Loss of red cell alloantibodies over time

The loss of red cell alloantibodies over time was analyzed in 160 patients with 209 antibodies retested 1 to 60 months after initial identification. The mean follow-up consisted of 3.6 specimens taken over 18 months. Twenty-nine percent of clinically significant and 72 percent of clinically insignificant antibodies were not detected on at least one follow-up screening. Anti-Jka and anti-C were lost in 59 and 45 percent of cases, respectively. No significant differences in overall antibody loss were found according to sex, diagnosis, or the presence of multiple antibodies. Patients under 20 years of age may be more likely to lose significant antibodies. Pretransfusion review of previous records is vital for the prevention of delayed hemolytic transfusion reactions, because of the high number of clinically significant antibodies that are undetected in subsequent routine screening.     

Long-term follow-up testing of red cell alloantibodies

BACKGROUND: In previous studies, 29 to 34 percent of potentially hemolytic red cell antibodies were not detected after short-term follow- up. STUDY DESIGN AND METHODS: To examine long-term detection, records were reviewed for 44 consecutive patients who were tested more than 5 years after their potentially hemolytic red cell antibodies were first identified in this hospital. RESULTS: After 5 to 10 years, 14 (39%) of 36 Rh, Kell, and Duffy system antibodies were not detected on at least one occasion. Twenty-two other such antibodies were sought again after more than 10 years; 10 (45%) were not detected. When restimulation by pregnancy was excluded, these rates were 42 and 48 percent, respectively. CONCLUSION: Clinically significant red cell antibody formation is probably more common than previously realized, because nearly half of these antibodies are undetected after long-term follow- up.     

Accelerated destruction of radiolabeled red cells due to anti-Coltonb

A patient who developed anti-Cob in response to transfusion was studied. The antibody was a warm-reactive, high-titered, IgG alloantibody that did not fix complement, and reacted strongly in the antiglobulin phase. During a period of transfusion the patient developed a positive direct immunoglobulin test with anti-Cob recoverable in the eluate. Reactions were stronger with enzyme-treated red cells. Survival studies with 51Chromium-labeled red cells showed: 1) normal survival of Co(b-) red cells, and 2) accelerated destruction of Co(b+) red cells; initially, cells were destroyed with a one-half disappearance time of 4 days, but after about 4 days, the rate of destruction increased. This study, together with reported suspected transfusion reactions attributed to anti-Cob, suggest that anti-Cob should be considered a clinically significant antibody.     

Induction of additional red cell alloantibodies after intrauterine transfusions

BACKGROUND: The aim of this study was to determine the frequency and origin of additional alloantibodies directed against red cells (RBCs) after intrauterine transfusion (IUT). STUDY DESIGN AND METHODS: Between March 1987 and December 1992, fetuses with severe hemolytic disease (n = 91) received a total of 280 ultrasound-guided IUTs of RBCs from unrelated donors. The specificity of alloantibodies to RBCs in maternal serum was determined both before and after each IUT. If additional alloantibodies directed against RBCs were detected, their origin was determined by phenotyping the fetal, donor, and paternal RBCs for each particular antigen. The study included a control group of 69 pregnant women who underwent either amniocentesis or fetal blood sampling. RESULTS: Production of additional alloantibodies directed against RBC antigens was detected in 24 women (26%). The source of the immunizing antigen, either donor or fetus, was identified in 14 patients. The additional alloantibodies were directed against fetal antigens in 11 women and against donor antigens in 3. One additional alloantibody directed against donor antigen clearly reduced the survival of donor RBCs. The fetus and the donor shared the immunizing antigen in four cases, and in one case, the antibody occurred naturally. In five cases, the source of the immunizing antigen was not determined. In the control group, additional antibodies were detected in two patients. CONCLUSION: IUT therapy is associated with a high incidence of additional alloantibodies. In the majority of patients, the use of maternal RBCs for IUT would not have prevented additional formation of alloantibody to RBCs.     

In vivo studies of the long-term 51Cr red cell survival of serologically incompatible red cell units

The long-term survival of serologically incompatible red cell units was measured in five patients with antibodies to high-frequency antigens. Initially, the survival of 1 ml of 51Cr-labeled incompatible red cells was measured over 1 hour. After demonstrating that the 1-hour survival times were successful (greater than 70%), each patient then received 5 ml of the same 51Cr-labeled red cells followed by the transfusion of the remainder of the red cell unit. The long-term T 1/2Cr survival for each case was patient 1 (anti-McCa), 15 days; patient 2 (anti-JMH), 12 days; patient 3 (anti-Kna), 31 days; patient 4 (anti-McCa), 12 days; and patient 5 (anti-Hya), 14 days. Each antibody tested in an in vitro homologous macrophage assay showed less than 5 percent phagocytosis. Anti-JMH was the only antibody to react with IgG subclass antisera and was determined to be IgG4. The macrophage assay, IgG subclass testing, and short-term (1 hour, 1 ml) 51Cr survival studies all indicated that the short-term survival was good. However, only the measurement of long-term survival with transfused units of serologically incompatible red cells was able to determine the actual survival, and "clinical significance" of the alloantibodies. Determining the actual long-term survival by the method described here can be of importance for patients requiring chronic red cell transfusion.     

Lack of clinical significance of "enzyme-only" red cell alloantibodies

In a retrospective study on samples from 10,000 recently transfused patients, 35 samples were found to contain an antibody that reacted with ficin-treated red cells but was not demonstrable by low-ionic- strength saline solution and indirect antiglobulin test (LISS-IAT). In those 35 patients, the specificity of the antibody was such that each patient would have been transfused with antigen-negative blood had the antibody reacted in LISS-IAT. Tests on red cells from the units already transfused showed that 19 patients had among them received, by chance, 32 antigen-positive and 74 antigen-negative units. The remaining 16 patients had among them received 57 units that were, again by chance, all antigen negative. One patient given antigen-positive blood suffered a delayed transfusion reaction; in two others the antibodies became LISS-IAT active after transfusion. However, similar changes to the LISS- IAT-active state were seen with two antibodies of patients given only antigen-negative blood. Also found in the 10,000 patients were 28 clinically insignificant antibodies, 77 sera in which the antibody was too weak to identify, and 216 autoantibodies that reacted only with ficin-treated red cells. These data support a belief, generally held in the United States but not necessarily elsewhere, that the use of protease-treated red cells for routine pretransfusion tests creates far more work than the accrued benefits justify.     

Predicting the clinical significance of red cell alloantibodies using a monocyte monolayer assay

Few data have been published that correlate in vitro monocyte monolayer assays (MMA) and red cell (RBC) survival in patients with alloantibodies of unknown significance. Over the past 6 years we gathered clinical correlations in 12 patients with the following antibodies: anti-Lan (three patients), -Ge (three patients), -Yta (five patients), and -Ytb (one patient). RBC survival was estimated using 51Cr studies in seven patients and follow-up of transfusion of incompatible blood in the other five. Six patients with no evidence of RBC destruction had negative MMA findings (anti-Lan [one patient], -Ge [two patients], and -Yta [three patients]). Five patients with evidence of in vivo RBC destruction had significant MMA results. The two clinically significant anti-Lans required fresh serum to give a meaningful MMA result. One patient (anti-Ytb) had an MMA result of borderline significance–normal 51Cr RBC survival at 1 hour–but a reduced T50Cr. The MMA we used appeared to predict the clinical outcome of transfusion in every patient with antibodies to high-frequency antigens whom we tested.     

The James Blundell Award Lecture 2007: Do we really understand immune red cell destruction?

summary We have learned a great deal about immune red blood cell (RBC) destruction since the elaboration of biochemical/immunological interactions of antibodies, complement and macrophages during the past 50 years. We first learned about the direct lysis of RBCs involving complement. We then learned of the role of the macrophage (particularly in the spleen and the liver) in initiating phagocytosis and antibody‐dependent cytotoxicity of antibody‐coated RBCs. Later, as the complexities of the human complement system were unravelled, we learned that complement‐coated RBCs that were not directly haemolysed could interact with macrophages and that specific complement molecules on the RBC membrane could lead to a phagocytic event or the RBC (although heavily coated with complement) could survive normally. The application of isotope‐labelling procedures (e.g. ⁵¹Cr) for RBC survival (starting in the 1950s) advanced our knowledge considerably. Advances in knowledge in immunology helped us understand the complexity of the immunoglobulins (e.g. subclasses) and the specific receptors on macrophages and their role in immune haemolysis. Nevertheless, after more than 30 years researching this area, I am sometimes embarrassed to realize how much I cannot explain. Why do some patients have severe haemolytic transfusion reactions because of antibodies that are only detectable by one technique or not detectable by any? How do we explain autoimmune haemolytic anaemia with negative direct antiglobulin tests (DATs)? Why do RBCs strongly coated with immunoglobulin (Ig)G1 or IgG3 sometimes have normal survival? Are cells, other than macrophages, involved in immune RBC destruction? Could the relative amount of cytotoxicity vs. phagocytosis explain different clinical findings and response to treatment? How do we explain ‘hyperhaemolysis’ in sickle cell disease? Could novel mechanisms involving IgG glycosylation, CD47, ‘armed’ macrophages, bystander lysis, antibody activated reactive oxygen species, natural killer cells or antibody perturbation of RBC membrane be involved? Why do RBCs die after circulating for 100–120 days in healthy individuals? How should we define a ‘clinically significant’ antibody; how do we evaluate this? So many questions, so little time!     

Multiple or uncommon red cell alloantibodies in women: association with autoimmune disease

BACKGROUND: Patients with multiple or uncommon red cell (RBC) alloantibodies require special efforts in the blood bank. This study investigated whether such persons had other immune-related conditions that might help to explain or predict their propensity for RBC antibody formation. STUDY DESIGN AND METHODS: Charts were retrospectively reviewed of 29 men and 83 women with multiple (> or = 3) RBC antibodies of potential clinical significance, uncommon RBC antibodies (anti-e, - Kpb, -Jkb, -Fyb, -S, -U, -Yta, -Dib, -Ata), or both. The clinical features in 43 women with multiple antibodies were compared to those in two equal-sized control cohorts of women matched for transfusion- related diagnoses, but having either one RBC antibody or none. RESULTS: Women with uncommon RBC antibodies had a 33-percent (18/54) prevalence of autoimmune disease. Twenty-eight percent of the 43 women with multiple antibodies had autoimmune disease, compared to 14 percent of women in the cohort with one RBC antibody (p = 0.09) and 7 percent of those in the cohort without RBC antibodies (p = 0.01). Only one of the 29 men had autoimmune disease. CONCLUSION: Autoimmune disease is a common underlying factor in women who make multiple or uncommon RBC alloantibodies of potential clinical significance.     

In vivo red cell destruction by anti-Lu6

An example is presented of an IgG1, anti-Lu6, that reacted by indirect antiglobulin test and was capable of destroying antigen-positive red cells in vivo. Two methods for the measurement of red cell survival, 51Cr labeling and flow cytometry, gave the same result: 20 percent of the test dose of Lu:6 red cells was destroyed in the first hour after injection and 80 percent in the first 24 hours. The clinical relevance of the antibody was correctly predicted by an in vitro monocyte monolayer assay. The finding that this example of anti-Lu6 was clinically significant should not be taken to mean that all antibodies directed against high-incidence Lutheran and Lutheran system-related antigens will behave similarly. When such antibodies are encountered, in vivo and/or in vitro studies to assess their clinical significance are necessary before rare blood is used for transfusion.     

Comparison of the performance of four microtube column agglutination systems in the detection of red cell alloantibodies

BACKGROUND: The purpose of this study was to compare the performance of four currently available microtube column agglutination systems in the detection of red cell alloantibodies to that of the standard tube low-ionic-strength solution (LISS) indirect antiglobulin test (IAT) (tube LISS-IAT). STUDY DESIGN AND METHODS: In a comparative study, 172 sera, previously demonstrated to contain red cell alloantibodies, were tested in parallel by the tube LISS-IAT and three microtube column agglutination techniques (DiaMed-ID, Ortho BioVue, and Sanofi-Pasteur Scangel) and one affinity-adherence test system (Gamma ReACT). Tests were performed simultaneously by a single person on freshly thawed sera that had been frozen at -20 degrees C. RESULTS: The rate of detection of clinically significant alloantibodies (n = 154) in microtube column systems was very similar. One hundred forty-one sera (91.6%) reacted in the DiaMed-ID, 139 (90.3%) in the ReACT, 139 (90.3%) in the BioVue, and 142 (92.2%) in the Scangel. Only 117 (76.0%) of these sera reacted in the tube LISS-IAT. The detection rates for 18 antibodies of minor clinical significance (anti-M, -N, -P1, -Le(a), and -Le(b)) varied among the test systems: DiaMed-ID, 5 (28%); ReACT, 7 (39%); BioVue, 14 (78%); Scangel, 10 (56%); and tube LISS-IAT, 6 (33%). Antibody reactivity as determined by titer and score was very similar in all microtube column systems and higher in these systems than in the tube LISS-IAT. CONCLUSION: The sensitivity of all four microtube column systems in the detection of clinically significant red cell alloantibodies was similar and was markedly superior to that of the tube LISS-IAT. An individual cost-benefit analysis should be performed in every institution to decide whether a microtube column system should be applied. If so, the antibody screen in the microtube column agglutination system should ideally be performed in advance of the crossmatch to provide time to screen for compatible units.