Sensitivity of column agglutination technology in detecting unexpected red cell antibodies

Summary. This study was conducted to compare a new microtube column agglutination technology (CAT) with a previously described tube saline-indirect antiglobulin test (SIAT) with carefully defined performance in the detection of unexpected antibodies. On testing 117 sera from fresh and frozen stock containing antibodies detectable by SIAT, CAT failed to detect two examples of weak anti-K. All other discrepancies between the two techniques involved antibodies generally regarded as clinically insignificant. Titration studies with anti-D (concentration approximately 10 ng/ml) and 23 other antibodies, and studies with 10 weak antibodies of various blood group systems, showed the two techniques to be of similar sensitivity. Equivocal CAT results requiring repeated testing were found in 4·1% of specimens tested. We conclude that the CAT method adequately meets our requirements in sensitivity of detection of unexpected antibodies in pre-transfusion testing and offers opportunities for savings in technical staff time.     

Comparison of three microtube column agglutination systems for antibody screening: DG Gel, DiaMed-ID and Ortho BioVue

The aims of the present study were to evaluate the estimated diagnostic accuracy of a new microtube column agglutination system (DG Gel, Diagnostic Grifols, Barcelona, Spain), to analyse the antibody reactivity and to compare the data with the two well-established DiaMed-ID and Ortho BioVue systems. We collected 3024 consecutive samples from blood donors, transfusion recipients and pregnant women, and 100 samples containing antibodies of known specificity. All these samples were tested in parallel by the three microtube agglutination systems. The estimated sensitivity was 100% for DG Gel and Ortho BioVue and 97.58% for DiaMed-ID. The estimated specificity was 99.93% for Ortho BioVue and 100% for DiaMed-ID and DG Gel. The score mean and range of the antibody titration of DG Gel, DiaMed-ID and Ortho BioVue were 34.31 (5-119), 30.3 (3-121) and 37.38 (3-112), respectively. All three column agglutination systems work well showing a high estimated diagnostic accuracy.     

The routine use of Rh-negative reagent red cells for the identification of anti-D and the detection of non-D red cell antibodies

BACKGROUND: Before 1987, fewer than 50 patients per year at the authors' laboratory had a positive antibody detection test due to antepartum Rhesus immunoprophylaxis. However, after 1987, a marked increase was observed in the number of patients who had received Rh immune globulin (RhIG) during pregnancy as part of routine antepartum Rh immunoprophylaxis. In anticipation that an increased use of RhIG during pregnancy would increase the number of patients in whom anti-D was detected by this laboratory, a protocol was developed to abbreviate the process required to identify anti-D. Although this protocol was adopted primarily to address an anticipated increase in antenatal RhIG usage in women, it was also applied to alloimmunized Rh-negative males. STUDY DESIGN AND METHODS: When an Rh-negative patient (male or female) had a reactive screening test for unexpected antibodies and met certain other criteria, the patient's serum was tested with a three-vial set of Rh-negative reagent red cells (Rh-negative screening RBCs), instead of with panels of typed RBCs (panel RBCs), for the identification of anti- D or the detection of non-D antibodies. If the serum under test did not agglutinate or hemolyze Rh-negative screening RBCs, anti-D was identified and no further testing was performed. If the serum agglutinated or hemolyzed Rh-negative screening RBCs, conventional testing with panel RBCs was done to determine the antibody specificity. RESULTS: Rh-negative patients (n = 1174) who had reactive screening tests for unexpected antibodies were tested with Rh-negative screening RBCs; 1079 were found to have anti-D as a single antibody. Seven of these patients subsequently developed a non-D alloantibody, after transfusion or pregnancy, and one patient had anti-C that escaped detection at the time of initial testing with Rh-negative RBCs (a false- negative result). Ninety-two patients had anti-D in combination with a non-D antibody, and three patients had a non-D antibody but not anti-D. Use of the anti-D identification protocol actually reduced the laboratory workload by 176 College of American Pathologists workload units per month, in spite of a marked increase in the number of patients in whom anti-D was detected. No hemolytic transfusion reaction was attributed to the abbreviation of anti-D identification. CONCLUSION: The identification of anti-D may be abbreviated without jeopardizing patient safety. Such a protocol can reduce laboratory workload and might be particularly appealing to health care facilities that perform antibody detection testing on large numbers of Rh-negative pregnant women, especially if antepartum RhIG is administered routinely.     

A LISS spin enzyme method for the detection of red cell antibodies and its use in routine antibody screen procedures

A technique for performing the enzyme phase of the antibody screen on red cells suspended in low-ionic-strength-salt solution (LISS) is described. The reliability of this LISS spin-enzyme (LSE) technique was compared with the two-stage papain-tile method, in the detection of 62 previously identified enzyme reacting antibodies. All 62 antibodies examined were detected by the LSE method, and no false-positive reactions were found. Using LSE and papain-tile methods in parallel, further assessment was obtained by screening 2000 sequential blood samples under routine service conditions. Fifty-six blood samples contained alloantibodies, of which 43 reacted by both methods, eight by the LSE method only, and five by the papain-tile method only. It was concluded that the LSE method was comparable to the papain-tile method.     

新鲜混合血清用于乙型肝炎病毒DNA检测室内质量控制的效果评价

目的对自制新鲜混合血清用于乙型肝炎病毒（HBV）DNA定量检测室内质控的使用效果作出初步评价。方法收集HBVDNA定量检测值在10^7 copy／mL左右的阳性新鲜血清,分装-70℃保存,初定靶值后用于日常室内质控,并统计出恒定靶值,绘制室内质控图,观察精密度和使用情况。结果首批20个样本测定的均值（i）及批内精密度（OCV）％分别为7．44和1．62％;第1月、第1～2月、第1～3月的检测i及精密度（CV）％分别为7．46和2．93％、7．43和3．10％、7．47和3．09％,精密度均小于2OCV,稳定性良好;统计自制混合血清使用半年在Levey-Jennings质控图上的落点,在控率为92．50％（74／80）,警告率为7．50％（6／80）,失控率为0％（0／80）,使用效果良好。结论自制混合血清可作为室内质控品用于HBVDNA定量检测的质量控制。     

菏泽地区健康人群红细胞不规则抗体的分布与研究

目的 研究菏泽地区健康人群红细胞不规则抗体的分布规律,提高菏泽市临床输血前检查技术水平.方法 通过检测30294人份参加自愿无偿献血人群的血液标本,采用全自动微柱凝胶方法和凝聚胺方法相结合的方式,进行红细胞不规则抗体筛选试验,并对筛查阳性者进行抗体特异性鉴定,分类IgM/IgG性质、效价,统计不规则抗体在健康人群中的发生率,对比分析其是否存在地区、民族、性别、年龄等差异.结果 抗体筛选阳性标本共计44例,阳性率0.145%;7种不规则抗体分别分布于Lewis血型系统、自身抗体、MN血型系统、Rh血型系统四类,占比43%＞23%＞18%＞16%;女性高于男性,在18～24周岁和O型血献血者中发生率较低,未发现有民族差异性.结论 不规则抗体存在地区性差异,具有多态性分布特征;凝聚胺方法用于输血前检查是解决不规则抗体引起输血溶血性反应的有效方法.     

三种红细胞不规则抗体筛选试验方法的比较

目的寻找简便、快速、敏感的红细胞不规则抗体筛选的试验方法。方法采用蛋白水解酶法、抗球蛋白法(AGT)及聚凝胺三步法试验(TSPT)平行检测一批Rh、MNSs、Duffy、K idd等系统的不规则抗体效价,观察TSPT等的敏感性。结果在上述3种方法中,TSPT敏感性最高,其次为AGT,TSPT测出了所有IgG和IgM性质的不规则抗体,酶技术未检出抗-M、N、S、s、Fya、Fyb,TSPT和AGT的试剂性能稳定。结论TSPT操作简便迅速,敏感性高,无需特殊设备,TSPT适用于红细胞血型不规则抗体筛选的常规试验。     

The detection of cell-bound antibody on complement-coated human red cells

This study sought to elucidate the mechanism by which human red cells, in a variety of clinical settings, become coated in vivo with autologous complement components in the absence of anti-red cell autoantibodies demonstrable by standard methods. By means of a newly developed complement-fixing antibody consumption test, previously undetectable red cell-bound gammaG globulin could be detected and quantified. By this technique, the complement-coated red cells of 13 of 16 patients were shown to carry abnormally high numbers of gammaG molecules per cell, which were nevertheless below the level for detection by the direct antiglobulin test. Eluates were made from the red cells of seven of these patients and each eluate, when sufficiently concentrated, was capable of sensitizing normal human red cells (with gammaG antibodies) to give a positive indirect antiglobulin test with anti-gammaG serum. In the presence of fresh normal serum, six of the eluates so tested were capable of fixing complement to normal human red cells. The antibodies in the red cell eluates did not exhibit Rh specificity and did not react with nonprimate red cells. When studied by sucrose gradient ultracentrifugation, the gammaG antibodies to human red cells in these eluates sedimented in the 7S region. It is concluded that in many patients in whom direct antiglobulin tests reveal only cell-bound complement, the complement fixation is mediated in vivo by small quantities of "warm-reacting" erythrocyte autoantibodies of the gammaG class.     

New reference reagent for the quality assurance of anti-D antibody detection

A UK BTS-NIBSC freeze-dried anti-D preparation has been prepared and used to monitor the performance of routine antibody detection tests and of the test operators. With the day-to-day use of this preparation, adverse changes in test performance and in test operator may be detected and appropriate action taken before the effect becomes significant. Two dilutions of this preparation have been defined, one which should be detected unequivocally in every test; the other, more dilute, may not be detected in every test but is used to monitor changes in performance. Experience with the use of this preparation is reported from three test centres undertaking routine antibody detection tests. By monitoring results over a series of working days, significant differences were noted in operator performance within one test centre, as was a reduced sensitivity of a given test system within one test centre compared with the same system in the other test centres. These differences were detected only by monitoring the results obtained with this preparation.     

无偿献血者献血前梅毒螺旋体抗体快速检测结果分析及策略优化

目的回顾性分析无偿献血者献血前梅毒螺旋体(TP)快速检测的结果,优化献血前筛查策略。方法献血前采用TP金标试纸条对献血者进行快速检测,献血后采用TP-酶联免疫吸附测定(ELISA)法对血液进行检测。比较开展TP快速检测前、后血液TP阳性报废率。对TP快速检测阳性献血者的献血次数、献血间隔时间以及假性结果等内容进行分析。结果2014-2015年献血前TP快速检测73 990例,阳性529例,阳性率为0.71%,其中初次献血者阳性472例,占89.2%,献血间隔时间超过3年者阳性35例,占重复献血者阳性的61.4%。TP快速检测假阳性5例,假阴性15例。开展TP快速检测后,献血者血液检测抗TP阳性报废率由0.71%下降至0.17%。结论开展献血前TP快速检测能有效降低血液TP阳性报废率;结合献血者献血次数、献血间隔时间,优化献血前筛查策略,能提升献血服务效率及水平。     

血浆与红细胞先后加入顺序对检测抗体灵敏度的影响

目的比较按不同先后顺序加血浆与红细胞对检测抗体灵敏度的影响。方法采用戴安娜微柱凝胶与博唯优玻璃珠配血卡,测定Rh(D)抗体效价,比较两种加入法,即先加血浆后加红细胞和先加红细胞后加血浆对抗体灵敏度的影响,同时对凝集结果进行分析。结果微柱凝胶和玻璃珠配血卡检测抗体效价时,先加血浆的检测结果均比先加红细胞的好。结论血浆中含抗体成分,先加入反应体系时易均匀分散在介质中,容易与后加入的红细胞发生凝集,从而提高抗体检出率。     

大量输注多种抗体红细胞但无不良反应的输血分析

目的分析一例临床配血不合的不明抗体的成分和来源,探讨大量输注配血不合红细胞而没有导致输血不良反应的原因。方法对患者血浆做血型鉴定、直抗、抗筛、抗体鉴定和抗体效价测定等血清学试验。结果患者血浆中同时存在效价高达1∶1024的抗-I为主的多种抗体,包括特异性抗体和药物抗体且已激活补体,造成配血不合。结论大量配血不合红细胞的输注并没有引起明显输血不良反应,临床输血应根据患者病情灵活调整输血方案。     

血清抗CCP抗体和抗AKA抗体联合检测对类风湿关节炎的诊断价值

目的探讨抗环瓜氨酸肽抗体(抗CCP抗体)、抗角蛋白抗体(抗AKA抗体)检测对类风湿关节炎(RA)诊断的敏感度和特异性,以及二者联合检测在RA诊断中的应用价值。方法分别采用化学发光微粒子免疫检测法、间接免疫荧光法检测抗CCP抗体、抗AKA抗体,共检测80例RA患者,40例其他自身免疫性疾病患者和30例健康体检者。结果 RA组抗CCP抗体和抗AKA抗体的阳性率明显高于非RA组和对照组,差异具有统计学意义(P0.05)。单独检测抗CCP抗体的灵敏度为73.8%高于抗AKA抗体(41.3%),特异性为86.2%低于抗AKA抗体(95.7%),二者联合检测时敏感度和特异性均有所增高。结论抗CCP抗体对RA患者有很高的敏感度和特异性,并且与抗AKA抗体联合检测对于RA的临床诊断和预后判断具有很高的临床应用价值。     

The use of the antiglobulin ''gel-test'' for antibody detection

Four antibodies used routinely in-house for the assessment of antiglobulin reagents (anti-Fyb, anti-Jka, anti-S) were tested in parallel using tube and antiglobulin 'gel-test' low ionic strength antiglobulin techniques. In the latter the red cells are centrifuged following incubation through a dextran matrix incorporating an anti-human globulin reagent. The results show that the antiglobulin 'gel-test' was less sensitive than the tube technique in the detection of these difficult antibodies.     

Feasibility and cost-benefit of implementing pooled screening for HCVAg in small blood bank settings

To examine the accuracy, feasibility and benefits of screening for hepatitis C virus core antigen (HCVAg) using enzyme-linked immunosorbent assay (ELISA) test in pools. Many countries cannot afford to test blood donations for hepatitis C using molecular methods. Screening individual units using the ELISA HCVAg test is an acceptable, yet still expensive, alternative, especially for small blood bank settings. This study evaluated the option of screening for HCVAg in pools. The sensitivity (Se) and specificity (Sp) of HCVAg in pools of three and six antibody-negative samples were estimated and compared with polymerase chain reaction (PCR). The feasibility and cost-benefit of the assay was assessed on 960 routine samples collected at a hospital blood bank in Gaza. Based on results for 50 PCR-positive pools and 50 and 110 PCR-negative pools of three and six, the Se of testing in pools of three and six samples is 80-82% [95% confidence interval (CI): 66.3-91.4] and Sp >or=98% (95% CI: 89.4-100.0) compared with PCR. The incidence of antigen in donors in Gaza was 0.1% (95% CI: 0-0.56). Cost analyses suggested significant benefits from implementing screening blood donations for HCVAg when the incidence rate is >4.2/10,000, leading to reduction in the expenditures needed to treat patients infected with HCV. The risk of transfusion-transmitted hepatitis C in resource-deprived developing countries can be efficiently reduced by additional screening of antibody-negative blood donations for HCVAg in pools of six.     

献血者抗核抗体及抗可提取性核抗原抗体谱的检测

目的 探讨抗核抗体(ANA)及抗可提取性核抗原(ENA)抗体谱在健康献血者中检测的临床意义.方法 应用间接免疫荧光法检测ANA,同时采用免疫印迹技术检测抗ENA抗体,采用血液分析仪进行白细胞计数和分类.结果 93例健康献血者中ANA阳性14例(15.1%),其中45～55岁阳性者11例(39.3%),随着年龄增高,ANA的阳性检出率逐渐增高(χ2=94.01,P＜0.05);健康献血者中,不同性别ANA阳性率差异无统计学意义(P＞0.05).与ANA阴性组比较,ANA阳性献血者白细胞、中性粒细胞绝对值和中性粒细胞相对数差异均有统计学意义(P＜0.05).结论 健康人可能存在生理性自身免疫应答,但不一定能激活自身免疫系统,引起自身免疫损伤而导致自身免疫性疾病.     

抗人红细胞血型糖蛋白A非多态性表位单克隆抗体的制备和鉴定

目的制备抗人红细胞血型糖蛋白A(Glycophorin A,GPA)非多态性表位单克隆抗体并鉴定其特性。方法用小鼠B淋巴细胞杂交瘤技术获得分泌单抗-GPA非多态性表位的杂交瘤细胞株;鉴定抗体特异性和亚型;通过和各种酶处理细胞的反应确定单抗结合抗原位点的特性。结果得到的2株抗-GPA非多态性表位的单克隆细胞株Q6D7和Q7C9,均属IgG1亚类、Kappa型轻链,所针对的抗原位点均抗胰蛋白酶、胰凝乳蛋白酶处理,对无花果酶、木瓜酶、唾液酸酶敏感。结论制备获得2株抗-GPA非多态性表位单克隆抗体。     

The experience of flow cytometry for specific antibody against cisplatin-treated red blood cells: A case report

Background

Cisplatin-associated hemolysis is a rare but important adverse effect. Nonimmunological protein adsorption (NIPA) due to erythrocyte membrane modification has been reported as the leading cause of cisplatin-associated hemolysis. However, limited data exist on cisplatin-associated immunological hemolysis because of a lack of an established diagnostic method. Here, we used flow cytometry (FCM) to diagnose a patient with cisplatin-associated immunological hemolysis.

Study Design and Methods

A 55-year-old woman with uterocervical cancer was treated with weekly cisplatin monotherapy (40 mg/m²). She had no previous transfusion and medication history, nor any significant family history. On the 26th day after cisplatin administration, severe hemolysis was noted. Her red blood cells (RBCs) and sera were evaluated by direct antiglobulin test (DAT) and indirect antiglobulin test (IAT), respectively. To explore immunological reactions for cisplatin-treated RBCs, we attempted FCM using cisplatin-treated and -untreated RBCs. After incubating conditioned RBCs with the patient's serum or healthy donor serum, we evaluated their fluorescent intensity by fluorescein isothiocyanate (FITC)-conjugated anti-human immunoglobulin (Ig) G antibodies.

Results

The patient's DAT was positive, and an IAT using her plasma was positive for cisplatin-treated RBCs. FCM using cisplatin-treated RBCs revealed that the patient's serum had higher FITC intensity than the donor's serum, indicating the existence of cisplatin-treated RBC-specific IgGs in patient's serum.

Conclusion

Here, we report a rare case of a patient with hemolysis diagnosed using FCM to identify specific antibodies against cisplatin-treated RBCs. NIPA and immunological mechanisms may contribute to hemolysis onset during cisplatin treatment.