the haematoxylin mixture, mixing constantly.
7. Filter into Coplin Jar for use. Solution is ready for use immediately. It has
reliable storage life of 6 months at room temperature, and it will remain usable
for longer if stored at 4

Orange G 6:

Ingredients:

Orange G crystals 5 g
95% ethyl alcohol 950 ml
Phosphotungstic acid 0.15 g

Procedure:

1. Prepare 10% aqueous solution by dissolving 5 g orange G in 50 ml distilled
water. Shake well and allow the solution to stand for a week.

2. Add 950 ml of ethyl alcohol to make 1,000 ml of solution.

3. Add 0.15 g phosphotungstic acid and store the final solution in dark brown
glass bottles with stopper.

EA-65.

Ingredients:

Light green SF yellow, C.I. No. 42095
Bismarck brown, C.I. No. 21000
Eosin Y
174
Phosphotungstic acid
95% Ethyl alcohol
Distilled water

Procedure:

Preparati on of stock soluti ons-

Aqueous Stock Solutions for EA

A: 2% Light green SF yellow,: Dissolve 2 g of Light green SF yellow in 100 ml of Dist.
water.

B: 10% Bismarck brown: Dissolve 10 g of Bismarck brown in 100 ml of Dist. water.

Alcoholic Stock Solutions Made From the Aqueous Solutions

C: 0.1% Light green: 50 ml of Solution A + 950 ml of 95% ethyl alcohol.

D: 0.5% Bismarck brown: 5 ml of solution B + 95 ml of 95% ethyl alcohol.

E: 0.5% Eosin: 5 g eosin + 1,000 ml of 95% ethyl alcohol.

Preparati on of Eosi n-Alcohol-65 (EA-65) stain-

1. Mix 225 ml solution C with 100 ml of solution D. Then add 6 g of phosphotungstic
acid. To this solution add 450 ml of solution E. Finally add 225 ml of 95% ethyl
alcohol to prepare 1,000 ml of EA-65.

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