Quantitative Differential Agglutination Method Using the Coulter Counter to Measure Survival of Compatible but Identifiable Red Blood Cells

Abstract

The method described here using a centrifuge and Coulter Cell Counter for the quantitative differential agglutination of human red cells uses commercial anti-A and anti-B antisera for the ABO system, and for the Rh system a commercial anti-D serum, a low ionic strength solution and an anti-human IgG antiserum. We compared this Coulter Counter method with the Technicon Auto Analyzer method which utilizes bromelin and polyvinyl pyrrolidone, and anti-A, anti-B and anti-CD antisera, and found this new method to be the simpler of the two. The nonagglutinable count with the Coulter Counter was 1.07% for A(1) red cells, 2.26% for A(2) red cells, 1.06% for B red cells, and 1.78% for Rh-positive red cells, results similar to those seen with the Technicon Auto Analyzer. Results with the Coulter Counter method were consistently accurate whether the ACD red cells were studied on the day of collection, after 10 days of 4 °C storage, or after 4 °C storage for up to 6 days followed by cryopreservation with 40% (w/v) glycerol at -80 °C, thawing and washing. In this study, red cell samples obtained from recipients who had received compatible but identifiable donor red cells were frozen with 40% glycerol and stored at -80 °C for 10 months, thawed and washed. Survival measurements on these washed previously frozen red cells were similar to the values in liquid-stored red cells.

Characterization of reemergent anti‐B red blood cell antibodies in a patient with recurrent acute myeloid leukemia with ABO‐incompatible allogeneic peripheral blood stem cell transplantation

“…The RBC ABO chimerism was estimated by the quantitative differential agglutination method using a centrifuge and Coulter cell counter, as previously described. 2 Her haptoglobin, lactate dehydrogenase, and reticulocyte levels gradually recovered. The direct antiglobulin test with monoclonal antibodies against human immunoglobulins and Complement 4 was negative.…”

“…On Day 120, the RBC ABO grouping phenotype showed 97% B and 3% O, and in the serum ABO grouping the anti‐A antibodies were persistent (4+), but the anti‐B antibodies disappeared (Table ). The RBC ABO chimerism was estimated by the quantitative differential agglutination method using a centrifuge and Coulter cell counter, as previously described . Her haptoglobin, lactate dehydrogenase, and reticulocyte levels gradually recovered.…”

Blood doping in athletes—Detection of allogeneic blood transfusions by flow cytofluorometry

“…To detect allogeneic transfusions, currently available methods are based on the antigenic differences between the recipient's RBCs and the donor's RBCs. Originally, serological techniques were performed, such as detection of ''mixed field'' reactions when performing antigen typing and differential agglutination where percentages of agglutinated and unagglutinated RBCs were determined manually or by automated methods [9][10][11][12][13]. More sensitive quantitative techniques to characterize transfused RBCs are the enzyme-linked antiglobulin test [14] and flow cytofluorometry [15][16][17][18][19][20][21][22][23].…”

Detection and quantitation of ABO RBC chimerism by a modified coil planet centrifuge method

“…Differential agglutination methods have been applied to detect and quantitate agglutinated and nonagglutinated RBCs in a mixed cell population 1‐5 . The first differential agglutination procedure was introduced by Ashby 1 by using anti‐A, anti‐B, and anti‐H and microscopic counting of agglutinated and nonagglutinated RBCs, and later, this method was modified by Beattie, 2 where RBCs were mixed in a Petri dish and the agglutinates were removed from the nonagglutinated RBCs by pipette.…”

“…However, their methods were manual and very time consuming. Automated differential agglutination methods were introduced by Szymanski et al 3 with an analyzer (Auto‐Analyzer, Technicon Corporation, Research Park, Ardsley, NY) and by Valeri et al 4 with a centrifuge and a counter (Coulter Counter ZB, Coulter Electronics, Hialeah, FL). More recently, a method that uses computer image processing has been reported 5 .…”