The cross-match procedure determines whether donor blood is compatible (or incompatible) with recipient blood. A variant of this test is the mare-foal or mare-stallion crossmatch or incompatibility testing.

In relation to blood transfusions, a cross-match should be performed in the following situations:

  • Naturally occurring pathogenic antibodies to foreign blood group antigens are present. This occurs in the cat. In this species, a cross-match should be performed on the first and every transfusion, unless the blood types of both donor and recipient are known. In cat breeds in which there is a low percentage of type B cats (e.g. Siamese, DSH or DLH in the USA), transfusion of blood from an uncross-matched or untyped donor can be performed relatively safely. However, there is still a (albeit small) chance of a major transfusion reaction and this procedure is not recommended.
  • Sensitization of an animal to foreign red cell antigens, in a species without naturally occurring antibodies. This is the situation in the dog and horse, resulting in the production of acquired antibodies. In these species, a cross-match does not need to be performed on the first transfusion the animal receives (as long as you can be sure this is the first transfusion ever), but should be performed at subsequent transfusions (if the interval between the first and subsequent transfusions is more than 5 days).

Cross-matching will detect incompatibilities between the donor and recipient that will not be evident on blood typing (as blood typing is not available against every blood group, just the major ones). In addition, the cross-match procedure will not pick up low titer antibodies and thus will not prevent delayed-type hemolytic transfusion reactions (for more information, see adverse reactions).

There are two types of cross-matches:

  1. Major cross-match: This is the most important cross-match, comparing donor erythrocytes to recipient serum (i.e. you are checking for preformed (acquired or naturally occurring) antibodies in recipient serum against donor erythrocytes. For the major crossmatch, you need red blood cells from the donor (this can be whole blood from a donor animal or packed red blood cells) in EDTA or citrate and serum from the recipient (non-anticoagulant tube).
  2. Minor cross-match: This compares donor serum to recipient erythrocytes and checks for preformed antibodies in donor serum that could hemolyse recipient red cells. This cross-match is less important as usually the donor serum is markedly diluted after transfusion and is unlikely to produce a significant transfusion reaction. This type of cross-match could be important if transfusing small patients, in which hemodilution is less likely to occur. In the cross-match procedure, washed erythrocytes are incubated with serum. For horses, ruminants and camelids, a source of complement (to hemolyze the erythrocytes) is added, as the antibodies in horses are usually hemolysins that require a source of complement for their hemolytic action.
  • For the major cross-matchDonor erythrocytes are washed and incubated with recipient serum.
  • For the minor cross-matchDonor serum is incubated with washed recipient erythrocytes.

The mixture of erythrocytes and serum are then observed visually for hemolysis (especially in the horse), and microscopically for agglutination. Any evidence of agglutination or hemolysis indicates an incompatible cross-match. There are also commercial kits available to detect blood group incompatibilities. Many of these are based on gels, to which donor red blood cells and recipient serum is added (major crossmatch) or donor serum and recipient red blood cells (minor crossmatch). Positive reactions (incompatible crossmatches) yield a line of red blood cells in the middle of the gel, whereas in a negative (compatible) reaction, the red blood cells sediment to the bottom of the tube. Studies have shown that these rapid gel-based methods are not as sensitive as more time-consuming laboratory assays that assesses for agglutinins microscopically (or hemolysins visually for some species) and results do not always correlate between the assays (Guzman et al 2016).

When there is a strong incompatible reaction on the major cross-match, the donor blood should not be transfused under any circumstances. When there is an incompatible reaction on the minor cross-match, the transfusion can go ahead. However, if the donated serum is likely to contribute substantially to the plasma volume of the recipient, the serum should be removed from the donor whole blood. The packed cells (washed or unwashed) should be reconstituted in sterile isotonic saline before infusion. Note that if cross-match-incompatible blood is given to adult horses on the first transfusion, the transfused red blood cells will have markedly shortened survival and mild non-hemolytic reactions may be seen (Tomlinson et al 2015). As a consequence, the investigators recommend crossmatching horses on the first transfusion.

Further information is available on cross-match testing offered by the Animal Health Diagnostic Center of Cornell University.

Mare-foal or mare-stallion incompatibility test

The mare-foal or mare-stallion incompatibility test is a cross-match procedure that looks for incompatibility between the mare and the foal or the mare and the stallion. Specifically, the test compares whether there are antibodies in mare serum (or colostrum) to foal or stallion erythrocytes. The mare-foal incompatibility cross-match is a test for the confirmation or prevention of neonatal isoerythrolysis.

Neonatal isoerythrolysis (NI) is a hemolytic anemia that occurs in foals (that inherit their sire’s blood group antigens) born to mares of a different blood type to the stallion they were mated to. The mare is sensitized to the blood group antigen of the stallion through:

  • previous pregnancies (the most common situation)
  • previous blood or plasma transfusions
  • vaccination with products containing equine erythrocyte antigens

Sensitization occurs during pregnancy due to a phenomenon called retroplacental bleeding, in which the foal’s blood comes into contact with the mare’s circulation during the last few weeks of pregnancy. It can naturally occur earlier if there is any placental pathology. Therefore, NI occurs usually in multiparous mares and only after the foal has ingested colostrum. The most common blood types in the horse that produce NI are Aa and Qa antigens, although NI to other antigens (including Pa and Ua) has been reported. When the foal ingests colostrum containing antibodies from the mare against the sire’s red blood cell antigens, the foal (if the sire’s blood type has been inherited) suffers a hemolytic anemia, which can be severe and result in neonatal mortality. Most signs of NI occur within 24-36 hour; weakness, hemoglobinuria and hemoglobinemia can be seen in severe cases.

Furthermore, all horses lack an antigen that is unique to donkey red blood cells (so-called donkey factor). Thus, mule pregnancies are at risk of initiating NI (MacLeay, 2001Boyle et al., 2005).

Mare-foal incompatibility testing

To determine if NI has occurred in a foal that is already born, a cross-match can be performed on the mare and foal. This requires the submission of erythrocytes from the foal (EDTA blood) and separated serum from the mare. At Cornell University, we perform a mare-foal incompatibility test and look for both agglutination (microscopic assessment) and hemolytic reactions (the latter via adding a source of complement to lyse the red blood cells).

Alternatively, to diagnose NI in a foal, a direct Coombs test (looking for antibody or complement bound to foal’s red blood cells) can be done on the foal’s blood (this requires an EDTA tube from the foal). However, these tests are usually performed by referral laboratories and the results may only be available after several days.

There is a kit available, called the jaundice foal agglutination (JFA) test, that can be performed “stallside” (if you have a centrifuge). This test assesses for agglutination between mare colostrum or serum, and foal blood. Colostrum or serum is serially diluted and mixed with foal blood in test tubes. The tubes are centrifuged and the button is observed for agglutination. If agglutination is present, the button does not disperse when the tube is flicked. A positive reaction at a titer of > 1:16 is indicative of an incompatibility (at lower titers, false-positive reactions may occur) and indicates that colostrum should be withheld from the foal. This is a useful test to determine if a mare, that has previously had a foal with NI and has been mated to the same stallion that produced the NI foal or another stallion of unknown blood type, has antibodies to the current foal. However, the test does not look for hemolysis and is less sensitive than the routine cross-match procedure (which assesses for microscopic agglutination and hemolysis).

Coombs (direct) Cross-match (hemolytic) Jaundice foal agglutination test
Detects Antibody/complement bound to foal RBCs Mare serum alloantibody bound to foal RBCs Alloantibody from mare colostrum/serum on foal RBCs
Samples Foal: venous blood (EDTA) Foal: venous blood (EDTA and non-anticoagulant)
Mare: venous blood (non-anticoagulant)
Foal: venous blood (EDTA)
Mare: colostrum/serum
Procedure Washed foal RBCs are incubated with the equine polyvalent Coombs reagent (anti-IgG, anti-IgM and anti-C antibodies) at 37° C. Dilutions may be performed. Washed foal RBCs are incubated at 37°C with mare serum and mixed with exogenous complement. Two dilutions (1:4 and 1:16) are performed. Foal whole blood is centrifuged with serial dilutions of mare colostrum/serum
Positive result Agglutination Hemolysis at dilutions >1:16
Agglutination in dilutions 1:16 or greater


Mare-stallion incompatibility testing

Neonatal isoerythrolysis can potentially be prevented by testing a susceptible mare before parturition (mare-stallion incompatibility test) and withholding colostrum for 24 to 48 hours (by muzzling the foal and feeding an alternative source of colostrum). Ideally, the mare should be cross-matched with the stallion (stallion red blood cells and mare serum), especially in the last 1-3 weeks of pregnancy when exposure to the foal’s erythrocytes is likely to occur (if there is an incompatibility, the mare will have an anamnestic response to the antigen, resulting in a high antibody titer at this time). Some authors recommend repeat cross-matching every two weeks in the last month of pregnancy to look for a rising titer (often increases from < 1:4 to >1:256 in the last four weeks prior to foaling). A rising titer indicates a high likelihood of NI and reveals the need for withholding of colostrum from the foal.

Alternatively, the jaundice foal agglutination test can be used at parturition and colostrum can be withheld. Note that a single cross-match between a mare and a stallion may not reveal an incompatibility if the mare is tested before or early in pregnancy (due to low antibody titers that may not produce an incompatible reaction). Since the mare-stallion incompatibility test requires red blood cells from the stallion (EDTA blood), which may be difficult to get, an alternative approach to determine if the mare is likely going to develop antibodies against foal red blood cells (with the sire’s  blood type), the mare’s serum can be screened against a panel of RBC antigens for the presence of antibodies, which is done at specific centers (see related links). Again, this test should be done as close to foaling is possible, when the mare is likely to develop an anamnestic response (an enhanced response after re-exposure to the primary red blood cell antigen) from incompatible red blood cell type on the foal’s blood (see above). When a mare has had an NI foal and is mated again to the same stallion, there is a 50% chance the next foal will be at risk of NI (if it inherits the sire’s red blood cell type). In this case, it is always safer to withhold colostrum from the foal. If the mare is mated to a new stallion, typing of both the mare and stallion is ideal to determine if they have incompatible blood types. Indeed, once a mare has had a foal with NI, it is always good to determine the mare’s blood type and the likely offending antigen so that future matings can be managed appropriately. The only advantage a cross-match has against the specific antibody titer approach is that it may pick up blood group incompatibilities against antigens that are not in a screening panel. However, there is a low risk that an anti-RBC antibody will be missed because the serum is tested against the common offending erythrocyte antigens and reports of other antigens being associated with NI are rare. Both tests (cross-match and antibody titers) have the potential to miss  low levels of antibodies. This is why it is essential to test the mare as close to foaling as possible.


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