Qualitative test for determination of human anti-Ig G and anti-C3d
on red blood cells.
IVD
Store at 2-8ºC
PRINCIPLE OF THE METHOD
When used by the recommended techniques, the reagents will react with
immunoglobulins and/or complement attached to the red cell surface, resulting in
agglutination (clumping) of adjacent sensitised cells. Cells not sensitised will not be
agglutinated (See Limitations).
REAGENTS
Spinreact Polyspecific Anti-Human Globulin Elite Clear and Anti-Human Globulin Elite Green
reagents contain anti-IgG derived from rabbits with non-specific activity removed by absorption
and mouse monoclonal IgM anti-C3d, Clone BRIC-8. The antibodies are diluted in a buffered
solution containing bovine albumin. Each reagent is supplied at optimal dilution, for use with all
the recommended techniques stated below without need for further dilution or addition. For lot
reference number and expiry date see Vial Label.
Reagent Colour Dye Used
Anti-Human Globulin Green Green Patent Blue and Tartrazine
PRECAUTIONS
1. The reagents are intended for in vitro diagnostic use only.
2. If a reagent vial is cracked or leaking, discard the contents immediately.
3. Do not use the reagents past the expiration date (see Vial Label).
4. Do not use the reagents if a precipitate is present.
5. Protective clothing should be worn when handling the reagents, such as
disposable gloves and a laboratory coat.
6. The reagents have been filtered through a 0.2 µm capsule to reduce the bioburden. 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 contamination.
7. The reagents contain 0.1% sodium azide. Sodium azide may be toxic if
ingested and may react with lead and copper plumbing to form explosive metal
azides. On disposal flush away with large volumes of water.
8. No known tests can guarantee that products derived from human or animal
sources are free from infectious agents. Care must be taken in the use and
disposal of each vial and its contents.
9. For information on disposal of the reagent and decontamination of a spillage site
see Material Safety Data Sheets, available on request.
NOTES
1. It is recommended a positive control (weak Anti-D 0.1 IU/ml) and a negative control (an
inert serum) be test in parallel with each batch of tests. Tests must be considered
invalid if controls do not show expected results.
2. The antiglobulin techniques can only be considered valid if all negative tests react
positively with IgG sensitised red cells.
3. In the techniques, here mentioned, one volume is approximately 40µl when using the
vial dropper provided.
4. Use of the reagents and the interpretation of results must be carried out by properly
trained and qualified personnel in accordance with requirements of the country where
the reagents are in use. User must determine the suitability of the reagents for use in
other techniques.
STORAGE
Do not freeze. Reagent vials should be stored at 2 - 8ºC on receipt. Prolonged storage at
temperatures outside this range may result in accelerated loss of reagent reactivity.
MATERIAL REQUIRED
Glass test tubes (10 x 75 mm or 12 x 75 mm).
Test tube centrifuge.
Volumetric pipettes.
Phosphate Buffered Saline (PBS): NaCl 0.9%, pH 7.0 ± 0.2 at 22ºC ± 1ºC.
IgG sensitised red cells.
Inert antibody Serum.
Weak anti-D.
Water bath or dry heat incubator equilibrated to 37ºC ± 2ºC.
Coombs cell washer.
Low Ionic Strength Solution (LISS) , i.e..: Spinreact’s REF. 1700080.
SAMPLE
Samples should be drawn aseptically into EDTA to prevent in vitro complement binding and
tested within 24 hours. If EDTA is unavailable, samples drawn into ACD, CPD or CPDA-1 are
preferable to clotted ones. If only clotted samples are available, do not refrigerate them before
testing. All blood samples should be washed at least twice with PBS before being tested.
PROCEDURE
A. Direct Antiglobulin Technique (DAT)
1. Wash test red cells 4 times with PBS, taking care to decant saline between
washes and resuspend each cell button after each wash. Completely decant
saline after last wash.
2. Add 2 volumes of Anti-Human Globulin to each dry cell button.
3. Mix thoroughly and centrifuge all tubes for 20 seconds at 1000 rcf or for a
suitable alternative time and force.
4. Gently resuspend red cell button and read macroscopically for agglutination
B. Indirect Antiglobulin Technique (NISS IAT)
1. Prepare a 2-3% suspension of washed test red cells in PBS.
2. Place in a labelled test tube: 2 volumes of test serum and 1 volume of test red
cell suspension.
3. Mix thoroughly and incubate at 37ºC for 15 minutes.
4. Wash test red cells 4 times with PBS, taking care to decant saline between
washes and resuspend each red cell button after each wash. Completely decant
saline after last wash.
5. Add 2 volumes of Anti-Human Globulin to each dry cell button.
6. Mix thoroughly and centrifuge all tubes for 20 seconds at 1000 rcf or for a
suitable alternative time and force.
7. Gently resuspend red cell button and read macroscopically for agglutination
C. LISS Indirect Antiglobulin Technique (LISS IAT)
1. Prepare a 1.5-2% suspension of washed test red cells in LISS.
2. Place in a labelled test tube: 2 volumes of test serum and 2 volumes of test red
cell suspension.
3. Mix thoroughly and incubate at 37ºC for 15 minutes.
4. Follow steps 4 to 7 of NISS IAT above.
INTERPRETATION OF TEST RESULTS
Positive: Agglutination of test red cells constitutes a positive test result and within the
accepted limitations of the test procedure, indicates the presence of IgG and/or complement
(C3) on the test red cells.
Negative: No agglutination of the test red cells constitutes a negative result and within the
accepted limitations of the test procedure, indicates the absence of IgG and/or complement
(C3) on the test red cells.
Stability of the reactions
1. Washing steps should be completed without interruption and tests centrifuged and read
immediately after addition of the reagent. Delays may result in dissociation of antigenantibody complexes, causing false negative or weak positive results.
2. Caution should be exercised in the interpretation of results of tests performed at
temperatures other than those recommended.
LIMITATIONS
1. Red cells that have a positive DAT due to a coating of IgG cannot be typed by the
Indirect Antiglobulin Techniques.
2. A positive DAT due to complement sensitisation may not reflect in vivo complement
fixation if test cells are from a refrigerated clotted specimen.
3. Inadequate washing of red cells in the indirect antiglobulin techniques may neutralise
the anti-human globulin reagent.
4. Following completion of the wash phase excess residual saline may dilute the antihuman globulin, reducing its potency.
5. A negative direct antiglobulin test result does not necessarily preclude clinical diagnosis
of ABO Haemolytic Disease of the Newborn or Auto Immune Haemolytic Anaemia. It
also does not necessarily rule out HDN, especially if ABO incompatibility is suspected.
6. False positive or false negative results may also occur due to:
Contamination of test materials
Improper storage, cell concentration, incubation time or temperature
Improper or excessive centrifugation
7. The user is responsible for the performance of the reagents by any method other than
those here mentioned.
8. Any deviations from the techniques here recommended should be validated prior to
use9
.
PERFORMANCE CHARACTERISTICS
1. The reagents have been characterised by all the procedures here described.
2. Prior to release, each lot of Spinreact’s Anti-Human Globulin is tested, by the
techniques here mentioned, against red cells coated with Anti-D, Anti-K and Anti-Fya to
check suitable reactivity.
3. The anti-IgG and anti-C3d potencies have been tested against the following minimum
potency reference standard obtained from National Institute of Biological Standards and
Controls (NIBSC): Anti-AHG reference standard 96/666
4. Anti-C3d potency is demonstrated in tests employing cells coated with C3.
5. The presence of contaminating heterospecific agglutinins or antibodies to C4d has been
excluded in tests employing red cells of all ABO groups and cells coated with C4d.
6. The reactivity of any Anti-IgM, Anti-IgA or Anti-light chain components that might be
present has not been established.
7. The Quality Control of the reagents was performed using red cells that had been
washed twice with PBS prior to use.
8. The reagents comply with the recommendations contained in the latest issue of the
Guidelines for the UK Blood Transfusion Services.
