the AB blood group system has been recognized. It comprises the common types A and B as well as the rare type AB.1,2 The A and B antigens are glycolipids ...
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Comparison of various blood-typing methods for the feline AB blood group system Knut Stieger, Dr med vet; Hanna Palos, DVM; Urs Giger, PD, Dr med vet
Objective⎯To compare feline blood-typing results determined by use of the card (CARD), gel (GEL), tube (TUBE), University of Pennsylvania (Penn) tube, and Penn slide tests. Sample Population⎯Blood samples from 38 healthy cats. Procedures⎯Blood samples, anticoagulated with EDTA, were screened by use of each blood-typing method according to manufacturers’ protocols. Results⎯On the basis of the standard Penn tube and slide test results, 20, 11, and 7 cats were classified as type A positive, type B positive, and type AB positive, respectively. The same results were obtained with the anti-B and anti-B reagents of the TUBE test. Use of anti-A antibodies of original polyclonal and current monoclonal CARD tests resulted in mostly 2+ to 3+ (scale, 0 to 4+) agglutination reactions with blood samples from type A-positive cats; agglutination reactions with blood samples from type AB-positive cats were weak (1+). The anti-B lectin of the CARD test induced a 2+ to 4+ reaction with blood from all type B- and type AB-positive cats. Use of the GEL test allowed recognition of type A and type B blood samples; following addition of anti-A serum to control columns, type B blood was differentiated from type AB blood. Conclusions and Clinical Relevance⎯Use of the inpractice CARD test allows identification of type Aand type B-positive cats, but weak reactions of type AB blood with the anti-A monoclonal antibody raise concerns. The modified GEL and TUBE tests appear to be reliable clinical laboratory methods for feline blood typing. (Am J Vet Res 2005;66:1393–1399)
B
lood types result from genetically determined markers on the surface of RBCs, which can be antigenic and are specific to a given species. In cats, only the AB blood group system has been recognized. It comprises the common types A and B as well as the rare type AB.1,2 The A and B antigens are glycolipids and differ by an N-acetyl- versus N-glycyl-neuraminic acid, respectively.3,4 Because type A is dominant over B, cats Received August 11, 2004. Accepted November 23, 2004. From the Section of Medical Genetics, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104. Dr. Stieger’s present address is Laboratoire de Thérapie Génique, INSERM U649, 44035 Nantes, Cedex 1, France. Dr. Palos’ present address is the Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996. This manuscript represents a portion of a doctoral thesis submitted by Dr. Stieger to the University of Leipzig. Supported in part by a grant from the NIH (RR02512). Presented in part at the Forum of the American College of Veterinary Internal Medicine, Charlotte, NC, June 2003. Address correspondence to Dr. Giger. AJVR, Vol 66, No. 8, August 2005
with type A blood have the genotype a/a or a/b, and only homozygous b/b cats express the type B antigen on their RBCs.5 The rare type AB blood is caused by a third allele apparently recessive to the a allele and dominant to the b allele, leading to the expression of A and B antigens.6 Cats with type AB blood are not the result of mating of a type A- to a type B-positive cat, unless the A cat carries the rare allele for type AB blood.6 Type AB blood has been rarely found in purebred as well as domestic shorthair cats.5-8 The frequency of feline type A and type B blood varies geographically and among breeds. Blood type A is by far the most common type in domestic shorthair cats, whereas the frequency of type B can vary in purebred cats from none, as in the Siamese breed, to 30% to 40%, as seen in British shorthair and Devon Rex breeds, to approximately 50% in Turkish Angora and Van cats.5,7,9-15 Feline plasma generally contains naturally occurring alloantibodies against the blood type the individual cat is lacking.16,17 In particular, type B-positive cats develop strong anti-A antibodies (1:32 to 1:2,048) of the IgM class and, to a lesser extent, of the IgG class at a few weeks of age, whereas type A-positive cats have generally weak anti-B alloantibodies (1:2).17,18 Type ABpositive cats do not have any alloantibodies.6 Hence, a mismatched transfusion with type A blood given to a type B-positive cat results in a life-threatening acute hemolytic transfusion reaction,18,19 and type A and AB kittens born to a type B queen receiving anti-A alloantibodies through the colostrum are at risk for developing neonatal isoerythrolysis during the first days of life.20-22 In contrast, type B blood given to a type A-positive cat will result in shortened survival of the transfused RBCs with milder signs of acute hemolytic anemia.16,18 Type B kittens born to a type A queen are not affected by neonatal isoerythrolysis.8 Blood typing and crossmatching are considered essential tools in clinical feline practice to help select compatible donors and mates and to prevent serious transfusion and neonatal isoerythrolysis reactions. The principle of a positive serologic blood-typing reaction is the macroscopically visible agglutination of RBCs within a few seconds to minutes with known antibodies or special agglutinin reagents. If no agglutination reaction occurs, the test result is considered to be negative. The reagents for blood typing are either polyclonal or monoclonal antibodies or lectins.4,16 Antisera from type A- and type B-positive cats have been used to type cats for decades23; particularly, the anti-A serum has high RBC agglutination activity. The lectin from Triticum vulgaris has replaced the feline anti-B serum as it preferentially agglutinates type B-positive RBCs, whereas it agglutinates type A-positive RBCs only at higher concentrations.4,24 The transfusion laboratory at 1393
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the School of Veterinary Medicine University of Pennsylvania (Penn)a offers tube and slide agglutination assays that use the anti-A serum and T vulgaris lectin. This tube assay was developed for use in specialized laboratories. It is reliable and considered the gold standard and thus has been the technique applied as the reference standard in several surveys.6,10,25 The slide assay is a simplification of the tube assay and permits blood typing of whole blood and therefore serves as an in-office method for routine clinical application. On the basis of this technique, a commercial laboratoryb in collaboration with Kansas State University developed a blood-typing card method that has been in use since 1996 in which a polyclonal anti-A antibody and the T vulgaris lectin as the B agglutinin are used.25 Since 2002, the company has offered a new blood-typing card (CARD) testb with monoclonal anti-A antibodies replacing the polyclonal feline anti-A serum for the identification of type A-positive cats. A company in Switzerland has established a new blood-typing test technique that uses a gel matrix column to fix the agglutination reaction for blood typing of humans26,27 and has recently adopted the procedure to produce gel test columns for canine and now a gel (GEL) testc column for feline blood typing. A company in Japan recently introduced a laboratory tube (TUBE) testd that uses monoclonal antibodies recognizing the feline A and B antigens.28 The purpose of the study reported here was to compare feline blood-typing results of the Penn, CARD, GEL, and TUBE tests. Materials and Methods Animals and reagents⎯The 38 cats enrolled in our study were a mix of type A-, B-, and AB-positive cats selected in part because they were found to have the less common type B or type AB blood during routine screening at the Transfusion Center at the University of Pennsylvania. This group included 23 domestic shorthair, 7 Devon Rex, 3 Norwegian Forest, 3 Scottish Fold cats, 1 Birman cat, and 1 Ragdoll cat. About 2 mL of EDTA-anticoagulated blood was collected and stored at 4oC for < 3 days until analysis. Cats were healthy and nonanemic, were between 6 months to 9 years old, and represented equal numbers of female and male cats. For each sample, the control test result for autoagglutination was negative and alloantibody testing to assess the presence of anti-A alloantibodies was done to confirm the blood-typing results.7 Preparation of 2% to 5% washed RBC suspension⎯ One milliliter EDTA-anticoagulated whole blood was poured into a 12 X 75-mm glass tube and centrifuged for 2 minutes in a specific serologic centrifuge.e After this step, the plasma was separated into a separate glass tube for alloantibody testing. Thereafter, 5 mL of PBS solution was added to the RBC pellet and centrifuged for 2 minutes. After the supernatant was aspirated and discarded, another 5 mL of PBS solution was added to the pellet and the suspension was mixed and centrifuged for 2 minutes. This RBC washing step was repeated 2 more times. After the last wash, the appropriate volume of PBS solution was added to reach an approximately 50% RBC suspension. On the basis of the microhematocrit value, the suspension was further diluted approximately 1:10 to reach a final RBC volume of 2% to 5%. CARD test⎯The original polyclonal antibody blood-typing cardsb consisted of 6 wells as follows: 3 wells in the first column were labeled as type A and contained lyophilized anti1394
A serum from type B-positive cats and the 3 wells in the second column were labeled as type B and contained the lyophilized lectin from T vulgarisf (anti-B reagent). Control type A and type B blood samples provided by the manufacturer or the laboratory’s own control samples can be used, as was done during this study. The lyophilized reagents on the surface of each well were reconstituted with PBS solution at the time of blood typing and were equilibrated to room temperature (approx 18o to 25oC). The evaluation of autoagglutination was done on a separate autoagglutination card or on a glass slide. One drop (50 µL) of PBS solution was applied to each well and mixed with a stirrer to assist reconstitution of the anti-A antibody and the lectin. With separate pipettes, 50 µL of type A and type B control blood was applied to the wells of the first and second row, respectively, and 50 µL of the test sample was applied to the well for the patient’s blood samples. Suspensions were mixed with a separate stirrer for approximately 10 seconds by pressing the stirrer downward onto the well. The test card was gently rocked with a transaxial motion for 2 minutes, assuring the mixing of the suspensions in each well. Thereafter, cards were set at a 20o angle to allow excess blood to run to the bottom of the wells. Blood-typing results were read and interpreted according to the manufacturer’s instruction as follows: if the patient sample had macroscopic agglutination only in the type A well, the patient was type A positive; if agglutination was visible only in the type B well, the patient had blood type B; and if agglutination was seen in both wells, type A and type B, the cat had type AB blood. Herein we applied more discriminating interpretation criteria as follows: 0 (negative), no agglutination; 1+, small agglutinates form after 30 seconds; 2+, many small agglutinates visible within 15 seconds; 3+, large agglutinates seen within 10 seconds; and 4+, large agglutinates present within 5 seconds. Any fine granular agglutination after 2 minutes was disregarded. The currently available monoclonal antibody blood-typing cardsb included 3 wells and used only patient or control blood samples. Because the monoclonal antibody blood-typing cards became available during the study, blood samples from only 30 cats were evaluated by use of this method. The top offset well was labeled as the autoagglutination-salinescreen well and did not contain any reagents; the well labeled as type A contained lyophilized monoclonal anti-A antibodies, and the well labeled as type B contained the T vulgaris lectin. One drop of PBS solution was dispensed into the type B well and the autoagglutination-saline-screen well and 2 drops of PBS solution into the type A well. With a pipette, 50 µL of the patient sample was dispersed into each of the 3 wells, taking care that the pipette tip did not touch the well or PBS solution. The stirrer was pressed downward onto the well, and the suspension was mixed in each well with a separate stirrer for 10 seconds. The test card was then gently rocked with a transaxial motion for 2 minutes, assuring that the materials were mixed within each well. Thereafter, the cards were set at a 20o angle to allow excess blood to run to the bottom of the wells. The interpretation criteria were the same as described for the polyclonal antibody blood-typing cards. GEL test⎯Each GEL test cardc contained 6 gel columns filled with a polypropylene gel that allowed the blood typing of 2 cats. Columns labeled as AB and B contained the lectin of T vulgaris at a high and low concentration, respectively, whereas columns labeled as ctl did not contain any antibodies or lectins and served as a negative autoagglutination control. The diluent,g which was used to prepare the RBC suspension, was a modified, low ionic strength solution with antibiotics and was stored at 4oC but allowed to come to room temperature prior to use. First, 50 µL of whole blood or 25 µL of sedimented AJVR, Vol 66, No. 8, August 2005
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blood was diluted with 500 µL of the diluentg to achieve a 5% RBC suspension. Aluminum foil was removed from the top of the GEL test card, a 12.5-µL RBC suspension from a cat was added to the first set of 3 columns, and a 12.5-µL RBC suspension from another cat was added to the second set of 3 columns. The card was centrifuged at 80 X g for 10 minutes in a special centrifuge,h and the results were interpreted as follows: 0 (negative), all RBCs were at the bottom of the tube; 1+, few RBC agglutinates were dispersed in the gel, but most of the RBCs were at the bottom of the tube; 2+, all RBC agglutinates were dispersed in the gel; 3+, some RBC agglutinates were dispersed in the upper part of the gel, most of the RBCs form a red line on the surface of the gel; and 4+, all RBCs were agglutinated and form a red line on the surface of the gel. According to the manufacturer’s interpretation pattern, the AB-labeled column had to react with each sample, acting somewhat as a positive control. The ctl-labeled column must have a negative reaction with each blood sample to interpret results, and the B-labeled column could have a positive or negative reaction. If the B-labeled column had a negative reaction, the cat had type A blood, and if this column had a positive reaction, the cat had type B or the rare type AB blood. We found that samples from type AB-positive cats reacted like type B-positive cats; therefore, no differentiation could be made between type B- and type AB-positive cats with the prototype GEL test. Therefore, we modified the GEL test system to allow differentiation of all 3 blood types in this study. After carrying out the standard protocol, 3 µL of polyclonal anti-A serum from an adult type B-positive cat (equivalent to the volume used in the Penn tube test) was added onto the ctl-labeled column. Again, a 12.5-µL RBC suspension was pipetted into each column, and the card was centrifuged for 10 minutes. For type A- or AB-positive cats, the ctl-labeled column had a strong positive reaction; hence, we were able to differentiate all 3 blood types. This modification, with anti-A serum instead of the high concentration of the lectin in the ABlabeled column, is now standard in the new commercially available GEL test blood-typing kits.c Penn slide and tube tests⎯The Penn slide assaya was performed on glass slides by use of whole blood and 2 reagents. Serum, which was heated (56oC; 30 minutes) to inactivate complement and was filtered (0.45-µm-diameter pore), from an adult type B-positive cat was used as an antiA antibody reagent. The T vulgaris lectin was used as an antiB antibody reagent at a concentration of 64 µg/mL.4 Three slides were prepared for blood typing of each cat and were labeled as anti-A, anti-B, and autoagglutination-control (C). Fifty microliters of anti-A, anti-B, or PBS solution reagents were added to the specified slide, respectively. Then 25 µL of whole blood from a cat was added to each slide, taking care that the pipette did not touch the slide or solution. The slide was gently agitated for 2 minutes, and the agglutination reaction was recorded as for the CARD test. A 5% RBC suspension and the same reagents as for the Penn slide test, albeit at lower concentrations, were used in the Penn tube test.a A 1:8 dilution of serum from type B-positive cats was used as an anti-A antibody reagent and a lower concentration of the T vulgaris lectin (8 µg/mL)4 was used as the anti-B antibody reagent. Tubes (12 X 75 mm) were prepared for each sample and labeled anti-A, anti-B, and C, and 50 µL of anti-A, anti-B, and PBS solution reagents was added with separate pipettes to the respective tubes. Then, 25 µL of the cat’s RBC suspension was added to each tube without letting the pipette touch the tube or solution. The content was gently mixed by brief vortexing and allowed to incubate for 15 minutes at room temperature. Thereafter, tubes were centrifugede for 15 seconds. To read the test results, tubes were gently agitated to resuspend the nonagglutinating RBCs in AJVR, Vol 66, No. 8, August 2005
the cell button. The strength of the agglutination reaction was recorded as follows: 0 (negative), no agglutination, all RBCs were in suspension; 1+, some small agglutinates with many not agglutinated RBCs in suspension; 2+, a few moderate-size agglutinates mixed with some small agglutinates; 3+, a few large agglutinates and clear solution; and 4+, 1 large agglutinate with clear supernatant. TUBE test⎯This manufacturerd has apparently developed 2 monoclonal antibodies (A and B) that recognize the 2 RBC surface antigens in cats. A 2% to 5% RBC suspension was used in a small tube assay. The method is similar to the Penn tube test, except no autoagglutination control was recommended. Briefly, 50 µL of the RBC suspension from a cat was added to the appropriate anti-A and anti-B tubes followed by 50 µL of the anti-A or anti-B antibody solution, respectively. After gentle mixing, tubes were incubated at room temperature for 5 minutes and centrifugede for 10 seconds. Prior to reading the results, tubes were gently agitated in an attempt to resuspend the cell pellets. The interpretation used was close to that used with the Penn tube test as follows: 0 (negative), no agglutination, all RBCs were in suspension; +/-, a few tiny agglutinates, but most of the RBCs were in suspension; 1+, many small agglutinates besides RBCs in suspension; 2+, some larger agglutinates mixed with many small agglutinates; 3+, a few large agglutinates with clear solution; and 4+, 1 large agglutinate and clear supernatant. Samples with negative or +/– reactions were considered to be a negative result for that blood type, and 1+ to 4+ reactions were interpreted as a positive result. Alloantibody test⎯Because cats have naturally occurring alloantibodies, the blood type can be confirmed by observing an agglutination reaction after mixing a cat’s serum or plasma with known type A- or type B-positive RBCs. Briefly, 50 µL of plasma from a cat was placed in a tube and mixed with 25 µL of a 2% to 5% RBC suspension from either a type A- or type B-positive cat. After incubating the mixtures for 15 minutes at room temperature, tubes were centrifugede for 15 seconds and then agitated slightly to resuspend the loose RBCs in the cell pellet. All type B-positive cats, which are > 3 months old, have developed strong anti-A antibodies in their plasma that can cause a strong reaction with type A-positive RBCs.5,17 On the other hand, plasma from type A-positive cats generally agglutinates only weakly with type B-positive RBCs. Plasma from type AB-positive cats will not agglutinate with type A- or type B-positive RBCs. Results of the agglutination reaction were interpreted in the same manner as for the Penn tube test.
Results Samples⎯Blood samples from 38 cats were analyzed for the feline AB blood group system with each blood-typing method. None of the whole blood or RBC suspension samples had autoagglutination, thus allowing for unambiguous reading of the blood-typing results with every method (Table 1). Penn slide and tube tests⎯By use of the Penn tube and Penn slide tests, all blood samples had a strong agglutination reaction with at least 1 reagent, as no type 0 cats in this feline AB blood group system were found (type 0 cats would not carry any of the 2 blood group antigens on their RBC surface). Both methods use the same anti-A and anti-B antibody reagents but at different concentrations designed to match the differing numbers of RBCs in whole blood and RBC suspensions. With the Penn tube test, 25 samples had a maximal 4+ and 2 samples had a 3+ 1395
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Table 1⎯Blood-typing results for the feline AB blood group system by use of the University of Pennsylvania (Penn) tests,a card (CARD) tests,b gel (GEL) test,c and tube (TUBE) test.d Results Tests
No. of cats
Interpretation
Penn tube test for A or B antigens (n = 38) 0 (no agglutination) 1+ (some small agglutinates) 2+ (few medium agglutinates and some small agglutinates) 3+ (few large agglutinates and clear supernatant) 4+ (1 large agglutinate with clear supernatant) No. of cats reacting with both antigens
11 (A), 20 (B) 0 (A), 0 (B) 0 (A), 0 (B) 2 (A), 0 (B) 25 (A), 18 (B) 7 (AB)
Neg Pos Pos Pos Pos 20 Pos for type A 11 Pos for type B 7 Pos for type AB
Penn slide test for A or B antigens (38) 0 (no agglutination) 1+ (small agglutinates form after 30 s) 2+ (many small agglutinates form within 15 s) 3+ (large agglutinates form within 10 s) 4+ (large agglutinates present within 5 s) No. of cats reacting with both antigens
5 (A), 13 (B) 6 (A), 7 (B) 0 (A), 0 (B) 4 (A), 0 (B) 23 (A), 18 (B) 7 (AB)
Neg Neg Pos Pos Pos 20 Pos for type A 11 Pos for type B 7 Pos for type AB
CARD test for A or B antigens (polyclonal method; 38) 0 (no agglutination) 1+ (small agglutinates form after 30 s) 2+ (many small agglutinates form within 15 s) 3+ (large agglutinates form within 10 s) 4+ (large agglutinates present within 5 s) No. of cats reacting with both antigens
11 (A), 20 (B) 9 (A), 0 (B) 5 (A), 12 (B) 13 (A), 6 (B) 0 (A), 0 (B) 7 (AB)
Neg Pos Pos Pos Pos 20 Pos for type A 11 Pos for type B 7 Pos for type AB
CARD test for A or B antigens (monoclonal method; 30) 0 (no agglutination) 1+ (small agglutinates form after 30 s) 2+ (many small agglutinates form within 15 s) 3+ (large agglutinates form within 10 s) 4+ (large agglutinates present within 5 s) No. of cats reacting with both antigens
7 (A), 16 (B) 8 (A), 0 (B) 2 (A), 2 (B) 11 (A), 1 (B) 2 (A), 11 (B) 7 (AB)
Neg Pos Pos Pos Pos 16 Pos for type A 7 Pos for type B 7 Pos for type AB
1 (AB), 20 (B), 11 (anti-A) 5 (AB), 0 (B), 0 (anti-A) 2 (AB), 11 (B), 0 (anti-A) 4 (AB), 7 (B), 0 (anti-A) 26 (AB), 0 (B), 27 (anti-A) 11(B and anti-A)
Neg Pos Pos Pos Pos 20 Pos for type A 11 Pos for type B 7 Pos for type AB
11 (A), 20 (B) 0 (A), 0 (B) 0 (A), 0 (B) 0 (A), 1 (B) 9 (A), 5 (B) 18 (A), 12 (B) 7 (AB)
Neg Neg Pos Pos Pos Neg 20 Pos for type A 11 Pos for type B 7 Pos for type AB
GEL test for AB and B antigens and anti-A antibody (38) 0 (all RBCs are at bottom of tube) 1+ (few agglutinates dispersed in gel) 2+ (all RBCs in agglutinates and dispersed in gel) 3+ (most agglutinates form red line on gel surface) 4+ (all agglutinates form red line on gel surface) No. of cats reacting with B antigen and anti-A antibody
TUBE test for A or B antigens (38) 0 (no agglutination) +/- (few tiny agglutinates) 1+ (many small agglutinates) 2+ (some large agglutinates with many small agglutinates) 3+ (few large agglutinates with clear supernatant) 4+ (1 to 2 large agglutinates with clear supernatant) No. of cats reacting with B antigen and anti-A antibody
Neg = Negative. Pos = Positive.
reaction with the anti-A serum. Thus, 27 cats were considered to have the A antigen on their RBC surface, whereas 11 samples did not react with the anti-A serum. Blood samples from 18 cats had a 4+ agglutination reaction with the anti-B antibody reagent; therefore, these RBC samples were considered positive for the B antigen, whereas 20 samples did not agglutinate with the anti-B antibody reagent. Red blood cells from 7 cats reacted with both reagents in the Penn tube test. 1396
Considering that the agglutination reactions were unambiguous, the results indicate that 20 cats have type A, 11 have type B, and 7 have type AB blood. Furthermore, by use of the alloantibody test, every plasma sample from type B-positive cats induced a strong agglutination reaction with type A-positive RBCs (4+) as a result of the presence of strong anti-A antibodies, whereas plasma from type A-positive cats incubated with type B-positive RBCs had variably weak AJVR, Vol 66, No. 8, August 2005
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agglutination as a result of weak anti-B antibodies in the plasma of type A-positive cats. No agglutination was observed with plasma from type AB-positive cats when mixed with either type A- or type B-positive RBCs. Thus, these alloantibody test results are consistent with the Penn tube test results. Therefore, we considered the classification on the basis of the Penn tube test results as the reference for all other methods in this study. With the Penn slide test, 23 and 4 blood samples had strong 4+ and 3+ agglutinations, respectively, by use of the anti-A antibody reagent and thus were considered to carry the A antigen on their RBCs. With 6 whole blood samples, a weak 1+ reaction was observed when incubated with the anti-A antibody reagent, and with 5 samples, no agglutination was observed. After incubation with the anti-B antibody reagent, 18 blood samples had a maximal 4+ agglutination reaction and thus were considered to have the B antigen on the RBCs, whereas only weak (1+; n = 7) or no agglutination (13) was seen with the remaining 20 samples. These 20 samples were classified as either type B negative or type AB negative. Complete concordance was found between these 2 blood-typing methods at Penn, with respect to the blood-typing results and classification of all cats tested. CARD test⎯Two different blood-typing cards were used in this study for the CARD test. By use of the original polyclonal antibody CARD test, blood from type A-positive and type B-positive cats is run as controls, but the autoagglutination test is performed on a separate card. In the anti-A antibody well, 3+ (n = 13) or 2+ (5) agglutination reactions could be observed, whereas with 9 samples, only a weak 1+ reaction was seen. All these samples were classified as type A positive or type AB positive. Blood samples from 18 cats had either a 2+ (n = 12) or 3+ (6) agglutination reaction in the anti-B antibody well, whereas the remaining 20 samples had no reaction in the anti-B well. Therefore, 20 cats were classified as type A positive, 11 as type B positive, and 7 as type AB positive, the same classification with each cat as with the Penn tube and slide methods. The agglutination reactions of samples from cats classified as having type AB when using the anti-A antibody reagent were weak when compared to the agglutination of the same group of samples with anti-B lectin as well as the agglutination of type A-positive RBCs. This finding was consistent and repeatable, being seen with retesting and when using a test card from a different lot. In contrast, the newer monoclonal antibody CARD test does not contain type A-positive and type B-positive controls but only evaluates the test sample; however, they have an additional well to evaluate autoagglutination. Of the 30 blood samples tested with the new monoclonal antibody blood-typing cards, 23 blood samples had an anti-A antibody agglutination reaction with a wide range in the results as follows: 1+ for 8 samples, 2+ for 2 samples, 3+ for 11 samples, and 4+ for 2 samples. As with the polyclonal antibody cards, the anti-A antibody agglutination reactions of type AB-positive cats were always weak (1+). As expected, the agglutination results with the lectin of the 14 positiveAJVR, Vol 66, No. 8, August 2005
reacting samples on the anti-B antibody well were strong and similar to the results with the polyclonal CARD test as follows: 2+ (n = 2), 3+ (1), and 4+ (11). Therefore, 16 cats were classified as type A positive, 7 as type B positive, and 7 as type AB positive. GEL test⎯The lectin from T vulgaris at different concentrations is used in the original GEL test; a high concentration is used in the AB-labeled column, and a low concentration is used in the B-labeled column, whereas the ctl-labeled column is empty and serves as autoagglutination control. None of the RBCs were retained in the ctl-labeled column; hence, no autoagglutination was found. However, all but 1 sample had a positive reaction in the AB-labeled column that included 4+ (n = 26), 3+ (4), 2+ (2), and 1+ (5) reactions. The 1 negative result in the AB-labeled column was confirmed in another GEL test. Reviewing the results of the B-labeled columns, 18 samples were positive at 3+ (n = 7) or 2+ (11), whereas RBCs from 20 samples did not agglutinate. Therefore, 20 cats were identified as type A positive and 18 as type B positive or type AB positive. No differentiation between type B or type AB blood was possible when using this test method in accordance with the manufacturer’s protocol. However, an alloantibody test with known blood samples from type A-positive and type B-positive cats could differentiate between type B and type AB blood because only plasma from adult type B-positive cats would produce agglutination reactions in response to the presence of anti-A antibodies. In this study, following the regular test, the GEL test protocol was modified by adding anti-A serum from a type B-positive cat to the ctl-labeled column. By use of this variation, the reaction in the AB-labeled and B-labeled columns did not change, whereas the modified ctl-labeled column had a maximal 4+ reaction with blood samples from type A-positive or AB-positive cats. Thus, this modification permitted the differentiation of all 3 blood types, and the classification of cats was identical to the reference Penn tube test method. TUBE test⎯According to the manufacturer’s instructions, monoclonal antibodies for both feline RBC antigens are used in the TUBE test. With the antiA antibody reagent, 27 washed RBC samples were strongly positive with either a 3+ (n = 9) or 4+ (18) reaction and were considered to carry the A antigen on the RBC surface, whereas the remaining 11 samples did not react. Eighteen cats had varying degrees of agglutination reactions with the anti-B antibody reagent (2+ [n = 1], 3+ [5], and 4+ [12]), thus indicating the presence of the B antigen on the surface of the RBCs, whereas 20 samples did not agglutinate. Therefore, 20 cats were classified as type A positive, 11 as type B positive, and 7 as type AB positive, which is consistent with the results of the Penn tube test. Excellent concordance was found between all blood-typing methods with respect to all blood-typing results and classification of all cats tested. Discussion The feline AB blood group system is thus far the only RBC surface antigen system known to exist in cats, 1397
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and blood type mismatches are responsible for acute hemolytic transfusion reactions and neonatal isoerythrolysis.1-4 Feline blood typing has been offered in clinical practice since the late 1980s and is now considered good standard practice prior to transfusing or breeding any cat.8,21 In our study of blood samples from 38 healthy cats, when comparing the results of a standard laboratory technique (ie, Penn tube test) with various novel blood-typing methods, nearly identical blood-typing results were obtained, suggesting that all methods can be used to help assure blood type compatibility. However, our study was in part designed to emphasize the discrimination of the rare type B and type AB blood and included only blood samples that had no evidence of autoagglutination from nonanemic cats. Use of the original Penn tube test, which involves the use of an RBC suspension and feline anti-A serum and the lectin from T vulgaris as anti-B antibody reagent,3,4 resulted in unequivocally strong 3+ and 4+ RBC agglutination reactions, when either the A or B antigen was present on the RBC surface, respectively. Of the 38 cats we tested, we classified 20 cats as having type A blood, 11 as having type B blood, and 7 as having type AB blood. This laboratory method is considered the standard to which other methods were compared. Although the manufacturer of the TUBE testd proposes that 2 different monoclonal antibodies are used in their test, use of the TUBE test in our study resulted in the same 3+ to 4+ agglutination reactions as those of the Penn tube test (which uses anti-A serum and anti-B lectin from T vulgaris). Although a washed RBC suspension is used in the Penn tube test, which restricts the test to laboratory application, similar blood-typing results were obtained when whole blood, undiluted or diluted, was used in the Penn slide, CARD, or GEL tests. In the Penn slide test, the same anti-A and anti-B antibody reagents are used as in the standard Penn tube test but at higher concentrations. It is therefore not surprising that identical blood-typing results were obtained with these 2 blood-typing methods. Because of its simplicity, this method is preferred over the Penn tube test in general screening for feline blood typing.6,8 However, in the presence of autoagglutination, the Penn tube test, by use of washed RBCs, may allow discrimination of blood types, whereas the results from the Penn slide test will be obscured by autoagglutination. In any case, if true autoagglutination exists, which persists after washing the RBCs, blood typing cannot be performed. In such situations, alloantibody testing, which identifies the presence of alloantibodies, may be used to identify type B blood, as plasma or serum of a type Bpositive cat agglutinates type A-positive RBCs.2-8 However, because of the often weak anti-B alloantibodies, type A or type AB blood cannot be definitively determined by the alloantibody test method. The Penn slide agglutination assay uses anti-A and anti-B antibody reagents that are stored either long term at –20oC or temporarily at 4oC, whereas the corresponding reagents in the CARD test are lyophilized on specific cardboard. For our study, 2 different CARD tests were available, as the company was in the process of switching from polyclonal to monoclonal anti-A 1398
antibody reagents. For the production of the original CARD test, the lyophilized anti-A antibody reagent had to be prepared from the serum of type B-positive cats, which was of a limited resource and was sometimes variable in its strength as well as stability. By use of monoclonal anti-A antibodies, the new CARD test avoids these problems of consistency and stability. With the CARD test, both use of the original polyclonal and the new monoclonal anti-A antibodies resulted in mostly a 2+ to 3+ agglutination reaction with blood from type A-positive cats, whereas the agglutination with blood from type AB-positive cats was consistently weak (1+) by use of either reagent. With samples from type AB-positive cats, the weak agglutination reaction in the type A well was particularly concerning; if the type B field had a strong positive reaction, these cats may be misclassified as having type B blood, which could result in clinical complications. Although type AB-positive cats are rare in the general population, the findings of our study raise concerns. Therefore, when using the CARD test, it may be prudent to confirm type B-positive cats with an alloantibody test or crossmatch test, as both of these confirmatory tests should result in strong agglutination between RBCs from a control type A-positive cat with plasma or serum from an adult type B-positive cat but not with plasma or serum from an adult type AB-positive cat.6,8 It should be noted that the anti-A alloantibodies may be transferred from a type B-positive queen to its neonatal offspring via colostrum during the first day of life, and naturally occurring anti-A alloantibodies develop after 6 to 10 weeks of life.17,22 Furthermore, the AB-positive RBCs may result from different alleles, and thus the actual AB antigen on RBCs of type ABpositive cats may be slightly different.4 Because the monoclonal anti-A antibody recognizes specific epitopes, a surface antigen from type A-positive and various type AB-positive cats may not be equally well recognized, leading to different agglutination reactions. All of the methods mentioned have some variation in results, and it is difficult to apply a standardized interpretation of the agglutination reaction. Unwashed diluted whole blood samples, as used in the GEL test, were recently introduced in human medicine. Specific agglutination reagents hold back positive RBCs at the top of or within the gel column. This method allows for easy standardization and automation of test result reading and has therefore gained popularity in human blood banks.26,27 The dilution of the blood sample to a Hct of 4% generally allows dispersion of nonspecific autoagglutination, but each sample is also tested in an empty ctl-labeled column. The prototype of the columns of the GEL test for feline blood typing used only the T vulgaris lectin as the agglutinating reagent. At high concentration (AB-labeled column), the lectin bound type A-, type B-, and type ABpositive RBCs, whereas at the low concentration (Blabeled column), the lectin bound only type B- and type AB-positive RBCs.3,4 A control tube without any lectin acted as a negative control. The degree of agglutination in the AB-labeled column was variable among samples, with all but 1 having some degree of agglutination. The nonagglutinating sample of our study was eventually found to be type A blood by use of the GEL test. In addiAJVR, Vol 66, No. 8, August 2005
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tion to the variability of the agglutination in the ABlabeled column, type B and type AB blood samples could not be differentiated, as both reacted equally in the ABlabeled and B-labeled columns. By including anti-A serum in the ctl-labeled column in a second assay, an agglutination reaction occurs only with blood samples from type A- and AB-positive cats. This result together with the interpretation of the results from the B-labeled column permitted unequivocal separation between all 3 blood types, just as with the standard Penn tube and slide tests. The reaction in the modified ctl-labeled column with any type AB blood sample was equally strong to that observed with blood type A, in contrast to findings with the CARD test. The column with anti-A serum has replaced the AB-labeled high lectin in the commercially available new GEL test, which promises to be an easy and standardized clinical laboratory method for the blood typing of cats.i Although this method will likely be restricted to clinical pathology laboratories because of the need of a special centrifuge for the gel column sets, this gel column technique has the potential to be applied for simple blood crossmatching to recognize other blood groupsj and Coombs’ reaction (RBC antiglobulin) testing. In conclusion, various reliable clinical pathology laboratory methods are now used (the GEL test is the only commercially available clinical laboratory method) for feline blood typing, which give identical blood-typing test results when instructions are followed and autoagglutination is excluded. Currently, the only assay for use in clinical practice is the CARD test. Although these cards are simple to use and readily identify type Aand type B-positive cats, the results observed with type AB blood may be confused with type B blood. Thus, type AB and type B results should be confirmed either by alloantibody testing or in a laboratory by use of a technique other than the CARD test. Fortunately, cats with the type AB blood are rare in the general population. Additional comparative studies are warranted, which should include a larger group of blood samples from healthy and diseased (anemic) cats and should include the modified and now standard GEL test and the simplified TUBE test.i a. b. c. d. e. f. g. h. i. j.
Transfusion Laboratory, Section of Medical Genetics, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pa. DMS rapidVet-H (feline) blood-typing CARD test was a gift from DMS Laboratories Inc, Flemington, NJ. Vet ID Card A+B GEL test was a gift from DiaMed AG, Cressier sur Morat, Switzerland. Shigeta’s blood-typing kit was a gift from Shigeta Animal Pharmaceuticals Inc, Komoridani, Oyabe City, Toyama Pref, Japan. Serofuge II, Clay Adams Co, Morristown, NJ. Catalog #9640, Sigma Chemical Co, St Louis, Mo. ID-Diluent Vet 2, DiaMed Microtyping System, Cressier sur Morat, Switzerland. ID-Centrifuge 12 S II, DiaMed Microtyping System, Cressier sur Morat, Switzerland. Giger U, Blais MC, Weinstein NM. Assessment of a commercial gel column technique for feline AB blood typing and crossmatching (abstr). Am Soc Vet Clin Path 2005;in press. Weinstein NM, Blais MC, Greives K et al. A new blood group antigen in domestic shorthair cats: The feline Mik red cell antigen. J Vet Intern Med 2005;19:400–401.
References 1. Bell K. The blood groups of domestic mammals. In: Agar NS,
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Board PG. eds. Red blood cells of domestic mammals. Amsterdam: Elsevier, 1983;133–164. 2. Giger U. The feline AB blood group system and incompatibility reactions. In: Kirk RW, Bonagura JD, eds. Kirk’s current veterinary therapy Vol 11. Philadelphia: WB Saunders Co, 1992;470–474. 3. Andrews GA, Chavey PS, Smith JE, et al. N-glycolylneuraminic acid and N-acetylneuraminic acid define feline blood group A and B antigens. Blood 1992;79:2485–2491. 4. Griot-Wenk ME, Pahlsson P, Chisholm-Chait A, et al. Biochemical characterization of the feline AB blood group system. Anim Genet 1993;24:401–407. 5. Giger U, Bücheler J, Patterson DF. Frequency and inheritance of A and B blood types in feline breeds of the United States. J Hered 1991;82:15–20. 6. Griot-Wenk ME, Callan MB, Casal ML, et al. Blood type AB in the feline AB blood group system. Am J Vet Res 1996;57:1438–1442. 7. Giger U, Griot-Wenk M, Bücheler J, et al. Geographical variation of the feline blood type frequencies in the United States. Feline Pract 1991;19(6):5–11. 8. Giger U. Blood typing and cross matching to ensure compatible transfusions. In: Bonagura JD, ed. Kirk’s current veterinary therapy. Vol 13. Philadelphia: WB Saunders Co, 2000;396–399. 9. Haarer M, Grünbaum EG. Blutgruppenserologische Untersuchungen bei Katzen in Deutschland. Kleintierpraxis 1993;38:195–204. 10. Knottenbelt CM, Addie DD, Day MJ. Determination of the prevalence of feline blood types in the UK. J Small Anim Pract 1999;40:115–118. 11. Mylonakis ME, Koutinas AF, Saridomichelakis M, et al. Determination of the prevalence of blood types in the non-pedigree feline population in Greece. Vet Rec 2001;149:213–214. 12. Bagdi NM, Magdus M, Leidinger E, et al. Frequencies of feline blood types in Hungary. Acta Vet Hung 2001;49:369–375. 13. Jensen AL, Olesen AB, Arnbjerg J. Distribution of feline blood types detected in the Copenhagen area of Denmark. Acta Vet Scand 1994;35:121–124. 14. Hubler M, Arnold S, Casal ML, et al. Die Blutgruppenverteilung bei den Hauskatzen in der Schweiz. Schweiz Arch Tierheilkd 1993;135:231–235. 15. Arikan S, Duru SY, Gurkan M, et al. Blood type A and B frequencies in Turkish Van and Angora cats in Turkey. J Vet Med A Physiol Pathol Clin Med 2003;50:303–306. 16. Auer L, Bell K. Transfusion reactions in cats due to AB blood group incompatibility. Res Vet Sci 1983;35:145–152. 17. Bücheler J, Giger U. Alloantibodies against A and B blood types in cats. Vet Immunol Immunopathol 1993;38:283–295. 18. Giger U, Bücheler J. Transfusion of type-A and type-B blood to cats. J Am Vet Med Assoc 1991;198:411–418. 19. Giger U, Akol KG. Acute hemolytic transfusion reaction in an Abyssinian cat with blood type B. J Vet Intern Med 1990;4:315–316. 20. Hubler M, Kaeflin S, Hagen A. et al. Feline neonatal isoerythrolysis in two litters. J Small Anim Pract 1987;28:833–838. 21. Giger U. Feline neonatal isoerythrolysis: a major cause of the fading kitten syndrome, in Proceedings. 8th Am Coll Vet Intern Med Forum 1991;347–350. 22. Giger U, Casal ML. Feline colostrums—friend or foe: maternal antibodies in queens and kittens. J Reprod Fertil Suppl 1997;51:313–316. 23. Auer L, Bell K. The AB blood group system of cats. Anim Genet 1981;12:287–297. 24. Butler M, Andrews GA, Smith JE. Reactivity of lectins with feline erythrocytes. Comp Haematol Int 1991;1:217–219. 25. Kohn B, Niggemeyer A, Reitemeyer S. Blutgruppenbestimmung bei der Katze mit Hilfe einer neuen Testkartenmethode. Kleintierpraxis 1997;42:941–950. 26. Hitzler WE, Ulrich M, Daumer C, et al. The gel centrifugation test: comparison of antibody identification at 37oC with antibody identification at room temperature. Infusionsther Transfusionsmed 1993;20(suppl 2):61–63. 27. Bromilow IM, Adams KE, Hope J, et al. Evaluation of the ID-gel test for antibody screening and identification. Transfus Med 1991;1:159–161. 28. Kaoru A, Kyo K. Determination of canine and feline blood types using monoclonal antibodies. Provet 2001;10:12–16.
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