# Evaluation of Canine Erythrocyte Surface Antigens and Morphological Alterations Induced by Trypsin Treatment

## TL;DR

This study finds that trypsin treatment has mixed effects on dog blood cell antigens, suggesting it's not reliable for making universal blood products.

## Contribution

The study reveals trypsin's variable effects on canine erythrocyte antigens, challenging its use for universal blood product development.

## Key findings

- Trypsin increased antigenicity of DEA 1 and DEA 4, increasing agglutination risk.
- Trypsin reduced antigenicity of Dal, but not consistently across all antigens.
- Morphological changes were minimal, but fewer echinocytes were observed in trypsin-treated samples.

## Abstract

Blood transfusions are essential for treating severe anemia and critically ill patients. However, limitations such as an insufficient supply of blood products and the risk of fatal transfusion reactions hinder their use in veterinary critical care. As a result, various techniques to eliminate or reduce the antigenicity of erythrocyte surface antigens have been investigated. In veterinary medicine, trypsin has previously been suggested to reduce the antigenicity of canine erythrocytes. However, in this report, trypsin produced variable results across three erythrocyte surface antigens. To develop universal blood products for dogs, antigen-specific enzymes are needed, rather than broad-spectrum proteases like trypsin. Therefore, a thorough understanding of the biochemical structures and interactions of these antigens should be a priority.

Dogs have multiple blood type antigens, among which DEA 1, DEA 4, and Dal can induce severe acute hemolytic transfusion reactions. Various antigen modulation techniques have been developed to reduce immunogenicity and transfusion reactions. Recently, trypsin has been suggested as a potential tool for modulating the antigenicity of DEA 1 in veterinary medicine. Following this rationale, this study aims to evaluate the effects of trypsin on the antigenicity of these three antigens. A 50% RBC suspension treated with 1 mg/mL trypsin was incubated at 37 °C for 120 min. The antigenicity of DEA 1, DEA 4, and Dal was assessed using blood typing assays before and after trypsin treatment. As a result, trypsin did not reduce the antigenicity of DEA 1 and DEA 4; instead, trypsin significantly increased their antigenicity (p = 0.008) and promoted agglutination, whereas Dal exhibited a significant reduction in antigenicity (p = 0.008). Quantitative morphological parameters obtained from an automated hematology analyzer revealed no significant differences between trypsin-treated and negative control groups. However, morphological scoring under an optical microscope showed significantly fewer echinocytes in the trypsin-treated group (p = 0.008). Consequently, broad-spectrum proteases like trypsin are unsuitable for universal blood production due to their variable effects on erythrocyte surface antigens.