1434 Human antiglobulin

Direct and Indirect Coombs Tests

Blood groups
Qualitative determination of human anti-Ig G and anti-C3d in 3. Mix thoroughly and incubate at 37ºC for 15 minutes.
4. Wash the red blood cells 4 times in PBS, being careful to decant the saline solution in between.
red blood cells
washing and resuspending the cell pellet after each wash. After the last wash,
IVD completely decant the saline solution.
5. Add 2 volumes of human antiglobulin to each cell button.
Store at 2-8ºC 6. Mix thoroughly and centrifuge the tubes for 20 seconds at 1000 rcf (g) or at
an appropriate alternative force and time.
PRINCIPLE OF THE METHOD 7. Carefully resuspend the cell button y read macroscopically by
Using the recommended techniques, the reagents will react with immunoglobulin and/or agglutination.
complements linked to the surfaces of the red blood cells, causing the agglutination of the
C. Indirect antiglobulin technique LISS (LISS IAT)
adjacent sensitized cells. Unsensitized cells will not agglutinate (see
1. Prepare a 1.5 - 2% suspension of washed red blood cells in lytic solution.
Limitations.
2. Deposit in an identified tube: 2 volumes of the serum to be tested and 2 volumes of the
suspension of red blood cells to test.
CLINICAL SIGNIFICANCE
In 1945, Coombs, Mourant, and Race discovered the use of human anti-globulin for the 3. Mix thoroughly and incubate at 37ºC for 15 minutes.
detection of non-agglutinating antibodies of red blood cells. In 1957, Dacie et al. showed 4. Follow steps 4 to 7 of the technique (NISS IAT)
that the antibodies of an antiglobulin serum were directly against certain
INTERPRETATION OF THE RESULTS
components of the complement. Human antiglobulin reagents detect both molecules
1. Positive: The agglutination of the red blood cells to test is a positive result and
of non-agglutinating antibodies, such as complement molecules bound to red blood cells
within the accepted limitations for the test procedure, indicate the presence
following antigen-antibody reactions in vivo in vitro. IgG and/or complement (C3) in the red blood cells to be tested.
REAGENTS 2. Negative: The absence of agglutination of the red blood cells to test constitutes a result
The Spinreact poly-specific human anti-globulin reagents contain anti-IgG derived from negative and within the accepted limitations for the procedure of the technique,
rabbit, whose unspecific activity has been eliminated by absorption and mouse monoclonal IgM Indicates the absence of IgG and/or complement (C3) in the red blood cells to be tested.
anti-C3d, clone BRIC-8. The antibodies are diluted in a buffered solution that contains Stability of reactions
bovine albumin. Each reagent is supplied in the optimal dilution for its use in 1. The washing stages must be completed without any interruptions and the centrifugation and reading
all the techniques recommended here without the need for dilutions o additions It must be carried out immediately after the addition of the reagent. Delays may lead to the
supplementary. Check the batch and expiration date of each reference on the vial label. dissociation of the antigen-antibody complexes, causing false negatives or results
weak positives.
Reactive Color Coloring used 2. The results from tests conducted at other temperatures than those recommended here must be
interpreted with caution.
Human green antiglobulin Green Tartrazine and patented blue
LIMITATIONS
PRECAUTIONS 1. The red blood cells that are positive by DAT due to an IgG coating do not
1. The reagents are for in vitro diagnostic use only. They can be typed by the Indirect Antiglobulin techniques.
2. If the reagent vial is broken or cracked, immediately discard its content. 2. A positive DAT for sensitization with complement may not reflect the fixation.
3. Do not use expired reagents. (see vial label). live complement, if the cells to be filled come from a clotted sample
4. Do not use reagents that present precipitates. refrigerated.
5. The handling of the reagent must be done with the appropriate protective clothing, such as 3. Inadequate washes in indirect antiglobulin techniques can neutralize the reagent.
such as disposable gloves and lab coat. human antiglobulin.
6. The reagents have been filtered through 0.2 µm capsules to reduce the biological load. 4. After completing the washing phase, the excess residual saline phase can dilute the
Once the vial is opened, the contents must remain viable until the expiration date. human antiglobulin, reducing its potency.
as long as there is no marked turbidity which could be indicative of deterioration or
5. A negative result for direct antiglobulin techniques does not necessarily exclude a
reagent contamination.
7. The reagents contain < 0.1% of sodium azide. Sodium azide can be toxic if ingested. clinical diagnosis of ABO Hemolytic Syndrome of the Newborn o Anemia
and it can react with copper or lead from the pipes and form explosive metal azides. In Autoimmune Hemolytic. It also does not rule out HDN, especially if suspected.
In case of product disposal, do so with plenty of tap water. of ABO incompatibility.
8. No method can guarantee that products derived from human or animal sources 6. False positive or negative results may also occur due to:
are free from infectious diseases. Handle and dispose of vials and their •Contamination of the test materials

9.
content.
For more information about product disposal or decontamination in case of
•Inadequate preservation, cellular concentration, time or temperature of
incubation
spill, see the safety data sheets.
7.
•Inappropriate or excessive centrifugation
The user is responsible for the operation of the reagents in any other method.
NOTES
It is recommended to use a positive control (weak Anti-D 0.1 IU/mL) and a control different from those mentioned here.
negative (inert serum) to test in parallel in each batch of tests. The tests 8. Any deviation from the techniques detailed here should be validated before their
They must be considered invalid if the controls do not show the expected results. utilization9.
2. Antiglobulin techniques can only be considered valid if all tests CHARACTERISTICS OF THE METHOD
negatives react positively with red blood cells sensitized with IgG.
1. The reagents have been characterized for all the procedures here.
3. In the recommended techniques, a volume is approximately 40µl using the
mentioned.
supplied dropper.
2. Prior to its release, each batch of Spinreact Human Antiglobulin is tested for
4. The use of reagents and the interpretation of results must be carried out through the recommended techniques in front of a panel of Anti-D, Anti-K red blood cells and
qualified and trained personnel according to the requirements of the country where the
Anti-Fya, to ensure the proper reactivity.
reactives are being used. The user must determine the suitability of the
3. The anti-IgG and anti-C3d potency has been tested against the reference standard of
reagents for other techniques. minimum power, Anti-AHG 96/666, originating from the National Institute of Biological
CONSERVATION Standards and Controls (NIBSC).
Do not freeze. The reagent vials must be stored at 2-8ºC. Storage 4. The anti-C3d potency is demonstrated in tests that use coated cells with
prolonged exposure to temperatures outside this range can cause an acceleration of the C3.
loss of reactivity. 5. The presence of heterospecific contaminating agglutinins or anti-C4d antibodies
it has been excluded in tests that use red blood cells from all ABO groups and cells
NECESSARY MATERIAL covered with C4.
• Glass tubes (10 x 75 mm or 12 x 75 mm). 6. The reactivity of any Anti-IgM, Anti-IgA or Anti-components has not been established.
• Tube centrifuge. of light chain, which could be present.
• Volumetric pipettes. 7. The Quality Control of the reagents was carried out using washed red blood cells.
• Phosphate buffered saline (PBS): NaCl 0.9%, pH 7.0 ± 0.2 at 22ºC ± 1ºC. times in PBS, prior use.
• IgG sensitized erythrocytes 8. The reagents meet the recommendations of the latest version of the Guidelines for the
• Inert antibody serum. Blood transfusion services of the United Kingdom.
• Weak anti-D.
• Hot bath or balanced dry heat incubator at 37ºC ± 2ºC. BIBLIOGRAPHY
• Coombs cell washer 1. Coombs RRA, Mourant AE, Race RR. A new test for the detection of weak and 'incomplete' Rh
• Liss solution (LISS), e.g.: Spinreact REF 1700080.
2.
antibodies. Brit J Exp Pathol. 1945; 26:255.
Wright MS, Issit PD. Anti-complement and the indirect antiglobulin test. Transfusion 1979; 19:688-694.
3. Howard JE, Winn LC, Gottlieb CE, Grumet FC, Garratty G, Petz LD. Clinical significance of the anti-
SAMPLES complement components of anti-globulin antisera. Transfusion 1982: 22:269.
The samples must be treated aseptically in EDTA to prevent in vitro binding. 4. Howell P, Giles CM. A detailed serological study of five anti-Jk asera reacting by the antiglobulin
to complement and analyze within 24 hours. In case the EDTA is not available, it is preferable technique. Vox. Sang. 1983;45: 129-138.
samples treated in ACD, CPD or CPDA-1, which are coagulated samples, If only available 5. Issitt PD, Smith TR. Evaluation of antiglobulin reagents. A seminar on performance evaluation.
clotted samples, do not refrigerate before analyzing. All blood samples must Washington, DC. American Association of Blood Banks. 1976; 25-73.
6. The Department of Health and Social Security. Health Services Management Antiglobulin Test. False
must be washed with PBS at least twice before performing the test. negative results, HN (Hazard) (83) 625 Nov 1983.
7. Bruce M, Watt AH, Hare W, Blue A, Mitchell R. A serious source of error in antiglobulin testing.
PROCEDURE Transfusion 1986;26: 177-181.
8. The anti-complement reactivity low ionic methods as published by FDA. Recommended Methods for Anti-
A. Direct Antiglobulin Test (DAT) Human Globulin Evaluation (revision October 1984).
1. Wash the red blood cells to be tested 4 times in PBS, being careful to decant the saline solution. 9. Dynan PK. Evaluation of commercially available low ionic strength salt (LISS) solutions. Med Lab Sci
between washes and resuspending the cell button after each wash. After the last wash, (1981)381: 13-20.
completely decant the saline solution. 10. Voak D, Downie DM, Moore BPL, Ford DS, Engelfreit CP, Case J. Replicate tests for the detection and
2. Add 2 volumes of human antiglobulin to each cell button. correction of errors in anti-human globulin (AHG) tests: optimum conditions and quality control.
Hematology 1988;21(1): 3-16.
3. Mix thoroughly and centrifuge the tubes for 20 seconds at 1000 rcf (g) or to 11. Guidelines for the Blood Transfusion Service in the United Kingdom. H.M.S.O. Current Edition.
an appropriate alternative force and time. 12. British Committee for Standards in Haematology, Blood Transfusion Task Force. Recommendations for
4. Carefully resuspend the cell button y reading macroscopically by evaluation, validation and implementation of new techniques for blood grouping, antibody screening and
agglutination. cross matching. Transfusion Medicine, 1995,5, 145-150.

B. Indirect antiglobulin technique (NISS IAT) PRESENTATION

1. Prepare a 2-3% suspension of washed red blood cells in PBS.
2. Deposit into an identified tube: 2 volumes of the serum to be tested and 1 volume of the Human anti-globulin (Coombs) Ref: 1700051 10 ml
suspension of red blood cells to test.

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