凝胶微柱法检测酸放散液中的抗体

试管法作为红细胞(RBC)血清学试验中检测抗体的标准方法已有数十年,然而随着凝胶微柱抗体检测系统的大量运用,许多试验可以用凝胶微柱法代替试管法。凝胶微柱法已用于检查ABO、Rh血型、血清中的意外抗体、进行直接抗人球蛋白试验(DAT)。与试管法相比,凝胶微柱法只需少量的样本和试剂,结果判读简便且更易于自动化,因此许多血液中心及输血机构都采用此法。本试验的目的在于比较凝胶微柱法和试管法检测红细胞放散液中抗体的能力,放散液则来源于脐带血和外周血经上述两种方法检测DAT阳性或DAT阴性的红细胞。     

酸放散方法对弱D抗原的筛选研究

目的 目的确定用盐水抗D检测为D阴性的红细胞标本中是否存在Del型红细胞标本。方法 将待检红细胞样本与人血清抗D抗体混合吸收。采用酸放散方法将致敏在红细胞上的抗D抗体放散下来，用微柱凝胶抗人球蛋白试剂卡及试管法检测放散液中是否存在抗体。结果 51份由盐水抗D确定为D阴性的红细胞样本中有3人用传统试管法抗人球蛋白实验检测为弱凝集；经酸放散实验后，检测放散液，此3人放散液中均存在较强的抗D抗体，以此确定此3人红细胞抗原为Del型。结论 酸放散实验在临床输血中可做为确定红细胞上是否存在弱D抗原的敏感方法。     

直接抗人球实验中使用传统试管法与微柱凝胶和微柱亲和方法的比较

背景 用直接抗人球实验(DAT)检测结合在红细胞上的免疫球蛋白或补体在诊断免疫溶血性贫血方面很有价值。传统的DAT是通过使用抗IgG或抗C3d在试管内凝集来完成。该研究的目的是对体外致敏红细胞和病人血样的试管法DAT与微柱凝胶法、微柱亲和法DAT及流动细胞计数进行比较。设计与方法 84份病人红细胞用试管法DAT、一种微柱凝胶、二种微柱亲     

罕见的LW不规则抗体分析及输血策略——附1例报道并文献复习

目的准确分析鉴定患者抗体类型,为其制定更为安全科学合理的输血方案。方法使用试管法和微柱凝胶法分别进行血型正反定型,用Rh血型为ccdee红细胞进行吸收放散试验,再分别用二硫苏糖醇(DTT)处理和未经处理的RhD阳性红细胞与放散液进行反应,鉴定患者所含不规则抗体。结果未经DTT处理的RhD阳性红细胞与放散液反应呈阳性,经DTT处理的RhD阳性红细胞与放散液反应呈阴性。结论患者体内所含不规则抗体为抗-LW,而非抗-D,为其输注用抗球蛋白法筛选出的凝集最弱的RhD阳性红细胞安全有效。     

微柱凝胶法检测孕妇IgG抗-A(B)抗体效价结果的判定

目的 建立微柱凝胶法检测孕妇IgG抗-A(B)抗体效价的结果判定标准.方法 用微柱凝胶法和试管法平行测定126例孕妇IgG抗-A(B)抗体效价,分别统计试管法和微柱凝胶法1+、3+凝集结果;比较两种方法的阳性率,并对结果进行相关性分析,对微柱凝胶法IgG抗-A(B)抗体效价以1+判定、1∶256为决定值和以3+判定、1∶64为决定值结果的评价指标比较.结果 微柱凝胶法1+、3+凝集和传统试管法阳性例数和阳性率分别为90例(71.40%)、26例(21.40%)、24例(19.05%);微柱凝胶法3+凝集报告与试管法阳性率差异无统计学意义(P>0.05).微柱凝胶法以凝集1+、3+报告,与传统试管法结果相关系数分别为0.897、0.919,均高度相关.以微柱凝胶法1+凝集判定结果、效价1∶256为决定值和以3+凝集判定结果、效价1∶64为决定值,两者差异无统计学意义(P>0.05);两者相对于试管法的灵敏度、特异度及准确度分别为0.958、0.961、0.960、0.833、0.960和0.936.结论 以微柱凝胶法3+凝集判定的结果,可以直接报告结果.     

微柱凝胶卡式法Coombs试验在自身免疫性溶血性贫血诊断中的应用

目的 采用微柱凝胶卡式检测方法进行直接Coombs试验,探讨其在自身免疫性溶血性贫血( AIHA)诊断中的临床意义.方法 对临床怀疑为AIHA的128例患者标本同时用试管法和微柱凝胶卡式法进行直接Coombs试验,对两种检测方法进行比较.检测患者同期血红蛋白水平、网织红细胞比例、胆红素及游离血红蛋白水平,比较不同型别患者间的差异.结果 应用微柱凝胶卡式法进行直接Coombs试验阳性检出率为88.4％,试管法阳性检出率为37.7％,患者红细胞包被不同免疫球蛋白时溶血程度不同.结论 微柱凝胶卡式法较试管法敏感,操作简单,易标准化,结果判读客观,可长期保存.对AIHA的诊断具有临床意义.     

红细胞膜免疫磁珠检测成分血中抗A/抗B的研究

目的比较应用红细胞膜免疫磁珠法及传统方法进行成分血中抗A/抗B效价检测之间的相关性。建立应用红细胞膜免疫磁珠检测血浆标本中高效价抗体的方法。方法采用新鲜细胞试管法、微柱凝胶法及红细胞膜免疫磁珠法进行血浆样本抗体效价检测。将试管法检测效价≥128的样本10倍、15倍、20倍稀释后,用红细胞膜免疫磁珠检测,测定其凝集强度。结果微柱凝胶法最敏感,其次为试管法,红细胞膜免疫磁珠法低于试管法1个滴度。将试管法检测效价≥128的待检样本15倍稀释后,用红细胞膜免疫磁珠检测,其凝集强度≥2+。结论红细胞膜免疫磁珠可做为标准红细胞抗原应用于抗体效价检测,将样本进行15倍稀释,若凝集强度≥2+,则可视其带有高效价抗体。     

微柱凝胶在放散实验中应用

目的探讨微柱凝胶方法在放散实验中的应用及价值。方法对101例患者ILIAD阴性的标本同时用传统抗人球蛋白法（AGT）和微柱凝胶抗人球蛋白法（MGT）做吸收放散试验,并对两种不同鉴定方法进行比较分析。结果MGT法检出阳性标本33例,AGT法检出阳性标本30例。结论两种方法检测IgG抗D无显著差异,但MGT法检出率高于传统AGT法,抗原抗体的反应凝集强度也优于传统AGT法。     

ABO血型不合新生儿溶血病免疫三项模式分析

目的探讨ABO血型不合新生儿溶血病(ABO-HDN)免疫3项结果的模式。方法采用微柱凝胶法与试管法对疑诊ABO-HDN患者测定免疫3项(直接抗人球蛋白试验、游离试验和放散试验),观察结果模式并进行方法学对比分析。结果在75例疑诊患者中,采用微柱凝胶法确定ABO-HDN 42例(56%),阳性模式以DAT、游离、放散试验呈(-++)者最多(27例,36%),其次为(+++)模式(12例,16%);采用试管法确定ABO-HDN 30例(40%),最常见模式亦为(-++)(19例,25%),其次亦为(+++)模式(8例,11%)。采用微柱凝胶法和试管法分别有29例(39%)和22例(29%)DAT阴性、放散试验阳性而确诊为ABO-HDN。微柱凝胶法放散试验阳性率(56%)高于试管法(40%)(χ2=3.85,P<0.05)。结论 DAT阴性的ABO-HDN模式在临床常见,宜选用敏感度较高的微柱凝胶法进行免疫3项检测。     

自身免疫性溶血性贫血的微柱凝集技术诊断应用

目的探讨微柱凝集体技术在诊断自身免疫性溶血性贫血(AIHA)中的应用.方法采用BioVue系统微柱凝集技术对26例AIHA患者进行检测,同时用传统的试管法抗人球蛋白试验作比较.结果统计学上两种方法对AIHA检出率无显著性差别,但微柱凝集技术能检出试管法所不能检出的阴性血样,比试管法检测凝集强度高1+～2+.结论BiVue系统微柱凝集技术能快速准确检测出AIHA,具有更高的灵敏度,是直观可靠的方法.     

Detection of antibodies in acid eluates with the gel microcolumn assay

BACKGROUND: Gel microcolumns can be used to detect unexpected serum antibodies and to determine ABO blood group and Rh phenotype. DATs can also be performed with this system. The purpose of this study was to compare the gel microcolumn to the tube IAT using anti-IgG for the detection of antibodies eluted from RBCs. STUDY DESIGN AND METHODS: Acid eluates were prepared from 30 peripheral blood and 41 umbilical cord blood samples. Twelve of the 71 eluates made were from control samples (known DAT negative). Specificities of eluted antibodies were determined by both tube and gel assays with a three-cell screen plus A1 and B cells, as determined by blood type. RESULTS: Ten of 30 peripheral blood eluates were reactive in both assays. Eighteen were nonreactive in both assays, and two from patients with autoimmune hemolytic anemia were reactive by gel assays and nonreactive by tube assays. Thirty three of the 41 cord blood eluates were reactive in both assays. Eluates from 2 of the 35 DAT-positive samples reacted with A1 and B cells by the tube method but were nonreactive by the gel method. Of the 33 cord blood eluates that were reactive by both assays, antibody specificity differed for two samples. When tested by tube assay, these eluates reacted with both A1 and B cells, whereas the same eluates tested by gel assay showed one reacting with only A1 cells and the other with only B cells. CONCLUSIONS: Results of testing eluates in gel assays were similar to those obtained in tube assays. The gel assays may be better at detecting antibodies eluted from RBCs from patients with autoimmune hemolytic anemia, and tube assays may be better at detecting isohemagglutinins eluted from umbilical cord blood.     

Comparison of DATs using traditional tube agglutination to gel column and affinity column procedures

BACKGROUND: Detection of immunoglobulin or complement bound to RBCs by using the DAT is valuable in the diagnosis of immune-mediated hemolytic anemia. Traditionally, the DAT has been performed by tube agglutination using anti-IgG or anti-C3d. The purpose of this study was to compare the tube agglutination DAT to gel microcolumn, affinity microcolumn, and flow cytometric DATs on RBCs coated in vitro and on patient RBC samples. STUDY DESIGN AND METHODS: RBCs from 84 patients were assessed by tube agglutination DAT, one gel microcolumn DAT, and two affinity microcolumn DATs. One affinity microcolumn assay was unmodified and one was modified by the addition of polyspecific antiglobulin or anti-IgG as a secondary antibody. RBCs from 15 of the 84 patients underwent analysis by flow cytometry with fluorescence-labeled anti-IgG. The assays were also compared by using D+ RBCs sensitized with serially adjusted concentrations of anti-D. RESULTS: Both tube agglutination and gel microcolumn DATs were positive in 49 patient samples; both assays were negative in 20 samples, and the results were discordant in 15. Gel microcolumn DATs were more likely than were tube agglutination DATs to detect IgG on RBCs. Affinity microcolumn DATs were less likely than gel microcolumn or tube agglutination DATs to detect IgG on RBCs. Flow cytometry results were the same as gel microcolumn results in 12 of 15 patient samples and the same as tube agglutination results in 13 of 15. Tube agglutination and both affinity microcolumn assays reacted with RBCs coated with anti-D that was diluted 1-in-100. The gel microcolumn and flow cytometry assays reacted with RBCs coated with anti-D diluted 1-in-400. There was no correlation between tube agglutination and gel microcolumn DATs in detecting bound C3d. CONCLUSION: Detection of IgG bound to RBCs was not consistent with the methods described. Gel microcolumn DATs were more sensitive than tube agglutination and affinity microcolumn DATs. Given the varied results of these assays, reference laboratories should not rely on a single method for DATs. More comprehensive testing should be performed when the tube agglutination DAT is negative in a patient with suspected immune-mediated hemolytic anemia. Further comparisons are necessary to determine the proficiency of flow cytometric assays.     

The evaluation of a positive direct antiglobulin test in pretransfusion testing

The results of serologic studies on 879 blood samples with a positive direct antiglobulin test (DAT) are presented. All blood samples were from patients who were either anemic, for reasons other than blood loss, recently transfused, or had serum antibodies detected during routine pretransfusion tests. Blood samples from only 81 of the patients included in this study had serologically reactive eluates (64 autoantibodies, three antibodies to penicillin and cephalothin treated red blood cells, three passively acquired anti-A antibodies, and 11 transfusion-induced alloantibodies). The eluted antibodies were also detected in the serum by routine pretransfusion tests in 13 of the patients whose red blood cells eluted autoantibodies, and in five of the patients whose red blood cells eluted transfusion-induced alloantibodies. All but one of the 11 transfusion-induced alloantibodies were detected within 14 days posttransfusion. Based on these findings, a cost-effective and safe approach to the management of blood samples with a positive DAT would be to restrict the preparation and testing of eluates to those samples from recently transfused patients. It is the contention of the authors that the incorporation of the DAT in pretransfusion testing should primarily serve to detect alloantibody formation before such antibodies are evident in the serum, and should not be used to screen patients for unsuspected autoimmune hemolytic anemia. Furthermore, the authors question the necessity for blood banks to routinely perform an autocontrol on all blood samples from prospective transfusion recipients.     

Is it necessary to add the eluate testing to the direct antiglobulin test to improve the detection of maternal erythrocyte alloantibodies?

BACKGROUNDThe screening of umbilical cord blood samples by the Direct Antiglobulin Test (DAT) is the reference tool for the identification of maternal erythrocyte alloantibodies present in erythrocytes; however, its diagnostic usefulness is controversial.OBJECTIVETo evaluate the diagnostic validity, safety, and efficiency of the eluate testing (detection of antibody in erythrocyte eluates by the Indirect Antiglobulin Test/IAT) in cord blood samples for detection of maternal erythrocyte alloantibodies in comparison with the DAT.MATERIALS AND METHODSEvaluation study of diagnostic tests. DAT and eluate testing were performed in 306 cord blood samples from neonates born to mothers admitted at Clínica Somer in Rionegro, Colombia; then, antibodies present in the eluates were identified with erythrocyte panels. Percentage of positive results by DAT and IAT were compared with the Pearson's chi-square test and the agreement between both assays with the Cohen's kappa coefficient. The diagnostic sensitivity, specificity, safety, and efficiency of the eluate testing were calculated, taking into account the use of DAT as an imperfect reference test.RESULTSThe DAT detected alloantibodies in 6.21% of samples and the eluate testing in 14.1 %; the strength of agreement between both tests was moderate (k = 0.56) due to 25 discrepancies. The eluate testing showed sensitivity and specificity of 98.83 % and 92.31 % respectively, and a negative predictive value of 99.9 %. The diagnostic efficiency was sufficient for detection of maternal erythrocyte alloantibodies. The antibodies identified in the erythrocyte eluates were anti-A or anti-B (79.5 %), anti-D (136%), anti-C (2,3%), and anti-Fya (2,3%).CONCLUSIONThe eluate testing in cord blood samples is a valid, safe, and efficient test for the diagnosis of maternal erythrocyte alloantibodies.     

Comparison of conventional tube test technique and gel microcolumn assay for direct antiglobulin test: a large study

Gel microcolumn assay (GMA) is a modified serological technique that has been used for ABO and Rh typing, direct antiglobulin test (DAT), detecting alloantibodies, red cell phenotyping, and other applications. However, for DAT, the role of GMA is controversial. The purpose of this large study was to compare the performance of the conventional tube test (CTT) to GMA for detecting potentially significant antibodies coating red blood cells in vivo. From January 1996 to May 2002, we performed DATs by GMA and CTT on 9,862 blood samples submitted to our reference laboratory, using LISS/Coombs cards (DiaMed-Latino America, Lagoa Santa-MG, Brazil) for GMA and polyspecific and monospecific anti-IgG reagents for CTT. Acid eluates were prepared from all positive DAT samples. The specificity of eluates was determined by GMA. We detected nonconcordant results in 2,079 out of 3,163 positive DATs (65.7%). All of these tests were only positive in GMA. Sensitivity and specificity for DATs was 100% and 83.0% for gel, and 50.7% and 97.8% for tube, respectively. Based on this study GMA showed to be more sensitive than CTT for detecting potentially significant antibodies coating red blood cells in vivo.     

The role of the elution in antibody investigations

BACKGROUND: The direct antiglobulin test (DAT) commonly detects immunoglobulin G (IgG) molecules or complement fragments on the red blood cell (RBC) surface. If IgG antibodies are present then elution procedures can be performed to identify the specificity of these antibodies. Our reference laboratory performs elutions on the RBCs of those patients who have received cellular blood products in the past 30 days and have either a newly identified positive DAT with anti-IgG or the agglutination strength is increased over a previous DAT and if ordered by a clinician regardless of transfusion history. This study questioned how frequently elutions contributed novel serologic information under our reference laboratory's current policy or whether elutions should be performed in more selective serologic conditions.
STUDY DESIGN AND METHODS: Recipients whose RBCs underwent eluate testing were identified from the blood bank's database and information about the antecedent DAT and antibody detection test and eluate was recorded.
RESULTS: In total 648 eluates were evaluated and 82 of 648 (12.7%) revealed a novel antibody not present in the serum (an informative eluate). In 2 of 82 informative eluates non–anti-A/B alloantibodies that were not present in the serum were detected: one example each of anti-D and anti-E. Both were associated with a microscopically positive antecedent DAT. The rate of an informative eluate was higher when the antibody detection test was negative.
CONCLUSION: The strength of the DAT does not indicate the likelihood of an informative eluate. Performing an eluate when the antibody detection test is positive has limited value.     

False-positive eluate reactivity due to the low-ionic wash solution used with commercial acid-elution kits

BACKGROUND: During the use of commercial red cell (RBC) acid-elution kits for adsorption and elution (adsorption/elution) studies with anti- D, unexpected reactive eluates (anti-D) were obtained from D- RBCs. Such results were not obtained with a parallel xylene method or, historically, with heat and ether methods. STUDY DESIGN AND METHODS: Single-donor and commercial polyclonal anti-D samples were incubated with D+ and D- RBCs. Acid eluates were prepared by the manufacturers' directions. Variations in the wash step of the eluate preparation included the use of commercial kit wash solution versus phosphate- buffered saline versus solutions of various ionic strengths. RESULTS: Anti-D was eluted from 20 of 22 samples of D- RBCs after incubation with commercial polyclonal anti-D (titer 512) and from 2 of 3 samples of D- RBCs incubated with single-donor anti-D (titer 256). With a low- titer (16) single-donor anti-D, 0 of 4 eluates from D- RBCs reacted. When phosphate-buffered saline was substituted for the commercial wash solution, 0 of 11 D- RBC eluates reacted, as compared with 9 of 11 D- RBCs that yielded positive 1+(-)2+ eluates with the commercial wash solution. If the recommended initial phosphate-buffered saline wash was omitted before the use of the commercial wash solution, the eluate reactivity was stronger (2+(-)3+). When low-ionic-strength (< 0.03 M) saline was substituted, anti-D was eluted from D- RBCs. All last washes were nonreactive. Antiglobulin tests on all adsorbing D- were negative. CONCLUSION: Commercial wash solutions used for acid elution are at low ionic strength and commonly yield superior eluates, but in the presence of high-titer antibodies, false-positive eluates can result. It is our belief that the low-ionic-strength wash solution caused aggregation of IgG and nonspecific attachment of IgG on RBCs. Aggregates will contain IgG serum antibodies in proportion to the titer of the antibody. It is this nonspecifically bound antibody that is eluted from antigen- negative RBCs.     

Positive direct and indirect antiglobulin tests associated with oxaliplatin can be due to drug antibody and/or drug-induced nonimmunologic protein adsorption

BACKGROUND: Two patients were suspected of having immune hemolytic anemia (IHA) due to oxaliplatin. A related drug, cisplatin, is known to cause nonimmunologic protein adsorption (NIPA). Studies were performed to determine the presence of oxaliplatin-dependent antibodies in addition to oxaliplatin-induced NIPA.
STUDY DESIGN AND METHODS: Sera and eluates from the two patients were tested against red blood cells (RBCs) treated with oxaliplatin, cisplatin, and carboplatin (another platinum drug). Sera were also tested against untreated RBCs in the presence of the same drugs. Testing with pooled normal sera and anti-human albumin was used to demonstrate the presence of NIPA. Oxaliplatin-treated RBCs sensitized with the patients' sera and pooled normal sera were tested by a monocyte monolayer assay (MMA) to determine potential clinical significance.
RESULTS: Both patients had high-titer antibodies to oxaliplatin in their sera that reacted with oxaliplatin-treated RBCs and with untreated RBCs in the presence of oxaliplatin. RBCs treated with oxaliplatin, cisplatin, and carboplatin all demonstrated NIPA (pooled normal sera and anti-human albumin were reactive to low titers). NIPA was also detected in tests with untreated RBCs in the presence of oxaliplatin and cisplatin. Lower-titer reactivity of both patients' sera with cisplatin may have been due to NIPA and/or cross-reactivity of anti-oxaliplatin with cisplatin. MMAs were weakly positive due to NIPA and more strongly positive due to oxaliplatin antibodies.
CONCLUSION: Two patients with IHA were demonstrated to have oxaliplatin-dependent antibodies. Oxaliplatin was also shown to cause NIPA. The drug-dependent antibody and/or the drug-induced NIPA could have contributed to the patients' hemolytic anemia.     

Evaluation of the manual hexadimethrine bromide (Polybrene) technique in the investigation of autoimmune hemolytic anemia

The use of the direct manual hexadimethrine bromide (Polybrene) test (DPT) in the investigation of patients for autoimmune hemolytic anemia (AIHA) was evaluated. Seventy-nine blood samples from 68 patients were tested. A direct antiglobulin test (DAT) using monospecific reagents and the DPT were performed, and a concentrated ether eluate was tested. The DAT was positive in 62 (78%) of 79 patients and negative in 17 (22%). There is a good correlation among DAT, eluate, and DPT in demonstrating the presence of immunoglobulin on the red cell surface. In contrast, the DPT does not detect C3d and is often negative in cases of AIHA in which C3d alone is demonstrated by the DAT. In DAT-negative cases, DPT results correlated with reactive eluates. However, in four cases of steroid-responsive, DAT-negative hemolytic anemia, the DPT supported the diagnosis of AIHA when the eluate did not react. The DPT is a useful additional screening test for the investigation of AIHA, but it is not recommended as a replacement for either eluate testing or the DAT.     

Immune-mediated agglutination of cytoskeleton-free RBC microvesicles

BACKGROUND: The factors contributing to RBC agglutination are complex. The RBC cytoskeleton's participation in and contribution to this phenomenon are difficult to separate from those of the plasma membrane. Immunoreactive, cytoskeleton-free, band 3-enriched microvesicles can be generated from normal RBCs. Band 3 has been defined as an important antigen in autoimmune hemolytic anemia (AIHA). STUDY DESIGN AND METHODS: RBC microvesicles devoid of major cytoskeletal proteins were generated and sensitized with eluates obtained from AIHA patients, DAT-positive blood donors, and antisera to common RBC antigens. Monoclonal anti-human IgG was added and agglutination was investigated. Autoantibody-specific binding was evaluated by employing (125)I protein A. RESULTS: RBC vesicle agglutination with a 4+ anti-human globulin score was obtained with 10 autoantibody eluates from AIHA patients and anti-D (3+), but not with eluates from 20 DAT-positive blood donors or antisera directed to eight other common RBC antigens. Microvesicles sensitized with AIHA eluates bound 67 to 167 times as much (125)I protein A radioactivity as did those incubated with buffered normal saline and 18 to 45 times more than vesicles incubated with normal serum. CONCLUSION: The major proteins of the RBC cytoskeleton are not required for supporting IgG immune-mediated agglutination of RBC microvesicles.