Working Experience with Solid Phase Antibody Detection Technique

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1 Rounds [Blood Bank/Transfusion Medicine] Working Experience with Solid Phase Antibody Detection Technique Anjali A. Satoskar, MD, David Krugh, MT(ASCP)SBB, CLS(NCA), Melanie S. Kennedy, MD Department of Pathology, Division of Transfusion Medicine, The Ohio State University, Columbus, OH Solid Phase Antibody Detection Technique Antibody Detection Room Temperature Reactive Antibodies Immediate Spin Phase Antihuman Globulin Phase Newer, more sophisticated techniques have made their way into the clinical laboratories, and the blood bank is no exception. Cumbersome glass tubes have given way to microtesting using microtiter wells and solid phase technique for serologic antibody testing. The aim is to allow faster turn around time and batch testing which reduces labor costs, allow results to be read by more than 1 person, and reduce or eliminate insignificant room temperature reacting antibodies. Identification of room temperature antibodies can be extremely time consuming for the blood bank technologist, but ultimately not be clinically significant. However, when using newer serologic methods for antibody screening and identification, one must not just dwell on the strengths of the methods, but keep in mind the differences in methodology and resulting discrepancies. Clinical Background A 54-year-old male presented to the emergency department with dyspnea, hemoptysis, and fever (103 F). Chest radiograph showed bilateral infiltrates. The patient was known to have pulmonary hypertension and had been receiving epoprostenol (Flolan ) through a central venous catheter. He had been seen on several occasions at our medical center. His past medical history included essential thrombocythemia, resected pituitary adenoma, peripheral vascular disease, and splenectomy for sports-related injury. Within the past month, the patient had received multiple blood and blood components (9 units of red blood cells and 20 fresh frozen plasma). During the present admission, pneumonia or possibly central line infection was suspected. Blood cultures were sent. The patient was anemic, with hemoglobin level of 8.0 g/dl. A type and crossmatch for 2 units of red blood cells was ordered. Laboratory Findings: The patient s blood specimen typed as O Rh(D) negative (rr). He had a history of anti-d, which had been identified on an earlier admission. T1 shows a routine antibody screen using the patient s plasma and the Capture R 131

2 your lab focus Capture R Ready Screen T1 1 R1R Positive 2 R2R Positive Capture-R Ready ID T2 1 RzR Positive 2 R1R Positive 3 R2R Positive 4 Ror w Positive 5 r'r Weak Positive 6 r''r Negative 7 rr Negative 8 rrk Negative 9 rrfya Negative 10 rr Negative 11 rr Negative 12 rr Negative 13 R1R Positive Positive 3+ Positive Control 132 Ready Screen (Immucor M, Norcross, GA), a solid phase technique using microwell strips. This was followed by a Capture R Ready ID red cell panel for antibody identification [T2]. The patient s direct antiglobulin test (DAT) was negative with polyspecific antihuman globulin at both immediate spin and after 5 minutes incubation at room temperature. Using the manufacturer s antigram, we confirmed the presence of anti-d, using cells 1 through 4 and cell 13. The weak positive reaction with cell five (r r) raised the possibility of the presence of anti-c. However, anti-c was ruled out based on further serological workup. All other clinically significant alloantibodies were ruled out and the positive reaction with cell 5 was deemed an inconclusive reaction. We proceeded to a complete immediate spin anti-igg human globulin (polylaboratorymedicine> february 2002> number 2> volume 33

3 ethylene glycol) crossmatch to obtain compatible donor units. We chose three O Rh (D) negative red blood cell units, in keeping with the patient s blood type and respective alloantibody. The results are shown in T3. The crossmatch proved to be positive with 2 of the 3 red blood cell units tested at the immediate spin phase of testing. At this point, there are two critical items to consider. The weakly positive reaction seen on cell number 5 [T2] and the 2 positive crossmatches at immediate spin. Among the Rh(D) negative units of any ABO blood group type, the majority have an Rh genotype rr (cde/cde). They do not possess the C antigen. Hence, the positive agglutination reaction seen in T2 with cell 5 is unlikely to be caused by anti-c reacting at immediate spin phase. Anti-C, like other Rh antibodies, is typically a 37 C and antihuman globulin phase reactive antibody. More importantly, a cold reactive antibody to a commonly occurring red cell antigen is seen at the immediate spin phase of the crossmatch. A cold autoantibody can be ruled out because of the nonreactive autocontrol and the reaction pattern in the crossmatch (only 2 out of 3 positive crossmatches at the immediate spin phase of testing). A cold reacting alloantibody is likely, such anti-i and/or - H, but most probably anti-p1, -M, or -N. Settling The Issue With Conventional Tube Testing: A 3 cell immediate spin anti-igg human globulin (polyethylene glycol) antibody screen was completed [T4]. This pattern and the phase of reactivity pointed in the direction of more than 1 antibody specificity, one of which reacted at only the immediate spin phase and the other at only the anti-human globulin phase. It appeared that the known anti-d was reactive at anti-human globulin phase. The next step was to run an immediate spin and anti-igg human globulin (polyethylene glycol) antibody identification red cell panel [T5]. Using the manufacturer s antigram, anti-d was confirmed at the antihuman globulin phase of testing using cells 1 through 4 and 9 through 11. The pattern Crossmatches of positive reactions at the immediate spin phase was strongly suggestive of anti-m, reacting only at immediate spin. This was further confirmed by running additional rule-in and rule-out cells and antigen-typing the patient s red cells with anti-m reagent. The M antigen typing (Immucor ) clearly showed 2 cell populations (or a mixed field reaction), since the patient had been recently transfused. The two cell populations are as follows: M antigen positive transfused red blood cells, still circulating in the patient s blood from a previous transfusion, which resuspended as an agglutinated solid cell button, and M antigen negative patient s red blood cells, remaining as a residual ring of unagglutinated cells at the bottom of the tube. This is commonly referred to as the halo effect, in which a mixed field reaction can be seen macroscopically [F1]. your lab focus Donor Unit ABO/Rh(D)Type Results Interpretation Immediate Spin Antihuman globulin 1 O Rh(D) negative 0 0 Negative 2 O Rh(D) negative 3+ 0 Positive 3 O Rh(D) negative 3+ 0 Positive Gamma Biologicals Cell I R1R1 Cell II R2R2 Cell III rr Immediate spin Antihuman globulin Interpretation Positive Positive Positive T3 T4 Summary The patient had 2 alloantibody specificities in his plasma, the previously identified anti-d, and an unknown anti-m. Anti-M is a relatively common antibody reacting optimally at 4 C, and weakly or not at all at 37 C. 2,3,6 Anti-M reacts at the immediate spin phase of serological testing, and sometimes, only at this phase. 6,7 However, the solid phase Capture R Ready Screen does not detect antibodies at this phase. It only detects antibodies at the antiglobulin phase after 37 C incubation. Hence the presence of anti-m was undetected, until unmasked by an immediate spin and anti-igg human globulin (polyethylene glycol) crossmatch. The immediate spin testing in the major crossmatch involves mixing donor red blood cells and patient s plasma or serum at room temperature and reading immediately after centrifugation. The main purpose of the crossmatch is to rule out ABO incompatibility, the most serious cause of acute hemolytic transfusion reactions. The Standards of the American Association of Blood Banks 1 require crossmatch methods to demonstrate ABO incompatibility and clinically significant unexpected antibodies. If the patient has no clinically significant unexpected antibodies, only the test phase that demonstrates ABO incompatibility is required. Immediate spin crossmatch is required, unless additional steps are taken to allow an electronic crossmatch and verification of ABO. Since this patient did have a known anti-d, immediate spin anti-igg human globulin (polyethylene glycol), crossmatches were performed using D antigen negative red blood cell units. The crossmatches were positive with 2 of the 3 red blood cell units tested at only the immediate spin phase of testing. ABO typing of the patient and red blood cell units were checked and incompatibility 133

4 your lab focus Immucor Panocell I.S AHG T5 1 R1R Positive 2 R1R Positive 3 R2R Positive 4 Ror Positive 5 r'r Negative 6 r''r Positive 7 rrk Positive 8 rrfya Negative 9 R1R Positive 10 R2R Positive 11 R1R Positive Auto 0 0 Negative Control 134 was ruled out. At this point, a room temperature reactive antibody is a strong possibility. The solid phase antibody screen and red cell panel do not have an immediate spin reading; therefore, room temperature reactive antibodies will not be detected. Thus, crossmatching may prove to be the first detection point for cold reacting antibodies. Principle Of Solid Phase Serological Test Solid phase technique is based on the procedures of Plapp et al and Juji et al. 4,5 Polystyrene microtitration strip wells with bound and dried red blood cell membranes are used for serological testing. The membrane antigens serve to capture red cell specific IgG antibodies from patient or donor sera. Following a brief incubation period, unbound residual immunoglobulins are rinsed from the wells and a suspension of indicator red blood cells, coated with anti-human IgG, is then added. Centrifugation brings the indicator red blood cells to the bottom of the microwells. However, this is impeded if the patient or donor serum has IgG antibodies bound to the red cell membranes because anti-igg-igg complexes are formed on the surface of the immobilized reagent layer. This indicates a positive test result. As a consequence of antibody bridging, the indicator red blood cells adhere to the screening red blood cells as a second immobilized layer. In the absence of detectable antigen-antibody interactions, the indicator red blood cells will not be impeded during their migration and will pellet to the bottom of the wells as tightly agglutinated red blood cell buttons. This indicates a negative test result. The pattern of positive reactions obtained with Capture- R Ready Screen and Capture-R Ready-ID are compared with the antigen profile master lists provided with each product to identify the specificity(ies) of the antibody(ies) present. Anti-M Anti-M is a relatively common antibody reacting optimally at 4 C and weakly or not at all at 37 C. It is usually assumed to be naturally occurring and to consist of IgM; however, many have an IgG component. 2,3,6 Anti-M can also occur as a room temperature reactive alloantibody in M antigen negative individuals transfused with M antigen positive red blood cells. These antibodies do not bind complement. It has been suggested that such antibodies are not clinically significant when

5 your lab focus A B [F1] A. Mixed field agglutination giving the halo effect in patient s sample. Agglutinated cell button (thick arrow) of M-antigen positive transfused red cells and a rim of unagglutinated M-antigen negative patient s cells (thin arrow). B. For comparison, an agglutinated cell button of a single population of antigen positive red cells. No mixed field reaction seen. they do not react at temperatures greater than 30 C. 7 Several reports, however, describe anti-m causing a delayed hemolytic transfusion reaction (DHTR). 7,9,10 Conversely, other studies correlated the survival of 51 Cr-labelled M antigen positive and M antigen negative red blood cell units in vivo after transfusion therapy 8 with in vitro studies of monocyte-macrophage phagocytosis of red blood cells sensitized with the patient s anti-m. 9 This has been studied at hypothermic conditions. These studies, as well as Issitt, 6 suggest that ignoring room temperature reactive anti-m in red cell transfusion therapy does not result in an immediate or delayed hemolytic transfusion reaction. However, the presence of an IgG component and high thermal amplitude of anti-m cannot be ignored and requires crossmatch compatible or M-antigen negative red blood cells (if reacting weakly at 37 C or antihuman globulin phase of testing) to be transfused to the recipient. Conclusion A higher potential for discrepancies between antibody screen and crossmatch results may occur based on different techniques being used. The phase of testing incorporated in a particular serologic method of antibody screen must be kept in mind when solving such serological discrepancies, particularly with incompatible immediate spin crossmatches. 1. Meyer EA, Shulman IA. The Sensitivity and Specificity of the Immediate Spin Crossmatch. Transfusion. 1989;29: AABB Technical Manual, 13th ed. Bethesda, MD: American Association of Blood Banks, 1999; Harmening, DM. Modern Blood Banking and Transfusion Practices, 4th ed. Philadelphia, PA: F.A. Davis Company, 1999; Plapp FV, Sinor LT, Rachel JM, et al. A Solid Phase Antibody Screen. Am J Clin Pathol. 1984;82: Juji T, Kano K, Milgrom F. Mixed Agglutination With Platelets. Int Arch Allergy. 1972;42: Issitt PD. Applied Blood Group Serology. 3rd Edition. Miami, FL: Montgomery Scientific, 1985; Sancho JM, Soler M, Manzano P, et al. Delayed Hemolytic Transfusion Reaction Due to Anti-M Antibody. British Journal of Hematology. 1998;103: Kurtz SR, Ouellet R, Valeri CR, et al. Survival of MM Red Cells During Hypothermia in Two Patients with Anti-M. Transfusion. 1983;23: Alperin JB, Riglin H, Petz LD, et al. Anti-M Causing Delayed Transfusion Reaction. Transfusion. 1983;23: Furlong MB, Monaghan WP. Delayed Hemolytic Episodes Due to Anti-M. Transfusion. 1981;21: Maffei LM, Shulman IA, Steiner EA et al. Survey on Pretransfusion Testing. Transfusion. 1992;32:

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