Geda Health Center Laboratory

Standard Operating Procedure

For

Urine LF-LAM Testing

Compiler: Signature:

Approver: Signature:

Effective Date:July 01, 2017 Version No. 01

Document No: XX/ALS/SOP5.4-01 Copy No. _____

 Logo Geda Health Center Laboratory Document No: GHCL/ALS/SOP5.4-01
Version No:01
SOP for Processing Urinary Page 2 of 6
Lipoarabinomannan (LF-LAM) Effective Date: July 01, 2017

Revision and amendment

A. Annual Review of Document

Revision Reviewed by: Approved by:

Review Date
No. Name Signature Name Signature

B. Version Change History/Description

Version. Effective Description of Version Change Name & Name &

No. Date Signature of Signature of
Reviewer approval

C. Amendment

Rev. Page Description of Amendment Amendment Effective Name &

No. No Date Date Signature of
approval
1

Authority of Issue:GHCL Authorized Date: June 30,2021

This is a controlled document for internal use only
Logo Geda Health Center Laboratory Document No: GHCL/ALS/SOP5.4-01
Version No:01
SOP for Processing Urinary Page 3 of 6
Lipoarabinomannan (LF-LAM) Effective Date: July 01, 2017

1.Purpose
The purpose of this (SOP) To describe the procedures for processing urine samples
correctly performing, interpreting, and documenting valid results for TB LAM Ag
Lateral Flow Assay (LFA). TB LAM LFA is immunoassay for the detection of
lipoarabinomannan (LAM) antigen of Mycobacteria in human urine as an aid in
the diagnosis of active mycobacterial infection in HIV positive individuals with
clinical symptoms of tuberculosis.
2. Scope
The procedure applies to all facilities performing urine TB LAM Ag Lateral flow
Assay to assist in the diagnosis of TB in HIV infected adults with signs and
symptoms of TB (pulmonary or extra pulmonary) who have a CD4<100cells/ul or
who are seriously ill (WHO stage III/IV
Responsible staff: FIEBRE laboratory staff
Responsibility and Authorization
The persons responsible for performing this test are Laboratory Technologist and
trained non-laboratory personnel (e.g Nurses, HTC counsel)
Principle
Alere Determine TB LAM Ag is an immunochromatographic test for the
qualitative detection of lipoarabinomannan (LAM) antigen of Mycobacteria in
human urine. Alere Determine TB LAM Ag employs highly purified antibodies
specific to the major polysaccharide antigen of the genus Mycobacterium:
lipoarabinomannan (LAM). These antibodies are used for both the capture and the
detection tracer. The capture antibodies are adsorbed onto the nitrocellulose
membrane of the test strip. The detection antibody is labeled by conjugation to
colloidal gold particles. After a urine specimen is added to the sample pad, the
colloidal gold conjugated antibodies attach to the LAM antigen and are released by
the specimen from the conjugate pad. This immunological complex is then
captured by anti LAM antibodies immobilized on the nitrocellulose membrane and
made visible due to the presence of the colloidal gold label. A positive result (a
visible purple/gray line) indicates that LAM antigen of Mycobacteria is present in
the sample at or above the detection limit of the test; whereas a negative result (no

Authority of Issue:GHCL Authorized Date: June 30,2021

This is a controlled document for internal use only
Logo Geda Health Center Laboratory Document No: GHCL/ALS/SOP5.4-01
Version No:01
SOP for Processing Urinary Page 4 of 6
Lipoarabinomannan (LF-LAM) Effective Date: July 01, 2017

visible purple/gray line) indicates it is not present or below detection limit. To

ensure assay validity, a procedural control bar is incorporated in the assay device.
Supplies and Materials
 Sample logbook (paper and ODK)
 Alere Determine TB LAM test kit
 Positive control solution
 Quality assurance log (example in Appendix 1)
 Calibrated pipette and pipette tips, or single-use squeezable pipettes
 Sample labels with patient's QR code
 Gloves (single-use latex or vinyl)
 Biohazard disposal system
 Lab surface disinfectant (e.g. Virkon)
 Sharps bin
 Timer
6 Procedures:
6.1 Collecting urine sample
Collect urine sample as described in SOP F.04.
6.2 Storage of urine sample
6.2.1 Fresh urine samples can be used within 8 hours if kept at room temperature,
however, in order to generate the most reliable results the test should be completed
as soon as possible
6.2.2 Urine samples should be stored at 2-8oC if the test is delayed and to be run
within 3 days of collection. If testing is delayed more than 3 days, the samples
should be frozen (-20oC or cooler)
6.2.3 For frozen or refrigerated samples bring to room temperature one hour before
use
6.2.4 Frozen samples may contain aggregates; all thawed samples must be
centrifuged at 10,000g for 5 minutes at room temperature and the 60microlitre test
sample should be carefully collected from the clear supernatant. Avoid repeated

Authority of Issue:GHCL Authorized Date: June 30,2021

This is a controlled document for internal use only
Logo Geda Health Center Laboratory Document No: GHCL/ALS/SOP5.4-01
Version No:01
SOP for Processing Urinary Page 5 of 6
Lipoarabinomannan (LF-LAM) Effective Date: July 01, 2017

freeze/thaw cycles.Specimens that have been frozen and thawed more than 3 times
cannot be used
6.3 Quality assurance of urinary LAM test kits
If you have already quality assured the lot you are using, proceed to section 6.4.
6.3.1 For each new lot of Alere Determine TB LAM Ag test kits received, and for
each new delivery, quality assurance testing with positive control solution must be
undertaken. For each lot, document the
number on the lot card in the quality assurance log (example in Appendix 1)
6.3.2 Wear latex or vinyl gloves. Remove one test unit from the card strip by
bending and tearing at the perforation. Removal of the test units should start from
the right side of the test card to preserve the lot number which appears on the left
side of the test card. The control testing should be initiated within 2 hours after
removing the protective foil cover from each test
6.3.3 Remove the protective foil cover from the test
6.3.4 Apply 60μL of positive control solution (or 2 drops) to the sample pad (white
pad marked by the arrow symbol)
6.3.5 Wait a minimum of 25 minutes and two people should read the result.
Visualise the strip under standard indoor lighting conditions or in the shade. Do not
visualize the strip under direct sunlight.
Results are stable for up to 35 minutes after sample application. Do not read
beyond 35 minutes
6.3.6 The test should read positive i.e. purple/grey bars appear in both the control
window (labelled “Control”) and the patient window (labelled “Patient”) of the
strip. The test result is positive even if the patient bar appears lighter or darker than
the control bar
6.3.7 If the test reads positive, document that the batch has been quality assured,
write 'QA passed' on all packets in the lot and proceed to testing patient samples
6.3.8 If the test reads anything other than positive (see section 6.4.8), document on
quality assurance log and return to manufacturer

Authority of Issue:GHCL Authorized Date: June 30,2021

This is a controlled document for internal use only
Logo Geda Health Center Laboratory Document No: GHCL/ALS/SOP5.4-01
Version No:01
SOP for Processing Urinary Page 6 of 6
Lipoarabinomannan (LF-LAM) Effective Date: July 01, 2017

6.4 Processing urine sample

6.4.1 Wear latex or vinyl gloves. The desired number of test units from the 10-test
card can be removed by bending and tearing at the perforation. Removal of the test
units should start from the right side of the test card to preserve the lot number
which appears on the left side of the test card. Initiate the assay within 2 hours after
removing the protective foil cover from each test
6.4.2 Check the patient’s label on the urine cup. Open the urine cup, being careful
not to let anything touch the inside of the cup or the lid
6.4.3 Remove the protective foil cover from each test
6.4.4 Ensure each test is labelled with correct participant ID
6.4.5 Apply 60μL of sample (or 2 drops of urine) to the sample pad (white pad
marked by the arrow symbol)
6.4.6 Results must be read by 2 people. If the 2 readers interpret the test
differently, a third person must be available to make the decision. Each read should
be independent, without knowing the opinion of the other readers
6.4.7 Wait a minimum of 25 minutes and read result. Visualize the strip under
standard indoor lighting conditions or in the shade. Do not visualize the strip under
direct sunlight. Results are stable for up to 35 minutes after sample application. Do
not read beyond 35 minutes

Authority of Issue:GHCL Authorized Date: June 30,2021

This is a controlled document for internal use only
Logo Geda Health Center Laboratory Document No: GHCL/ALS/SOP5.4-01
Version No:01
SOP for Processing Urinary Page 7 of 6
Lipoarabinomannan (LF-LAM) Effective Date: July 01, 2017

Figure 1: Urinary LAM testing procedure

6.4.8 Read the test result; to assist with results reading and interpretation, use the
reference scale card (provided in the kit) by holding it alongside the patient
window. Possible results are:
POSITIVE: purple/grey bars appear in both the control window (labelled
“Control”) and the patient window (labelled “Patient”) of the strip. The test result
is positive even if the patient bar appears lighter or darker than the control bar
NEGATIVE: one purple/grey bar appears in the control window of the strip
(labelled “Control”) and no purple/grey bar appears in the patient window of the
strip (labelled “Patient”)
INVALID: if there is no purple/grey bar in the control window of the strip, even if
a purple/grey bar appears in the patient window of the strip, the result is invalid
and the test should be repeated
INDETERMINATE: one purple/grey bar appears in the control window of the
strip (labelled “Control”) with unclear or incomplete purple/grey bar in the patient
window of the strip (labelled “Patient”). For a better clinical-decision the test
should be repeated. Alternatively, collect a new urine sample in the following days
from the patient and test. Early morning urine is recommended.
6.4.10 Record the result in the patient's CRF (ODK or logbook)
6.4.11 Where there is a positive result, inform the clinical team looking after the
participant to guide patient management
Quality Control Testing
Conduct Quality control for TB LAM test weekly, before you test the first
specimen to be analyzed during that particular week. In the case where there are no
specimens to be test for TB LAM, Quality Control may not be Evaluated in that
week. Record quality control results in the TB LAM result log book.
7.3.1 Follow the following procedure to evaluate TB LAM Ag quality control:
TB LAM Ag positive control
 First label the test strip with TB LAM Positive control

Authority of Issue:GHCL Authorized Date: June 30,2021

This is a controlled document for internal use only
Logo Geda Health Center Laboratory Document No: GHCL/ALS/SOP5.4-01
Version No:01
SOP for Processing Urinary Page 8 of 6
Lipoarabinomannan (LF-LAM) Effective Date: July 01, 2017

 Add 60µL of the TB LAM Ag positive control on the labeled test strip.
 Read results after 25 minutes
TB LAM Ag Negative control
 Label the test strip, TB LAM negative control
 Add 2 drops of saline/distilled water
 Read results after 25 minutes

Reference
 Alere Determine TB LAM Ag package insert

Declaration
I, the undersigned laboratory personnel, certify that I am conducting every steps of the
procedures incorporated in this SOPs after a prior reading.
Name Signature and Date
…………………………………… …………………………………….
…………………………………… …………………………………….
…………………………………… …………………………………….
…………………………………… …………………………………….
…………….…………………….… ……………..…………………….
………………….…………….… ………….…..…………………….
……………………………..…… ..……………………………….….

Authority of Issue:GHCL Authorized Date: June 30,2021

This is a controlled document for internal use only