Primary Sample Collection Manual - English - Iss. 02 - Rev.01

Cover Page
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Document Number Document Title
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M/PEP/SCT/02 Primary Sample Collection Manual

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Reference to CAP / ISO Standard:

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Standard / Guidelines Clause No. Title

ISO 15189:2012 5.4 Pre-examination Processes
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ISO 9001:2015 8.5 Production & Service Provision

CAP (Version 22.08.2018) GEN.40100 Specimen Collection Manual
Elements
GEN.40125 Handling of Referred Specimens
GEN.40490 Patient Identification
GEN.40491 Primary Specimen Container
Labelling
GEN.40492 Specimen Label Correction
GEN.40501 Phlebotomy Adverse Reaction
GEN.40502 Chain-of-Custody Procedures
GEN.40511 Specimen Tracking/Labelling
Primary Sample Collection Manual
GEN.40530 Specimen Tracking

GEN.40535 Specimen Transport QM
GEN.40750 Requisition Elements
GEN.40930 Authorized Requestor
COM.06000 Specimen Collection Manual
COM.06100 Primary Specimen Container
Labelling
COM.06200 Secondary Specimen Container
Labelling
COM.06300 Specimen Rejection Criteria
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CHM.12133 Aliquoting
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Doc. No: M/ PEP/SCT/02 Primary Sample Collection Manual

Issue No: 02 Issue Date: Effective from: Rev. No: 01 Rev. Date: Page 2 of 35
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Prepared by GM-QA Issued by GM-QA
operations Approved by CD operation

RELEASE AUTHORISATION
This Primary Sample Collection Manual
is
released under the
authority of
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Dr. Sanjeev Shah
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&
is the property of
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Sterling Accuris Diagnostics, a unit of ‘‘Sterling Accuris
Wellness Pvt. Ltd.’’

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Dr. Sanjeev Shah
Clinical Director
Place: Ahmedabad

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Table of Contents
Cover Page ....................................................................................................................................... 1
RELEASE AUTHORISATION ............................................................................................................ 1
Table of Contents .............................................................................................................................. 2
1.0 REVISION HISTORY ............................................................................................................. 4
2.0 READ & UNDERSTOOD FORM ............................................................................................ 5
3.0 Overview................................................................................................................................ 6
4.0 Phlebotomy – in general......................................................................................................... 6
A. Test Requisition Form ............................................................................................................ 7
B. Patient Preparation / Instructions for various Tests ................................................................. 7
 Blood Specimens ............................................................................................................... 7

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24 hours Urine Collection ................................................................................................... 7
 Urine collection for Urine Routine Examination ................................................................... 8
 Obtaining urine sample from catheter ................................................................................. 8

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Stool specimen:.................................................................................................................. 9
 Semen specimen:............................................................................................................... 9

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Glucose Tolerance Test: .................................................................................................... 9
 Timed Collection: ............................................................................................................. 10
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 Single Specimen: ............................................................................................................. 10

 Therapeutic monitoring of patients on medication: ............................................................ 10
 Molecular, infectious disease serology & genetic testing ................................................... 11
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C. Blood Specimen Collection / Phlebotomy ............................................................................. 11


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Basic steps for Drawing a Blood Specimen....................................................................... 11

 Selection and preparation of Vein site for Blood Collection:............................................... 12
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 Procedure for vein selection ............................................................................................. 12

 Disinfections of the Venipuncture site ............................................................................... 13
 Applying the Tourniquet.................................................................................................... 13
 Blood collection by Closed System (Vacutainer) ............................................................... 14
 Winged Collection (Butterfly) with Evacuated Tubes or Syringe (Open draw system) ........ 14
 Blood Culture Collection ................................................................................................... 15
 Central Line Catheter ....................................................................................................... 16
 Preventing hematoma during venipuncture ....................................................................... 17
 Tube Chart & Order of Draw ............................................................................................. 17
 Collection of blood from pediatric patients:........................................................................ 18
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 Procedure to be followed post blood collection: ................................................................ 18

 In case of continued bleeding ........................................................................................... 18
 Dispose of puncturing unit ................................................................................................ 18
 Troubleshooting hints for blood collection: ............................................................................ 19
5.0 Body fluids ........................................................................................................................... 22
6.0 Tissue biopsy for histopathology/ IHC................................................................................... 23
7.0 Microbiological Specimens ................................................................................................... 23
7.1 Blood culture .................................................................................................................... 23
7.2 Cerebral Spinal Fluid (CSF).............................................................................................. 23
7.3 Sputum ............................................................................................................................ 24
7.4 Bronchoscopy, Endotracheal Aspirates, Transtracheal Aspirates: ..................................... 24
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7.5 Pus .................................................................................................................................. 24
7.6 Urine ................................................................................................................................ 25
7.7 Stool ................................................................................................................................ 25
7.8
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Throat swab ..................................................................................................................... 26
7.9 Cutaneous (Fungal only) .................................................................................................. 26
8.0 Specimen Collection for Influenza / COVID-19 ..................................................................... 27
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8.1 Nasopharyngeal Swab collection ...................................................................................... 27
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8.2 Deep nasal swab collection .............................................................................................. 28

8.3 Combined nasal & throat swab ......................................................................................... 29
9.0 Specimen Transportation ..................................................................................................... 30
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9.1 Blood, Urine / stool & specimens for microbiology:............................................................ 30

9.2 Histopathology & Cytopathology specimens: .................................................................... 30
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9.3 Frozen Section: ................................................................................................................ 31

9.4 Renal biopsy specimens: .................................................................................................. 31
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9.5 For Influenza / Covid-19 specimens.................................................................................. 32

10.0 Specimen rejection criteria ................................................................................................... 32
10.1 Rejection criteria Clinical specimen in general .................................................................. 32
10.2 Rejection criteria for flu specimens ................................................................................... 33
11.0 References: ......................................................................................................................... 33

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1.0 REVISION HISTORY
Sr. Page Section Revision Reason for Date of Rev. Sign of CD

No. No. No. details Revision Revision No.
1. 16 4.0 (C) Central Line Findings 12.04.2022 01
Catheter during the
blood draw NABL
procedure assessment
added
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2.0 READ & UNDERSTOOD FORM

I hereby declare that I have read and understood this document.
Sr. No. Name Emp. ID Designation Date Sign
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3.0 Overview
Phlebotomy – the drawing of blood – has been practiced for centuries and is still one of the most
common invasive procedures in health care. However, practice varies considerably between countries,
and between institutions and individuals within the same country. These differences include variations
in blood-sampling technique, training (both formal and “on-the job”), use of safety devices, disposal
methods, reuse of devices and availability of hepatitis B vaccine. Valid laboratory results are dependent
upon proper specimen collection and handling prior to the arrival of the sample in the laboratory.
The following is a basic phlebotomy procedure, followed by procedures specific for each type of
collection.
4.0 Phlebotomy – in general

1. Wash hands thoroughly before beginning any phlebotomy procedure. Be sure to check
expiration dates on tubes before proceeding. DO NOT USE EXPIRED TUBES.
2. Confirm the identity of the patient by checking at least two identifiers before collecting the
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specimen(s). This can be done by asking the patient to state their full name and
requesting to see the patient's ID wrist band.
3. Explain the procedure, including small risk of hematoma, slight pain, and some light-
headedness. Inquire whether the patient has a history of fainting or dizziness with
phlebotomy procedures so that blood can be collected in a reclining position. Explain that
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loss of vacuum or a collapsed vein may necessitate another draw.
4. On a table or desk, assemble all necessary equipment: cotton balls and/or gauze, tubes,
safety needle, alcohol swab, tourniquet, gloves, and bandage. Wearing safety gloves is
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MANDATORY. Wear additional protective equipment if contamination is expected.
Safety needles should always be used; the only exception is if the patient is very hard to
draw then a butterfly needle set may be used.
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5. Position the patient so that they are seated comfortably in a chair with their arm extended
on an armrest, desk, or table to form a straight line from the shoulder to the wrist. The
patient’s arm and elbow should be firmly supported, and not bent at the elbow.
6. Check both arms to select the larger and fuller veins. Palpate and trace the path of the
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veins several times with your index finger. Tap the vein at the site of the draw with your
index finger and second finger. This will cause the vein to dilate.
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The following factors should be considered in site selection:

i. Extensive scarring. Healed burn areas or scar tissue should be avoided.
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ii. Specimens collected from an area with a hematoma may yield erroneous test
results. If another vein site is not available, the specimen should be collected distal
to the hematoma.
7. Apply the tourniquet.

8. Ask the patient to open and close his/her fist so their veins become prominent. Vigorous
hand pumping is not necessary to activate blood flow and should be avoided.
9. Clean the venipuncture site with the alcohol swab in a circular motion from the centre of
the area to the outside. Allow the area to air-dry to prevent haemolysis and a burning
sensation to the patient. Insert the stopper of the first tube to be drawn into the adaptor.
Do not push too far to avoid premature loss of vacuum via puncture of the needle.
10. Insert the needle into the vein with the bevel facing upward. Puncture the stopper on the
tube by pushing it onto the end of the needle, and grasp the edge of the adaptor to provide
stability once the blood flow has begun. Have the patient open his/her fist.

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11. Fill the tube until the vacuum is exhausted. Remove the tube from the adaptor and insert
subsequent tubes. Be sure that all tubes are completely filled to ensure sufficient blood
sample for laboratory analysis.
12. Place a cotton ball or 2 x 2 square piece of gauze over the site. All used needles must
be disposed of in a puncture proof biohazard receptacle. Never recap a needle.
Recapping, purposeful bending, breaking, removing from disposable syringes, or other
manual manipulations of needles is prohibited. Apply pressure to the site for 2-5 minutes.
Place a bandage over the puncture site.
13. Again, verify that the information on the sample tubes match the consent/requisition form.
14. Remove gloves and dispose of in a properly identified biohazard bag or container. Wash
hands thoroughly after phlebotomy.
A. Test Requisition Form

Test requisition form should be filled with the following information:
1. Patient details
2. Specimen details
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3. Referring doctor details
4. Clinical History (where applicable)
Vacutainers and other containers should be labelled with:

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Patient’s full name, Accession number (generated after collecting the sample from software), patient’s
location & Date.
Specimen Details as to type and site of collection - whether collection is done under fasting, postprandial
condition or any other specification.
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B. Patient Preparation / Instructions for various Tests

There are few lab investigations in which consent from patient has to be taken before collecting the
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blood specimen for those tests. The counselling for the same will be done by patient’s
physician/pathologist.
Investigations Consent form

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HIV: HIV Counselling and Consent form (F/PEP/03)

FNAC FNAC Consent form (F/PEP/06)
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Bone marrow Bone marrow aspiration Consent Procedure (F/PEP/HIS/03)
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Blood Specimens
1. Fasting Plasma Glucose:
Fasting period of 8-10 hours is required. Blood & first voided midstream morning urine for urine
glucose are to be tested.
2. Postprandial Plasma Glucose:
Blood and urine specimens collected at 2 hours after completion of meals.
3. Lipid Profile:
8 – 10 hours of fasting required.
• 24 hours Urine Collection

Instruct the patient about the preservative and restrict patient from discarding the same.
To start collection: - Ask the patient to discard the first urine passed in the morning. Note down the
exact time (i.e. 7:00 am).
From this time onwards collect all subsequent urine samples in the container provided. Collection
should be continued till same time the next day (i.e. last void at 7:00 am next day).
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The patient should return the 24 hours’ urine sample containers within 1 hour to the pathology
department.
•24 hours’ collection shall be done in clean can in which the preservative is added wherever
necessary.
• Patients should not collect the first urine sample in the can thereafter all urine has to be
collected in the container for the next 24 hours. The container should be refrigerated till
submission on the next day to the laboratory.
HCL – 15 ml, Use this as preservative in following tests
• VMA
• Metanephrines
Plain container for urinary calcium, magnesium, phosphorus, copper and uric acid should be used.
Toluene – 10 ml, Use this as preservative in following tests
• Protein
• Creatinine
• Sodium
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• Potassium
• Chloride
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Urine collection for Urine Routine Examination
Specimen: Urine specimen must be first voided mid-stream morning Urine. It must be collected in the
clean plastic container.
Instruction given to the patient: Patient must be instructed to void directly in to the container. During
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the collection the initial portion of urine stream is allowed to escape while the mid-stream portion is
collected.
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Preservation: In case of delay in testing, urine must be stored at 2 to 8 °C (Lower cooling compartment
of the refrigerator).
• Obtaining urine sample from catheter

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The process of obtaining a sample of urine from a patient with an indwelling urinary catheter must be
obtained from a sampling port. The sample must be obtained using an ascetic technique.
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The port is usually situated in the drainage tubing, proximal to the collection bag which ensures the
fresh net sample possible. Avoid the use of drainage systems without sampling port. Specimens should
not be collected from the tap from the main collecting chamber of the catheter bag. Follow below
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procedural steps to collect the sample.
1. If no urine is visible in the tubing, apply a non-traumatic clamp/gate clip a few centimetres distal
to the sampling port.
2. Once sufficient urine has collected in the tube, wipe the sampling port with an alcohol-
impregnated swab.
3. Stabilising the tube below the sampling port, insert the needle into the port at an angle of 45.
4. In a needle-free system, insert the syringe into the sampling port according to the manufacture’s
recommendations.
5. Aspirate the required amount of urine (minimum 10 ml).
6. Remove syringe/needle.
7. Dispose of sharps in sharp collector.
8. Inject the urine into urine container.
9. Wipe the sampling port with an alcohol-impregnated swab, allow it to dry.
10. Unclamp the catheter tubing as required.

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NOTE:
Urine/Sputum specimen must be collected in the 50- ml capacity non-sterile screw top plastic container.
Urine specimen must be collected in the 50- ml capacity sterile screw top plastic container for urinary
Sodium / Potassium / Chloride / Osmolality / myoglobulin.
Urine specimen must be collected in the 50- ml capacity dark sterile screw top plastic container for
urinary porphobilinogen.
Stool specimens (preferably morning samples) must be collected in leak proof containers with the help
of spatula provided within the container. A spoon-full of stool specimen is enough for routine
examination.
• Stool specimen:
No special preparation is required before collecting a stool specimen (sample). Patient must be given
a sterile container with spatula to collect the specimen. The containers can be requested from pathology
laboratory by sending “specimen container requisition slip’.
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Following procedure must be explained to patient for collecting the stool specimen.
1. Urinate before collecting the stool so that you do not get any urine in the stool sample. Do not
urinate while passing the stool.
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2. Stool can contain material that spreads infection, so wash your hands before and after you
collect the specimen.
3. Pass stool (but no urine) into a cleaned plastic cup / disinfected clean bedpan. Cleaned plastic
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cup can be placed in toilet seat to catch the stool sample.
4. Either solid or liquid stool can be collected with help of spatula attached with stool container
cap.
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5. Do not mix toilet paper, water, or soap with the sample.

6. Place the lid on the container and label it with your name and the date the stool was collected.
7. Deliver the stool specimen immediately to the pathology department.
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• Semen specimen:
The semen specimen should be collected after at 3-7 days of sexual abstinence unless otherwise
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instructed by physician.
Give following instructions to patient for collecting semen specimen.
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1. Collect the specimen by masturbation into a sterile container (containers are provided by the
laboratory). Collecting semen in a condom or by coitus interruptus is not acceptable.
Since the volume of semen produced may be significant to diagnosis, it is important to submit
the entire specimen. The container lid should be closed tightly.
2. Label the container with patient’s name, date of collection, and exact time of collection.
3. Keep the sample near body temperature (25°-40° C or 77°-104° F) during transportation.
• Glucose Tolerance Test:

Specimen: Fasting Blood Specimen must be collected in the grey top sodium Fluoride vacutainer
to a full draw and fasting urine specimen must be collected in clean urine container.
The patient must be given 75 grams of Glucose (anhydrous) or 82.5 gm of Glucose (monohydrate). The
time is noted down for intake of glucose. For pregnant patient, 100 gm of glucose (anhydrous) or 110
gm of glucose (monohydrate) shall be given. Two Blood specimen and urine specimen are collected at
one hourly interval for subsequent two hours for GTT 2 samples & at 30 minutes’ interval for subsequent
five hours for GTT 5 samples. At every interval one blood specimen and urine specimen is collected.

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Each of these specimens is labelled appropriately indicating specimen type and time of collection.
Fasting Blood Specimen must be collected in the grey top sodium Fluoride Vacutainer to a full draw
and fasting urine specimen must be collected in clean urine container.
Instruction to be given to the patient:

The patient should be on a balanced diet (containing normal daily requirement of carbohydrates
approximately 300 mg) at least for 2 to 3 days prior to the test.
Patient must come to the laboratory after adhering to 8 to 10 hours fasting period.
Patient must collect a fasting midstream urine specimen in a clean dry container and submit it.
Since subsequent samples have to be collected at an interval of one hour/30 minutes each, the patient
has to be made aware of the waiting period of 2 to 3 hours in fasting condition at the pathology
department.
• Timed Collection:
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For some hormonal studies specimens are collected at specific timing e.g. cortisol test. The blood
specimens are collected at 8:00 am and 4:00 pm. Fasting condition is recommended in tests like PTH,
folic Acid, Vitamin B12.
The timing of blood collection as clinical significance and hence must be clearly specified on the
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vacutainer. The serum / Plasma must be separated at the earliest.
Fresh sample is required in case ABG, Lactate (Arterial/Venous), Ammonia, Alcohol Level, PTH.
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Single Specimen:
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Here are some instances in which timed single specimens may be required.
Fasting plasma glucose alone or in conjunction with a random glucose determination (fasting here is
defined as no caloric intake for at least 8 hours.)
Post Prandial Glucose may be performed 2 hours after a meal for a Timed Test that is helpful in diabetes
detection.
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Blood Glucose Determination may be ordered at a specific time to check the effect of insulin treatment.
Blood Cultures may be ordered for a specific time if a blood stream bacterial infection is suspected.
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• Therapeutic monitoring of patients on medication:

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Multiple Specimens:
Here are some instances in which timed multi specimen tests may be ordered.
The most common timed procedure is a glucose tolerance test. First, a blood specimen is drawn from
a fasting patient. Then, the patient is given glucose orally and blood specimens are drawn at fixed
intervals, beginning with a 1 our specimen.
To test the effect of a certain medication, a physician may order the same tests to be obtained on
consecutive days, before, during, and after the patient has received medication.
Collection of an acute and convalescent Serum to aid in the diagnosis of a Viral Infection when culturing
is not feasible.
Other examples include such test as occult blood, ova and parasites, and blood cultures.
Serial Monitoring:
Monitoring a patient over time for a specific condition is a variation of sequential sampling. Many tumour
markers (tests used to follow the patient’s response to treatment for cancer) are monitored over the
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course of several years. Specific instructions for serial monitoring are found with the test being
monitored.
• Molecular, infectious disease serology & genetic testing

Clinical molecular genetic testing provides for detection of genetic variations that offer the promise of
revolutionizing personalized medicine and altering the way physicians diagnose and treat illness. At
present, clinical molecular diagnostic technologies focus on
• identifying whether an individual has a certain genetic disease
• identifying whether an individual has an increased risk of a particular disease
• classifying an individual’s genetic makeup to determine whether a drug and dosage is suitable
for that particular patient
• examining the whole genome to discover genetic alterations that may cause disease.
Before specimen collection, essential information must be obtained. The patient’s demographics are
required and an informed consent form must be signed. Sterile whole-blood specimens for molecular
genetic testing are generally collected in the lavender-top EDTA tubes or tubes specially designed for
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this type of diagnostic study, that is, white-top gel tubes. Other anticoagulant tubes used for genetic
testing could contain ACD (Anticoagulant Citrate Dextrose Solution), sodium citrate or sodium heparin.
When a test for RNA (Ribonucleic acid) is ordered, the blood specimen must be sent for testing
immediately or collected in a special stabilizing reagent. Blood specimens will be rejected if frozen,
haemolyzed, or clotted. Ideal handling is to ship immediately at ambient temperature for overnight
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delivery. In hot weather a cool pack can be enclosed. Specimens for molecular genetic testing can be
refrigerated up to 7 days before shipping. In this particular area of laboratory testing, major advances
are being made daily. It is important for the Healthcare workers involved in collection and handling of
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this type of specimen stay current in facility protocol.
The term serology means the study of serum. Serology tests deal with the body’s response to the
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presence of bacterial, viral, fungal, or parasitic diseases stimulating antigen– antibody reactions that
can easily be demonstrated in the laboratory. Autoimmune reactions, in which autoantibodies produced
by B lymphocytes attack normal cells, are becoming more prevalent and can be detected by serologic
tests. Testing can be done by polymerase chain reaction (PCR), enzyme-linked immunosorbent assay
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(ELISA), fluorescent antibodies, agglutination, or precipitation to determine the antibody or antigen

present and to assess its concentration or titer.
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C. Blood Specimen Collection / Phlebotomy

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The phlebotomist/nursing staff should be well trained in blood collection with syringe and needles as
well as Vacutainer System. He/she is informed and shown that the vacutainer system, which consists
of, a vacutainer needle, a needle holder and a vacutainer tube instead of the syringe barrel and plunger.
In vacutainer system, once the vein is punctured, the requisite quantity of blood flows automatically into
vacutainer tube so that the needle to pull the plunger is obviated. Appropriate anticoagulants are pre-
added in appropriate quantities in vacutainer so that all is required are a clean venipuncture and
collection of blood to a full draw. Vacutainers are simpler to use and safer. The Vacutainers system is
a cleaner system, as blood does not come in contact with the atmosphere as it flows straight from the
vein though the sterile needle into the sterile tube. Contamination from spilled blood is entirely removed.
• Basic steps for Drawing a Blood Specimen

Check and confirm with the patient details given on the test requisition form.
Ascertain whether the patient is fasting and record the same on the Test Requisition Form.

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Assemble the vacutainer, needle holder, alcohol/spirit swab, cotton swab, tourniquet etc. required for
phlebotomy. The phlebotomist should select the appropriate type of needle based on patient’s physical
characteristics and amount of blood to be drawn. Keep vacutainer/s in order to draw.
Reassuring the patient: The phlebotomist must gain the patient’s confidence and assure him that
although the venipuncture is slightly painful, it would be of a short duration. Panic or anxiety of the
patient will lead to difficulty in collecting the specimen.
Positioning the Patient: The patient should be made to sit comfortably in a chair and should
position his/her arm on a slanting arm rest, extending the arm straight from the shoulder without bending
at the elbow.
Unless contra-indicated if the patient wants to lie down, let the patient lie comfortably on the back. The
patient should extend the arm straight from the shoulder. For support, a pillow may be placed under the
arm.
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Check the prescription, test requisition form, vacutainer type and their labels to ensure that all the details
are matching and are appropriated for the test ordered, before proceeding for collection.
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• Selection and preparation of Vein site for Blood Collection:
Selecting vein site: For most venipuncture procedures on adults, veins located on the arm are used.
The Median Cubital Vein is most commonly used for the patient. If the venipuncture of this vein is
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unsuccessful, one of the cephalic or basilic veins may be used.
The following conditions should be avoided while selecting a Vein:

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1. Vein from area having extensive scarring.

2. Phlebotomy should not be performed on any size of haematoma.
3. Specimens should not be collected from the arms having intravenous infusions.
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Patients on IV therapy for extended periods often have veins that are palpable & visible but partially
occluded or sclerosed. Avoid using the arm with IV line. Instead, use the other arm/site. Where both
arms have IV devices, collection should be distal (upstream), from the point of infusion. If proximal
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(“downstream”) site cannot be avoided, the IV line can be disconnected after obtaining permission from
the Physician or nurse in charge of the patient. After a minimum of 30 minutes delay and with the first
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drawn tube discarded, samples can then be collected. In case, when blood sample is to be collected
from C.V.P. line, the infusion has to be stopped. 10 ml N.S. should be flushed and 20 ml of blood should
be withdrawn with new syringe. The sample should now be collected with another syringe in appropriate
collection tubes.
The withdrawn blood should be reintroduced to the C.V.P. line. The C.V.P. line should be reconnected
with infusion.
• Procedure for vein selection
Locating veins: To locate veins it is necessary to palpate and trace the path of the veins several
times with index finger.
Alternate site: Sight such as dorsal wrist or hand and ankles or lower extremities may be required
for patients with difficult veins.
If a vein is difficult to find, it may become easier to see after massaging the arm from the wrist to elbow,
which forces blood into the vein. You may need to examine the patient’s other arm if you are having
difficulty finding a vein. You may select a dorsal hand or wrist vein and collect with a smaller gauge
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needle (22g or 23g).

Release the tourniquet. Hemoconcentration will occur after one minute.
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• Disinfections of the Venipuncture site

The puncture site must be cleansed to prevent microbiological contamination of the specimen and
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infection at the venipuncture site.

Cleansing should be done with gloved hands. Spirit or 70% iso-propyl alcohol is used for disinfections.
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A cotton ball should be soaked in the spirit excess should be dripped away the cleaning should start
from the vein and move out in a circular motion towards the other surface. Allow the area to air dry to
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prevent haemolysis of the specimen give enough contact time for the alcohol to bring out the
disinfections of venipuncture and prevent the patient from experiencing a burning sensation when the
venipuncture is performed. Once disinfected this site should not be touched with bare hands.
• Applying the Tourniquet
A tourniquet is used to increase venous filling. This makes the vein more prominent and easier to enter.
Precautions when using a tourniquet: The tourniquet should be released after no more than
one minute. Local stasis can occur with hemo-concentration and the possible formation of a haematoma
due to infiltration of blood into tissue. This may result in erroneously high values for tests like PT, APTT,
PCV, K+ etc and other Cellular elements.
Tourniquet tying Location: Apply the tourniquet around the arm 3 to 4 inch above the
venipuncture site. The tourniquet must be tied, should never be left on the arm for more than 2 minutes
because a tourniquet prevents blood from flowing freely.
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• Blood collection by Closed System (Vacutainer)
1. Screw the vacutainer needle into the vacutainer holder.

2. Introduce the Vacutainer needle apparatus with the bevel up at a 15- to 30-degree
angle to the skin and parallel to the vein.
3. Once the needle is properly positioned in the vein, anchor the needle by grasping the
holder with thumb on top and other fingers under the holder, resting securely on the
patient’s arm. Push the appropriate Vacutainer tube into the holder with gentle pressure
in order to puncture the cap. The tube will automatically fill with blood.
4. Watch the blood as it flows into the Vacutainer tube until collection is complete.
5. For draws requiring multiple tubes, the tubes must be drawn as per order of draw (Refer
Table 2):
6. Gently invert all tubes 5 - 10 times after filling (as per company’s Vacutainer mixing
chart).
7. Label specimen tubes before leaving the patient with barcode or name, age, date &
time.
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8. Compare the labelled specimen(s) to the patient’s identification number,
requisition or ask the patient to confirm the tube is properly labelled with the
correct spelling of first and last name.
9. Wash hands thoroughly after removing gloves.
LL
• Winged Collection (Butterfly) with Evacuated Tubes or Syringe (Open draw
system)
O
This method is used primarily for difficult draws, hand draws, infant draws and blood culture
collections. For pediatric and neonatal patients, the choice of site and procedure (venous
TR
site, finger-prick or heel-prick – also referred to as “capillary sampling” or “skin puncture”)

will depend on the volume of blood needed for the procedure and the type of laboratory test
to be done.
1. Prepare patient as defined under “Patient Identification & preparation” section. Perform
phlebotomy with winged butterfly set using a Vacutainer holder or remove the lure lock
N
at the end of the butterfly tubing and attach a syringe. Use a winged steel needle,
preferably 23 or 25 gauge.
O
2. Wash hands. Put on properly fitting gloves.

3. Select the puncture site for paediatric or neonatal patients – heel or finger. In heel-
C
pricks, the depth should not go beyond 2.4 mm. For premature neonates, a 0.85 mm
lancet is available. The distance for a 7-pound (3 kg) baby from outer skin surface to
bone is: a) medial and lateral heel – 3.32 mm; b) posterior heel – 2.33 mm (this site
should be avoided, to reduce the risk of hitting bone); c) toe – 2.19 mm. The
recommended depth for a finger-prick is: a) for a child over 6 months and below 8 years
– 1.5 mm; b) for a child over 8 years – 2.4 mm.
4. Immobilize the baby or child with the help of another phlebotomist and parents. Ask the
immobilizer to stretch an arm across the table and place the child on its back, with its
head on top of the outstretched arm; pull the child close, as if the person were cradling
the child; grasp the child’s elbow in the outstretched hand; use their other arm to reach
across the child and grasp its wrist in a palm-up position.
5. Holding the wings of the butterfly with your dominant hand, smoothly insert the needle
with the bevel up, parallel to the vein, at approximately a 10- to 15-degree angle.
6. Once the needle is properly positioned in the vein, hold one wing of the winged
collection set and insert evacuated tubes using the vacutainer holder according to the
order of draw. For syringe draws, gently pull on the plunger to allow blood to flow into
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syringe. Pulling on the plunger too fast may cause possible collapse of the vein and
restrict blood flow into the syringe and/or haemolyze the sample.
7. When sufficient blood has been collected, release tourniquet within one minute.
8. Place gauze over the site, and remove the needle from the patient’s arm. Activate the
safety feature of the device.
9. Apply pressure to the site until bleeding has stopped.
10. Discard the holder and butterfly device into a sharp’s container.
11. If a syringe was used with the butterfly, properly discard butterfly device into a sharp’s
container, and attach a transfer device to the syringe. Fill the appropriate tubes without
applying force on the plunger.
12. The order in which vacutainer tubes are filled from the syringe is the same as for the
vacutainer system.
13. Inspect the puncture wound. When the bleeding has stopped completely, apply a
bandage. If bleeding continues, apply pressure for an additional three-five minutes.
Prolonged bleeding may be related to the patient’s disease or medication.
Exception: A bandage should never be applied to patients younger than 2 years unless
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the patient is under close observation by an adult until the bandage is removed. Small
children may choke on bandages. If a bandage is applied, instruct the adult to remove
it within two hours.
14. Label specimen tubes before leaving the patient as defined in the labelling section of
this procedure.
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15. Compare the labelled specimen(s) to the patient’s identification number,
requisition or ask the patient to confirm the tube is properly labelled with the
correct spelling of first and last name.
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•
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Blood Culture Collection

NOTE: Each set of blood cultures requires a separate venipuncture.
1. Prepare patient as defined in “Patient Identification & preparation” section.
2. Wash hands. Put on properly fitting gloves.
N
3. Cleanse the rubber stoppers on the blood culture bottle(s) with 70 % isopropyl alcohol.
Do not use Betadine. Allow to air dry completely.
4. Apply tourniquet and select venipuncture site.
O
5. Remove tourniquet, Cleanse the area for venipuncture in a circular motion from the
centre to outward with a 70% isopropyl alcohol pad. Cleanse the site again with iodine
C
in a circular motion from the centre outward and followed by 70% isopropyl alcohol.
Allow to air dry for 1 minute.
6. The site must dry completely. Do not palpate or blow on the vein after it has been
disinfected. For new-born’s, allow the iodine to dry at least one minute and then remove
with sterile water prior to venipuncture.
7. Reapply tourniquet and perform the venipuncture following procedures described on
preceding page using a winged collection needle and sterile syringe obtaining up to 20
ml blood. For new-borns, draw 0.5 to 1.0 ml blood. Refer table for required blood
draw volume on blood culture vial.
8. Perform the venipuncture without retouching the site.
9. If absolutely necessary to palpate the site of venipuncture, put on sterile gloves after
swabbing the site.
10. If multiple specimens for testing need to be obtained at the same time, draw blood
cultures first with the syringe attached, or attach a second syringe to collect blood for
other samples. A vacutainer adapter may be attached if necessary to draw the
remainder of the samples.
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11. Release the tourniquet (within one minute), place gauze square over the puncture site
and withdraw the needle. Activate the safety device of the butterfly.
12. Remove safety activated winged collection set from the syringe and put into the sharp’s
container.
13. Apply a blood transfer device to the syringe. Inoculate the anaerobic bottle first (do not
allow any air to enter the anaerobic bottle), then inoculate the aerobic bottle.
14. Divide the specimen equally, approximating the amounts. If less than 1 ml is obtained
for paediatric or infant blood cultures, inoculate only the aerobic bottle unless otherwise
instructed by the physician.
15. Discard the syringe and transfer device into the sharp’s container.
16. Apply pressure to the site until bleeding has completely stopped. Inspect the puncture
wound.
17. When the bleeding has stopped, apply a bandage. If bleeding continues, apply pressure
until bleeding has stopped. Prolonged bleeding may be related to the patient’s disease
or medication.
18. Label specimen bottles before leaving the patient as defined in the labelling section of
ED
this procedure.
19. For blood cultures, also note on the specimen label: series number (ex. 1 of 2 or 2 of
2) of the blood culture, site on the patient where drawn, volume (ml) in each bottle.
20. Compare the labelled specimen to the patient’s identification bracelet, requisition or ask
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the patient to confirm the tube is properly labelled, especially the spelling of the names
and date of birth.
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• Central Line Catheter
1. Peripheral-midline catheters should not be used for routine blood drawing.
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2. Blood draws via central line catheters may be performed only by nurses trained in
central line.
3. This procedure shall be done using aseptic technique.
4. Protective gloves shall be worn during this procedure and consideration given to
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wearing goggles.
5. Place a new primed needleless connector and extension tubing on the catheter after
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the blood draw, or follow manufacturer recommendations.

6. When drawing blood samples from catheters with more than one lumen, all infusions
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being administered through the catheter should be stopped for a full minute prior to
obtaining the blood sample.
7. Prior to blood draws from patients on continuous infusions of TPN, the infusion
shall be stopped for one full minute and the catheter must be flushed with 20ml of 0.9%
sodium chloride (USP), using two 10ml syringes.
8. Put on gloves. Prime new needleless connector and extension tubing.
9. Cleanse needleless connector with 3 alcohol swabs, wiping for one full minute.
10. Attach blood draw device or syringe to needleless connector and aspirate 5ml of blood
and discard.
11. Attach a new syringe (or tube to Vacutainer®) and aspirate the total amount of blood
required.
12. Fill appropriate tubes with blood as per sequence mentioned in “Order of draw”.
13. For hub to hub technique:

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a) cleanse the catheter/needleless connector junction with 3 alcohol swabs, wiping

for one full minute, or use other appropriate disinfectant. Clamp the catheter
(unless it is a Groshong®) and remove the needleless connector.
b) luer lock a 10ml syringe onto the catheter hub.
c) release the clamp and aspirate 5ml of blood into the syringe; clamp the catheter
(if applicable), remove the syringe and discard.
d) attach the syringe for drawing the blood sample, release the clamp (if applicable)
and aspirate the total amount of blood required for the tests.
14. Flush the catheter with 10ml of 0.9% sodium chloride. Repeat using second 10ml
syringe of sodium chloride, if appropriate. (This may be done after the new primed
needleless connector and extension tubing have been attached to the catheter).
15. Attach a new primed needleless connector and extension tubing to the catheter.
16. Attach a transfer safety device to the syringe containing blood for tests and allow the
vacuum to draw blood into the tube while directing flow toward the wall of the tube.
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17. Label the tubes and transport to the lab per specific laboratory requirements.
• Preventing hematoma during venipuncture LL

- Puncture only the upper most wall of the vein.
- Remove the tourniquet before removing the needle.
- Use major veins.
- Do not make partial penetration with needle.
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- Apply a small amount of pressure to the area with cotton after blood collection.
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• Tube Chart & Order of Draw

Tube Type Common Tests Collection Instructions
and Order
Microbiology: Septicemia, Toxaemia and Aerobic (green) bottle MUST
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bacterial culture. be collected first then

Anaerobic (Orange).
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Pediatric Collection - Yellow

Blood Cultures bottle only.
Mix the specimen with
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gentle agitation.
Label bottles with Patient’s full
name, date of birth, date &
time of collection and barcode.
Store at Room Temperature.
Haematology: Prothrombin times, Note: Correct volume critical.
Coagulation Studies, INR, Factor VIII, See marker level on tube
Lupus Anticoagulant, D-Dimer, Protein INVERT tube GENTLY 6-8
Sodium Citrate C+S, APC, AT3, PFA100 (Platelet times after collection.
(BLUE) Function Test) etc.
Biochemistry: Therapeutic Drugs and INVERT tube GENTLY 6-8
Antibiotics, Serum Copper times after collection
Plain (RED)
Biochemistry: Lipids, LFT’s U/E, Collect extra tube for Hepatitis
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Tube Type Common Tests Collection Instructions

and Order
Creatinine, SUA, Cardiac Enzymes, Serology or HIV Collect extra
Amylase, Calcium, Phosphate, Ionized tube for Endocrinology Collect
Calcium, Protein Assays, TFT, βHCG Iron dedicated tube for Ionized
Studies, C Reactive Protein, B12, PSA, Calcium- Seal and do not
Insulin Endocrinology – Hormones open prior to testing.
SST (GOLD) Serology / Immunology: Hepatitis A, B, For Creatinine Clearance:
C, Allergy, Rubella, Rheumatoid Factor, Collect tube when 24hr
EPG, HIV, ANA, Viral antibodies, Syphilis. urine is collected.
Cytogenetics, Biochemistry: INVERT tube GENTLY 6-8
Cholinesterase, Red cell & Insecticide - times after collection
Organochlorines
Lithium
Heparin
(GREEN)
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Haematology: FBC, Blood Film, Hb, INVERT tube GENTLY 6-8
WCC, Diff, Platelets, blood group, Hb times after collection
Electrophoresis (EPG), Glycated Hb
(HbA1C), T&B Cells, ESR, Malaria
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Parasites (Thick & Thin Films), IM
(Infectious Mononucleosis) Biochemistry:
EDTA Red cell Folate, Carboxy - Hb,
(PURPLE) Manganese, Ammonia, Homocysteine,
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Troponin / Beta HCG (AQT90 – Regional
and Perth peripheral labs)
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Biochemistry: INVERT tube GENTLY 6-8

Glucose, Alcohol, Lactate times after collection
Fluoride
Oxalate
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(GREY)
• Collection of blood from pediatric patients:
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Paediatric collection should be done with the help of scalp vein needle (butterfly needle) and the blue
top vacutainer needle (leur adapter). The end of the butterfly needle rubber tubing has to be fixed on to
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the blue top needle that is fitted on to the holder and used with the required vacutainer tubes.
• Procedure to be followed post blood collection:

A cotton ball is held firmly over the venipuncture site as soon as the needle is removed. After checking
that there is no blood flow, micropore tape is applied at the site of venipuncture and the patient is given
a cotton ball to be held at the site of venipuncture that has to be removed after 5-10 minutes.
• In case of continued bleeding

Apply pressure to the site with a gauze pad until the bleeding stops. Hold cotton firmly for sometimes
on the site and then put a bandage. Tell patient to keep it for 15 minutes
• Dispose of puncturing unit

Burnt the used needle in needle Burner and dispose the needle promptly in container containing sodium
dichloroisocyanurate (NaDCC) (Add 2 tablets in 2000 ml i.e. 140 ppm (parts per million)).
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• Troubleshooting hints for blood collection:

Table 1 No blood seen or too little blood flow into the tube
Causes Solutions
Needle may not be placed at the centre of the Remove and reposition the tube correctly
stopper causing blockage
Needle bevel may be positioned against the wall Rotate the needle ¼ turn clockwise*
of the vein causing blockage
Tourniquet applied too lightly or too long Remove tube from the holder & discard.
stopping blood flow Release tourniquet and allow vein to recover.
Re-apply tourniquet to continue in new tube.
Tube may have been previously punctured or Replace tube
tube may have previously been opened
The needle has transfixed the vein (going Pull back slightly on the needle holder* Be
through the back wall of the vein) alert to hematoma
The needle is not completely in the vein or has Advance the needle forward until you feel the
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not reached the vein “give” as the needle penetrates the vein*
The vein has collapsed Remove tube. Allow vein to recover by
releasing tourniquet. Re-apply tourniquet &
re-insert same tube.
LL
* The tubes may be removed from the holder during the repositioning process. Re-insert the same
tube in the holder when the needle is repositioned.
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Table 2 Under filling of tubes
Causes Solutions
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Premature removal of tube Ensure tube remains in place until ‘vacuum’ is

exhausted and blood flow ceases
‘dead space’ in tubing of winged collection set Draw “discard tube” first to ensure accurate
test results. Particularly important with small
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draw (e.g. 2.7ml & 1.8 ml Sodium citrate tubes)

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Table 3 Blood stops flowing during blood collection

Causes Solutions
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Vein may have collapsed Remove tube. Allow vein to recover by

releasing tourniquet. Re-apply tourniquet & re-
insert same tube.
Needle may have been repositioned outside the Reposition needle or repeat venipuncture at
vein during venipuncture different site if hematoma occurs.
Table 4 Occurrence of Hematoma

Causes Solutions
Needle not completely in vein Release tourniquet and remove the needle.
Needle transfixed the vein Apply firm pressure over swollen area (or
elevate affected arm). Reassure patient that
the bruise will resolve. Repeat venipuncture at
a different site (opposite arm or distal to original
site).

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Table 5 Volume draw too large

Causes Solutions
Draw volume of tubes may be inappropriate for Change to smaller tubes or use Micro
patient e.g. paediatric patients vacutainer for paediatric patients
Table 6 Possible causes of hemolyzed specimens

Causes Solutions
Vigorous mixing of tubes Mix tubes by gentle inversions
Under filling of tubes Redraw specimens as indicated with trauma –
free venipuncture
Transferring from syringe & needle to evacuated Safely remove needle from syringe and
tube remove stopper of tube before transferring
specimen
ED
Alcohol contamination (skin preparation) Allow disinfected site to “air-dry” before
venipuncture procedure
Prolonged tourniquet application (> 1 minute) Release tourniquet, re-apply and perform the
appropriate palpation again
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Table 7 Complications associated with blood collection
Complications Description / Causes Management
O
Fainting Many patients become The phlebotomist should be aware of the
(Syncope) dizzy and may faint at the patient’s condition throughout the collection
thought / sight of blood procedure. This can be done by asking
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ambulatory patients if they feel faint or if they

have ever fainted during previous blood
collections. If so, they should be moved from a
seated to a lying position. If a patient faints during
N
the procedure, the phlebotomist should try to

terminate the venipuncture procedure
immediately and make sure that the patient does
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not fall. The phlebotomist should seek assistance

but remain with the patient who should be placed
C
in a ‘head down’ position. Patients who faint must

be allowed to recover fully before being allowed
to leave and should be instructed not to drive a
vehicle for at least 30 minutes.
Hematoma This complication can If hematoma begins to form, the tourniquet and
occur when the needle the needle should be removed immediately, and
has gone completely firm pressure should be applied to the area for at
through the vein. The least 5 minutes. If bleeding continues, a nurse /
bevel opening is partially doctor should be notified.
in the vein. Or not enough Conscious patients should be instructed to
pressure is applied to the maintain firm pressure on the puncture site
site after puncture. beyond this minimum time.
Susceptibility to Phlebotomists should not leave an unconscious
hematoma may be patient until they are satisfied that the hematoma
increased in patients with is stabilized or responsibility has been passed to
bleeding disorders and a nurse or doctor.
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those receiving certain
drugs (aspirin, warfarin,
and cortisone). Strenuous
use of the arm following
venipuncture may also
induce hematoma.
Thrombosis Thrombi are solid masses Avoid collecting blood from thrombosed veins.
(clots) that reside in blood
vessels. Veins with
thrombi are rigid / bumpy
and lack elasticity.
Petechiae Small red spots appearing Patients displaying this complication may bleed
on patient’s skin, excessively. Always make sure blooding has
indicating minute amount stopped after venipuncture before leaving
of blood escaping into the patient’s side.
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skin epithelium. This
complication may be a
result of coagulation
defects such as platelet
abnormalities or other
LL
blood disorders.
Excessive Patients on Remember to apply pressure to the venipuncture
bleeding anticoagulation therapy, site until bleeding stops. Phlebotomist must not
O
and/or those taking other leave the patient until bleeding stops or a nurse
medication, may bleed for takes over to assess the patient’s situation.
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a longer period
Seizures This is a rare complication If seizure occurs, the phlebotomist should
that may occur during immediately release the tourniquet & needle,
blood collection. attempt to maintain pressure over puncture site
N
& call for help from the doctor. No attempt should

be made to place anything in the patient’s mouth.
The phlebotomist should remain with the patient.
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Mastectomy Patients who have Venipuncture should not be performed on the

undergone mastectomy same side as that of mastectomy.
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may have lymphostasis

(no lymph flow). This
increases the risk of
thrombophlebitis.
Collapsed veins If the syringe plunger is Where a needle and syringe technique are used,
withdrawn quickly during the syringe plunger should be pulled slowly
venipuncture, or vacuum during collection.
of a tube is too great, the Use smaller draw volume or ‘partial draw’
vein may collapse. evacuated tubes for patients with smaller veins
Smaller veins and veins of and geriatric patients.
geriatric patients are most Following venous collapse, release tourniquet
susceptible. and remove tube from holder. Re-apply
tourniquet and once vein has re-dilated, replace
tube in holder. Do not probe a collapsed vein.

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Burned or This complication involves Burned areas are very sensitive and susceptible
scarred area areas that have been to infection. Veins under scarred areas are
burned or scarred, e.g. difficult to palpate. Avoid these areas.
burn scars, graft sites, etc.
Edema Abnormal accumulation of Blood collection should be avoided from these
fluid in the intercellular sites because veins in these areas are difficult to
spaces of the body. palpate and puncture. Specimens may become
contaminated with tissue fluid.
Obesity Obese patients generally Take extra time locate suitable veins, e.g.
have veins that are difficult massage arm, with arm hanging down, use of
to visualize & palpate. ward towel etc. take care not to probe
excessively.
Intravenous Patients on IV therapy for Avoid using the arm with IV line. Instead, use the
therapy extended periods often other arm / site. Where both arms have IV
(Damaged, have veins that are devices, collection should be distal (‘upstream’)
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sclerosed or palpable & visible but from the point of infusion. If a proximal
occluded veins) partially occluded or (‘downstream’) site cannot be avoided, the IV line
sclerosed. can be disconnected (obtain permission from the
physician or nurse in-charge of the patient). After
LL a minimum of 3 minutes delay and with the first
drawn tube discarded, samples can then be
collected.
Hemo Increased concentration Avoid the following.
O
concentration of larger molecules and Prolonged tourniquet application*
formed elements in the Excessive massaging / squeezing / probing a site
TR
blood. Several factors can Sclerosed / occluded veins

cause this complication. *Maximum time 1 minute
Hemolysis Hemolysis can be caused Avoid the following:
by improper phlebotomy Use of small gauge needles (21 g
N
technique and may also recommended).

be the result of Where a needle and syringe collection technique
physiological are used, do not draw or expel syringe plunger
O
abnormalities. too rapidly. Never transfer specimen from

syringe to tube via the needle.
C
Shaking / missing tubes vigorously.

Failure to allow alcohol skin preparation to dry.
Allergies Some patients are allergic If a patient indicates this symptom, all efforts
to povodine iodine should be made to use an alternative product.
(Betadine) or plasters.
5.0 Body fluids

Body fluids, peritoneal fluid and cerebrospinal fluid are to collected with a hypodermic syringe and
needle as per the normal procedure (collection to be done by doctor). After surface disinfection of yellow
top plain vacutainer, the specimen must be injected through the rubber septum of the vacutainer. The
vacutainer tube must be suitably labelled indicating patient’s name & identity no.

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6.0 Tissue biopsy for histopathology/ IHC

Quantity of formalin added to preserve the tissue specimen in transit, must be just enough to cover the
tissue biopsy.
The containers must be recapped tightly.
7.0 Microbiological Specimens

All diagnostic information from the microbiology laboratory is contingent on the quality of specimen
received. Consequences of a poorly collected and/or poorly transported specimen include failure to
isolate the causative microorganism and recovery of contaminants or normal microbiota, which can lead
to improper treatment of the patient. Often, direct specimen smears are utilized to determine the quality
of the specimen, to provide rapid information for diagnosis and therapy, and to allow the physician to
determine if additional, better quality specimens should be collected. This procedure addresses
instructions for physicians, nurses, and laboratory assistants on collecting and transporting samples.
7.1 Blood culture
ED
• Try to collect blood culture before antibiotic therapy to be started. In patients receiving
antibiotics, timing of the sample collection is just before the next dose.
• Never collect the sample from an indwelling peripheral venous catheter or a central venous
line.
• Never collect the sample from an indwelling peripheral venous catheter or a central venous
LL
line.
Follow below procedural steps for blood culture specimen collection.
1. Choose the vein to be drawn by touching the skin before it has been disinfected.
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2. Wear sterile gloves. Cleanse the skin over the venipuncture site in a circle about approximately
5 cm diameter with 70% alcohol/spirit, rubbing vigorously. Allow to air dry.
TR
3. Starting in the center of circle, apply 2% tincture of iodine/providone in ever widening circle until
the entire circle has been saturated with iodine. Allow the iodine to dry on skin for at least one
minute. Site to be cleaned with 70% alcohol/spirit followed by iodine/providone and lastly 70%
alcohol/spirit again & air dry. Remove soiled gloves.
4. Rub hands with 70% alcohol. Wear sterile gloves. The gloved finger can be used for palpation.
N
5. Sterile disposable BD blood collection set “Safety-Lok” (23Gx3/4”x12”) should be used for blood
collection, with precautions to avoid touching & re-contaminating the venipuncture site.
O
6. Detach & safely discard the collection set. Then fit a fresh sterile needle & inoculate the sample
into blood culture bottle & mix immediately.
7. Label bottle with patient name & id. Number. Transfer in Bactec system within 10 min of
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collection. Bottle code should be entered in blood culture register.
Volume of sample to be collected:
• Aerobic plus / anaerobic / fungus culture bottle: 5-10 ml per bottle

• Peds plus bottle: minimum 2-5 ml
• 20 ml i.e. 1 set has to be collected for aerobic blood culture.
• If patient is in critical care unit – 2 set at the interval of 30 min must be collected.
7.2 Cerebral Spinal Fluid (CSF)
Examination of CSF is an essential step in the diagnosis of any patient with evidence of meningeal
irritation or affected cerebrum. Almost 3-10 ml of CSF is collected and part of it is used for biochemical,
immunological and microscopic examination and remaining for bacteriological or fungal examination.
The following important precautions need to be taken for CSF collection and transportation:
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1. Collect CSF before antimicrobial therapy is started.

2. Collect CSF in a screw – capped sterile container and not in an injection vial with cotton plug.
3. Do not delay transport and laboratory investigations.
4. Transport in a transport medium if delay in processing is unavoidable.
5. CSF is a precious specimen, handle it carefully and economically. It may not be possible to get
a repeat specimen.
6. Perform physical inspection immediately after collection and indicate findings on laboratory
requisition form.
7. Store at 37°C, if delay in processing is inevitable.
7.3 Sputum
Sputum is processed in the laboratory for aetiological investigation of bacterial and fungal infections of
the lower respiratory tract. It is of utmost importance in the diagnosis of pulmonary tuberculosis.
1. Select a good wide-mouthed sputum container, which is preferably disposable, made of clear
thin plastic, unbreakable and leak proof material.
ED
2. Give the patient a sputum container with the laboratory serial number written on it. Show the
patient how to open and close the container and explain the importance of not rubbing off the
number written on the side of the container.
3. Instruct the patient to inhale deeply 2-3 times, cough up deeply from the chest and spit in the
sputum container by bringing it closer to the mouth.
LL
4. Make sure the sputum sample is of good quality. A good sputum sample is thick, purulent and
sufficient in amount (2-3 ml).
O
Give the patient an additional container with laboratory serial number written on it for an early morning
specimen. Explain to the patient to rinse his/her mouth with plain water before bringing up the sputum.
TR
7.4 Bronchoscopy, Endotracheal Aspirates, Transtracheal Aspirates:
1. Bronchoscopy specimens include brushing, transbronchial biopsies, or bronchial secretions

N
that are aspirated through the inner channel of the bronchoscope with or without an irrigating
solution. In the transtracheal aspiration procedure, a large-bore intravenous catheter is inserted
through anesthetized skin and the cricothyroid membrane into the trachea. After the catheter is
O
advanced several centimetres into the trachea, the needle is carefully withdrawn leaving the
catheter in place. Material is obtained by applying suction to the catheter with a syringe.
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2. Specimens will be cultured regardless of cellular components. Anaerobic culture will be set up
on properly collected and transported transtracheal aspirate specimens. A separate order
should be entered for an anaerobic culture.
3. Clearly indicate all other collection methods (e.g., transtracheal aspirates, etc.). These will be
prompted for when an order for respiratory culture is ordered.
7.5 Pus
Note: Do not apply antiseptic before taking the specimen.
1. Two swabs are generally collected, one is for direct microscopic examination and the other is
for culture from the wound.
2. If the infection is suspected to be due to anaerobe, aspirate the draining pus into the sterile
syringe and immediately put it into a Thioglycolate broth.
3. Specimen collected during operation or curetting from infected sinuses should be homogenized
with the little nutrient broth under aseptic conditions. It is treated as exudates.

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7.6 Urine
Prevention of contamination by normal vaginal, perineal and anterior urethral flora is very vital. It is the
responsibility of the laboratory to provide the patient with sterile, wide-mouthed glass or plastic, jars,
beakers or other suitable receptacles which should have tight-fitting lids.
Though urine collected by suprapubic aspiration is the gold standard, it is not a practical method.
Alternatively, mid-stream urine or clean catch urine is collected. Whenever possible, urine specimen
should be collected in the morning, before the patient has voided urine.
Specimen: First morning midstream urine is the ideal specimen for microbiological testing.
But in case of urgency random urine specimen can be collected.
Under normal circumstances urine is sterile. The lower part of the urethra and the genitalia are normally
colonized by bacteria, many of which may also cause urinary tract infection. Since urine is a good
growth medium for all sorts of bacteria, proper and aseptic collection assumes greater importance for
this specimen.
For microbiological examination urine must be collected as a "clean catch-mid-stream" specimen.
ED
Instruction to be given to the patient:
Collect about 50 ml of urine sample in provided urine container.

LL
Females:
1. Wash hands with soap and warm water. Collect mid-stream urine.
2. Spread the labia (folds of skin) apart with one hand and wipe with the tissue paper provided.
O
Wipe from front to back.
3. Continue holding the labia apart. As you start to urinate, allow a small amount of urine to fall
into the toilet bowl. (This clears the urethra of contaminants) Do not touch the inside of the cup.
TR
4. After the urine stream is well established, urinate into the cup. Once an adequate amount of
urine fills the cup (the cup only needs to be half-full), remove the cup from the urine stream.
5. Pass the remaining urine into the toilet.
6. Screw the lid on the cup tightly (do not touch the inside of the cup or lid). Give the cup to the
N
Lab collection centre.

Males:
O
1. Wash hands with soap and warm water. Collect mid-stream urine.
2. If uncircumcised, retract foreskin.
3. Clean the end of penis. As you start to urinate, allow a small amount of urine to fall into the
C
toilet bowl. (This clears the urethra of contaminants). Do not touch the inside of the cup.
4. After the urine stream is well established, urinate into the cup. Once an adequate amount of
urine fills the cup (the cup only needs to be half-full), remove the cup from the urine stream.
5. Pass the remaining urine into the toilet.
6. Screw the lid on the cup tightly (do not touch the inside of the cup or lid). Give the cup to the
Lab collection centre.
Urine specimens should be transported to the laboratory within one hour for bacteriological examination,
because of the continuous growth of bacteria in vitro thus altering the actual concentration of organisms.
7.7 Stool
Faecal specimens for the aetiological diagnosis of acute infectious diarrhoea should be collected in the
early stage of illness and prior to treatment with antimicrobials.
A stool specimen rather than a rectal swab is preferred.
1. Refer to stool specimen collection procedure in “Patient preparation for various tests” (Page 4).

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2. Collect the specimen during the early phase of the disease and as far as possible before the
administration of antimicrobial agents.
3. 1 to 2 gm quantity is sufficient.
4. If possible, submit more than one specimen on different days.
5. The fresh stool specimen must be received within 1-2 hours of passage.
6. Store at 2-8°C.
7.8 Throat swab
1. Depress the tongue with a tongue blade.

2. Swab the inflamed area of the throat, pharynx or tonsils with a sterile swab taking care to collect
the pus or piece of membrane.
3. Transport in sterile transport tube.
7.9 Cutaneous (Fungal only)
ED
Hair
1. Scrape the scalp with a blunt scalpel.
2. Place specimen in a dry sterile container.
3. Transport at ambient temperature.
4. The following specimens are also acceptable: 1. Hair stubs 2. Contents of plugged follicles 3.
LL
Skin scales 4. Hair plucked from the scalp with forceps.
5. Cut hair is NOT an acceptable specimen.
O
Nails
1. Cleanse the nail with 70% alcohol.
2. Remove the outermost layer by scraping with a scalpel.
TR
3. Place specimen in a dry, sterile container.

5. The following specimens are also acceptable:
• Clippings from any discoloured or brittle parts of nail.
N
• Deeper scrapings and debris under the edges of the nail

O
Skin
1. Cleanse the skin with 70% alcohol.
2. Collect epidermal scales with a scalpel, at the active border of the lesion.
C
3. Place specimen in a dry sterile container. Do not tape specimen to slide.

Tissue
1. Tissue collection is an invasive procedure and requires surgery by a trained physician/ surgeon.
2. Collect tissue aseptically. Include material from both the centre and the edge of the lesion.
3. Place the specimen in a sterile container on sterile gauze moistened with sterile non-
bacteriostatic saline.
4. Transport in less than an hour at ambient temperature, in a manner to ensure recovery of
anaerobic organisms. For virology cultures, do not allow the tissue to dry and transport in viral
transport media (M4RT).
5. Do not submit tissue in formalin.
6. Do not jam the tissue into a Culturette using the swab; this is not an acceptable transport device.

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8.0 Specimen Collection for Influenza / COVID-19

8.1 Nasopharyngeal Swab collection
Verify patient’s identity with reference to the test request form.
8.1.1 Materials: Sterile Dacron / Nylon swab, Viral transport media tube (should
contain 1 – 3ml sterile viral transport medium)
1. Tilt patient’s head back 70 degree.
2. Insert swab into nostril. (Swab should reach depth equal
ED
to distance from nostrils to outer opening of the ear.)
Leave swab in place for several seconds to absorb
secretions.
LL
3. Slowly remove swab while rotating it. (Swab both nostrils
O
with same swab.)
TR
N
4. Place tip of swab into sterile viral transport media tube and
O
snap/cut off the applicator stick.

C

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8.2 Deep nasal swab collection

8.2.1 Materials: Sterile Dacron / Nylon swab, Viral transport media tube
(should contain 1 – 3ml sterile viral transport medium)
2. While gently rotating the swab, insert swab less than one inch
into nostril (until resistance is met at turbinates).
ED
3.
LL
Rotate the swab several times against nasal wall and repeat in
other nostril using the same swab.
O
TR
N
O
C

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8.3 Combined nasal & throat swab

8.3.1 Materials: 2 dry sterile polyester swabs (aluminium or plastic shafts
preferred), Viral transport media tube (should contain 1 – 3ml sterile viral
transport medium)
2. While gently rotating the swab, insert swab less than one inch into
nostril (until resistance is met at turbinates’).
ED
LL
3. Rotate the swab several times against nasal wall and repeat in
other nostril using the same swab.
O
TR
N

O
C
5. For throat swab, take a second dry polyester swab, insert into
mouth, and swab the posterior pharynx and tonsillar areas. (Avoid
the tongue.)

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6. Place tip of swab into the same tube and cut off the applicator tip.
9.0 Specimen Transportation
9.1 Blood, Urine / stool & specimens for microbiology:

9.1.1 Specimens are checked for any leakage or absence of identity.
9.1.2 Verify that TRF is filled up properly.
9.1.3 Ensure that specimens are tightly capped.
ED
9.1.4 Arrange blood samples in upright position in rack with cool packs packed in
plastic bag (wherever required).
9.1.5 The samples to be transported on ambient temperature shall be kept in separate
compartment of bag.
9.1.6
LL
Place the TRFs separate from sample in order to prevent soiling due to sample
leakage.
9.1.7 The racks are placed in thermocol / carton box and closed.
9.1.8 Place the cool pack in plastic bag and keep it aside of thermocol / carton box.
O
Carry this box in sample carrier hand bag.
9.1.9 Transport the specimen to laboratory at earliest.
TR
9.2 Histopathology & Cytopathology specimens:

9.2.1 Small biopsies may be put in a small bottle containing formalin. The size of the
container should be adequate show as the biopsy must be accommodated
properly with formalin.
N
9.2.2 Quantity of formalin added to preserve the tissue specimen in transit, must be
ten times volumes of the tissue biopsy.
O
9.2.3 The container should be sealed tightly and additionally fortified by brown tape.
9.2.4 The specimen container / Plastic bag must then be packed in a corrugated
C
cardboard box along with the request form labelled with biohazard sticker and
transported in the laboratory.
9.2.5 If slides and blocks are being sent, these should be placed in cardboard slide
boxes/envelope.
9.2.6 As far as possible both fixed and air-dried smears of FNA/ Non gynaec cytology
must be sent.
9.2.7 Pap smears are transported in containers with 80% alcohol.
9.2.8 Smears fixed with cytofix must dry before being wrapped in paper and then
transported in an envelope.
9.2.9 Cytology smears including FNAC are to be transported in a tray when dry fixed.
9.2.10 For outside samples- for air dried smears, these smears must be dried before
being wrapped in paper and then placed in an envelope.
9.2.11 All cytology smears which are wet fixed are to be transported in containers with
80% alcohol.
9.2.12 The fluids are transported in plain container.

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9.3 Frozen Section:

Sample for frozen section is to be transported in plain container (without any preservative)
immediately to the laboratory.
9.4 Renal biopsy specimens:

Renal biopsy specimens are to be transported in saline (phosphate buffered) or MICHEL’S
media at room temperature (16 - 24°C) within 24 hours.
Table 8 Test specific instructions

Sr. Test Instruction
No.
1. Cortisol Cortisol blood tests may be drawn at about 8 am, when cortisol
should be at its peak, and again at about 4 pm, when
the level should have dropped significantly. Sometimes urine is
tested for cortisol; this usually requires collecting all of the urine
ED
produced during a day and night (a 24-hour urine) but sometimes
may be done on a single sample of urine collected in the morning.
2. Beta HCG Last menstrual period date of patient needs to be recorded.
3. FNAC (Fine needle This test involves final needle aspiration procedure to be conducted
aspiration cytology)
LL
on specific body part and hence written consent for the same is
required from the patient / guardians.
4. Urine for porphyrins Sample must be collected in dark container. Exposure to light shall
(porphobilinogen) be prevented and sample must be immediately transported to the
O
lab.
5. Double / triple / Duly filled “Maternal marker patient information form” and
quadruple marker Sonography report shall be collected from the patient for accurate
TR
result interpretation.
6. Biopsy Relevant clinical details, radiological reports and blood reports shall
be collected from patient.
7. HIV Pre-test and post-test counselling shall be done. Written consent
N
from patient is required before sample collection.

8. Body fluids for Clinical information on medication is required to be collected.
O
Cytopathology
9. Bone marrow / Duly filled “Bone marrow / Flowcytometry workup requisition form”
Flowcytometry by physician is required to be collected.
C
workup
10. Culture and drug Clinical information on antibiotic treatment is required to be
sensitivity collected.
11. Blood gas analysis, Fresh collected sample is required.
Lactate
(Arterial/Venous),
Ammonia, Alcohol
Level, PTH (i-PTH),
Electrolytes (Sodium,
Potassium, Chloride,
Bicarbonate)
12. P.T. (Prothrombin Clinical information on medication is required to be collected.
time) & a.P.T.T.
(Activated Partial
Thrombin Time)
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9.5 For Influenza / Covid-19 specimens
9.5.1 Packing:
1. Label the specimen on viral transport media tube and ensure cap on tube is tightly sealed. (Do
not use a pencil or pen for labeling, as they can rub off or smear. Instead, use a bar code or
permanent marker). Place the tubes in zip lock bag / kangaroo bag.
2. Include a cool pack with the specimen(s).
3. Fill out paperwork in accordance with state health department guidelines. Attach TRF on outer
box of clinical specimen for reference. Label the box with shipping address.
4. Seal the outer box using adhesive tape properly.
5. Ensure that there is at least three-layer packing of clinical specimen while transporting.
9.5.2 Storing:
1. Specimens should be placed into sterile viral transport media and immediately placed on
refrigerant gel packs or at 4 degrees Celsius (refrigerator) for transport to the state public health
laboratory.
ED
2. Keep specimens refrigerated (2-8 degrees Celsius, 26-46 degrees Fahrenheit) prior to
shipping.
9.5.3 Shipping:
LL
1. Ship specimens for testing as soon as possible.
2. If delivery will be delayed for more than 3-4 days, specimen should be frozen at -70 degrees
Celsius (-94 degrees Fahrenheit).
3. Ensure specimen will be received by the laboratory during normal business hours.
O
9.5.4 Considerations:
TR
1. A nasopharyngeal (NP) swab is the optimal upper respiratory tract specimen collection method
for influenza testing. However, such specimens cannot be collected from infants and many
older patients may not allow an NP specimen to be collected. Alternatively, a combined nasal
and throat swab specimen or aspirate specimens can provide good influenza virus yield.
N
2. Some influenza tests are approved only for use with certain kinds of respiratory tract
specimens, so follow guidelines provided by test. Also, some tests (e.g., rapid influenza
diagnostic tests) are only approved for certain kinds of respiratory tract specimens.
O
3. For best results (i.e., highest influenza virus yield), collect respiratory tract specimens within
four days of illness onset.
C
4. Most sensitive and accurate tests for influenza virus detection are molecular or nucleic acid
amplification tests (RT-PCR).
5. Negative test results obtained from rapid influenza diagnostic tests (RIDTs) that detect
influenza viral antigens do not exclude influenza virus infection in patients with signs and
symptoms of influenza. A negative test result could be a false negative and should not preclude
further diagnostic testing (such as RT-PCR) and starting empiric antiviral treatment.
10.0 Specimen rejection criteria
10.1 Rejection criteria Clinical specimen in general

The specimen rejection criteria have been developed by the pathology department on the basis of which
the processing of a specimen will not be done by the laboratory.
The criteria for rejection of specimen are listed below.
1. Unlabelled samples
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2. Contaminated samples or leaked samples

3. Inadequate information on test request form (as per need of lab investigation)
4. Haemolyzed (EDTA, Serum, Plasma)/ clotted Samples (EDTA, Citrate, fluoride, Heparin,
blood culture etc.)
5. Suspected dilution of specimen with IV fluids
6. Improper container,
7. Wrong specimen submitted
8. Insufficient or excessive quantity with respect to the sample container specifications
9. Inappropriate transportation condition
10. Mismatch name on sample and TRF.
NOTE: Clinical specimens like biopsies, bone marrow, cytological specimens, CSF or body fluids,
arterial blood for blood gas analysis are precious and hence cannot be rejected.
All attempts are made to resolve inadequacy of specimen telephonically or by other means. Specimens
must be sent to testing area for processing. Reporting must be done only after the inadequacy of
ED
specimen is resolved with appropriate remarks wherever required.
10.2 Rejection criteria for flu specimens

LL
10.2.1 Samples not received in at least three-layer packing;
10.2.2 Samples not received in cold chain (2 to 8℃);
10.2.3 Leakage found;
10.2.4 Samples not transported in viral transport medium;
O
10.2.5 TRF is missing or inadequate patient information as required by ICMR or
statutory guideline;
TR
10.2.6 Labelling on specimen is missing or illegible;

10.2.7 Samples collected using other than nylon / Dacron / polyester swabs (e.g.
Cotton).
11.0 References:
N
1. http://www.nursingcenter.com/journalarticle?Article_ID=843503 - LPN2009, April 2009,

O
Volume: 5 Number 2, page 46- 47.

2. WHO guidelines on drawing blood: Best practices in phlebotomy; 2010.
3. https://www.who.int/
C
4. https://www.cdc.gov/
5. https://cpcb.nic.in/covid-waste-management/

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Cover Page

M/PEP/SCT/02 Primary Sample Collection Manual

Reference to CAP / ISO Standard:

Standard / Guidelines Clause No. Title

ISO 9001:2015 8.5 Production & Service Provision

GEN.40530 Specimen Tracking

Doc. No: M/ PEP/SCT/02 Primary Sample Collection Manual

operations Approved by CD operation

This Primary Sample Collection Manual

released under the

Sterling Accuris Diagnostics, a unit of ‘‘Sterling Accuris

Wellness Pvt. Ltd.’’

Dr. Sanjeev Shah

Doc. No: M/ PEP/SCT/02 Primary Sample Collection Manual

operations Approved by CD operation

 Single Specimen: ............................................................................................................. 10

C. Blood Specimen Collection / Phlebotomy ............................................................................. 11

Basic steps for Drawing a Blood Specimen....................................................................... 11

 Procedure for vein selection ............................................................................................. 12

operations Approved by CD operation

 Procedure to be followed post blood collection: ................................................................ 18

8.2 Deep nasal swab collection .............................................................................................. 28

9.1 Blood, Urine / stool & specimens for microbiology:............................................................ 30

9.3 Frozen Section: ................................................................................................................ 31

9.5 For Influenza / Covid-19 specimens.................................................................................. 32

Doc. No: M/ PEP/SCT/02 Primary Sample Collection Manual

operations Approved by CD operation

1.0 REVISION HISTORY

Sr. Page Section Revision Reason for Date of Rev. Sign of CD

Doc. No: M/ PEP/SCT/02 Primary Sample Collection Manual

operations Approved by CD operation

2.0 READ & UNDERSTOOD FORM

Sr. No. Name Emp. ID Designation Date Sign

Doc. No: M/ PEP/SCT/02 Primary Sample Collection Manual

operations Approved by CD operation

4.0 Phlebotomy – in general

The following factors should be considered in site selection:

7. Apply the tourniquet.

Doc. No: M/ PEP/SCT/02 Primary Sample Collection Manual

operations Approved by CD operation

A. Test Requisition Form

Vacutainers and other containers should be labelled with:

B. Patient Preparation / Instructions for various Tests

Investigations Consent form

HIV: HIV Counselling and Consent form (F/PEP/03)

Bone marrow Bone marrow aspiration Consent Procedure (F/PEP/HIS/03)

• 24 hours Urine Collection

operations Approved by CD operation

• Obtaining urine sample from catheter

procedural steps to collect the sample.

Doc. No: M/ PEP/SCT/02 Primary Sample Collection Manual

operations Approved by CD operation

5. Do not mix toilet paper, water, or soap with the sample.

• Glucose Tolerance Test:

Doc. No: M/ PEP/SCT/02 Primary Sample Collection Manual

operations Approved by CD operation

Instruction to be given to the patient:

• Therapeutic monitoring of patients on medication: