FOR IN VITRO LABORATORY USE ONLY.
WARRANTY: VMRD, Inc. warrants that this product is as described in the quantity and contents stated on the label at the
time of delivery to the customer. NO OTHER WARRANTIES, EXPRESS OR IMPLIED, ARE MADE BEYOND THE LABEL
DESCRIPTION, INCLUDING WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR USE. Remedy
is limited to replacement of the product or refund of the purchase price. VMRD, Inc. is not liable for property damage,
personal injury, or economic loss caused by the product. The information listed in this information sheet is provided for
reference only, and should not be substituted for the user’s own incoming material quality control.
H:
\
Quality VMRD
\
QC
\
CofA
\
Coombs
\
Equine Coombs
\
492-2 P091005-001 140222.docx
25 February 2012
VMR
D
Telephone: 509-334-5815
Fax: 509-332-5356
E-mail:
vmrd@vmrd.com
PO Box 502, Pullman, WA 99163 USA
Web site:
http://www.vmrd.com
Certificate of Analysis
EQUINE COOMBS REAGENT
CATALOG NO.:
492-2
VOLUME:
2 ml
LOT:
P091005-001
EXPIRATION DATE:
22 February 2014
INTRODUCTION: The equine Coombs test, also called direct antiglobulin test, is designed to detect immune-mediated
erythrocyte destruction which occurs in autoimmune hemolytic anemia, and in some cases with infections and neoplastic
disorders, in neonatal isoerythrolysis. Hemolysis in these diseases is caused by the erythrocytes being coated with antibody
(IgG, IgM) and/or complement components (C3). Coated erythrocytes are lysed in the bloodstream and/or removed by
phagocytes.
The Coombs reagent is an antiserum to equine IgG, IgM, and C3 prepared in goats. After obtaining the antiserum,
complement is inactivated at 56C for 30 minutes and then the antiserum is absorbed repeatedly with washed normal equine
erythrocytes. These treatments ensure that the Coombs reagent will not react with normal equine erythrocytes. However,
equine erythrocytes that are coated with IgG, IgM, and/or C3 will be agglutinated by the Coombs reagent because it contains
antibodies to equine IgG, IgM, and C3.
QUALITY CONTROL METHOD: Washed sheep red blood cells (SRBC) were sensitized with the Equine Coombs
Positive Control (catalog no. 472-2). The procedure is performed according to the Coombs Positive Control Procedure. The
Equine Coombs Reagent was tested according to the Equine Coombs Reagent Procedure.
Specific Reaction:
The Coombs reagent produced agglutination on sensitized SRBCs of 1+ agglutination at
1/2, 1-2+ at 1/4 and +/- agglutination at 1/8. No agglutination on unsensitized SRBCs.
Other Comments:
The subagglutinating dose for the equine positive control was determined to be 1/8.
When using the positive control this dose may vary under your laboratory conditions and
especially with your source of sheep red blood cells. Therefore, we recommend that you
titer the positive control with your own SRBCs before using it. Please refer to Section A
of the Coombs Positive Control procedure.
INDICATIONS FOR TEST: Horses with anemia (including that caused by intravascular and extravascular hemolysis) of
unknown origin are reasonable candidates for evaluation by Coombs testing. Foals with neonatal isoerythrolysis are often
Coombs positive.
PRECAUTIONS: Use the reagent at the dilutions described in the procedure to avoid nonspecific and prozone effects.
STORAGE: Store at <–10ºC until expiration date or at 2-7ºC if used within 6 months of opening.
REFERENCES:
McGuire, T.C., et al. Complement (C3)-coated red blood cells following infection with the virus of equine infectious
anemia. J. Immunology 103:239-299 (1969).
Anderson, I.J. Idiopathic autoimmune haemolytic anemia in the horse. New Zealand Vet. J. 22:102 (1974).
2
PROCEDURE:
A. Erythrocytes for testing can be obtained a number of ways and are listed in order of preference:
1. Blood collected in ethylenediamine tetraacetic acid (EDTA).
2. Blood collected in heparin.
3. Erythrocytes teased from clotted blood, being careful to remove clumps.
Note: Whenever possible, blood from a healthy non-anemic horse should be evaluated along with blood
from the anemic horse. Blood from the normal horse will serve as a negative control.
B. Washing of erythrocytes.
1. Centrifuge blood (standard tabletop centrifuge for 5 minutes at room temperature).
2. Remove 0.1 ml of packed erythrocytes and add to 4.9 ml phosphate buffered saline (PBS) or
normal saline solution. (NOTE: Other solutions may influence results.)
3. Mix the erythrocytes and PBS. Centrifuge the mixture as above and remove the supernatant.
Resuspend the erythrocyte pellet in 4.9 ml of PBS.
4. Repeat the washing procedure in the previous step three more times. This provides for four
washings of the erythrocytes.
5. At the end of the last wash remove the supernatant and resuspend the pellet in 4.9 ml of PBS.
This provides a 2% suspension of erythrocytes.
C. Dilution of the Coombs reagent.
1. Label four test tubes (12 x 75 mm) 1, 2, 3, 4 consecutively.
2. Add 0.1 ml PBS to all four tubes.
3. Add 0.1 ml of Coombs reagent to tube 1, mix well and transfer 0.1 ml of this mixture to tube 2.
Mix tube 2 well and then transfer 0.1 ml to tube 3. Mix tube 3 well, then remove and discard 0.1
ml.
4. At the end of this process, tube 1 should contain 0.1 ml of a 1/2 dilution of the Coombs reagent,
tube 2 a 1/4 dilution, and tube 3 a 1/8 dilution. Tube 4 should contain only PBS.
5. Steps C-1 to C-4 should be repeated for each sample to be tested, including the negative control.
D. Coombs test.
1. Add 0.1 ml of washed resuspended erythrocytes from the horse to be tested to tubes 1 through 4.
Gently mix.
2. Incubate for 30 minutes at 37C.
3. Centrifuge for 1 minute.
4. To dissociate any nonspecific agglutination, hold each tube at a 45 angle and tap firmly on a
table top 15 times just prior to step 5.
5. Evaluate the contents of each tube by placing a small amount of the solution on a slide and
viewing with a microscope (100X magnification is suitable).
E. Test interpretation.
Negative—erythrocytes are not clumped or agglutinated.
Positive—there are clumps and large aggregates of erythrocytes. The clumps should not be
present in the control cells. Occasional clumps (3 or 4 per slide) may occur in test and control
erythrocytes and should be disregarded. Hemolysis should not be considered a positive reaction.
Horses with active equine infectious anemia (EIA) have C3-coated erythrocytes and will produce a
positive Coombs test. Such horses will also have a positive Coggins test.
