凝胶微柱法与试管法检测放散液中抗体的比较

目的比较凝胶微柱法和试管法检测红细胞释放液抗体的能力。方法将30例外周血和41例脐血红细胞用两种方法测定直接抗人球蛋白试验(DAT)并进行酸放散,将放散液与三种谱细胞、A1、B细胞反应检测抗体的特异性。71例中12例作为阴性对照样本(DAT均为阴性),其中6例为健康献血者外周血,6例为不发生新生儿溶血病(Heamolytic disease of thenewborn,HDN)的婴儿脐血样本。结果24例外周血放散液中有10例两种方法均阳性,12例均为阴性,2例自身免疫性溶血性贫血(AIHA)患者样本凝胶微柱法阳性而试管凝集法阴性,6例健康捐献者的样本DAT试验阴性且两种方法检测放散液均为阴性;41例脐血样本中33例放散液两种方法均反应,2例放散液在试管法中与A1和B细胞反应而同样的放散液做凝胶微柱法时一个和A1细胞反应,另一个只与B细胞反应。结论两种方法检测放散液结果基本是一致的,检测AIHA患者红细胞释放液抗体时用微柱法比较好,检测脐血细胞释放的同种血凝素时试管凝集法更好—些。     

A preliminary comparison of flow cytometry and tube agglutination assays in detecting red blood cell-associated C3d

This study compared flow cytometric analysis with tube agglutination assays for the detection of red blood cell (RBC)-associated complement and immunoglobulins (Igs). RBCs from 20 patients with reactive tube direct antiglobulin tests (DATs) were evaluated by flow cytometry with anti-C3d, anti-IgG, anti-IgM and anti-IgA. Serial samples were also tested from a patient at risk of passenger lymphocyte haemolysis. Results of flow cytometry and tube assays for anti-IgG were as follows: 12 of 20 samples reactive in both; six of 20 nonreactive in both; two of 20 discordant with a reactive tube and a nonreactive flow cytometry assay. Anti-C3d results showed nine of 20 reactive in both and 11 of 20 discordant with a nonreactive tube and a reactive flow cytometry assay. In the IgM flow cytometry assay, three samples were reactive with anti-IgM. Samples from a group A woman who was transplanted with stem cells from a group B donor showed that on days 3 through 6 post-transplant, the flow cytometry assays for anti-IgG and/or anti-C3d were reactive, whilst the tube assays were nonreactive. In conclusion, flow cytometric analysis is more sensitive than the tube assay for the detection of RBC-associated C3d. Further studies are needed to determine the correlation of C3d levels with clinical sequelae.     

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.     

Correlation between in vivo hemolysis and the amount of red cell-bound IgG measured by flow cytometry

A flow cytometry method was used to compare the amount of red cell (RBC)-bound IgG in 73 patients with and without immune hemolytic anemia (IHA). The positive results in 10 of the direct antiglobulin tests (DATs) were idiopathic, and those in 25 were due to methyldopa therapy; 38 of the 73 DAT-positive patients were babies born to women with IgG alloantibodies of potential clinical significance. Normal blood donors with (n = 30) and without (n = 121) positive DATs were also tested. RBCs that had been strongly sensitized (4+ indirect antiglobulin test) in vitro with different quantities of IgG anti-D, but that had similar antiglobulin test (AGT) titration scores, could easily be differentiated by flow cytometry. The mean percent fluorescence of RBCs, incubated with fluorescein-labeled anti-IgG, from neonatal patients with IHA was higher than that of RBCs from those without IHA, but there was no statistical difference in the other groups. There was considerable overlap in the respective ranges of percent fluorescence of RBCs from patients with and without IHA in all groups. It was not possible to define a clear quantitative threshold differentiating patients with IHA from those without. Although flow cytometry was more precise and reproducible than standard serology (e.g., AGT titration scores), correlations of the amount of RBC-bound IgG and in vivo hemolysis were similar.     

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.     

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。     

两种方法测定孕妇 IgG 型抗-A（B）效价的比较

目的：对微柱凝胶技术和流式细胞术两种检测 IgG 型抗 ABO 抗体的方法进行比较。方法以 O 型孕妇为实验组, A/B 型孕妇作为对照组,分别用微柱凝胶技术和流式细胞术检测 IgG 型抗 ABO 抗体,分析实验组和对照间阳性率的差别以及两种方法间相关性。选取不同滴度样品在不同日期进行检测以比较两种方法的重复性。结果共收集实验组300例,对照组300例。微柱凝胶技术和流式细胞的检测结果表明,实验组阳性率高于对照组,差异有统计学意义（P ＜0．05）。微柱凝胶检测和流式细胞术检测相关系数 rs ＝0．694。微柱凝胶技术检测的变异系数小于流式细胞检测变异系数（P ＜0．05）。结论 ABO 血型不合多见于 O 型孕妇,流式细胞检测与微柱凝胶技术具有很好的相关性,流式细胞术重复性好于微柱凝胶技术。     

Microwave dissociation of antigen-antibody complexes: a new elution technique to permit phenotyping of antibody-coated red cells

To evaluate the effectiveness of microwave irradiation in dissociating IgG from red cells (RBCs), the use of chloroquine diphosphate (CDP) was compared to that of microwaves. Fifteen paired samples of RBCs from 15 patients with positive direct antiglobulin tests (DATs) were treated with both CDP and microwave radiation. Total microwave exposure times ranged from 20 to 100 seconds. Posttreatment DATs were performed, and the reaction grades of the posttreatment DATs were compared. RBC phenotyping was also performed on repeatedly microwaved RBCs to demonstrate possible effects on RBC antigen expression. Microwaves successfully reduced the reaction grade of the DAT in 14 of 15 samples; CDP reduced the reaction grade in 12 of 15 samples. In samples with a DAT of 2+ or greater (n = 13), the microwave method yielded a greater reduction in DAT strength in six cases (results in the other 7 cases were identical with both methods) (p = 0.01). Five of eight cases with a DAT of 3+ showed a greater reduction in the DAT with microwave treatment than with CDP treatment; results in the remaining three cases were identical (p = 0.03). RBC antigenicity remained unchanged after exposure to microwave radiation (A, B, C, c, D, E, e, Fya, Fyb, Jka, Jkb, K, k, S, and s). Microwave treatment required less than 10 minutes per sample, while CDP treatment required 30 to 120 minutes per sample (mean, 88 min). The microwave technique of antigen-antibody dissociation from RBCs provides a rapid and accurate method of facilitating the phenotyping of RBCs coated with warm autoantibodies and is superior to other methods, which destroy RBC antigens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hemolytic anemia after kidney transplantation: a prospective analysis

BACKGROUND: Hemolysis may occur in 9% to 40% of patients after solid organ transplantation and be caused by the passenger lymphocyte syndrome (PLS). STUDY DESIGN AND METHODS: We have prospectively examined 217 kidney transplant recipients before (Day ?1) and after (up to Days +10, +20, and +30) surgery. ABO‐identical transplant was performed in 180 (82.9%) patients, while 37 (17.1%) individuals received ABO‐compatible nonidentical grafts. Direct antiglobulin tests (DATs) were performed by tube technique (polyspecific anti‐human globulin [IgG + C3d]), positive DAT samples were further tested by gel agglutination (monospecific anti‐IgG, ‐IgM, ‐IgA, or ‐C3), and eluates were prepared from DAT‐positive red blood cells (RBCs) by the dichloromethane elution test. RESULTS: We observed that 34 of 217 (15.7%) patients developed a positive DAT up to Day +30. The percentage of patients with positive DATs was significantly higher in those having ABO‐compatible nonidentical transplants compared to those that received ABO‐identical grafts (10/37 = 27.0% vs. 24/180 = 13.3%; p = 0.037). Specific RBC antibodies (anti‐A or anti‐B) were found in only 5 of 37 (13.5%) patients having ABO‐compatible nonidentical transplants who presented with clinical hemolysis. We found only three reactive eluates from 24 patients with positive DATs who received ABO‐identical transplants but had no hemolysis. CONCLUSIONS: Our data collected prospectively demonstrated that: 1) positive DATs occurred in 15.7% of all patients up to Day +30 after a kidney transplant; 2) the DAT positivity occurred up to Day +10 in 9.7% of all transplanted patients; 3) the majority of the transplant recipients with a positive DAT had a nonreactive RBC eluate; and 4) PLS was the cause of a positive DAT in 13.5% of patients submitted to ABO‐compatible nonidentical kidney transplants.     

Serologic findings in autoimmune hemolytic anemia associated with immunoglobulin M warm autoantibodies

BACKGROUND: Autoimmune hemolytic anemia (AIHA) associated with immunoglobulin M (IgM) warm autoantibodies is unusual but often severe, with more fatalities than other types of AIHA. Diagnosing this type of AIHA can be difficult because routine serologic data are not always informative.
STUDY DESIGN AND METHODS: Forty-nine cases of IgM warm AIHA in 25 years were studied by serologic methods.
RESULTS: Routine direct antiglobulin tests (DATs) detected red blood cell (RBC)-bound C3 in 90 percent of cases (65% had C3 but no immunoglobulin G [IgG] on their RBCs) and IgG in 24 percent. IgM was detected on 29 of 47 (62%) patients' RBCs; RBC-bound IgM was detected in 14 of 47 cases by a tube DAT method and in an additional 15 of 21 (71%) cases using fluorescein isothiocyanate anti-IgM and flow cytometry. Eighty-one percent of eluates from patients' RBCs reacted. Warm autoagglutinins were present in 94 percent of serum samples; untreated and enzyme-treated RBCs were hemolyzed at 37°C by 13 and 65 percent of serum samples, respectively. Most agglutinins were optimally reactive at 30 to 37°C. Patients' RBCs were spontaneously agglutinated in 78 percent of cases; washing with 37°C saline or treating RBCs with dithiothreitol resolved this problem. Clear specificity of autoantibody was defined in 35 percent of serum samples.
CONCLUSION: IgM warm AIHA can be confused with cold agglutinin syndrome and "mixed/combined"-type AIHA; a serologic workup by a specialist reference laboratory can help with the diagnosis.     

A newly developed gel centrifugation test for quantification of RBC-bound IgG antibodies and their subclasses IgG1 and IgG3: comparison with flow cytometry

BACKGROUND: Novel gel centrifugation test (GCT) cards were evaluated with respect to their ability to estimate the quantity of IgG on RBCs and the determination of the IgG subclasses IgG1 and IgG3. STUDY DESIGN AND METHODS: In 65 patients with a positive DAT, the amount of IgG-gamma-, IgG1, and IgG3 on RBCs was examined by use of GCT cards and flow cytometry (FC) in parallel. The results were correlated with the presence or absence of hemolysis. In addition, D+ RBCs were studied after sensitization with anti-D sera from 22 alloimmunized pregnant women. RESULTS: The amount of IgG on the RBCs as determined by GCT dilution cards correlated with FC (r=0.70, p < 0.0001). IgG subclass results as determined by GCT IgG subclass cards were confirmed by FC in 14 cases with an anti-IgG-gamma-chain titer > or =300, whereas IgG subclass cards were not suitable in cases with anti-IgG-gamma-chain titers less than 300. In 44 patients with 2+ or 3+ DAT in the GCT and anti-IgG-gamma-chain titer < or =30, no hemolysis was observed, whereas hemolysis occurred in 13 of 14 patients with an anti-IgG-gamma-chain titer > or =300. GCT data obtained by IATs with anti-D sera were concordant with FC results. CONCLUSION: There is a correlation between the amount of RBC-bound IgG and immune hemolysis. The GCT cards that detect the anti-IgG-gamma-chain may be useful to predict hemolysis in patients with a 2+ or 3+ DAT in the GCT. The diagnostic value of GCT cards for IgG subclass testing should be investigated further.     

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.     

IgG1 and IgG3 anti-D in maternal serum and on the RBCs of infants suffering from HDN: relationship with the severity of the disease

BACKGROUND: Anti-D IgG antibodies that are responsible for severe cases of HDN belong chiefly to IgG1 and IgG3 subclasses. The relationship between the concentrations of IgG1 anti-D and IgG3 anti-D in maternal serum and the amount bound to the surface of infants' RBCs is not known. In addition, the contribution of the two subclasses to the severity of HDN is not well established. STUDY DESIGN AND METHODS: Blood samples from 40 infants suffering from severe forms of HDN due to anti-D were collected before transfusion together with sera from their respective mother. The amount of total anti-D IgG as well as IgG1 anti-D and IgG3 anti-D on infants' RBCs and the concentration in maternal sera were determined by ELISA. RESULTS: The median percentages of IgG1 anti-D and of IgG3 anti-D in maternal sera were 90 and 10 percent, respectively, whereas on infants' RBCs they were 97 and 3 percent, respectively. The differences between maternal and infantile percentages were significant (p < 0.001). IgG1 and IgG3 anti-D bound to infants' RBCs increased concomitantly with the concentration of IgG1 and IgG3 anti-D in maternal sera. The severity of HDN correlated positively with the concentration of IgG1 anti-D in maternal sera, but negatively with the amount of IgG3 anti-D bound to infants' RBCs. In addition, the existence of a high proportion of IgG3 anti-D in maternal serum was associated with a delayed risk of fetal anemia. CONCLUSION: The proportion of IgG3 anti-D relative to the total anti-D IgG on infants' RBCs is only one- third of the proportion present in maternal serum. The study of the correlations between the amount of IgG1 anti-D and IgG3 anti-D and the severity of HDN suggests that IgG1 anti-D are more important than IgG3 anti-D in the pathogenesis of fetal anemia.     

RBC-associated IgG in patients with visceral leishmaniasis (kala-azar): a prospective analysis

BACKGROUND: Despite the fact that anemia is one of the most striking clinical features of visceral leishmaniasis (kala-azar), the factors involved in its pathogenesis are not fully understood. Although the cause of the anemia seen in these patients is often multifactorial, sequestration and destruction of the RBCs in the enlarged spleen, immune mechanisms, and alterations in RBC membrane permeability have been implicated. STUDY DESIGN AND METHODS: To investigate whether RBCs of patients with kala-azar were coated with IgG, blood samples of 67 patients were tested, prospectively, before (Day 1), during (Day 30), and after (Day 90) antimonial therapy, to examine the presence of RBC-associated IgG, circulating immune complexes (CICs), and rheumatoid factor (RF). RESULTS: The prevalence of a positive DAT on Day 1 was significantly greater than the prevalence of a positive DAT performed on Day 30 and on Day 90 (32.8 vs. 4 vs. 0%, p < 0.001). With an enzyme-linked antiglobulin test (ELAT) to measure the number of IgG molecules per RBC more accurately, it was found that the amount of IgG molecules per RBC was increased (mean, 298 molecules of IgG per RBC) in the group of patients with kala-azar tested before antimonial therapy, but was considered normal (<50 molecules of IgG per RBC) in all patients tested 90 days after treatment. The prevalence of a positive eluate test was low (15.0%) in DAT (anti-IgG)-positive patients and the positivity of DATs and ELATs correlated with the presence of either RF or CICs, respectively. CONCLUSIONS: These data suggest that a nonspecific adsorption of CICs on the RBC surface is probably the most important factor involved in the increased amount of RBC-associated IgG in patients with untreated visceral leishmaniasis; however, further prospective studies are required to establish the exact role of the RBC-associated antibodies, CICs, and RF as contributing factors of the anemia seen in these patients.     

Quantification of IgG anti-D bound to D-positive red cells infused into D-negative subjects after intramuscular injection of monoclonal anti-D

SUMMARY. A flow-cytometric method was developed to determine the number of molecules of IgG bound to D-positive red blood cells (RBC) when sensitized with low plasma concentrations of IgG anti-D in the presence of an excess of D-negative RBC. D-positive RBC were infused into 12 D-negative male volunteers 2 days after intramuscular injection of monoclonal anti-D (BRAD-3, IgG3 or BRAD-5, IgG1). Blood samples were taken immediately before, 3 min and 3 h after injection of the RBC, incubated for 1 h at 37°C, washed, then incubated sequentially with FITC-conjugated anti-IgG, IgM monoclonal anti-D, biotin-conjugated anti-IgM, and R-phycoerythrin-conjugated streptavidin. To prepare a standard curve, O R₁R₂ RBC were incubated in duplicate with varying dilutions of BRAD-3 or BRAD-5, and RBC from one set were mixed with an excess of D-negative RBC (1:100) and labelled as above, while cell-bound IgG on the other set was quantified by ELISA. The test samples and standards were analysed by flow cytometry and the mean channel (FITC) fluorescence was plotted against molecules IgG/cell, from which the sensitization level of D-positive RBC in the test samples was determined. The use of IgM anti-D enhanced the discrimination between D-positive and D-negative RBC, especially when fewer than about 3000 molecules IgG/cell were bound. The assay was sensitive to about 1000 molecules IgG/cell. The sensitization levels of the D-positive RBC in samples taken 3 h after injection were found to be in the range 1500-10,000 molecules IgG anti-D per cell.     

流式细胞术应用于Kidd血型Jka抗原检测的建立

目的:建立流式细胞术鉴定Kidd血型Jka抗原的实验方法,并验证其准确性。方法:随机选取96(人)份无偿献血者血液样本,将红细胞与IgG性质抗-Jka混合孵育,随后结合荧光素Alexa Fluor 647标记的羊抗人IgG二抗,采用流式细胞术检测样本红细胞表面Jka抗原。通过流式细胞术获得每个样品的荧光直方图。利用血清学凝集试验比较衡量流式细胞术检测Jka抗原强度的准确性,利用PCR-SSP与基因测序分型验证流式细胞术检测Jka抗原阴阳性的正确性。结果:流式细胞术对于红细胞Jka抗原强度的检测与血清学鉴定结果相一致,血清学凝集强度越高,流式细胞术检测荧光强度也越高,Jka抗原强度也越高。流式细胞术检测灵敏度优于血清学方法,Jka抗原阳性包括弱阳性样本与阴性样本的荧光强度差别很大,极易区分。全部样本流式细胞术检测结果与基因分型结果完全一致,证实了流式细胞术的准确性。结论:成功建立了流式细胞术鉴定红细胞Kidd血型Jka抗原的方法,灵敏度优于传统的血清学方法,可以准确区分不同强度的Jka血型抗原。     

Higher affinity human D MoAb prepared by light-chain shuffling and selected by phage display

BACKGROUND: In blood banks, D MoAbs are routinely used to phenotype donors and patients. However, most D MoAbs do not agglutinate RBCs that weakly express D. The use of higher affinity MoAbs could overcome this problem. In this work, an attempt has been made to increase the affinity of the human clone 43F10, an IgG anti-D, by light (L)-chain shuffling followed by selection using phage display. STUDY DESIGN AND METHODS: PBMNCs of three polyimmunized individuals were used to construct the kappa L-chain repertoire that was recombined with the 43F10 heavy chain in a phagemid vector system (pComb3H, Scripps Institute, La Jolla, CA). L-chain-shuffled 43F10-F(ab) phages were selected on intact RBCs and characterized by ELISA, indirect agglutination, and sequence analysis. RESULTS: L-chain shuffling combined with phage display permitted the selection of a 43F10 MoAb variant (p3.17) with improved reactivity with weak D RBCs in agglutination assays. Nucleic acid sequence analysis showed that p3.17 and wild-type (wt) 43F10 L chains are encoded by different VL segments of the Vk1 family and different J segments, thus showing a relatively low degree of homology (86.4%). CONCLUSION: The use of a variant such as p3.17 could permit a further increase of the potency of existing anti-D reagents. The low homology between p3.17 and wt 43F10 sequences further exemplifies the predominant role of the heavy chain in determining the specificity of the anti-D.     

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.     

Analysis of factors affecting quantification of fetomaternal hemorrhage by flow cytometry

BACKGROUND: An analysis was carried out to determine the sources and extent of errors encountered in the quantitation of the volume of fetomaternal hemorrhage (FMH) by flow cytometry. Different assay conditions were compared, to define the simplest, most accurate protocol. STUDY DESIGN AND METHODS: D-, D+, and artificial FMH (mixtures of D+ and D- RBCs) were stained either by a direct method (using FITC-conjugated IgG3 D MoAb [BRAD-3]), with or without dual labeling with PE-conjugated anti-GPA, or by indirect methods (using polyclonal anti-D followed by FITC- or biotin-conjugated anti-IgG reagents). Cells were selected for flow cytometric analysis on the basis of either forward or side scatter (log FSC/log SSC) characteristics or of GPA+ labeling or were unselected. The numbers of events labeled with anti-D were determined from histograms. For some samples, 10 replicates of 500,000 events each were analyzed. RESULTS: Background fluorescent events in 10 directly labeled gated D- samples ranged from 0.007 to 0.023 percent, equivalent to 0.15- to 0.51-mL FMH. Both the use of a gate on log FSC/SSC or the selection of GPA+ events only resulted in a reduction in FMH of 0.3 mL or less. The intra-assay variation in FMH, or sampling error, was found to be approximately 10 percent at low artificial FMH (<10 mL) but greater (< or =50% with a CV of 15%) with D- samples. Direct staining was quicker and produced a lower background than indirect staining. CONCLUSION: The inherent sampling error that is due to the random distribution of rare events throughout the blood sample contributed greatly to the variation in the volume of FMH calculated by flow cytometry. The FMH should not be underestimated. For a routine assay, a simplified protocol and calculation will be sufficiently accurate to determine the dose of prophylactic anti-D that should be given to the patient.     

Anti-D quantification by flow cytometry: an alternative to the Auto Analyser?

SUMMARY. A flow cytometry method for quantifying levels of anti-D is described. The method is based on the indirect antiglobulin test and uses an FITC Fab anti-human IgG reagent for detection of bound anti-D. A standard curve is generated from the fluorescence measured by flow cytometry using the British Standard anti-D preparation 73/515. The fluorescence obtained from test samples by flow cytometry is converted into IU/mL anti-D, using the standard curve. In a limited study with 42 serum samples the assay showed good correlation with results obtained using the AutoAnalyser for sera with anti-D levels less than 100 IU/mL. The method is simple to perform and has potential as a replacement for the AutoAnalyser.