The jaundiced foal agglutination

Wednesday, February 14, 2007

Mare-foal Incompatibility

The mare-foal incompatibility test is a crossmatch procedure that looks for incompatibility between the mare and the foal. Specifically, the test compares whether there are antibodies in mare serum (or colostrum) to foal erythrocytes. The mare-foal incompatibility crossmatch is a test for the confirmation or prevention of neonatal isoerythrolysis (NI). Neonatal isoerythrolysis 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 or 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 cell antigens, the foal (as long as it has inherited the sire's blood type) suffers a hemolytic anemia, which can be severe and result in neonatal mortality. 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.

To determine if NI has occurred in a foal, a crossmatch can be performed on the mare and foal. This requires the submission of erythrocytes from the foal (EDTA blood) and serum from the mare. Alternatively, a direct Coombs test can be done on the foal's blood (this requires an EDTA tube). 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 test, that can be performed "stallside" (if you have a centrifuge). This test assesses for mare-foal incompatibility by testing 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 crossmatch procedure (which assesses for hemolysis and microscopic agglutination).

Neonatal isoerythrolysis can be prevented by testing a susceptible mare before parturition and withholding colostrum. Ideally, the mare should be crossmatched with the stallion (stallion red cells and mare serum), especially in the last few weeks of pregnancy when exposure to the foal's erythrocytes is likely to occur (thus boosting the antibody titer, if there is an incompatibility). Some authors recommend repeat crossmatching every two weeks in the last month of pregnancy to look for a rising titer (often increases from <>1:256 in the last 4 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. Note that a single crossmatch 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).

Saturday, June 10, 2006

Neonatal Isoerythrolysis

Neonatal isoerythrolysis (NI) is an immunogenic disease that affects foals within the first week of life. The principle behind NI is very simple. A mare generates an antibody response against a foreign antigen. In this case, the target antigen is a red blood cell surface molecule, which she lacks on her own cells. These antibodies gain access to the circulation of her foal through ingestion of colostrum. If the foal has on its red cells the target antigen inherited from the sire, the antibodies attach to the red cells and cause their destruction or removal from the circulation. Mares become sensitized or immunized as a result of exposure to blood containing the foreign antigens from transfusion or exposure to blood of a fetus with incompatible blood type as a result of placentitis or at parturition.

For NI to develop, a sequence of events must occur. (1) The mare must be exposed to a red cell factor, the antigen, which she lacks on her own cells, and she must produce antibody to it. (2) She must be bred to a stallion that has this factor on its cells, and (3) the sire must pass the gene for that factor to the fetus, which then makes the factor on its cells. (4) The mare must make colostrum, which contains these antibodies. (5) The foal must ingest and absorb sufficient quantities of these antibodies. (6) The antibodies must combine with the factor on the red cells, which results in their rapid destruction or removal from the circulation leading to anemia.

The two red cell factors or antigens that appear to be most immunogenic and are most commonly associated with problems in horses are Aa and Qa. Only mares that lack these factors can produce antibody to them and are thus at risk. Mares that lack these factors can be identified by blood typing. When examining the blood typing report (Fig. 1), clinicians must look at the list of factors detected for the A system and Q system. The list amounts to a series of lower-case letters from "a" to "g" for the A system and "a" to "c" for the Q system and minus sign. If "a" is not listed under the system, the mare does not have that factor and is considered a higher risk for production of antibodies to that factor and subsequently an NI foal. This assumes that the factor actually occurs in the breed and thus stallions to which the mare may be bred have the factor. For example, factor Qa does not occur in Standardbreds at all, and thus although all mares appear to be at risk, that is they lack Qa, no "incompatible" Standardbred stallions exist to which they may be bred

NI has occasionally been associated with other red cell factors in horses. Theoretically, any factor lacked by the mare and present in the foal and stallion can be a problem, but the prevalence is so low that it does not warrant taking preventive measures for incompatibilities in these other factors. Virtually all mule pregnancies are incompatible for a red cell factor called donkey factor, which has been associated with NI. Not all mares at risk actually become sensitized and produce foals that develop NI.

Horses that lack factor Ca frequently produce anti-Ca antibody. This antibody does not appear to produce NI, and in fact it may confer some degree of protection against sensitization by other factors by rapidly eliminating foreign cells from circulation.

If a mare is known to be at risk either based on knowledge of her blood type or because of previous production of an affected foal, her sera can be tested for the presence of anti-red cell antibodies during the last month of gestation or her colostrum can be tested against the foal's or stallion's red cells before allowing the foal to nurse. If antibody is detected before ingestion of colostrum, the foal can be prevented from ingesting the "tainted" colostrum and an alternate source can be provided.


Typically, signs of disease occur in the first 2 to 24 hours but may occur as late as 7 or 8 days of age. Clinical signs are referable to destruction of red blood cells and resulting anemia and other complications of red cell destruction. Peracute cases may die within several hours and show only pallor, hemoglobinemia, and hemoglobinuria. Clinically, these are often weak, depressed, "floppy" foals. Foals that survive for several days or that have a slower and later onset are generally icteric. Tachycardia, tachypnea, and pallor may also be noticeable. Secondary signs resulting from anoxia may include those of central nervous system injury such as depression or seizures. Nephropathy associated with organ failure may result from renal vascular changes and excretion of hemoglobin. Mules suffering from NI frequently manifest thrombocytopenia as well as anemia, presumably because of the presence of anti-platelet antibody as well as anti-red cell antibody.

Delayed onset, after more than 6 days, of acute hemolysis is uncommon and may be due to continued presence of antibody of some class other than IgG. In these cases, the milk gives a positive result on the jaundiced foal agglutination (JFA) test (Table 1), suggesting the continued presence of agglutinins. In these cases it may be necessary to remove the foal from mare's milk.


Diagnosis may be suspected based on signs of pallor or icterus and concurrent anemia, hemoglobinemia, or hemoglobinuria. Septicemic foals may clinically share many of the features including depression and icterus, but infected foals are not usually anemic.

Diagnosis is confirmed by demonstrating antibody on the surface of red cells using a direct antiglobulin test (Coombs' test). This test is run on ethylenediaminetetraacetic acid (EDTA) anticoagulated samples and requires species-specific reagents. Human Coombs' test reagents do not work. Diagnosis is supported by demonstrating anti-red cell antibodies in the colostrum or serum of the mare using agglutination or hemolytic assays.


Treatment after the ingestion of colostrum and the onset of signs depends on the severity of clinical changes and the speed with which red cells are destroyed. In mild cases when the hematocrit (PCV) remains above 15% and is not decreasing rapidly, little except restriction of exercise may be indicated. In more severe cases, minimizing exertion is imperative because these foals may die while being manipulated or attempting to follow their dams. When the PCV is less than 15%, replacement blood cells that are resistant to the circulating maternal antibody may be required. Although transfusion may not be necessary if the PCV remains above 12%, if it is at 15% and decreasing, preparation should begin to have the blood ready if the PCV drops to 12% as collecting and processing blood for transfusion takes time.

Selection of a Donor

The key to donor selection is to provide a red blood cell that will not react with the maternal antibodies that the foal absorbed through the colostrum. at this age, the foal has only maternal antibody and no autologous antibody about which the clinician needs to be concerned. From a standpoint of speed and ease, the best donor lacks the factor against which the antibodies are directed (e.g., has the same blood type as the mare with regard to that factor, and has no anti-red cell antibodies in its serum). Whole blood can be used from these horses without additional processing. Depending upon the breed, however, the number of individuals that lack the offending factor and are thus suitable donors varies. For example, in Thoroughbreds, only about one in 50 horses lack factor Aa. This means that only one in 50 Thoroughbreds is a suitable red cell donor to treat a foal affected with NI due to anti-Aa antibody. The odds of randomly selecting a suitable donor are obviously slim.

With advance screening, horses that are Aa and Qa negative and free of antibody can be identified for donors (Table 2). Among the more common breeds in the United States, a suitable Aa and Qa negative donor would most likely be found among Quarterhorses. Because pregnancy is often associated with sensitization against red cells, the use of geldings as blood donors has been suggested. This is a consideration relating to the presence of antibodies in the transfused blood, but has no effect on which red cell factors are present.

If colostrum from the mare is available, a potential donor can be screened in the field by performing the JFA test (see Table 1) using the mare's colostrum and blood from potential donors. The mare's own blood is run as a control and the donor's blood cells should not react at any higher dilutions than the mare's own blood cells.



1. Centrifuge capable of centrifuging blood types at moderate speeds (300 x 600 x g)
2. Test tubes, either 13- x 100 mm disposable tubes or blood collection tubes.
3. Test tube rack.
4. Pasteur pipettes and rubber bulbs or other pipette system to deliver 1.0-ml volumes.
5. Room temperature 0.9% NaCl
6. Colostrum from mare.
7. Presuckle blood from the foal, preferably in EDTA anticoagulant.
8. Blood from the mare, preferably in EDTA anticoagulant, can be used for a control.


1. Add approximately 1 ml of 0.9% NaCl to each of 8 tubes.
2. Label the first tube SALINE CONTROL.
3. In the remaining 7 tubes, prepare serial dilutions (1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128) of the colostrum by adding 1 ml of colostrum to the tube labeled 1:2, then transferring 1 ml of the mixture to the second tube labeled 1:4, and so on until reaching the tube labeled 1:128. Discard 1 ml from the tube labeled 1:128 so that the total volume in each tube is approximately 1 ml.
4. Add one drop of the foal's whole blood to each tube and mix the samples.
5. Centrifuge the tubes for 2 to 3 minutes at medium speed (300-500 x g).
6. Invert each tube, one at a time, pouring out the liquid contents; observe the status of the button of the red cells at the bottom of the tube.

Complete agglutination causes the cells to remain tightly packed in the button; strong agglutination causes the cells to remain in large clumps. For weaker agglutination, the cells are in smaller clumps as they run down the side of the tube. When no agglutination exists, the cells easily flow down the side of the tube. The titer is defined as the highest dilution that gives strong agglutination.

If the blood agglutinates in the SALINE CONTROL tube, this may indicate that the foal has already absorbed antibodies and the cells are already coated at the time of collection. Colostrum can be tested on the dam's own cells to be certain that it is not the condition of the test or viscosity of the colostrum that are causing the agglutination.

Positive reactions at 1:16 or greater in horses and 1:64 or greater in mules are considered significant.

One reliable source of red cells that is not affected by maternal antibody is the dam. The dam's red cells will not be affected by her antibody, but the problem with using the dam's red cells is that they must be washed free of antibody to prevent administration of more offending antibody to the foal. The process of washing allows the red cells to sediment from anticoagulated blood and removes as much of the plasma as possible, followed by addition of several volumes of isotonic saline. The red cells are allowed to sediment again, after which time as much of the saline-diluted plasma as possible is removed. The red cells are then resuspended in isotonic saline at about a 50% suspension. This procedure takes several hours.

The sire of the foal is absolutely not a suitable red cell donor because his red cells share the same red cell antigen as the foal. Administration of more red cells that share the same antigens as the foal will only add to the load of damaged red cells that must be cleared by the reticuloendothelial system and hemoglobin that must be cleared by the kidneys.

In the case of mules with NI, virtually any horses can provide suitable red cells because the factor involved appears to be a uniquely donkey antigen (i.e., a xenoantigen), and thus it is necessary only to exclude horses with anti-donkey antibody in their serum as donors.

Administration of Erythrocytes

The red cells or whole blood must be administered through a blood administration set with a filter. Failure to do so may result in acute fatal reaction, probably anaphylactoid in nature. The volume of washed red cells or whole blood that can be given is somewhat limited by the size of the patient. In general, as much as possible is given without overloading the blood volume of the patient. The estimated blood volume of a 50-kg foal is 3.5 L., which is 7.5% of body weight. Usually 1 to 2 L of washed cells or whole blood is given initially, with an additional 1 to 2 L per day given over the next day or two if needed. This volume can be expected to increase the PCV about 10 percentage points. The PCV tends to gradually decrease again over the following several days. This is most likely attributable to the continued destruction and removal of the foal's cells, not the transfused cells, if they were compatible with the maternal antibody. If the decline in PCV is gradual, additional transfusions may be unnecessary, even if the levels decrease to around 15%. It may take several weeks for the PCV to return to levels expected for age, keeping in mind that the PCV level of normal foals tends to decrease, often into the 20's, over the first several weeks of life.


Two general methods exist for prevention of NI. One is to prevent incompatible matings and the other is to prevent the offending antibodies from entering the foal. A compatible breeding is to mate a mare and stallion lacking the same factors, therefore precluding the inheritance of those factors by the foal. Compatibility can be determined by routine blood typing of the mare and stallion. When examining the blood typing reports for the mare and stallion (Fig. 1), the list of factors detected for the A system and Q system should be examined. As described earlier, the list amounts to a series of lower-case letters from "a" to "g" for the A system and "a" to "c" for the Q system. If "a" is not listed under the A system for the mare, a compatible stallion would also lack the "a" factor under the A system. If "a" is not listed under the Q system for the mare, a compatible stallion would also lack the "a" factor under the Q system. It is the lack of a factor in the mare that creates the "at risk" situation, not the converse, and thus the presence of the factor in the mare and the absence in the stallion is of no consequence.

The presence of anti-red cell antibodies in the serum of mares late in gestation is presumptive evidence of an impending problem, although because the blood type of the fetus is not known, this does not predict absolutely that a problem will occur with the current pregnancy. Postpartum tests for antibody can be done using either the sire's blood or blood from several horses that are known to possess a spectrum of red cell antigens, known as a panel. Thus, blood from the sire is not necessary to detect problematic antibodies. If antibodies are detected, provision should be made to either check colostrum for specific reaction with the foal's cells at birth by means of the JFA test (see Table 1), or to arbitrarily withhold the dam's colostrum and provide an alternate source.

The presence of colostral antibodies against red cells can be detected using the JFA test. Mare colostrum is reacted with foal's blood collected before the foal is suckled and a titer determined (see Table 1). Titers of 1:16 or greater are considered significant in horses. Titers of 1:64 or greater of anti-donkey factor are significant in mules. The JFA does not differentiate antibodies against one factor from another; it simply detects the presence of some anti-red cell antibody. If the foal has already nursed and has anti-red cell antibody on its cells, the cells may agglutinate in the JFA test saline control and at all dilution even in the absence of colostrum; thus, it is important to use presuckle blood to accurately assess the test or the tendency will be to overestimate the titer.

Colostral titers as determined by JFA test generally decrease quite rapidly. It is safe to allow the foal to nurse once the titers decrease below 1:16. This may occur in as little as 4 hours. Recommendations to withhold milk for 24 to 72 hours are probably overly conservative. If colostral antibodies are ingested in the first few hours, the antibody levels may continue to increase in the blood for 24 hours, but probably not because new absorption is occurring but rather because the antibody already in the pipeline continues to reach the blood stream. By milking out the mare and repeatedly testing the milk using the JFA test with the foal's presuckle blood, the foal can safely be returned to the mare as soon as the titer drops, thus saving considerable time and effort required to feed a foal every several hours if it is being withheld from the mare for periods of 1 to 3 days.