533568073
Document type: SOP
Document code: TB 03-03
SPECIMEN COLLECTION MANUAL
Confidentiality: none
TABLE OF CONTENTS
1.
PURPOSE ...................................................................................................................... 2
2.
SCOPE ........................................................................................................................... 2
3.
RESPONSIBILITIES....................................................................................................... 2
4.
PROCEDURES .............................................................................................................. 2
4.1. Testing performed at the _________________TB Laboratory .................................................... 2
4.2. Primary samples for tuberculosis diagnosis ................................................................................. 2
4.3. Collection of primary specimens .................................................................................................. 3
4.3.1. Patient preparation ................................................................................................................... 3
4.3.2. Sample collection timing ........................................................................................................... 3
4.3.3. Specimen/sample containers .................................................................................................... 4
4.3.4. Sputum Collection Procedure .................................................................................................... 4
4.3.5. Collection of other specimens ................................................................................................... 4
4.4. Labeling of specimens................................................................................................................... 5
4.4.1. Laboratory Request Form .......................................................................................................... 5
4.5. Transportation of specimens to the _______________TB Laboratory ........................................ 5
4.5.1. Storage of specimens prior to transportation ........................................................................... 5
4.5.2. Safety precautions ..................................................................................................................... 5
4.5.3. Packaging ................................................................................................................................... 6
4.5.4. Transportation ........................................................................................................................... 6
5.
URGENT SAMPLES ...................................................................................................... 6
6.
REFERENCES ............................................................................................................... 7
7.
CHANGE HISTORY ....................................................................................................... 7
This SOP template has been developed by FIND for adaption and use in TB laboratories
Release date: ddMMMyy
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1.
PURPOSE
This manual is intended to assure that the quality of specimens received at the
_____________________TB Laboratory is of the required standard. The quality of the results of
laboratory testing is strongly influenced by the manner in which the primary samples were collected
and transported to the laboratory.
2.
SCOPE
This manual will describe proper methods for collection and transportation of sputum, tissue aspirates
and biopsies suitable for TB diagnosis. This manual will also describe patient preparation and
instructions prior to sample collection where necessary. This manual applies to all health service
providers at organizations, hospitals and health units involved in collection of specimens for
tuberculosis diagnosis at the _____________________TB laboratory.
3.
RESPONSIBILITIES
The Head of the _________________TB Laboratory is responsible for ensuring that this manual is
made available to all clinical sites and laboratories involved in providing specimens for work to be
carried out at __________________TB Laboratory.
The Medical Superintendents/in-charges of health facilities are responsible for ensuring that contents
of this manual are understood by personnel in their units involved in the actual collection of
specimens.
4.
PROCEDURES
4.1. Testing performed at the _________________TB Laboratory
Tests performed at the ______________________TB Laboratory may include the following:
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Direct and concentrated smear microscopy
ZN and fluorescent staining
Lowenstein-Jensen and MGIT 960 culture
MPT64 antigen test for species identification
Line probe assay (LPA) for MDR-TB screening
First line drug susceptibility testing on solid and/or MGIT culture
Second line drug susceptibility testing on solid and/or MGIT culture
Xpert MTB/Rif
4.2. Primary samples for tuberculosis diagnosis
The primary sample (also called specimen) is that part of a patient whether a fluid, sputum or tissue
biopsy that is submitted for examination to obtain a diagnosis. The following primary samples are
recommended for the diagnosis of tuberculosis:
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
Pulmonary tuberculosis
Sputum (preferably naturally produced or expectorated); this is the most suitable specimen for
diagnosis of pulmonary tuberculosis. Efforts should be made to avail sputum before resorting to
alternative samples.
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Other alternative samples
Lung aspirates
Gastric lavage (patients in coma or children who cannot produce sputum or swallow it)
Laryngeal swabs (especially for babies who cannot cough sputum)
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Extra-pulmonary tuberculosis
Choice of specimen for extra-pulmonary tuberculosis diagnosis will depend on the suspected
organ/tissue affected and the doctor’s clinical examination. Common samples collected include:
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Lymph node aspirates
Pus (wounds and boils)
Synovial aspirates
Urine
Other body fluids such as blood, bone marrow, pericardial and spinal fluid
Biopsies (pieces of tissue, necrotic or not, but suspected of harboring mycobacteria)
4.3. Collection of primary specimens
For efficient demonstration of the presence of AFBs in primary samples and optimal results, sputum
specimens should be collected before initiation of chemotherapy. Drug therapy even for a few days
may kill or inhibit sufficient numbers of AFBs to leave confirmation of tuberculosis in doubt. However,
samples for primary culture can still be collected up to one week of initiation of treatment.
Mycobacterium has capacity to affect all organs of the body but most cases of tuberculosis in disease
endemic settings are still pulmonary, requiring sputum specimen for diagnosis.
4.3.1. Patient preparation
No special preparation of the patient is required but prior to collection of the sputum the patient must
be advised to rinse his/her mouth thoroughly with water (preferably sterile to exclude Non Tuberculous
Mycobacteria; NTMs) before coughing out sputum for laboratory examination.
4.3.2. Sample collection timing
The early morning sputum collected within two hours after the patient rises from bed is the best.
Mycobacteria concentrations are highest in the early morning sputum (having accumulated in the night
secretions) making it easy to see in microscopy and easy to isolate on culture. However on-spot
collection of sputum is also acceptable but whenever possible efforts should be done to collect a
second early morning sputum sample from such patients if the first one is negative.
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4.3.3. Specimen/sample containers
NTRL advises the use of large mouthed screw-capped leak-proof conical shaped, 50 ml graduated
disposable plastic containers (e.g. 50ml Falcon tubes from Becton Dickson [BD]). These tubes are
large enough to allow processing of sputum in the same containers to increase recovery of bacilli as
well as minimize sputum transfer to other processing containers. However use of other available
sputum containers/mugs is acceptable but they must be robust, screw capped, transparent and leakproof. See Appendix II for pictorial examples.
4.3.4. Sputum Collection Procedure
Safety precaution:
DO NOT COLLECT SPUTUM SAMPLES IN THE LABORATORY, CLINIC EXAMINATION ROOMS
OR IN THE PATIENT/CLIENT WAITING AREA.
ALWAYS INSTRUCT PATIENT TO GO OUTSIDE IN OPEN AIR AWAY FROM OTHER PERSONS
TO MINIMISE RISK OF TRANSMISSION AND INFECTION OF OTHERS.
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Give each patient a new sputum container. If two samples are required, give two separate
containers. Sputum containers or mugs should never be re-used.
Give the patient instructions and demonstrate how she or he can produce and collect good
sputum; do up to three deep inhalations and exhalations and on the third exhalation or when a
strong cough reflex arises, accompany it with a cough from deep inside the chest as much as
possible (do demonstrative actions for the patient).
Demonstrate to the patient how to open and close the sputum container so as there are no leaks
or smearing on the exterior of the container.
Emphasize the need for the patient to supply the most useful specimen, the normally thick,
yellowish (sometimes blood-streaked), purulent material brought up from the lungs after a deep,
productive cough.
Emphasize that saliva produced by spitting is not sputum. However, if the only sample the patient
can produce is salivary, do submit it to the laboratory as it can still yield useful information.
Encourage the patient to bring the collected specimen back to the unit as quickly as possible.
Fill in all details on the National TB Program (NTP) request form and submit along with the
specimen to the laboratory.
4.3.5. Collection of other specimens
Other forms of samples other than sputum will need specialized techniques in collection techniques
from a qualified medical officer or clinician. Gastric lavage, biopsies, synovial fluid, bone marrow and
others, will need to be collected sterile and sometimes require transportation media or saline during
delivery to _________________TB Laboratory. Refer to reference materials and literature for safe,
sterile collection of these samples.
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4.4. Labeling of specimens
Label/identify the sample container with at least a patient’s name, facility name, date of collection and
other information as required by the relevant NTP request form. These parameters must at all times
be 100% identical to the ones written on the laboratory request form brought together with the
specimen, otherwise the specimen may be rejected.
4.4.1. Laboratory Request Form
The following information is usually included on the Laboratory Request Form:
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The health facility or research organization requesting examinations.
The name, sex, age of the patient from whom the sample was collected.
Address of the patient.
Clinical history of the patient (on chemotherapy or not etc.).
Laboratory procedure(s) being requested for.
Type of sample collected or the site from where the sample was collected.
Amount of the sample collected.
Date and time of collection of sample.
Name/signature /initials of person collecting the specimen (if applicable).
Signature of person requesting for laboratory investigation and date.
The laboratory request form must be completed fully at all times. It is the duty of the health facility incharges or principal investigators to ensure that personnel under their supervision fully complete the
request forms.
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Fill in the request form using a blue or black pen only. DO NOT USE RED PENS.
Complete the request form as fully as possible or at minimum, patient name or equivalent, date
and time of collection, name/initials and signature of person requesting the examinations and
name/initials and date of person collecting sample (especially for samples other than sputum) and
address of health facility.
Make sure that the patient names, date of collection recorded on the specimen container match
with those on the request form, otherwise specimen may be rejected.
4.5. Transportation of specimens to the _______________TB Laboratory
4.5.1. Storage of specimens prior to transportation
Suitable storage and transportation of clinical specimens is critical to ensure good results, especially
for specimens that are being submitted for culture. Specimens should be stored as soon as possible
after collection in a fridge.
4.5.2. Safety precautions
The shipment of diagnostics samples such as urine, sputum, tissue biopsies, blood and other body
fluids and cultures containing aetiologic agents, which includes all Mycobacterium spp, is of potential
public health risk and must be done in compliance with shipment of biological specimen regulations of
the country and abroad. Compliance to transportation standards is the responsibility of the shipper.
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4.5.3. Packaging
Diagnostic samples must be packaged to withstand leakage of contents, shocks, pressure changes
and other incidents of ordinary handling. Packing samples for international shipment may require the
services of someone certified in international shipment.
Materials required
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Plastic re-sealable bags
Sample (collected in appropriate container)
Ice packs (where possible)
Cushion material (air bags, cotton wool, styrofoam pellets etc)
Biohazard labels
Impact resistant transportation/shipping box (cooler box where possible).
Large envelopes
Procedure
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Wrap sample bottles/containers with cotton wool.
Seal sample containing sample bottles/tubes with cotton wool in plastic re-sealable bags. This
helps to contain leakage.
Place a layer of ice packs around the transportation box leaving enough space in the middle (a
must for biopsies and samples spending long hours in transit, optional for transportation over short
distances).
Arrange plastic bags containing the samples in the space between ice packs.
Fill any empty spaces with cushioning material e.g. cotton wool.
Tightly close the transportation container.
Stick biohazard/Biological Material labels on the top of the transportation box.
Enclose specimen laboratory request forms in an envelope outside the transportation box.
Specimens are ready for transportation.
4.5.4. Transportation
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5.
It is advisable to transport specimens in a separate means of transport from passengers. Use
motor cycles or a separate vehicle or hire services of a courier company.
Carry the envelope containing the laboratory request forms along with the sample but separate
enough to avoid contamination of forms.
Deliver samples as soon as possible from the time they were collected to the laboratory.
Upon arrival at the __________________TB laboratory, hand over the envelope and the
transportation box to a personnel in the laboratory. Do not unpack the contents as samples may
require specialized safety precautions.
The courier or transporting person should wait to leave until the sample reception process is
complete.
URGENT SAMPLES
In addition to the procedures outlined for routine samples, the following apply to the receipt, labeling
and processing of urgent samples:
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Samples collected at healthcare facilities must reach the laboratory in a timely manner. Special
arrangements may be required to inform sample collection personnel and couriers of procedures
to be followed for getting urgent samples in a timely manner.
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6.
All urgent samples must be marked as “Urgent”. Preferably, healthcare facilities must be issued
with brightly colored stickers that identify samples as urgent.
In the event that a sample is received that has been written as “Urgent”, but has no sticker, the
laboratory must place a sticker on both the sample container and the laboratory testing requisition
form.
Urgent samples must be sent from sample receiving areas to the processing laboratory in a timely
manner. Where batching of samples is required, the urgent sample can substitute a routine
sample, thus insuring that it is processed with the next batch.
The data from urgent samples must be captured in a timely manner. Data input personnel must be
informed of procedures to insure that urgent samples take precedence over routine samples.
Urgent samples must be processed as soon as possible.
The results of urgent samples must be reported in a timely manner. If possible, these should be
communicated telephonically to the requester of the urgent test.
Verbal request for testing
Verbal request for testing is not encouraged. In the case that the laboratory receives a verbal request
for additional testing of a sample already received, the laboratory must:
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Get name of person the urgent result is reported to search the database or worksheets for the
sample already received
Determine whether the request for additional testing is feasible (e.g. it would not be possible to
perform a drug susceptibility test on a culture negative sample). If it is not clear, a supervisor
should be consulted.
Reply to the requester with the following information:
a. Give Patient Name and Patient ID of sample
b. Confirm that the request test will be performed
c. Get name of person requesting the additional testing
Add the test to the laboratory database or relevant worksheet.
REFERENCES
Patricia T. Kent, B.S., George P. Kubica. 1985. Public Health Mycobacteriology; A guide for the level
III Laboratory. U.S Depart. Of Health, Centers for Disease Control .
Bobby E. Strong, M.S., George P. Kubica. Isolation and Identification of Mycobacterium tuberculosis;
A guide for the Level II Laboratory. US. Dept. of Health, Division of Human Services, Centers for
Disease Control.
8.
CHANGE HISTORY
New version #
/ date
Old version #
/ date
No. of
changes
Description of changes
Source of
change request
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