Immunohematology and Hemostasis


4
Immunohematology and Hemostasis


Karen V. Jackson


School of Veterinary Science, University of Queensland, Gatton, Queensland, Australia


Acronyms and abbreviations that appear in this chapter include: RBC, red blood cell; NI, neonatal isoerythrolysis; pRBC, packed red blood cells; EDTA, ethylenediamine tetraacetic acid; ACD, acid citrate dextrose; PBS, phosphate buffered saline; DS, 2% dextrose in 0.9% saline; IMHA, immune‐mediated hemolytic anemia; JFA, jaundiced foal agglutination; MAC, membrane‐attack complex; IgG, immunoglobulin class G; IgM, immunoglobulin class M; IgA, immunoglobulin class A; EIA/EIAV; equine infectious anemia/equine infectious anemia virus; CBC, complete blood count; UA, urinalysis; PCV, packed cell volume; DIC, disseminated intravascular coagulation; PGI2, prostacyclin; PCR, polymerase chain reaction; NO, nitric oxide; FV/FVa, factor V/activated factor V; FVIII/FVIIIa, factor VIII/activated factor VIII; TFPI, tissue factor pathway inhibitor; TF, tissue factor; tPA, tissue plasminogen activator; vWF, von Willebrand factor; PAI‐1, plasminogen activator inhibitor‐1; PAF, platelet activating factor; PDGF, platelet‐derived growth factor; PF4, platelet factor 4; ADP, adenosine diphosphate; ATP, adenosine triphosphate; GPIb, glycoprotein Ib; GPIIb‐IIIa, glycoprotein IIb‐IIIa; FXII/FXIIa, factor XII/activated factor XII; FXI/FXIa, factor XI/activated factor XI; HMWK, high molecular weight kininogen; PK, prekallikrein; FIX/FIXa, factor IX/activated factor IX; FX/FXa, factor X/activated factor X; FII, factor II or prothrombin; FIIa, activated factor II or thrombin; PL, phospholipid; FDPs, fibrin(ogen) degradation products; ATIII, antithrombin III; TAT, thrombin‐antithrombin complex; EPCR, endothelial cell protein C receptor; aPC, activated protein C; TAFI/TAFIa, thrombin‐activatable fibrinolysis inhibitor/activated thrombin‐activatable fibrinolysis inhibitor; BMBT, buccal mucosal bleeding time; TBT, template bleeding time; PT, prothrombin time; aPTT, activated partial thromboplastin time; ACT, activated clotting time.


4.1 Immunohematology Testing


Blood types are surface red blood cell (RBC) antigens that are genetically coded and therefore inherited. Benchtop blood typing uses specialized antisera to identify these RBC antigens. In the horse, their clinical significance has historically lain in parentage testing but now parentage testing via blood type is genetic in basis and benchtop blood typing is more often used in the transfusion setting for blood donor and recipient typing and in the investigation of neonatal isoerythrolysis (NI) cases. Cross‐matching is used to detect potential transfusion incompatibilities between donor sera and RBCs and recipient sera and RBCs to ensure optimal RBC survival post transfusion. It is often used directly prior to transfusion for selection of a compatible donor for packed RBC (pRBC) and plasma transfusions. Antibody screening and the jaundiced foal agglutination (JFA) test are most often used combined with blood typing for investigation or prediction of NI. Antibody screening alone can also be used in the transfusion setting for blood donor screening and patients at risk for previous development of antibodies (i.e., previous transfusions or pregnancies).


4.1.1 Blood Typing


4.1.1.1 Blood Types and Clinical Relevance


Horses have eight RBC groups or systems: A, C, D, K, P, Q, U, and T. The first seven are recognized by the International Society of Animal Blood Grouping Research and the remaining T blood group is mainly of research interest [1]. Recently, new nomenclature has been proposed for standardization, with the two letters ‘EA’ for erythrocyte antigen preceding the traditional groups above. Within these groups there are more than 30 factors, which are designated by a lowercase letter that follows the uppercase group/system. A summary of the horse blood groups and factors is presented in Table 4.1. As the equine blood group systems are complex with many factors, identification of a blood donor with an identical blood group profile is difficult, if not impossible. This is why blood typing and/or antibody screening is recommended to select blood donors with a high likelihood of being compatible to other horses although cross‐matching is always recommended prior to transfusion.


Although there are many blood groups and factors in horses and these can be overwhelming when viewed together, the majority of clinically significant transfusion reactions and/or NI cases relate to factor incompatibilities associated with blood groups EAA and EAQ. Incompatibilities occur when RBC antigens on the surface of one horse’s RBC (e.g., anemic patient, NI foal) encounter plasma antibodies from another horse against these same antigens (e.g., antibodies from donor plasma or colostrum). In practice, blood donors are blood typed to best predict compatible patient–donor pairings for cross‐matching and mares are blood typed to determine if they lack Aa and/or Qa as they are then considered at risk of having a foal with NI.


4.1.1.2 Methodology


Blood typing in horses is a time‐intensive process requiring 2.5–3.0 hours to complete. It is also specialized and only performed by select laboratories as the reagents are not commercially available. A list of some laboratories which perform equine blood typing is provided in Table 4.2; however, please note other veterinary laboratories may offer equine blood typing if contacted. Either an ethylenediamine tetraacetic acid (EDTA) (purple top) or acid citrate dextrose (ACD) (yellow top) whole blood sample is recommended for submission to the laboratory.


Table 4.1 Internationally recognized horse blood groups, factors, and alleles.




































Blood groups Factors Allelesa
EAA a b c d e f g Aa, Aadf, Aadg, Aabdf, Aabdg, Ab, Abc, Abce, Ac, Ace, Ae, A−
EAC a Ca, C−
EAD a b c d e f g h i j k l m n o p q r Dadl, Dadlnr, Dadlr, Dbcmq, Dcefgmq, Dcegimnq, Dcfgkm, Dcfmqr, Dcgm, Dcgmp, Dcgmq, Dcgmqr, Dcgmr, Ddeklr, Ddeloq, Ddelq, Ddflkr, Ddghmp, Ddghmq, Ddghmqr, Ddkl, Ddlnq, Ddlnqr, Ddlqr, Dq, D−
EAK a Ka, K−
EAP a b c d Pa, Pac, Pacd, Pad, Pb, Pbd, Pd, P−
EAQ a b c Qabc, Qac, Qa, Qb, Qc, Q−
EAU a Ua, U−

a The absence of a factor is denoted as a dash (−).


Table 4.2 Equine blood typing laboratories (United States).











University of California, Davis
Hematology Laboratory
Room 1012, Veterinary Teaching Hospital
One Garrod Drive
University of California, Davis
Davis, CA, 95616
+1–530–752‐1303
www.vetmed.ucdavis.edu/hospital/support‐services/lab‐services/clinical‐laboratory‐services/equine‐blood‐type‐and‐antibody‐screen
University of Kentucky
Animal Genetic Typing and Research Laboratory
108 Gluck Equine Research Centre
University of Kentucky
Lexington, KT, 40546
+1–859–218‐1212
www.ca.uky.edu/gluck/AGTRL.asp
Rood and Riddle Veterinary Laboratory
2150 Georgetown Road
Lexington, KY, 40511
+1–859–233‐0331
www.roodandriddle.com/laboratory.html
Hagyard Equine Medical Institute
4520 Iron Works Pike
Lexington, KY, 40511
+1–859–259‐3685
www.hagyard.com/

Briefly, the procedure involves incubating aliquots of 2% washed RBC saline suspension with aliquots of each specific antisera at 37 °C. A positive reaction, visualized as either hemolysis or agglutination depending on the known reaction of each antisera, indicates the presence of a blood factor. A negative reaction, visualized as a lack of hemolysis or agglutination, indicates the absence of a blood factor. This has to be performed for each blood factor individually. The hemolytic reactions require addition of complement before incubation. This complement is from rabbit serum that has been adsorbed at 4 °C with equine RBCs to remove nonspecific hemolysins.


More recent blood typing methods include a rapid agglutination method for equine blood typing for equine RBC blood types/antigens Aa and Ca which requires only 15–30 minutes and is performed a room temperature [2] and an immunochromatographic method for Ca which requires approximately three minutes at room temperature [3]. These may be more practical for pretransfusion testing but particularly as they cannot assess for blood group EAQ, as a hemolysin reagent (not agglutination) is used for this blood group, these methods cannot replace cross‐matching or complete blood typing and antibody screening for donor–patient compatibility testing before transfusion.


4.1.2 Cross‐matching


4.1.2.1 Clinical Relevance


Cross‐matching is used to detect incompatible antibodies in plasma/serum of the donor and patient that will react with RBC antigens/blood types of the patient and donor, respectively. Cross‐matching does not specify to which RBC blood types/antigens the incompatibility occurs and does not predict all transfusion reactions (e.g., platelet or leukocyte antibodies, pRBC bacterial contamination, urticarial reactions, hypocalcemia secondary to excessive citrate administration); however, even in patients that have been blood typed, cross‐matching should be performed prior to transfusion to minimize the likelihood of a transfusion reaction secondary to antibody incompatibilities.


All horses can develop antibodies to RBC antigens/blood types they do not have themselves if they are sensitized; however, sensitization even with incompatible blood transfusions is uncommon [4]. These antibodies are called acquired alloantibodies. They develop if a horse is exposed to RBC antigens/blood types it does not recognize as self (e.g., incompatible transfusion, mare with Qa or Aa foal when they are Q− or A− themselves). Approximately 10% of Thoroughbred horses and 20% of Standardbred horses have naturally occurring RBC alloantibodies (i.e., without sensitization they have antibodies present in their plasma that target RBC antigens that they do not have themselves) [5]. Whether these acquired or naturally occurring alloantibodies cause transfusion reactions or NI depends on their action and strength. Antibodies can be either agglutinins (i.e., causing agglutination of RBCs) or hemolysins (i.e., causing hemolysis of RBCs), or both. Anti‐Aa and anti‐Ca alloantibodies are both agglutinins and hemolysins whereas anti‐Qa alloantibodies are solely hemolysins. This is important to understand mainly for interpreting blood typing and cross‐matching results.


The majority of alloantibodies present in horses are either anti‐Aa or anti‐Ca, with fewer anti‐Qa alloantibodies [5]. Anti‐Aa and anti‐Qa alloantibodies are likely to cause clinically significant transfusion reactions and/or NI whereas anti‐Ca alloantibodies cause blood typing and cross‐match reactions that may not cause a significant transfusion reaction and have not been reported to cause NI. Other alloantibodies that have been reported to rarely cause NI include anti‐Ab, Qb, Qc, Qrs, Da, Db, Dc, Dg, Ka, Pa, and Ua Based on this information, blood typing and antibody screening for Aa, Ca, and Qa blood types/antigens and anti‐A, anti‐C, and anti‐Q antibodies should be performed as a minimum on blood donors and when managing NI cases or at‐risk pregnancies. Cross‐matching should, wherever possible, be used prior to transfusion as transfused RBC life span is markedly reduced with tube cross‐match incompatible transfusions (5d vs 11d vs 35d for >2+ incompatible, >1+ incompatible and compatible cross‐match reactions respectively) [4].


4.1.2.2 Methodology


As horses have naturally occurring and acquired alloantibodies that are both hemolysins and agglutinins, cross‐matching should be performed with both a saline‐agglutinating technique and a technique that can detect hemolysis. The hemolysis technique requires the addition of complement usually from rabbit serum that has been adsorbed at 4 °C with equine RBCs to remove nonspecific hemolysins. Although the saline‐agglutinating technique is often performed by many veterinary practices and is a good screening test prior to transfusion, a hemolytic technique should also be performed as clinically significant transfusion reactions often occur secondary to the presence of anti‐Q antibodies that are hemolysins and will not be detected with an agglutinating cross‐match alone.


Patient and donor(s) anticoagulated blood (either EDTA or ACD samples) and serum are required for cross‐matching. It has recently been shown that the patient and donor blood samples need to be fresh rather than stored for accurate cross‐matching and the best chance of finding a compatible donor [11]. If blood typing cannot be performed prior to donor selection, consider untransfused geldings of the same breed as the recipient as the best donor options. As blood types are hereditary, being of the same breed will help to minimize blood type incompatibilities whilst being untransfused and a gelding avoids any acquired alloantibodies (i.e., transfusion or pregnancy‐induced alloantibodies). Donkeys should not be used as donors for horses as they have a donkey‐specific RBC antigen that will cause sensitization [12].


The cross‐match procedures are outlined in Table 4.3. Briefly, in the agglutination cross‐match, aliquots of saline washed RBCs and serum from both the recipient and donor are mixed for the major and minor cross‐matches and incubated at 37 °C for 15 minutes. For the hemolysin cross‐match, an aliquot of rabbit complement is also added and the incubation is at 37 °C for longer, 90 minutes. Autocontrols are also performed for both the agglutination and hemolysin cross‐matches. The tubes are then centrifuged and assessed for agglutination (macroscopically and microscopically) and hemolysis. The agglutination can be graded (0–4+ for macroscopic [see Table 4.4], and positive or negative for microscopic) whereas hemolysis is either positive or negative [2, 13]. It has recently been shown that there is unlikely to be a need for concurrent micro‐ and macroscopic assessment for agglutination as the two findings are highly correlated [3]. The same study also evaluated a novel gel column cross‐matching method which requires some specialized equipment, whose results were well correlated with the agglutination but not the hemolysis component of the gold‐standard tube cross‐match [3]. Another study also correlated this novel gel method with a stall‐side gel method, showing adequate correlation and concluding that if a reference laboratory was not open to test, the stall‐side method is recommended [14]. Further studies have yet to be performed to determine the clinical accuracy of these new methods as these studies did not evaluate posttransfusion clinical information.


Table 4.3 Cross‐matching procedure.













  1. Obtain anticoagulated blood (EDTA or ACD, i.e., purple top or yellow top) and serum (coagulated blood, i.e., red top) from the recipient and donor(s).
  2. Centrifuge and separate the samples into multiple tubes:a

    1. Patient RBCs
    2. Patient serum
    3. Donor(s) RBCs
    4. Donor(s) serum

  3. Wash patient and donor(s) RBCs separately by adding saline, phosphate buffered saline (PBS), or 2% dextrose in 0.9% saline solution (DS) to a small amount of pRBCs from each. Mix, then centrifuge and pour off supernatant. Repeat at least 3 times.
  4. After the last wash, resuspend the pRBCs in saline, PBS, or DS to a 2–4% RBC suspension. This can be subjectively judged on color (i.e., “Kool‐Aid” or weak tomato juice in color) or calculated (i.e., 0.05 mL pRBCs in 2.4 mL saline gives a 2% suspension).
  5. Have a source of complement that has been adsorbed at 4 °C on equine RBCs to remove nonspecific hemolysins
  6. Make the following mixtures in appropriately labeled tubes (label abbreviations given in parentheses).
Agglutination cross‐match Hemolysin cross‐match


  1. Major agglutination (MaA) cross‐match: 2 drops patient serum, 2 drops donor 2–4% RBC
  2. Minor agglutination (MiA) cross‐match: 2 drops donor serum, 2 drops patient 2–4% RBC
  3. Autocontrol 1 (CTL1A): 2 drops patient serum, 2 drops patient 2–4% RBC
  4. Autocontrol 2 (CTL2A): 2 drops donor serum, 2 drops donor 2–4% RBC


  1. Incubate at 37 °C for 15 min
  2. Centrifuge for 15–20 sec (3400 rpm/1000× g)
  3. Read and record results.


    1. First examine tubes for hemolysis and record if present. Then gently rotate and shake the tube to cause RBCs to swirl off the RBC “button” at the bottom of the tube. Evaluate the suspension for the presence of aggregates/agglutinates:

      1. Macroscopically: Grade 0–4+ (see Table 4.4) depending on the strength of the agglutination.
      2. Microscopically: Place a drop of the mixture on a slide with a coverslip and examine unstained. If rouleaux is present, recentrifuge the original sample and try again with the pRBC and saline. This is either negative or positive.


  1. Major hemolysin (MaH) cross‐match: 2 drops patient serum, 2 drops donor 2–4% RBC, 2 drops complement
  2. Minor hemolysin (MiH) cross‐match: 2 drops donor serum, 2 drops patient 2–4% RBC, 2 drops complement
  3. Autocontrol 3 (CTL1H): 2 drops patient serum, 2 drops patient 2–4% RBC, 2 drops complement
  4. Autocontrol 4 (CTL2H): 2 drops donor serum, 2 drops donor 2–4% RBC, 2 drops complement
  5. Incubate at 37 °C for 90 min
  6. Centrifuge for 15–20 sec (3400 rpm/1000× g)
  7. Read and record results.

    1. Positive or negative for hemolysis.

aTubes are 12 × 75 mm glass test tubes.


Note: If autocontrols are positive for either hemolysis or agglutination, that portion of the cross‐match is invalidated.


4.1.3 Antibody Screening and Jaundiced Foal Agglutination Test


Antibody screening and the JFA test are modified cross‐match procedures. Antibody screening uses patient serum added to washed RBCs of known blood types (i.e., stock samples kept at the laboratory) to determine if the patient serum causes agglutination or hemolysis of the known blood type RBCs (as per the cross‐match procedure detailed above). Which of the mixtures are positive indicates the antibodies that are present within the patient’s serum. Horses without antibodies to EAA, EAC, and EAQ are good plasma donors and if they are negative for these antibodies within the final four weeks of pregnancy, they are unlikely to have a foal with NI.


Table 4.4 Macroscopic agglutination.





















0 No visible agglutination
Weak Few small aggregates of RBCs
1+ Many small aggregates of RBCs
2+ Large aggregates with some smaller aggregates of RBCs
3+ Several large aggregates of RBCs
4+ Single solid aggregate of RBCs

The JFA test is a modified agglutination cross‐match procedure mixing colostrum from the mare in various saline dilutions with RBCs from the foal. Anticoagulated blood (e.g., EDTA or ACD samples) from the foal and colostrum from the mare are required for this test. Colostrum is used as it contains the mare’s antibodies that are transferred to the foal for immunity, which may include an alloantibody for the foal’s RBCs. If there is a blood type mismatch between the mare and foal and the mare either has naturally occurring alloantibodies or has acquired alloantibodies (from a previous pregnancy or transfusion), then one of the antibodies transferred could also act as an agglutinin to the foal’s RBCs and cause NI. This test is considered clinically significant if there is agglutination at or greater than a dilution of 1:16 in horse foals and 1:64 in mule foals [15, 16]. NI will be further discussed in the next section.


4.2 Immune‐Mediated Hemolytic Anemia


Immune‐mediated hemolytic anemia (IMHA) is the premature destruction of RBCs due to the presence of antibodies directed against RBC antigens. This can be either a primary (i.e., autoimmune) or secondary (e.g., to RBC parasites, infection, neoplasia, drugs) process. As opposed to dogs and cats, IMHA in horses is not considered common and when it occurs, it is most often a secondary disease process. Excluding NI cases, approximately 45 cases of IMHA have been reported in the horse [17–38] and the most common causes were clostridial infection in approximately 20–29% of reported cases [22–25, 28, 30, 33, 36], penicillin administration in approximately 13% of reported cases [28, 29, 31, 32], and lymphoma in approximately 13% of reported cases [20, 21, 34].


Regardless of whether the cause is primary or secondary, the mechanism of destruction of the RBCs is the same and occurs through antibody binding to the surface of the RBCs causing either intravascular or extravascular hemolysis. Extravascular hemolysis is most common and occurs when the RBC‐bound antibody binds to the Fc receptors on tissue macrophages, predominantly in the spleen but also liver and other organs, causing premature removal and breakdown of the RBCs within the macrophages. Intravascular hemolysis is less common and a more severe clinical entity that requires the binding of complement to the antibody‐bound RBC surface with subsequent activation of the complement cascade and formation of the membrane‐attack complex (MAC) on the RBC surface, causing lysis within the vascular space. Extravascular hemolysis is clinically associated with icterus, hyperbilirubinemia, and bilirubinuria due to the excessive hemoglobin breakdown. Intravascular hemolysis is clinically similar except there is also hemoglobinemia and hemoglobinuria as the RBCs are lyzed within the vascular space and the free hemoglobin is cleared directly by the kidneys. Most often, the antibody type responsible for IMHA in the horse is immunoglobulin class G (IgG) with rare cases involving immunoglobulin class M (IgM) and as yet no immunoglobulin class A (IgA) reported but this is potentially due to the difficulty in identifying this antibody class in horses [28].


Primary and secondary IMHA cannot be clinically or diagnostically distinguished except if no secondary causes of IMHA can be found then primary IMHA is assumed. Common clinical signs associated with IMHA are due to the anemia and RBC breakdown and include fever, depression, pallor, icterus, hemoglobinuria (if intravascular), tachycardia, and weakness. IMHA is diagnosed if multiple of the following criteria are present: (i) an anemia (often regenerative); (ii) autoagglutination after RBC washing (i.e., persistent autoagglutination); (iii) a positive Coombs test; (iv) RBC morphological changes reported with equine IMHA (e.g., spheroechinocytes, type III echinocytes, agglutination) [36]; and (v) elimination of other causes (e.g., blood loss, oxidative damage).


Laboratory testing in cases of suspect IMHA should start with complete blood count (CBC), biochemical panel, and urinalysis (UA). If the CBC is supportive of an immune‐mediated process (i.e., anemia with concurrent hyperbilirubinemia without evidence of oxidative damage and with agglutination, spheroechinocytes, or type III echinocytes) then consider Coombs’ testing. Although IMHA is most often regenerative, this is difficult to determine on a single CBC in horses as reticulocytes are only very rarely noted in the peripheral blood of horses, even in severe anemia. Regeneration is therefore confirmed either through bone marrow aspiration or rising packed cell volume (PCV) with stable plasma/total protein.


Coombs’ testing (otherwise called direct antiglobulin testing) is used to confirm the presence of anti‐RBC antibodies and it does so by incubating washed patient RBCs with antiserum specific to equine IgG, IgM, and/or complement. If IgG, IgM, or complement is present on the washed RBCs, there will be agglutination of the RBCs and a positive result. This same method of incubating washed RBCs with specific antiserum has also been reported successful using flow cytometry instead of positive agglutination to detect the bound antibody; however, there were too few cases to determine if this is a more sensitive or specific method than the traditional Coombs’ test and this test is not easily accessible clinically. Flow cytometry was shown to accurately document reducing antibody in a foal with NI, so it may also have a use in disease monitoring [28].


Although a positive result often indicates an immune‐mediated process, negative results via conventional Coombs’ testing are relatively common in cases in which all other causes of anemia are ruled out or another method is used to confirm IMHA (e.g., flow cytometry, antipenicillin antibodies) [20, 39, 40]. Unfortunately, a defined sensitivity for Coombs’ testing is not known in equine IMHA given the low number of overall cases; however, this test is still recommended, as a positive result indicates immune‐mediated anemia is the most likely cause.


Other testing to consider is based on assessment for secondary causes of IMHA and would include targeted investigation of body systems for infection or neoplasia depending on clinical signs and presentation (e.g., abdominal or thoracic imaging, rectal examination, aspiration of lymph nodes, fluid analysis, any drug administration history) and serology and/or polymerase chain reaction (PCR) for infectious diseases with known associations with IMHA (e.g., equine infectious anemia [EIA], PCR for leptospirosis, PCR/culture for Streptococcus equi/Clostridium spp.). Differential diagnoses for anemia in horses include blood loss, oxidative hemolysis (e.g., red maple toxicity, hereditary diseases), infectious diseases (e.g., EIA, Babesia spp., ehrlichiosis, leptospirosis), and anemia of chronic or inflammatory disease.


4.2.1 Neonatal Isoerythrolysis


Neonatal isoerythrolysis is the most common cause for icterus and hemolytic anemia in the neonatal foal and is caused by the reaction of antibodies transferred in the colostrum of the mare that are directed against antigens present on the foal’s RBCs that are not present on the mare’s RBCs (i.e., immunogenic RBC antigens inherited from the stallion). Studies have shown that RBC incompatibilities between mare and foal are common (up to 14%) [41] and that antibody development by the mare is also common and breed specific, with 10% of Thoroughbred and 20% of Standardbred mares having detectable antibodies in serum [5]. However, despite this, the occurrence of NI is much lower and a simple answer for why this is the case has not yet been found. In the same study that documented the presence of serum antibodies against RBC antigens in 10% and 20% of Thoroughbred and Standardbred horses, respectively, there were only 1% in Thoroughbreds and 2% in Standardbreds of incompatibilities between mare and foal that were associated with NI [5]. The rate of NI is much higher in mules (donkey sire with horse dam) at 8–10% as donkeys have a unique RBC antigen to which the mare becomes sensitized during prior pregnancies [12].


4.2.1.1 Pathogenesis


Neonatal isoerythrolysis is caused by alloantibodies produced in the mare that are directed against RBC antigens that are only present on the foal’s RBCs. These alloantibodies are produced during pregnancy or delivery if there is leakage of blood across the placenta (e.g., placentitis or difficult delivery) or produced secondary to a previous mismatched blood transfusion. Alloantibodies can persist for years and are often strongest in late‐term pregnancy, so if antibody screening is to be performed to predict NI, it is recommended in late‐term pregnancy (i.e., the final four weeks). These alloantibodies are usually IgG and do not cross the placenta but rather are transmitted to the foal in colostrum.


Passive transfer of immunity is the transfer of immunoglobulins from the mare’s colostrum to the foal’s plasma via uptake of whole immunoglobulin through the gastrointestinal mucosa, which can occur only in the first 24 hours of life and provides the foal with immunity to environmental pathogens until its own immune system is fully functional. Unfortunately, if the mare has developed antibodies to foal‐specific RBC antigens (i.e., through this pregnancy, previous pregnancies, or blood transfusion) then these will also be transmitted in the colostrum and could cause hemolysis and/or hemagglutination, depending on whether the antibody is a hemolysin or agglutinin or both. Anti‐Aa alloantibodies are agglutinins and hemolysins whereas anti‐Qa alloantibodies are solely hemolysins. There are eight known blood groups and within these groups 35 known RBC antigens (see Table 4.1) to which alloantibodies could be developed, yet the RBC antigens and serum antibody incompatibilities that commonly cause NI are mostly related to blood groups EAA and EAQ. Anti‐Aa and anti‐Qa alloantibodies are most commonly implicated in NI with anti‐Ab, Qb, Qc, Qrs, Da, Db, Dc, Dg, Ka, Pa, and Ua also reported rarely


4.2.1.2 Clinical Features


As the alloantibodies are transferred in colostrum, the foals are normal at birth with signs developing after alloantibody absorption. Hemolysis and associated clinical signs can develop as early as five hours after birth but more commonly within 12–48 hours and as late as 12 days after birth [7, 9]. Clinical presentation is associated with the severity of anemia and rapidity of hemolysis and varies from lethargy and icterus to weakness, tachypnea, tachycardia, hemoglobinuria, and hypovolemic shock that can result in death through multiorgan failure and disseminated intravascular coagulation (DIC). Liver failure ± kernicterus have been noted as the major cause of death in NI patients with complications of sepsis noted as the other common cause [9, 42]. The development of liver failure ± kernicterus was found to be statistically associated with the volume of blood products administered (>4.0 L resulted in a 19.5 times higher likelihood of liver failure) and the maximum total bilirubin (>27.0 mg/dL resulted in a 17.0 times higher likelihood of kernicterus) during hospitalization [42]. A recent publication has shown the success of plasma exchange as an intervention to decrease marked hyperbilirubinemia and avoid kernicterus in two foals [43].


4.2.1.3 Diagnosis


As other causes of IMHA are considered very uncommon in the neonate, any neonate (i.e., less than 2 weeks old) presenting with a hemolytic anemia should be considered to have NI until proven otherwise. The CBC should be considered essential as a first step in diagnosis and if an anemia ± hyperbilirubinemia/hemoglobinemia with normal total/plasma protein is found, this is considered supportive. Definitive tests include cross‐matching (between the mare serum and foal RBCs – submit serum from the mare and EDTA/ACD from the foal); JFA test (submit EDTA/ACD from the foal with colostrum from the mare); direct Coombs’ test of the foal (submit EDTA/ACD from the foal); and/or antibody screening of the mare with concurrent blood typing of the mare and foal or stallion if prefoaling (submit EDTA/ACD from mare and foal with serum from the mare). See earlier in this chapter for methodologies, but recognize that the alloantibodies associated with NI are often stronger hemolysins than agglutinins, so if cross‐match or antibody screening is performed, they should include an incubation with complement to assess for hemolysins so need to be performed by a reference laboratory (see Table 4.2). Confirmation of NI occurs if there is proof of mare alloantibodies of sufficient titer directed against foal RBC antigens.


4.2.1.4 Prevention


Prevention is best achieved by identifying mares that are at risk of producing NI‐inducing alloantibodies and breeding them accordingly; however, if this is not possible or the mare is already pregnant, then prevention of NI involves determining if the foal is at risk and protecting the foal from exposure to alloantibody.


Blood typing of the mare prior to mating can help to choose appropriate stallions (i.e., if the mare is Qa or Aa negative then only breed the mare to stallions that are also negative for these blood group factors) and this is recommended in any mare with a history of NI. Blood typing during pregnancy, if not performed before, can be used to identify mares that are at risk and would benefit from antibody screening during the last 3–4 weeks of pregnancy. Antibody screening will identify whether the mare has alloantibodies to RBC antigens she does not carry (either developed during this/previous pregnancies or due to previous blood transfusion); however, determination of whether this will cause NI will require cross‐matching and/or blood typing of the foal or stallion with the mare. Antibody screening ± blood typing of the stallion is recommended prefoaling in mares with a history of NI. Unless hemolysis‐based cross‐matching is available where the foal is delivered, cross‐matching the foal and mare is unlikely to be clinically useful for prevention as it would need to be performed after delivery but before colostrum ingestion (i.e., within the first 2–3 hours of life). More often, cross‐matching is performed to diagnose NI as the cause for anemia and illness in a foal. The JFA test, however, can be used stall‐side and has been shown to correlate well with the hemolysis‐based cross‐match if performed by trained personnel [16]. As this is stall‐side, it can be used before the foal ingests colostrum to predict if the foal would develop NI as well as being used as a confirmatory test in diagnosing NI as the cause for a foal presenting hyperbilirubinemic and anemic.


If the foal and mare have incompatible RBC antigens and alloantibodies, it is recommended that the foal be muzzled to prevent colostrum ingestion for the first 36–48 hours postpartum. The mare should be stripped to ensure milk production for when the foal is allowed to return to nursing. The foal needs to receive colostrum from another source to ensure passive transfer of immunity. If a transfusion is required in a clinical NI case, washed pRBCs from the mare is considered the transfusion of choice. Blood from the stallion should never be used as it is his RBC antigens, which the foal has inherited, that are causing the mismatch.


4.2.2 Infection‐Associated (Clostridial, EIA, Rhodococcus equi, S. equi)


4.2.2.1 Pathogenesis


The mechanisms of clostridial‐associated, R. equi‐associated, and S. equi‐associated IMHA are unknown. Proposed mechanisms in clostridial‐associated IMHA in horses include that the clostridial toxins damage the RBC membrane, exposing new antigens to which the body develops autoantibodies [33], and/or that the clostridial toxins (specifically alpha toxin, a phospholipase) cause RBC damage, echinocyte/spheroechinocyte formation, hemolysis, and autoagglutination [36].


Equine infectious anemia is caused by a retrovirus for the Lentivirus genus that infects tissue macrophages. Infection occurs through transmission of blood from horse to horse by biting flies. After infection, the virus replicates in tissue macrophages, causes intermittent viremia, and infects the patient lifelong, allowing for further transmission. The mechanism of anemia in EIA is threefold. There is rarely intravascular immune‐mediated hemolysis in acute disease, more commonly there is extravascular immune‐mediated hemolysis in acute and chronic disease paired with an impaired bone marrow response. The immune‐mediated hemolysis is associated with complement binding to the surface of RBCs causing activation of the intravascular complement cascade (intravascular hemolysis) and phagocytosis by tissue macrophages (extravascular hemolysis). The complement binding in EIA is thought to be secondary to a hemagglutinin, one of the surface proteins of EIAV, or circulating virus–antibody immune complexes attaching to the RBCs and attracting and activating complement [44].


4.2.2.2 Clinical Features

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Oct 30, 2022 | Posted by in EQUINE MEDICINE | Comments Off on Immunohematology and Hemostasis

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