TM

ACCUCARE
DIRECT AND INDIRECT COOMB’S TESTS
Blood Grouping

Qualitative test for determination of human anti-IgG and anti-C3 on  The reagents have been filtered through a 0.2μm capsule to
red blood cells. For In Vitro Diagnostic Test use only. reduce the bio-burden. Once a vial has been opened the contents
should remain viable up until the expiry date as long as there is no
marked turbidity, which can indicate reagent deterioration or
ORDER INFORMATION contamination.
REF Pack Size  The reagent contains preservative. Do not swallow. Avoid contact
AHG 10 1 X 10mL with skin and mucous membranes.
AHG 100 10 X 10 mL  Consider Blood specimen as potentially infectious, handle and
AHG 1000 1 X 1000mL dispose it as per national applicable guideline.
 For information on disposal of the reagent and decontamination of
a spillage site see Material Safety Data Sheets, available on
CLINICAL SIGNIFICANCE request.
In 1945, Coombs, Mourant and Race described the use of anti-human
globulin serum for detecting red cell-bound non-agglutinating
antibodies. In 1957, Dacie et al showed that the antibodies present in WASTE MANAGEMENT
antiglobulin sera were directed against certain components of Please refer to local legal requirements.
complement.
Antihuman globulin reagents detect non-agglutinating antibody MATERIALS REQUIRED BUT NOT PROVIDED
molecules as well as molecules of complement attached to red cells
following in vivo or in vitro antigen-antibody reactions. Accordingly, Anti-  Test tubes (8X50mm),
Human Globulin is used for compatibility testing, antibody detection,  Pipettes,
antibody identification, testing for the variant of the Rho (D) antigen (DU  Centrifuge
tests), and umbilical cord red blood cell testing.  (0.9% NaCl) saline.
 General laboratory equipment
METHOD
Hemagglutination technique. SAMPLE COLLECTION AND PRESERVATION
 Blood should be drawn by an aseptic technique with an
PRINCIPLE anticoagulant. The specimen should be tested as soon as possible
The procedures used with this reagent are based on the principle of after collection.
heteroagglutinins directed against components of human serum as  If delay in testing should occur, the specimen must be stored at
originally described by Moreschin and agglutination as described by 2°C to 8°C. Bacterial contamination may cause false test results.
Landsteiner. Normal human red blood cells, in the presence of antibody  Blood drawn into EDTA should be use within 24 hours. If EDTA is
directed toward an antigen they possess, may become sensitize but fail unavailable, samples drawn into ACD, CPD or CPDA-1 are
to agglutinate due to the particular nature of the antigen and antibody preferable to clotted ones. If only clotted samples are available, do
involved. Anti-Human Serum will react with immunoglobulins and/or not refrigerate them before testing. All blood samples should be
complement attached to the red cell surface, resulting in agglutination washed at least twice with PBS before being tested.
(clumping) of adjacent sensitized cells. Cells not sensitized will not be
agglutinated (See Limitations). ASSAY PROCEDURE
Direct Antiglobulin Technique (DAT)
REAGENT 1. Wash test red cells 4 times with PBS, taking care to decant saline
Anti-Human Globulin Elite Green reagents contain anti-IgG derived from between washes and resuspend each cell button after each wash.
rabbits with nonspecific activity removed by absorption and mouse Completely decant saline after last wash.
monoclonal IgM anti-C3d, Clone BRIC-8. The antibodies are diluted in a 2. Add 2 volumes of Anti-Human Globulin to each dry cell button.
buffered solution containing bovine albumin. Each reagent is supplied at 3. Mix thoroughly and centrifuge all tubes for 20 seconds at 1000rcf
optimal dilution, for use with all the recommended techniques stated or for a suitable alternative time and force.
below without need for further dilution or addition.
4. Gently resuspend red cell button and read macroscopically for
Reagent Colour Dye Used agglutination
Coomb’s Sera (AHG) Green Patent Blue + Tartrazine
Indirect Antiglobulin Technique (NISS IAT)
REAGENT PREPARATION 1. Prepare a 2-3% suspension of washed test red cells in PBS.
The reagent supplied is ready to use. Protect from Bright Light. 2. Place in a labeled test tube: 2 volumes of test serum and 1 volume
of test red cell suspension.
REAGENT STORAGE AND STABILITY 3. Mix thoroughly and incubate at 37oC for 15 minutes.
This product will be well-preserved within utility limit till the expiry date, if 4. Wash test red cells 4 times with PBS, taking care to decant saline
stored at temperature between +2C and +8C. between washes and resuspend each red cell button after each
wash. Completely decant saline after last wash.
Caution: Do not freeze.
5. Add 2 volumes of Anti-Human Globulin to each dry cell button.
6. Mix thoroughly and centrifuge all tubes for 20 seconds at 1000rcf
WARNING AND PRECAUTIONS or for a suitable alternative time and force.
 For in vitro diagnostic use. 7. Gently resuspend red cell button and read macroscopically for
 Do not use components beyond the expiration date. agglutination
 If the reagent vial is cracked or leaking, discard the contents
immediately. LISS Indirect Antiglobulin Technique (LISS IAT)
 Do not use the reagents if a precipitate is present. 1. Prepare a 1.5-2% suspension of washed test red cells in LISS.
 Exercise the normal precautions required for handling all 2. Place in a labeled test tube: 2 volumes of test serum and 2
laboratory reagents. volumes of test red cell suspension.
Art No.: IFU/SR/AHG
Effective Date: 10/04/2020
Revision No.: 01
 TM
ACCUCARE
DIRECT AND INDIRECT COOMB’S TESTS
Blood Grouping

3. Mix thoroughly and incubate at 37oC for 15 minutes. LIMITATION OF THE PROCEDURE
Follow steps 4 to 7 of NISS IAT above.  Red cells that have a positive DAT due to a coating of IgG cannot
NOTES be typed by the Indirect Antiglobulin Techniques.
1. It is recommended a positive control (weak Anti-D 0.1 IU/ml) and a  A positive DAT due to complement sensitization may not reflect in
negative control (an inert serum) be test in parallel with each batch vivo complement fixation if test cells are from a refrigerated clotted
of tests. Tests must be considered invalid if controls do not show specimen.
expected results.  Inadequate washing of red cells in the indirect antiglobulin
2. The antiglobulin techniques can only be considered valid if all techniques may neutralize the anti-human globulin reagent.
negative tests react positively with IgG sensitized red cells.  Following completion of the wash phase excess residual saline
3. In the techniques, here mentioned, one volume is approximately may dilute the anti-human globulin, reducing its potency.
40μl when using the vial dropper provided.  A negative direct antiglobulin test result does not necessarily
4. Use of the reagents and the interpretation of results must be preclude clinical diagnosis of ABO Hemolytic Disease of the
carried out by properly trained and qualified personnel in Newborn or Auto Immune Hemolytic Anemia. It also does not
accordance with requirements of the country where the reagents necessarily rule out HDN, especially if ABO incompatibility is
are in use. User must determine the suitability of the reagents for suspected.
use in other techniques.
 False positive or false negative results may also occur due to:
Stability of the reactions
 Improper storage, cell concentration, incubation time or
1. Washing steps should be completed without interruption and tests temperature
centrifuged and read immediately after addition of the reagent.
 Improper or excessive centrifugation
2. Delays may result in dissociation of antigen-antibody complexes,
 The user is responsible for the performance of the reagents by any
causing false negative or weak positive results. Caution should be
method other than those here mentioned.
exercised in the interpretation of results of tests performed at
temperatures other than those recommended.  Any deviations from the techniques here recommended should be
validated prior to use Contamination of test materials.
INTERPRETATION
Positive: Agglutination of test red cells constitutes a positive test result BIBLIOGRAPHY
and within the accepted limitations of the test procedure, indicates the 1. Coombs RRA, Mourant AE, Race RR. A new test for the detection
presence of IgG and/or complement (C3) on the test red cells. of weak and “incomplete” Rh antibodies. Brit J Exp Pathol. 1945;
26:255.
Negative: No agglutination of the test red cells constitutes a negative 2. Wright MS, Issit PD. Anti-complement and the indirect antiglobulin
result and within the accepted limitations of the test procedure, indicates test. Transfusion 1979; 19:688-694.
the absence of IgG and/or complement (C3) on the test red cells. 3. Howard JE, Winn LC, Gottlieb CE, Grumet FC, Garratty G, Petz
LD. Clinical significance of the anti - complement components of
PERFORMANCE CHARACTERISTICS antiglobulin antisera. Transfusion 1982: 22:269.
1. The reagents have been characterized by all the procedures here 4. Howell P, Giles CM. A detailed serological study of five anti-Jka
described. sera reacting by the antiglobulin technique. Vox. Sang. 1983; 45:
2. Prior to release, each lot of Accucare’s Anti-Human Globulin is 129-138.
tested, by the techniques here mentioned, against red cells coated 5. Issitt PD, Smith TR. Evaluation of antiglobulin reagents. A seminar
with Anti-D, Anti-K and Anti-Fya to check suitable reactivity. on performance evaluation. Washington, DC. American
3. Potency of anti-IgG and anti-C3d have been tested against the Association of Blood Banks. 1976; 25-73.
following minimum potency reference standard obtained from 6. The anti-complement reactivity low ionic methods as published by
National Institute of Biological Standards and Controls (NIBSC): FDA. Recommended Methods for Anti - Human Globulin
Anti-AHG reference standard 96/666 Evaluation (revision October 1984).
4. Anti-C3d potency is demonstrated in tests employing cells coated
with C3. GLOSSARY OF SYMBOL
5. The presence of contaminating heterospecific agglutinins or
antibodies to C4d has been excluded in tests employing red cells Consult Instruction for Use Lot Number
of all ABO groups and cells coated with C4d.
Catalog Number Date of Manufacturing
6. The reactivity of any Anti-IgM, Anti-IgA or Anti-light chain
components that might be present has not been established.
Store between Use By or Expiration Date
7. The Quality Control of the reagents was performed using red cells
that had been washed twice with PBS prior to use. Manufacturer For in vitro Diagnostic use only
8. The reagents comply with the recommendations contained in the
latest issue of the Guidelines for the UK Blood Transfusion Keep away from sunlight Content of the kit
Services.
LAB-CARE DIAGNOSTICS (INDIA) PVT. LTD.
DISCLAIMER C1 Type, Shed No.: 3225, Chemical Zone,
GIDC Sarigam – 396155, Dist. Valsad, Gujarat, India.
 Each facility should verify the optimum spin time for the specific Tel.: +91 22 2554 2109 /1558
centrifuge in use. Email: accucarediagnostics.com; Website: www.labcarediagnostics.com

 Manual techniques are to be performed according to the

manufacturer’s instructions.
 Each deviation from these instructions is the sole responsibility of
the user.
 Used tests must be discarded as hazardous material. Manage
waste according to local, state and national regulations.

Art No.: IFU/SR/AHG

Effective Date: 10/04/2020
Revision No.: 01