Letter regarding “Validation of a cage-side agglutination card for <i>Dal</i> blood typing in dogs” – Validation of agglutination card kit or simple comparison to gel column method for canine <i>Dal</i> blood typing?

Drs. Verón and Blais from Montreal University recently compared typing results of blood samples from 150 anemic and nonanemic dogs1 that were obtained with a novel commercial Dal typing agglutination card kit (DMS Laboratories Inc., Flemington, NJ) and a previously reported laboratory gel column method.2 To further evaluate the ability of the card kit to assess Dal positivity in anemic dogs, they diluted 3 normal samples with their plasma. We would like to comment on requirements for method validation as well as practical aspects of Dal blood typing. In human and veterinary medicine, the process of validating diagnostic kits and laboratory methods is well established, and guidelines have been published.3-6 Validation of a new method requires comparing the expected error using the new method, such as an in-clinic typing kit, with a validated reference laboratory method. Validation studies include establishment of accuracy, confidence intervals, sensitivity and specificity, positive (PPV) and negative (NPV) predictive values, which take into account the expected prevalence of the condition (Dal+ and Dal− dogs in specific breeds), as well as imprecision, repeatability, possible interferents (e.g., autoagglutination, anemia), total error, effectiveness of quality control in detecting errors, and ease of performing the test. Pending manufacturer instructions, the sample requirements (e.g., acceptable anticoagulant, hematocrit range, storage time of samples), ease and adequacy of standard operating procedure, accuracy of testing based upon method, repeatability of results over time, required operator training, variation in interpretation of test results by untrained and trained operators, and several statistical analyses should be included. Unfortunately, these authors did not perform the required experiments to claim validation of the new Dal typing kit nor did they use a validated laboratory gel column typing method for comparison. Instead, they simply compared 2 methods by testing each clinical sample once using the kit and once using the laboratory method. Results were interpreted by 2 individuals. During validation, assessment of sensitivity and specificity as well as PPV and NPV should be restricted to samples in specified acceptable ranges. Based upon the manufacturer's kit inserts, this Dal and other card typing kits for dogs and cats have already been validated, albeit not in peer-reviewed publications. The titer and affinity of different batches of polyclonal antibody-containing sera collected from sensitized dogs at different times can vary. Additional studies carried out at a different department at Montreal University characterized a monoclonal anti-Dal antibody in 2020. Because monoclonal antibodies are more easily standardized, its use would have been ideal for comparison with the polyclonal alloantibodies and would likely better reflect a gold standard method.7 For optimal performance of the gel column test to be considered a gold standard, the antibody (sera) should be in and not only on top of the gel. Furthermore, centrifugation at 1000g for 10 minutes seems too harsh. The positive control wells on the typing card kits contain a likely monoclonal antipanerythrocytic antibody, and thus may not reflect the stability and degree of agglutination reaction to the polyclonal anti-Dal alloantibody used. Blood typing card kits against DEA 1 and AB (ABC) have been commercially available for nearly 30 years. Indeed, the commercially available veterinary blood typing kits would be better called point-of-care, rather than “cage-side” or “bedside” tests, because they require a laboratory protocol and bench, as well as expertise. As for any immunological assay, excessive red blood cells (RBCs) or antibody concentrations hamper agglutination reactions, and, therefore, the ratio of RBCs to antibody reagent must be optimized. Here, these authors used clinical samples with a hematocrit range of 5% to 70% whereas their reference method uses washed RBCs at a very specific dilute concentration (0.8% RBC suspension). The comparison of the laboratory gel test with a fixed hematocrit to the card kit using samples with highly varied hematocrit appears inappropriate. These authors excluded an unknown number of autoagglutinating blood samples, because they could result in false Dal+ results. Assessment of autoagglutination before clinical application of agglutination-based tests, be they typing, crossmatching, or direct antiglobulin (Coombs') testing, is always recommended for proper interpretation of results. However, ×3 washing of EDTA blood with saline most often will remove any autoagglutination, thereby improving performance of any agglutination-based tests, including the Dal card typing kit. Surveys from North America and Europe have discovered Dal− dogs in a few breeds. Thus, currently commercially available Dal typing is recommended in Lhasa Apsos, Cane Corsos, Dalmatians, Dobermann Pinschers, and Shih Tzus, as well as for blood donors or anemic patients (hematocrit adjusted before typing).2 Likewise, in the presence of an incompatible major crossmatch of a previously transfused dog, Dal typing may identify the cause of the incompatibility and thereby allow targeted searching for a compatible Dal− donor (e.g., littermate). A first delayed hemolytic transfusion reaction in a Dal− Lhasa Apso that accidently received Dal+ blood was recently reported (Walton J, Euler CC, Tappin S, Hale AS, Giger U. Dal-blood type in breeds beyond Dalmatians: A challenge in a previously transfused Lhasa Apso. Abstract BSAVA Congress proceedings; 2022). Prior surveys gave generally strong agglutination reactions for Dal typing, whereas these authors' results had, in part, weaker card and gel column agglutination reactions. This may be related to either the method (quantity of anti-Dal antibody) or the operator or could instead indicate varied Dal antigen expression, as reported for DEA 1.2, 8 Apparently weaker reactions were seen in breeds with a higher prevalence of Dal− dogs, suggesting that Dal antigen concentrations may be lower in some Dal+ dogs in these breeds. Clinically, the weaker agglutination reactions may affect interpretation and thus classification into Dal+ and Dal− dogs. The possibility of variable Dal expression indicates that further testing near the cutoff concentration triggering agglutination should be investigated as part of the validation process. In their comparative study, these authors only performed each test once for each blood sample and had 2 trained individuals visually interpreting the same test. Repeatability of performing and evaluating the tests within and between operators should be investigated. Furthermore, interpretation of results between evaluators occurred between 0.5 and 2 min, during which time the degree of agglutination is known to vary. In conclusion, a clinical comparison of 2 methods should not be confused with a formal validation study. If using nonautoagglutinating (or washed RBCs) blood samples from dogs that are not severely anemic (or adjusted to an appropriate hematocrit), the currently commercially available Dal typing card kit appears to accurately determine the Dal blood type. Therefore, it can be recommended for use in breeds where Dal− dogs have been recognized. Dr. Urs Giger has been a scientific advisor to various companies involved in blood typing and compatibility testing including Alvedia, DMS Laboratories Inc., and some lectures on veterinary transfusion medicine at conferences have been supported by companies.

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