Work Instruction for Rapid Diagnostic Kit Test

Date of Issue: --
Document No: RDK/HWC-SC/…../…
16/10/2023-----
Effective Date: --
Version/Issue No: 01
21/10/2023----------

Work Instruction
for Rapid Diagnostic Kit Test

ABHWC-HSCs

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 The signatures below certify that this procedure has been reviewed and approved, and
demonstrate that the Signatories are aware of all the requirements contained herein and are
committed to ensuring their provision.

Name Position Signature

Prepared By Riru Baghel CHO

Approved By Dr Amrit Lakra BMO

Amendment Note:

Page Context Revision Date

No

Note: Prior to use, ensure this document is the most recent issued
This procedure is reviewed to ensure its continuing relevance to the systems and process that it
describes. A record of contextual additions or omissions is given below:

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 Family Planning and Reproductive Health Care Services
Purpose
Overall purpose of this work instruction is to ensure that HWCs staff use standard procedure for
conducting screening and tests using rapid kit based diagnostic Tests in the catchment area of
the Health and Wellness Centre.
Scope:- It applies to the Health and Wellness team which includes (ASHA, ANM and CHO).
in completing the standard process of following rapid diagnostic kits available at HWCs-SC.
1. Blood Sugar
2. Haemoglobin
3. Urine Dipstick test for Sugar and protein
4. Urine Pregnancy test
5. Malaria
6. Dengue
7. HIV
8. Filariasis
9. Syphilis
10. Hepatitis-B
11. Test for fecal water contamination
12. Test for water chlorination
13. Test for Iodine in salt

Responsibility

Staff Member Responsibility

CHO (Community Health Officer) Prescribing and performing the rapid diagnostic test

ANM Performing the rapid diagnostic kit

Process and Activities:

 CHO examines the patient and prescribe the needed diagnostic tests and explains the entire
procedure to patient.
 ANM/MPW-M or CHO performs the kit based rapid tests.
 CHO note down the findings of tests on patient’s prescription and decides further treatment
plan and communicate the same to patient.

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 Blood Sugar Testing by Glucometer

Steps
1 Preparatory Steps of performing blood sugar examination:
 ∙ Single step testing using 75 g oral glucose and measuring plasma glucose 2 hour after ingestion.
 ∙ 75g glucose is to be given orally after dissolving in approximately 300ml water whether the PW
comes in fasting or non-fasting state, irrespective of the last meal. The intake of the solution
has to be completed within 5 minutes.
 ∙ A plasma standardised glucometer should be used to evaluate blood glucose 2 hours after the
 oral glucose load.
 ∙ If vomiting occurs within 30 min of oral glucose intake, the test has to be repeated the next
day, if vomiting occurs after 30 minutes, the test continues.
 ∙ The threshold plasma glucose level of ≥140 mg/dL (more than or equal to 140) is taken as cut off for
diagnosis of GDM.
2 Preparing Lancing Device for Blood sample:
Wash your hands and the sample site with soap and warm water. Rinse and dry thoroughly.
Unscrew and remove the adjustable tip.
Insert a new disposable lancet firmly into the carrier.
Twist off and set aside the protective cover of the disposable lancet and replace the adjustable tip.
 Choose a depth of penetration by rotating the top portion of the adjustable tip until the setting
number matches the arrow.
 To cock the lancing device, hold the tip in one hand. Pull the sliding barrel on with the other hand.
The lancing device is cocked when you feel a click
3 Using lancing device at fingertip sampling site
 Place the cocked lancing device against the pad of the finger.
 The best puncture sites are on the middle or ring fingers.
 Choose a different site each time you test.
 Repeated puncturing at the same sample site may cause pain or skin calluses.
 Press the release button. Remove the device from the finger. Wait a few seconds
4 Testing your blood glucose level
 Insert a test strip with contact bars facing up into the test strip port.
 Push the strip gently until the meter beeps.
 Then the symbol will appear on the screen. Use your lancing device to get blood sample. ∙
 A minimum volume of micro litres is needed to fill the confirmation window. After the symbol
appears on the screen, apply the blood sample to the narrow end of the test strip until the meter beeps.
5  Normal reference values
 RBS:70-110 mg/dl
 FBS: 70- 99mg/dl
 PPBS:<140mg/dl

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6 Viewing test results in meter memory.
The test result will appear after the meter counts down from 5 to 1. The result will be
automatically stored in the meter’s memory. If the test strip is removed after the test result is
displayed, the meter will automatically switch off after 3 seconds. Discard used test strips safely
in biohazard containers.
The meter memory can store up to ten (10) results at one time. The results will be saved in
the order it was measured, and you can view stored results by pushing the button. When the
button is pushed, number ‘1’ will appear on the screen for one second, and then the result will
be shown.
This is the most recent test result that is saved in the memory. To go on to the second recent
result, press the button again. Now number ‘2’ will appear for one second and the next result
will be displayed. By repeating this process, you can go through to result number 10 which is
the oldest data saved in the memory.
To exit from the memory mode, press and hold down the button for about two (2) seconds.
The meter will turn off

Haemoglobin estimation

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Steps
1 Keep all the necessary items ready: Digital hemoglobinometer with batteries or charger, micro cuvettes
or strips, lancets, alcohol swab/sprit cotton, sterile gloves, tissue paper, biohazard container to dispose
the used lancets, micro cuvettes/strips.
2 Turn “ON” the hemoglobinometer: In cuvette based hemoglobinometer After the monitor shows three
dashes, pull the cuvette holder in its loading position. Fill the microcuvette in one continuous process.
The correct amount of blood (10 μl) is drawn into the microcuvette. The microcuvette should be
completely filled. Wipe away any excess blood on the outside of the microcuvette tip. Check for air
bubbles in the filled microcuvette. If present, use a new microcuvette. Place the filled microcuvette in
the cuvette holder (within 40 seconds after filling the cuvette). Push the cuvette holder to its measuring
position. Read and record the result. Remove and discard the microcuvette in the appropriate bio-hazard
container. Push the cuvette holder back into the instrument. If strip based hemoglobinometer is used, the
system will undergo an auto-check and auto calibration after which the battery level, date, time and
strip’s batch code are displayed within 2 seconds. Enter the code mentioned on the strip bottle. The
meter would flash ‘strip’ symbol on the display. Insert a fresh test strip into the meter with the arrows on
the strip facing up and pointing towards the display. Ensure the correct positioning of the strip with the
guiding V notch. The meter would flash ‘drop’ symbol on the display. Allow the second drop of the
blood to fall to completely cover the white coloured test area. Read and record the result. Remove the
used test strip from the meter and dispose in appropriate biohazard container.
3 Wash hands and wear loose gloves.
4 Choose the third (middle) or fourth (ring) finger of the non-dominant hand of the beneficiary for the
finger prick. Avoid the thumb and little finger. Avoid fingers with thick calluses. Avoid fingers with
tight rings as they may constrict blood flow.
5 Ask the subject to rub their hands to promote blood flow.
6 Wipe the fingertip with the alcohol pad and let it air dry completely. Do not blow on the finger to dry
the alcohol. Do not wipe off the alcohol. Do not perform the finger prick until alcohol has completely
evaporated.
7 Hold the finger firmly just below the centre of the fingertip
8 Press and trigger the lancet flat and firmly against the finger at the puncture sites away from the midline
9 Discard lancet in biohazard container
10 Release pressure and allow a full drop of blood to collect on finger
11 Once a drop of blood has collected on the finger, use the cotton or tissue to wipe away the first drop of
blood.
12 Use the second or third drop of blood for estimation of haemoglobin

Urine testing for Sugar and Albumin

Steps
1 Keep all the necessary items ready: urine specimen collection bottles/containers and dipsticks.

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2 Check the expiry date on the kit and carefully read the instructions before use
3 Remove one strip from the bottle and screw the cap tightly.
4 Completely immerse the reagent area of the strip in the urine and remove it immediately
5 Remove the strip of the urine and tap at the edge of container to remove excess urine.
6 For glucose: compare the blue reagent area against the colour chart area on the bottle and
record the finding (time as per manufacturer’s instruction).
7 For urine albumin: compare the yellow reagent area against the colour chart area on the bottle
and record the finding (time as per manufacturer’s instruction).
8 Dispose of strip and urine as per GoI protocol.

Urine Pregnancy Test

Put three drops of urine sample in well of the test kit

See Result after 5

Minutes

Rapid Diagnostic Test (RDT) for Malaria

Steps to prepare the thick and thin smear

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1  Select the ring finger of the left hand.
2  Clean with antiseptic or sterile wipes.
3  Dispose of the cotton swab as per GoI protocol.
4  Allow the finger to airdry.
5  Puncture at sides of the flesh pad of the finger avoiding the centre and the tip of the
finger.
6  Allow the blood to come up automatically
7  Don’t squeeze the finger.
8  Hold the slide by the edges.
9  Touch the drop of blood with a clean slide
10  Collect 3 drops to prepare a thick smear and 1 drop for a thin smear. Place the thin and
thick smear at either end of the slide
11  To prepare a thin smear, touch a single drop of blood with the edge of the slide
12  Keep the slide in front of the second drop and allow the blood to spread.
13  Hold it at an angle of 45 degrees and spread with a rapid but not brisk movement.
15  Write the slide number on the same side as the thin smear
16  Spread the drop of blood with the corner of the slide to make a circle or a square of
approximately 1 cm in diameter.
17  Wrap and send the slide to the laboratory for staining and to be examined under the
microscope
Steps for malaria testing using the rapid diagnostic test kit (RDT)
1  Store the kits at the recommended temperature
2  Check that the RDT kit is not damaged.
3  Check the expiry date on the kit.
4  Remove the RDT packaging and take the dropper from the foil pouch and place it on a
flat, dry surface.
5  Label the RDT with the patient’s ID and the date the test was performed.
6  Allow the reagents to reach room temperature if kept in cold chain.
7  Select the finger for puncture, clean with spirit swab and allow to air-dry.
8  Puncture the finger with a sterile lancet.
9  Slowly add 1 drop of blood to the sample well and add 2 drops of the assay diluents
10  As the test begins to work, a purple colour will be seen moving across the result
window in the centre of the test device
11  Interpret test* result at 5-20 mins (do not interpret after 20 mins) as per the
manufacturer’s instructions

Interpretation of the result for monovalent RDT kit:

Negative result If only one line (band) appears, the test has worked and the patient is negative

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 for malaria.
Positive result If 2 lines (bands) appear within15-20 mins, the person is suffering from P.
falciparum malaria.
Invalid result If no line appears within 15-20 mins, discard and repeat the test.
Interpretation of the result for bivalent RDT kit:
Negative result If only 1 line (band) appears at C (control),the test has worked and the patient
is negative for malaria
Positive result If 2 lines (bands) appear within 15-20 mins at C (control) and T1, the person is
suffering from P. falciparum malaria.
Positive result If 2 lines (bands) appear within 15-20 mins at C (control) andT2, the person is
suffering from P. vivax malaria.
Positive result If 3 lines (bands) appear within 15-20 mins at C (control), T1 and T2, the
person is suffering from both P. falciparum and P. vivax malaria.
Invalid test If no line appears within 15-20 mins, discard and repeat the test.

Rapid Diagnostic Test for Syphilis

Steps

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 1 STORAGE: The test syphilis test kit should be kept in the refrigerator to maintain the
temperature at 2°C to 8°C. If a refrigerator is not available at the sub-centre, then the kits
should be kept at the PHC to maintain cold chain and be brought to the VHSND or
outreach sessions while ensuring that cold chain is maintained.

2 PROCEDRE: Remove test device from foil pouch. ∙ Add 20 uL whole blood to the
sample well and 3-4 drops of assay diluent. ∙ Interpret test results within 10 minutes. ∙
Negative result: presence of only one purple colour band visible within the result window. ∙
Positive result: Presence of both ‘T’ and ‘C’ bands visible within the result window. ∙
Invalid result: No purple band visible within the result window.

Rapid Diagnostic Test for Hepatitis B

Steps

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 1 SAMPLE/SPECIMEN COLLECTION & STORAGE
 Test should be performed on human serum or plasma only immediately after collection.
 If not tested immediately, specimen should be refrigerated at 2-8°C up to 3 days following
collection.
 If testing within 3 days is not possible, specimen should be stored frozen at 20°C.
 Haemolysed specimen or specimen with microbial contamination should be discarded and
fresh aliquot should be collected.
2 TEST PROCEDURE: Procedure should be followed as per kit manual. Briefly, the procedure is as
follows:
 Bring the required number of test foil pouches and specimen to room temperature prior to
testing
 Take out device from the foil pouch.
 Label the test card with patient’s name or identification number. ∙
 Add 2 drops (70 μl) of human serum/plasma specimen into the sample well using the
dropper provided (use separate dropper/microtip for each specimen).
 Allow reaction to occur during the next 20 minutes.
 Read results at 20 minutes.
 Discard the test kit immediately after reading result at 20 minutes, considering it to be
potentially infectious.
3 INTERPRETATION OF RESULT
REACTIVE: Appearance of pink coloured line, one each in test region “T” and control region “C”
indicates that the sample is REACTIVE for HBsAg.

NON-REACTIVE: Appearance of one distinct pink line in the control region “C” only, indicates
that the sample is “NON-REACTIVE” for HBsAg.

INVALID: When neither control line nor the test line appears on the membrane, the test should be
treated as invalid which maybe because of following reasons:
a. Improper storage at temperature other than the recommended temperature.
b. Wrong procedure.
c. Long atmospheric exposure of the test device after opening the pouch. The test should be repeated
using a new test card and test sample.

Rapid Diagnostic Test for HIV/AIDS

Steps
1 Materials and reagents: HIV TRI-DOT test device, buffer solution, Protein- A conjugate. Negative
control, positive control, sample dropper

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2 Storage: Store the entire kit at 2-8°C in the coolest and driest area available. Do not use the kit beyond
the expiry date. Do not freeze the kit components.
3 Specimen/ sample collection
 Collect blood in a clean dry sterile vial and allow to clot
 It is recommended that fresh sample should be used if possible.
 If serum is not to be assayed immediately, it should be stored at 2-8°C or frozen at minus 20°C
(-20°C).
 Hemolyzed specimen or specimen with microbial contamination should be discarded and fresh
aliquot should be collected.
4 Procedure: Procedure should be followed as per kit manual. Briefly, the procedure is as follows:
 Add 3 drops of Buffer Solution to the centre of the device
 Hold the dropper vertically and add 1 drop of patient’s sample 50μl (serum or plasma) using
the sample dropper provided (use a separate sample dropper for each specimen to be tested).
 Add 5 drops of Buffer Solution.
 Add 2 drops of Protein-A Conjugate directly from the conjugate vial.

 Add 5 drops of Buffer Solution and read results.

 Read results immediately and discard the device considering it to be potentially infectious
5 Interpretation of results:
NON-REACTIVE: If only one Dot (only the Control Dot) appears, the specimen is non-reactive for
antibodies either to HIV-1 or HIV-2. Interpret sample as non-reactive.
REACTIVE:
∙ If two Dots, one for the control and the other for HIV-1appear, the specimen is reactive for antibodies
to HIV-1.
∙ If two Dots, one for the control and the other for HIV-2appear, the specimen is reactive for
antibodies to HIV-2.
∙ If all the three Dots, one each for control, HIV-1 & HIV-2 appear, the specimen is reactive for
antibodies to HIV-1 & HIV-2.
INVALID TEST:
If no Dot appears after the test is complete, either with clear background or with complete
pinkish/purple background as, the test indicates ERROR. This may indicate a procedural error or
deterioration of specimen/reagents or particulate matter in the specimen. The specimen should be
tested

Rapid Kit Test for Dengue

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Test for Faecal Water Contamination

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Observe the Result After 48 Hours

Test for Water Chlorination

Directions for use

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 The water which is chlorination and to be tested is to be drawn
from well or to be collected from tap or a storage tank in a clean
glass tube or a bucket.

Take out the two tubes and thoroughly wash them before with
the same water that is collected and is to be tested. Fill up the
test tube with water and insert it into the left handhole.

Fill the right-hand side tube with water and add carefully three
to four drops of solution (Reagent) by means of the dropper.

Close the top of the test tube to mix with the water
homogenously by inverting the tube several times by putting the
thumb on the top of the test tube till a uniform (yellow color) is
developed. Insert the tube through the hole on the right-hand
side and fix the cap on the top.

Now it is ready for measuring the residual chlorine.

Method to Measure Residual Chlorine

 Hold the chloroscope at the level of your eyes with the front side facing you in
moderately bright light.
 Starting from below with 0.1 reading observe simultaneously at each pair of colors on
either side of the marking. Find out the pair in which the colors are distinctly matched
and note the reading between the pair.
 The reading gives you the exact amount of residual chlorine in parts per million (PPM)
 If no color develops after the reagent is added and mixed it means that there is no
residual chlorine in the water and it is safe for drinking.
 If the yellow color developed in the water is darker in shade then the color disc of 1.0 it
means that the Residual chlorine is more than 1.0 PPM even though it is safe, the light
chlorine content may irritate the mucosa of mouth and stomach and is not desirable for
drinking. It requires dilution with unchlorinated water.
 The optimum reading should be 0.2 PPM with a range of 0.1 PPM to 0.5 PPM.
Maintenance
 After every use throws away the water in the test tubes and thoroughly wash them with
clean water the brush to clean the inside of the tubes.
 Keep the empty test tube after shaking away the attached drops of water in their places.
Fix the cap to prevent collection of dust.
 It is essential that the glass tubes should be cleaned and dried so that optical color
matching is not interfered.

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 Test for Iodine in Salt by SPOT testing Kit
 In the spot testing method, estimation of iodine content in salt is done with the help of a
standard starch solution provided in the Spot Testing Kit (STK).
 The STK consists of a test solution (A), and a recheck solution (B).
 One drop of test solution (A) will be added to a pinch of salt sample.
 The change in colour of salt sample will range from white to dark violet depending on
the iodine content of salt, i.e. Nil, less than 15ppm, and 15 ppm and above, which will
be compared to the standard colour chart provided with the kit.
 If on addition of test solution (A), no change in colour of salt is observed, the recheck
solution (B) will be added.
 This will be done to make the salt medium acidic, in case the salt has alkaline
constituents, then the test solution (A) will be added again. The intensity of the blue
colour will be directly proportionate to the iodine content of salt

Procedure for use of Spot Testing Kit

Take a spoonful of iodated salt to be checked and

spread it flat.

Open the seal of the ampule (white cap) by making a

pin hole.

Discharge a drop of the test solution on the surface of

the salt by gently pressing the ampule.

The salt will turn light blue to dark violet depending on

the iodine content of the salt.

Use the colour chart given on plastic box to compare

and determine the iodine range in the iodated salt.

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References
1. Training Manual on Care During Pregnancy and Child Birth for Community
Health Officer at Ayushman Bharat - Health and Wellness Centres

2. Training Manual on Management of Communicable Diseases for Community

Health Officer at Ayushman Bharat – Health and Wellness Centres

3. National Iodine Deficiency Disorders Control Program (NIDDCP),

https://saltcomindia.gov.in/nidccp_estimation&content.html

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