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.     

Accelerated immune red cell destruction in the absence of serologically detectable alloantibodies

Two patients are described in whom clinically significant red blood cell alloantibodies could be demonstrated only by in vivo 51chromium (51Cr) survival studies. The first patient had experienced a severe delayed hemolytic transfusion reaction to four units of crossmatch compatible blood. Serial phenotype studies suggested the presence of a serologically undetectable anti-c (hr') antibody. 51Cr survival of c- positive red blood cells was one per cent at 24 hours, while survival of c-negative red blood cells was 80 per cent at 24 hours. The second patient had multiple red blood cell alloantibodies. An anti-c antibody was suspected but could not be convincingly demonstrated by in vitro techniques. 51Cr survival of c-positive red blood cells, however, was 57 per cent at 24 hours and 17 per cent at 48 hours. 51Chromium red blood cell survival studies should be considered whenever an unexplained hemolytic transfusion reaction occurs, or when an expected red blood cell alloantibody cannot be demonstrated by in vitro laboratory studies.     

Standardization of IgG subclass antiserums for use with sensitized red cells

The only IgG subclass antiserums standardized for use with red cells are those sold by the Netherlands Red Cross. Their recommended method is a sedimentation antiglobulin test (AGT) not generally used in the USA. Some years ago we tested the antiserums for sensitivity and specificity using the routine centrifugation AGT and found the methods comparable. More recently, we have become concerned since the antiserums are being used by a variety of methods other than the recommended procedure. It is well-known that it is difficult to prepare subclass antiserums without contaminating antibodies to other IgG markers. Thus, we thought it worthwhile to standardize the antiserums with particular emphasis on specificity using several different technics. We found that some methods that have been used to define the subclass of red cell-bound IgG were inappropriate, and led to many false positive results.     

红细胞血型系统IgG型不规则抗体固相凝集法检测试剂盒的研发

目的研发基于固相凝集技术的红细胞血型系统IgG型不规则抗体检测试剂盒并评估其性能。方法采用单克隆抗人-RBC抗体包被96孔微板,按100μL/孔加入红细胞悬液形成细胞单层,经过裂解后形成红细胞膜抗原分子单层,加入保护液冷冻干燥后制备成检测用的反应微板;用梯度倍比稀释的多克隆羊抗球蛋白试剂(IgG+C3d/4)与IgG抗-D致敏的O型红细胞反应,筛选构建最佳指示系统;用不同批次的试剂盒检测低效价的IgG抗-D和抗-E,评估试剂盒在保存期内的抗原稳定性;与微柱凝胶抗球蛋白卡、进口同类试剂盒检测不同效价的抗体以测试3者的灵敏度;与进口同类试剂盒对350例血样标本进行检测,以明确2者的检测效能是否一致。结果 20μg/mL的抗人-RBC抗体溶液能够很好的固定红细胞膜抗原分子单层;稀释16倍的多克隆羊抗球蛋白试剂与致敏红细胞构建的指示系统指示效果最佳;试剂盒中冻干的红细胞膜抗原在6个月的保存期内保持稳定;本研究的试剂盒检测灵敏度高于Capture-R Ready Screen试剂盒和抗球蛋白卡;试剂盒与Capture-R Ready Screen对血样标本检测的阳性一致性百分率为98.0%,阴性一致性百分率为99.66%,总一致性百分率为99.43%。结论成功研发了基于固相凝集技术的用于红细胞IgG型同种免疫性不规则抗体检测的试剂盒,该试剂盒具有灵敏度好、保存期长、性能稳定等优势,与进口同类试剂盒等效。     

Coexistence of autoantibodies and alloantibodies to red blood cells due to blood transfusion

BACKGROUND: Autoantibodies (AUTO) to red blood cells (RBCs) are frequently associated with alloantibodies (ALLO). The mechanism for the coexistence of these antibodies is obscure. STUDY DESIGN AND METHODS: Between August 1998 and June 2006, all in- and outpatients of the Charité University Hospital, Berlin, with detectable AUTO were included in this study. Serologic examination was performed with standard techniques for the detection of RBC antibodies. RESULTS: A total of 717 patients were found to have AUTO, with ALLO observed in 200 of these patients (28%). The history of antibody production could be evaluated in 98 of the 200 patients. Both AUTO and ALLO were due to RBC transfusion in 73 cases (75%) and peripheral blood progenitor cell transplantation in 6 cases (6%). Nine (9%) patients were primarily alloimmunized and subsequently developed AUTO. The remaining 10 (10%) patients were primarily autoimmunized and later developed ALLO. Only 6 of these patients had chronic idiopathic autoimmune hemolytic anemia. CONCLUSION: The majority of AUTO associated with ALLO appears to be due to RBC transfusion that must be recognized as a major cause for autoimmunization.     

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.     

流式细胞术检测抗体致敏红细胞方法的建立及初步应用

目的　建立应用流式细胞术 (FCM)测定被抗体致敏红细胞的方法。方法　用荧光素标记的绵羊抗人免疫球蛋白抗体 ,与致敏红细胞上的免疫球蛋白结合 ,以绵羊抗鼠IgG RITC标记的红细胞作同形对照 ,设定mark ,得测定管阳性细胞百分数。结果　Coombs试验在对比试验中 ,只能测出经 2 7稀释的抗D血清所致敏的红细胞 ,而FCM则能测出经 2 9稀释的抗D血清所致敏的红细胞。实际应用中 ,15例ABO新生儿溶血病标本 ,Coombs阳性率13 3% ,FCM阳性率 86 7% ,5例Rh新生儿溶血病标本 ,Coombs阳性率 4 0 % ,FCM为 10 0 %。结论　FCM技术比传统Coombs试验灵敏度高 ,定量更准确     

红细胞的低温冷冻保存及临床意义

血液的冷冻保存研究始于20世纪40年代。1949年Polge和Smith等发现甘油对牛精子的冷冻保存有保护作用，1950年Smith应用甘油作保护剂冷冻红细胞获得成功，但未能解决去除保护剂的问题，1963年Huggins发现糖溶液可洗涤去除冷冻保护剂一甘油，从而使冷冻红细胞长期保存并成功地     

B-lymphocytes activation by the Fc region of IgG

Strong stimulation of DNA synthesis (up to 150-fold) and blast transformation can be induced in mouse spleen cells by Fc fragments of human IgG. The mitogenic response is optimal on day 5 of culture and is dependent on the concentration of Fc fragments with a sedimentation rate of 3-5S. Intact IgG is also stimulatory, but only when modified by heat aggregation, and produces only a 10-fold increase in [3H]thymidine uptake. The stimulation by aggregated IgG is dependent on the Fc portion, since aggregated (or soluble) Fab or F(ab')2 fragments are inactive. The results show that the response is T-cell independent and that it is a function of nylon wool adherent, surface Ig-positive, Fc receptor-bearing B lymphocytes. Fc fragments do not induce plaque-forming cells to human IgG in normal mouse spleen cell cultures, but rather trigger polyclonal antibody synthesis (anti-goat erythrocytes, anti-2,4,6-trinitrophenyl). It is postulated that the Fc region of antibodies plays a role in the regulation of the humoral immune response by triggering clonal expansion of B lymphocytes.     

Use of ektacytometry to determine red cell susceptibility to oxidative stress

To define a more sensitive and reliable method to determine changes in the overall cellular characteristics of erythrocytes after oxidative damage, we used a viscodiffractometric method (ektacytometry) to measure the effect of oxidative stress. Erythrocytes were incubated in the presence of hydrogen peroxide, t-butyl hydroperoxide, or cumene hydroperoxide in phosphate buffer. This treatment resulted in decreased cellular deformability of the intact erythrocytes. In addition, deformability and fragility measurements of the erythrocyte ghost membranes indicated an increased membrane dynamic rigidity and altered-mechanical stability as a consequence of oxidant stress. These changes were observed before the onset of hemolysis. The observed decrease in deformability was accompanied by oxidation of hemoglobin, alterations of membrane proteins, and lipid peroxidation. To continuously measure the time course of the decrease in deformability in intact erythrocytes under oxidative stress, a new ektacytometric method was developed. Erythrocytes were oxidatively challenged within the viscometer at a constant osmolality and shear stress. The change in deformability was monitored and a typical range was defined for erythrocytes from normal individuals. Comparison of erythrocytes from patients with sickle cell disease with those from normal individuals demonstrated a higher susceptibility of sickle red cells toward oxidative stress.