传统试管法与达亚美微柱凝胶法检测抗-D效价的比较

目的：为了评价微柱凝胶技术（MGCT）检测抗-D效价。方法：用MGCT和试管抗球蛋白实验（TCT）同时检测38例孕妇血清抗-D效价。结果：MGCT检测抗-D效价高于TCT 2～6滴度（平均提高3～4个滴度）,MGCT检测抗-D效价灵敏度显著高于TCT,差异有统计学意义（P〈O．01）结论：MGCT法检测抗-D效价是一种灵敏度较高的方法。     

Comparison of conventional tube test with diamed gel microcolumn assay for anti‐D titration

Summary Anti‐D titration is the first step in the evaluation of the RhD‐sensitized patient. Traditionally, anti‐D titration has been performed by tube agglutination. Gel microcolumn assay is a method that has gained widespread usage throughout the world, mainly for ABO/Rh typing, unexpected antibody screening and direct antiglobulin tests. As gel assay has become widely used as a routine method to detect red blood cell alloantibodies, a critical anti‐D titer needs to be established. Seventy‐nine known blood samples with anti‐D (titers 1–32) were titrated simultaneously by the conventional tube test and the gel microcolumn assay. Red blood cells (R₀r phenotype) were used, with a final concentration of 3% for tube and 0.8% for gel. Serial twofold dilutions (2–2.048) were prepared for each technique, followed by reading in antiglobulin phase. Anti‐D titration in the gel microcolumn assay showed significantly higher titers (mean 3.4‐fold) than the conventional tube test in all samples studied. Based on these data, it was not possible to determine a critical titer for anti‐D titration by the gel microcolumn assay.     

微柱凝胶法与试管法检测新生儿溶血病免疫性抗体的比较

目的：比较微柱凝胶法与试管法对母婴血型不合新生儿溶血病（HDN）免疫性抗体的检出率。方法：对临床表现为高胆红素血症、疑似HDN的患儿血标本同时用微柱凝胶法和试管法进行直接抗人球蛋白试验、抗体游离试验及抗体放散试验,并对患儿阳性血标本进行血型不规则抗体特异性鉴定及其效价测定。结果：在275例临床表现为高胆红素血症、疑似HDN的患儿血标本中,用试管法检出直接抗人球蛋白试验阳性180例（65.5%）,抗体游离试验阳性197例（71.6%）,抗体放散试验阳性210例（76.4%）;用微柱凝胶法检出直接抗人球蛋白试验阳性224例（81.5%）,抗体游离试验阳性238例（86.5%）,抗体放散试验阳性251例（91.3%）。微柱凝胶法比试管法的凝集强度高1＋～2＋。结论：微柱凝胶法的敏感性略高于试管法,具有操作简便,影响因素少,易于标准化,结果客观及保存时间长等优点。     

Comparative Evaluation of Five Different Methods of Anti‐ABO Antibody Titration: An Aid for ABO‐Incompatible Organ Transplants

The accurate estimation of ABO antibody titers is of the utmost importance in organ transplants involving ABO incompatibility. We aim to compare five different methods of titration and analyze the data. Samples of 48 O group blood donors who donated during the month of December 2015 to January 2016 in our institution were subjected to ABO antibody titration by five different methods: immediate spin (IS) tube titer, antihuman globulin phase tube titer, Coomb's gel card titer, gel card titer after dithiotreitol (DTT) treatment of plasma, and the solid phase red cell adherence method. The mean number of titer serial dilution steps in the different titer estimation methods was compared using the paired t‐test and McNemar test. A correlation between the methods was tested using Spearman's rho and kappa statistics. The median antiglobulin (AHG) phase tube titers were found to be the highest anti‐A (128) and anti‐B (192) titers. Significant differences in the ABO antibody titer readings among the five different methods were noted. Titers were reduced by DTT treatment in nearly 50% samples tested for both anti‐A and anti‐B titers. Average agreements between the DTT‐applied AHG phase gel card titers and the solid phase red cell adherence (SPRCA) titers was observed for anti‐A (κ = 0.473) and anti‐B (κ = 0.530). The AHG phase tube and gel cards titers showed poor agreements. There are differences in the interpretability of the ABO antibody titer among different techniques. Consistent and uniform application of the method for titration throughout the treatment of a patient is highly essential.     

43例待产孕妇Rh血型抗体筛查阳性分析

目的：研究多次妊娠妇女体内Rh血型抗体与新生儿溶血病的关系。方法：运用微柱凝胶免疫法鉴定Rh抗体和分型,抗人球试验法测定抗体效价。结果：待产孕妇16845例,检出意外抗体48例,其中43例为Rh血型抗体且夫妇ABO顺式相容。分析抗体特异性种类和效价,抗D抗体8例,效价8～512;抗c抗体5例,效价16～128;抗E抗体16例,效价8～64;抗C抗体12例,效价4～8;抗e抗体2例,效价8。由此得出Rh血型抗原性强弱为D〉c〉E〉C〉e。结论：多次妊娠妇女体内Rh相关抗体效价强弱是引起新生儿溶血的原因之一。     

新生儿溶血病血清学检查2种方法的对比研究

目的：对比研究微柱凝胶法和抗人球蛋白试验在新生儿溶血病（HDN）血清学检查中的应用。方法：选择母婴ABO血型不合的新生儿血液标本80份。每份标本均用微柱凝胶法和抗人球蛋白试验进行HDN“三项检查”：直抗试验、游离试验和释放试验。并将数据进行统计学处理。结果：直抗试验中2种方法均无阳性结果；游离试验中2种方法比较差异无统计学意义（P〉0．05）；释放试验中2种方法比较差异有统计学意义（P〈0．05）。结论：HDN“三项检查”中微柱凝胶法优于试管法，快速、简便，标本量少，重复性强，结果判定直观，敏感性强。     

Development of Multiple Antibodies in an Apparently ''Protected'' Rh D Negative Mother

A case is described of a group O Rh (D) negative woman who received a blood transfusion after the delivery of her first infant, blood group O Rh (D) negative. In her second pregnancy anti-S was detected, presumably due to the prior transfusion. The second infant was D+S+ and the direct antiglobulin test on this infant's cells was positive. As no anti-D antibodies were detectable in this pregnancy, the positive antiglobulin test was presumably due to the anti-S. Anti-D immunoglobulin was administered after this pregnancy, but in spite of this the mother developed a strong anti-D antibody. The significance of this 'failure of protection' is discussed in relation to the augmenting affect of antibody development during pregnancy.     

Evaluation of the Microcolumn Technology for Pretransfusion Testing in Multiple Myeloma Patients

Objective: Microcolumn tests are useful for serological investigations, although because of their high sensitivity, false-positive results might be expected, e.g. in hypergammaglobulinemia. The aim of this study was to evaluate these tests in multiple myeloma. Methods: Pretransfusion testing was done in 80 patients with multiple myeloma using microcolumn and traditional tube tests. Results: All sera were negative in microcolumn indirect antiglobulin test and enzyme test, positive in 58% of samples in the enzyme tube test. The microcolumn direct antiglobulin test was positive in about 40% of samples but never in the tube direct antiglobulin test. This was not due to the presence of autoantibodies but to nonspecific binding of immunoglobulins related to their concentration in sera. Conclusion: Microcolumn tests appeared to be useful for pretransfusion testing in multiple myeloma in spite of positive autocontrols.     

Micro-Column Affinity Test and Gel Test: Comparative Study of Two Techniques for Red Cell Antibody Screening

BACKGROUND AND OBJECTIVES: We describe the results of a comparative evaluation of a gel test (ID Micro Typing) and a micro-column affinity test (MCAT, Cellbind Screen) for red cell antibody screening and identification under routine conditions. MATERIALS AND METHODS: 3,000 serum samples of patients from the Mannheim University Hospital were tested in parallel by means of the gel test and the MCAT, using the low-ionic-strength-saline indirect antiglobulin test and the protein G affinity technique, respectively. Test cells used were the same in all tests. In addition, we performed titration studies with all detected antibodies as well as with 59 frozen sera containing antibodies of known specificity. RESULTS: A total of 154 antibodies (5.1%) were detected, 149 by gel test and 147 by MCAT. The overall sensitivity and specificity of the gel test was 96.8 and 96.5% and of the MCAT 95.5 and 97.2%. No significant differences between the gel test and MCAT were found when the titer scores of all 213 (fresh and frozen) antibodies were used to check the results. The mean scores for the gel test and the MCAT were 26.8 and 28.5, respectively. For anti-Fy(a) and anti-Kell, a significantly higher titration score could be obtained in the MCAT, whereas anti-Lu(a) showed a significantly higher score with the gel test. CONCLUSION: For the screening of unexpected red blood cell antibodies, the MCAT is as sensitive as the gel indirect antiglobulin test. The sensitivity and specificity of the two systems are more or less the same although it seems that IgM antibodies are better detected by the gel test.     

The value of gel test and ELAT in autoimmune haemolytic anaemia

Summary Three methods for detection of warm type IgG autoantibody were evaluated using 400 blood samples from 147 patients suspected of autoimmune haemolytic anaemia (AIHA). Three direct antiglobulin techniques (DAT) were used: conventional tube DAT, gel-DAT by micro-method and gel-DAT enzyme linked antiglobulin test (ELAT). Eluate examinations confirmed the presence of autoantibodies on red cells. These tests were compared directly using 126 selected blood samples from 85 patients with IgG molecules on their red cells detected by the gel test. In 106 of these samples, collected from 65 patients with clinical symptoms of AIHA, the presence of autoantibody was confirmed by acid elution. The ELAT was positive in 100 samples (94%), 87 samples for tube DAT (82%). The ELAT as well as the tube DAT was negative in 20 samples with non-reactive eluates by gel test. The gel-DAT was therefore not fully specific and detected IgG on red cells of patients with hypergammaglobulinaemia. However, due its higher sensitivity it proved useful as a screening test. The ELAT allowed changes in the number of IgG molecules per red cell to be monitored quantitatively. Both methods play a part in the diagnosis and monitoring patients with warm type IgG autoantibody.     

The Direct Antiglobulin Test: Still a Place for the Tube Technique?

OBJECTIVES: Antibodies of different immunoglobulin isotypes, or complement, may coat red blood cells in vivo. They are detected by the direct antiglobulin test (DAT), usually performed by the conventional tube technique. The purpose of this study was to compare the latter technique with the gel test. METHODS: Three hundred and ninety-eight consecutive samples, sent to our laboratory for direct antiglobulin testing, were analyzed with the tube technique and the gel test, using reagents from different manufacturers. Eighty-seven samples had been collected from newborns and 23 from fetuses. Results were expressed as positive or negative. RESULTS: In 162 out of 398 cases, the DAT was negative with both methods, whereas in 178 out of 398 cases, the DAT was positive with both techniques using polyspecific antibodies (observed agreement: 84.5%; kappa = 0.71). Discrepancies between the two methods were observed in 58 cases: 51 samples appeared as DAT positive using the tube method and negative with the gel test, whereas only 7 were positive exclusively with the gel test. Among the 178 samples that were positive with both techniques, 93 samples showed discordant results when evaluated with monospecific antisera (11 with anti-IgG and 82 with anti-C3d, respectively). The sensitivity of the DAT performed by the gel test, in comparison with the conventional tube technique, was 75.4% (95% confidence interval (CI): 69.4-80.8). 96. 8% (95% CI: 92.8-99.0), and 16.3% (95% CI: 9.8-24.9) with polyspecific, anti-IgG and anti-Cd3 reagents, respectively. CONCLUSIONS: The gel test appeared to be less sensitive than the conventional tube technique when utilized for DAT, particularly when C3d was present on red blood cells. These results emphasize that before implementing a new technique in the laboratory, comparison with existing techniques, using different reagents, is mandatory.     

Intravenous anti-D as a treatment for immune thrombocytopenic purpura (ITP) during pregnancy

This pilot study assessed the safety and efficacy of intravenous anti-D in eight Rh(D)-positive women with immune thrombocytopenic purpura (ITP) during the second and third trimesters of pregnancy. The median pretreatment platelet count was 28 x 109/l. The patients received one to seven anti-D infusions at a mean dose of 62.7 micro g/kg, and the response rate to anti-D was 75%. A haemoglobin decrease of > 2.0 g/dl occurred only once. Fetal hydrops was not identified by ultrasonography. The direct antiglobulin test was positive in three out of seven Rh+ newborns, none of whom was anaemic or jaundiced. Anti-D is effective and appears to be safe for both mother and fetus.     

ADCC (K-Cell) Lysis of Human Erythrocytes Sensitized with Rhesus Alloantibodies III. COMPARISON OF IgG ANTI-D AGGLUTINATING AND LYTIC (ADCC) ACTIVITY AND THE ROLE OF IgG SUBCLASSES

Conventional manual (enzyme and antiglobulin titres) and AutoAnalyser quantitation of anti-D sera were compared with the ability of the same sera to mediate lysis of Rh(D) positive red cells in an ADCC assay. AutoAnalyser (AA) quantitation correlated significantly with manual titration methods, although there were wide discrepancies between individual sera. The ADCC activity of the anti-D sera did not correlate with any of the conventional assays (AA, enzyme, antiglobulin). There were significant differences between anti-D sera obtained from females immunized during pregnancy and male volunteers immunized by deliberate injection. The female sera contained significantly less anti-D when assessed by AA, yet were significantly more active in ADCC activity at equivalent concentrations. These functional differences in anti-D activity could not be attributed to the absence of IgG subclasses (IgG1 and IgG3) known to induce ADCC. However, there was a relationship between ADCC and IgG1/IgG3 titres in that high ADCC activity was seen where the IgG3 titre was higher than the IgG1 titre. In the male anti-D sera IgG1 titres were greater than or equal to IgG3 titres and ADCC activity was very low.     

The significance of anti-C sensitization in pregnancy

A study of maternal blood samples from 280,000 pregnancies in an 8-year period has shown 38 examples of anti-C (without anti-D) sensitization. This frequency (0.14/1000 pregnancies) was lower than that previously found for anti-c (0.63/1000 pregnancies) and for anti-D (2.55/1000 pregnancies). Although most of the 38 babies born to mothers with anti-C were unaffected by haemolytic disease of the newborn and none was anaemic at birth, two required exchange transfusion for hyperbilirubinaemia and one a top-up transfusion. Five cord blood samples had a positive direct antiglobulin test.     

Correlation of serological, quantitative and cell-mediated functional assays of maternal alloantibodies with the severity of haemolytic disease of the newborn

Serum samples containing IgG red blood cell (RBC) antibodies were collected without reference to clinical information from 131 pregnant alloimmunized women. Anti-D and anti-K were present in sera from 75 and 20 patients respectively. Antibody titres were determined by indirect antiglobulin test (IAGT), anti-D levels were measured by AutoAnalyzer, RBC-binding IgG was quantified using an enzyme-linked immunosorbent assay (SOL-ELISA), and functional activities were measured using the monocyte chemiluminescence (CL) test, antibody-dependent monocytemediated and K cell-mediated cytotoxicity (ADCC) assays, and rosette formation with U937 cells.
Details of clinical outcomes were obtained retrospectively from 104 pregnancies. Forty-one babies were 'antigen-negative', and of the remainder, four required top-up transfusions, 12 required exchange transfusions, three received intrauterine transfusions, and two died in utero. A comparison of test results with severity of haemolytic disease of the newborn (HDN) showed that, provided sera tested were collected within 8 weeks of the expected delivery date, the CL test and the monocyte-mediated ADCC assay differentiated those D-positive babies which required exchange transfusions from those unaffected or only mildly affected. The usefulness of results from the AutoAnalyzer and IAGT in predicting disease severity was compromised by the wide range of results from mothers of unaffected babies. This variability was less apparent in the SOL-ELISA which predicted severe HDN with greater precision. Results from the U937 rosette assay and the K cell-mediated ADCC assay failed to correlate with disease severity.     

应用微柱凝胶技术筛查新生儿ABO溶血病及结果分析

目的：探讨微柱凝胶技术在新生儿溶血病（HDN）筛查中的应用以及结果分析。方法：采用微柱凝胶技术对633例疑似HDN患儿进行检测,用经典抗球蛋白法做对照。结果：633例筛查实验微柱凝胶技术阳性率为30.5%,28%为ABO-HDN,2.5%为RH-HDN,其中直接抗球蛋白实验阳性率为6.8%,患儿红细胞抗体放散实验阳性率为30.5%（抗A抗体阳性89例,抗B抗体阳性45例,抗AB抗体阳性43例,抗D抗体阳性10例,抗E抗体阳性6例）,血清游离抗体鉴定阳性率为13%,而经典抗球蛋白法的直接抗球蛋白实验阳性率为3%,患儿红细胞抗体放散实验阳性率为仅为24.7%。结论：在温州地区的HDN以ABO-HDN最为常见,抗体分布特征为抗A抗体〉抗B抗体〉抗AB抗体〉抗D抗体〉抗E抗体。新生儿溶血微柱凝胶技术能快速准确检测出HDN,具有很高的灵敏度,并能够检出经典抗球蛋白法所不能检出的阴性标本,是直观可靠的方法。     

Prevalence,Specificity and Titration of Red Cell Alloantibodies in Multiparous Antenatal Females at a Tertiary Care Centre from North India

Screening and detection of clinically significant antibodies among antenatal women plays an important role in transfusion safety and preventing hemolytic disease of fetus and newborn. Routine screening of antenatal women for antibodies is not done in all blood centres of our country and so immunization rates are not known in pregnant women. We studied the prevalence of alloantibodies and titration of Anti D among antenatal multiparous women in Jammu region. In present prospective study, 750 antenatal multiparous women attending antenatal clinics were typed for ABO and D antigens. Alloantibody screening was done, if positive, specificity of alloantibody was ascertained by using commercially available red cell panel by tube method. Rate of alloimmunization was correlated with Rh D status, gravida, previous transfusion history and bad obstetric history. Titration of alloantibody D was done in first and third trimester of pregnancy. In present study most common blood group detected was B positive (38.4 %). Rh D negative cases constituted 7.6 % of total cases. Rate of alloimmunization was 2 %. A significant correlation was seen between Rh D-negative and alloimmunization (21 % in D-negative and 0.45 % in D-positive). There is significant increasing degree of alloimmunization with increase in Gravida. Alloimmunization in females with bad obstetric history was high (4.41 %) as compared to females with no bad obstetric history showing only 1.76 %. Alloantibodies detected were Anti-D, Anti-E, Anti-C and Anti-K. Anti-D constituted 80 % of all alloantibodies detected. Six women in their third trimester had raised titers of anti-D. Most common alloantibody detected was anti-D (80 %). Alloantibodies to other Rh antigens and Kell blood group systems were also identified. To minimize alloimmunization in Rh D negative women, proper Anti D immunoprophylaxis should be implemented.     

Antibody elutions in Thai patients with a positive direct antiglobulin test

Background

The direct antiglobulin test is performed to determine whether an anaemic patient with evidence of haemolysis has autoimmune or alloimmune haemolytic anaemia.

Materials and methods

We determined the antibody specificity of eluted IgG antibodies from patients’ blood samples with a positive direct antiglobulin test. Overall, 134 Thai patients were included in this study. EDTA blood samples were obtained from recently transfused patients, patients with unexplained anaemia and patients who had serum antibodies detected during routine pre-transfusion tests from different hospital blood banks. These complicated samples were sent to the National Blood Centre of the Thai Red Cross Society for investigation and to find compatible blood components. Each blood sample underwent a direct antiglobulin test with the gel technique using polyspecific antihuman globulin and mononospecific anti-IgG and anti-C3d. Acid eluates were prepared from the samples for which the direct antiglobulin test was positive and the specificities of the eluted antibodies were determined by the gel technique.

Results

Of the samples tested, 101 showed a positive direct antiglobulin test result (75.4%) using polyspecific antihuman globulin sera whereas only 95 samples (70.9%) were positive with anti-IgG or anti-IgG and anti-C3d. Moreover, 54 of 95 eluates (56.8%) were positive for antibody screening and tested with the reagent panel cells. Twenty-one eluates had specific alloantibodies, which were concordant with the findings in the patients’ sera and all patients had a history of blood transfusion. Additionally, 33 eluates contained pan-agglutinins. Interestingly, alloantibodies could be determined using titration studies in 5 of 26 eluates with pan-agglutinins.

Conclusion

Although the direct antiglobulin test is not routinely performed in pre-transfusion screening, this test and elution studies would be useful in patients with a history of previous transfusions, and in those for whom compatible blood cannot be found.     

微柱凝胶法检测Rh血型免疫性抗体的分析

目的：探讨Rh血型免疫性抗体筛查及特异性鉴定在临床输血和新生儿溶血病预防中的意义,观察微柱凝胶法检测Rh血型免疫性抗体的敏感性。方法：应用微柱凝胶法对患者输血前和孕妇产前做Rh血型免疫性抗体筛查,对筛查阳性者进一步做抗体特异性鉴定及效价测定。结果：在9878例患者中检出Rh血型免疫性抗体阳性86例（0.87%）,其中抗D12例（14.0%）,抗E25例（29.1%）,抗C11例（12.8%）,抗c9例（10.5%）,抗e4例（4.7%）,抗Ce7例（8.1%）,抗CE5例（5.8%）,抗CD6例（6.9%）,抗cD3例（3.5%）,抗cE4例（4.7%）。结论：在输血前和产前对拟输血患者和夫妇血型不合的孕妇进行Rh血型免疫性抗体筛查、特异性鉴定及效价测定,对确保输血安全及优生优育有重要的临床意义。     

5种不规则抗体筛查方法的检测阈值比较

目的:了解各种不规则抗体筛查方法的检测阈值,以避免不规则抗体漏检而引起输血不良反应。方法:选取献血者不规则抗体筛查阳性样本3例和配血不合疑难样本9例,采用盐水法、酶介质法、凝聚胺法、抗人球蛋白法、微柱凝胶法平行检测,比较5种方法的检测阈值。结果:盐水法只能检出IgM类的不规则抗体;木瓜酶法在检测IgG抗体效价时敏感性略低,而且有漏检的可能;凝聚胺法检测结果不易判断;抗人球蛋白法操作较繁琐;微柱凝胶法对IgG类抗体检测效价较高,高于凝聚胺法和抗人球蛋白法。结论:微柱凝胶法操作简单,检出率较高,是值得推广的不规则抗体筛查方法。