Ann X ls o \ C »n »¥ X l & LX] orX tory S¥ »T n¥T , vol. 30, no 4, 2000 379 R T v »T w: Cell Typing the Sensitized Transfusion-Dependent Patient Maria Rios, Kim Hue-Roye, Jill R. Storry, and Robert F. Reiss New York Blood Center, New York, New York Abstract. Extended red cell typing is required for the management of transfusion-dependent patients to confirm the identity of suspected alloantibodies or determine the specificity of potential additional antibodies that may be formed in the future. Typing may be complicated by the presence of circulating allogeneic cells or a positive direct antiglobulin test. Phenotyping such individuals by hemagglutination is dependent on the separation of a reticulocyte-enriched fraction by differential centrifugation. Flow cytometric typing of reticulocytes is also possible. The effectiveness of these techniques is limited in those who are heavily transfused or have low reticulocyte counts. Heavily transfused patients with sickle cell anemia may be typed, however, following hypotonic lysis of allogeneic cells. In patients with a positive direct antiglobulin test, sensitized cells are usually typed with either direct agglutinating antisera and/or IgG antisera following elution of the autoantibody. Inactivation of some antigens during the elution process or the lack of some antisera specificities limit such typing. By designing appropriate oligonucleotide primers, polymerase chain reaction (PCR) amplification of target gene sequences for most blood group systems and the identification of a large number of their allelic specificities is now possible. Peripheral blood leukocytes can be used as the DNA source. Restriction fragment length polymorphism determination is widely adopted for the identification of allelic specificity of the amplified target sequence. Alternate strategies, including allele-specific PCR, are often employed if the genetic basis of the polymorphism is more complex than a single nucleotide substitution, or if it does not create or ablate a restriction endonuclease cleavage site. These techniques may permit genotyping of sensitized transfusion-dependent patients, and can improve transfusion safety and efficacy. ( re ceived 2 7 A pril 2000, accepted 20 June 2000), Keywords: Red cell antigens, phenotyping, genotyping, polymerase chain reaction, transfusion Introduction The classical method of testing for blood group antigens and antibodies is hemagglutination. Hemagglutination is conceptually simple and easy to perform, does not require highly qualified technical skills or equipment, is inexpensive and, when done correctly, has a specificity and sensitivity that is appropriate for the clinical care of the vast majority of patients. In most cases, if antisera specificities are available, cell typing by hemagglutination is straight forward. On the other hand, performance of extended cell typing for transfusion-dependent patients who have Address correspondence to Robert F. Reiss, M .D., New York Blood Center, 310 East 67th Street, New York, NY 10021, USA; tel 212 570 3142; fax 212 570 3195; e-mail rreiss@ccmail.nybc.org. developed alio- or autoantibodies remains a difficult laboratory problem for the transfusion medicine specialist. Transfusion-dependent patients are defined as those who require transfusion on a consistent basis. Patients included in this category are those with thalassemia, selected patients with sickle cell anemia who are managed with chronic transfusion therapy, some patients with aplastic anemia and myelodysplasia who are ineligible for stem cell transplantation, and patients with autoimmune hemolytic anemia. Red cell typing is essential in sensitized patients to confirm the identity of suspected alloantibodies and to facilitate the identification of antibodies that may be formed in the future. Typing cells from patients who have been heavily transfused is complicated because it is difficult to distinguish between donor and patient cells. While it would be optimal to perform 0091-7370/00/0400-381 $2.00; © 2000 by the Association of Clinical Scientists, Inc.