UV-visible spectrophotometric approach to blood typing II: phenotyping of subtype A2 and weak D and whole blood analysis

BACKGROUND: A recently introduced quantitative blood typing approach uses antibody-induced changes in the UV-visible spectra of blood. Changes in the blood spectra's slope, caused by RBC agglutination, are translated into a numerical agglutination index (AI). Comparing the AI value against an established threshold yields a "yes and/or no" output from which to determine the phenotype. The efficacy and flexibility of this approach with whole blood use and the ability to analyze weak D, A2, and A2B were examined. STUDY DESIGN AND METHODS: Two hundred randomly selected blood bank donor samples were coded and forward typed directly from whole blood by using the spectrophotometric analysis. Reverse grouping on plasma from each sample was carried out with a new modified procedure by using higher ratios of plasma to RBCs. Results were compared to typing by an FDA-cleared automated typing system. Twenty-seven weak D samples, 15 A2 and 12 A2B, were similarly analyzed from whole blood. PEG improved detection of weak D, A2 and A2B subtypes. RESULTS: All two hundred coded samples were accurately typed, yielding identical results to the blood bank analysis in both forward and reverse grouping. All the weak D samples and A2 and A2B samples were clearly identified, having AIs above the type threshold indicator value. CONCLUSION: Spectrophotometric blood typing successfully phenotyped ABO and D in 200 whole blood samples. Reverse grouping of plasma was equally successful. The same method can identify weak D and A2 and A2B subtypes.     

An ABO blood grouping discrepancy: Probable B(A) phenotype

In B(A) phenotype, an autosomal dominant phenotype, there is a weak A expression on group B RBCs. We herein report a case of a probable B(A) phenotype in a first time 20-year old male donor. The cell and serum grouping were done using tube technique and also with blood grouping gel card (Diaclone, ABD cards for donors, BioRad, Switzerland). The antisera used were commercial monoclonal IgM type. To check for the weak subgroup of A, cold adsorption and heat elution was performed. The cell grouping was A_weakB RhD positive while the serum grouping was B. There was no agglutination with O cells and the autologous control was also negative. It was a group II ABO discrepancy with or without group IV discrepancy. Results for both the eluate and last wash were negative. Hence, the possibility of weak subgroup of A was unlikely. Blood grouping gel card also showed a negative reaction in the anti-A column. One lot of anti-A was showing ‘weak +’ agglutination while the other lot was showing ‘negative’ reaction with the donor RBCs by tube technique. There was no agglutination observed with anti-A1 lectin. Our case highlights the serological characteristics of a B(A) phenotype. This case emphasizes the vital role of cell and serum grouping in detecting such discrepancies especially in donors which can lead to mislabeling of the blood unit and may be a potential risk for the transfusion recipient if not resolved appropriately.     

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。     

双精子嵌合体致ABO血型正反定型不符1例简

目的 对1例正定型呈混合凝集的ABO正反定型不符样本进行鉴定.方法 采用微柱凝胶法进行ABO、RhD血型鉴定、直接抗球蛋白试验和不规则抗体筛查,PCR-SSP法进行ABO基因分型,并分析外周血染色体核型.结果 先证者ABO正定型为红细胞混合凝集,反定型检出抗-A1;母亲和父亲分别为O型和AB型.先证者ABO基因座位存在O1、A201和B三个等位基因,其中O1来自母亲,A201和B来自父亲.核型分析显示先证者血液中存在46,XX和46,XY两种细胞,而父母核型正常.结论 先证者为孤雌分裂产生的两个相同的卵子被两个精子受精后所产生的嵌合体.     

Solid-phase ABO grouping and Rh typing

A solid-phase adherence method (SPAM) for ABO grouping and Rh typing of red cells (RBCs) has been developed. Adherence reactions were read spectrophotometrically and interpreted by a computer. The SPAM had a 99.6 percent correlation with conventional microplate agglutination methods for ABO grouping and Rh typing. The increased sensitivity of the SPAM was demonstrated because it directly detected Du-positive RBCs and weak subgroups of A and B.     

The value of automated gel column agglutination technology in the identification of true inherited D blood types in massively transfused patients

BACKGROUND: Massive transfusion of D− trauma patients in the combat setting involves the use of D+ red blood cells (RBCs) or whole blood along with suboptimal pretransfusion test result documentation. This presents challenges to the transfusion service of tertiary care military hospitals who ultimately receive these casualties because initial D typing results may only reflect the transfused RBCs. After patients are stabilized, mixed-field reaction results on D typing indicate the patient's true inherited D phenotype. This case series illustrates the utility of automated gel column agglutination in detecting mixed-field reactions in these patients.
STUDY DESIGN AND METHODS: The transfusion service test results, including the automated gel column agglutination D typing results, of four massively transfused D− patients transfused D+ RBCs is presented. To test the sensitivity of the automated gel column agglutination method in detecting mixed-field agglutination reactions, a comparative analysis of three automated technologies using predetermined mixtures of D+ and D− RBCs is also presented.
RESULTS: The automated gel column agglutination method detected mixed-field agglutination in D typing in all four patients and in the three prepared control specimens. The automated microwell tube method identified one of the three prepared control specimens as indeterminate, which was subsequently manually confirmed as a mixed-field reaction. The automated solid-phase method was unable to detect any mixed fields.
CONCLUSION: The automated gel column agglutination method provides a sensitive means for detecting mixed-field agglutination reactions in the determination of the true inherited D phenotype of combat casualties transfused massive amounts of D+ RBCs.     

新生儿血型检测的结果分析

目的：了解新生儿 ABO 正反定型符合率,研究提高新生儿血型测定准确性的方法。方法用传统试管法检测当地新生儿 ABO 血型,以微柱凝胶卡和改良试管法、增强剂法及常规试管法、纸片法、微板法、聚凝胺法等7种方法检测抗-A、抗-B、抗-D 抗体的效价,并进行新生儿血型检测比较。结果新生儿 ABO 正反定型符合率为83％（913/1100）;检测15份抗-A、抗-B、抗-D 抗体的效价,敏感性最高是改良试管法和增强剂法,最差的是纸片法,新生儿标本检测结果类似。结论ABO 正反定型应增加血浆（血清）量并延长孵育时间或选用增强剂,从而提高新生儿血型检测的准确性。     

Low rates of anti-recipient isohemagglutinins in ABO incompatible hematopoietic stem cell transplants

IntroductionIsohemagglutinins occur naturally and form in an 'opposite' (antigen-negative) pattern to a patient’s ABO blood type. Patients undergoing minor and bidirectional ABO incompatible hematopoietic stem cell transplantation (HSCT) may demonstrate detectable antibodies against their native blood type. In this study, we sought to characterize the rates of such antibody formation and evaluate the clinical significance of our findings.Materials and MethodsAn internal database of HSCT patients at an academic medical center was queried for ABO incompatible transplant patients from 2009-2019. Serum typing results, clinical histories, and laboratory data were compiled and reviewed.ResultsA total of 182 minor and bidirectional ABO incompatible HSCT patients were identified. Anti-recipient isohemagglutinins were found in 9% (16/182) of the HSCT patients. The rate was higher in patients with minor incompatibility (12%: 15/127) versus bidirectional ABO incompatibility (2%: 1/55) (p = 0.04). No anti-recipient isohemagglutinins were identified in umbilical cord HSCT patients (0%: 0/7). Serologic agglutination reactions of recipient isohemagglutinins were overall mostly weak (13/16 weak + to 1+). There was a trend towards a higher rate of acute graft-versus-host-disease in patients with anti-recipient isohemagglutinins compared to those without (75% vs. 53%; p = 0.12), though not statistically significant. Rates of alloimmunization to minor red cell antigens were similar between the two groups. Few patients showed laboratory evidence of hemolysis at 12 months follow up.Discussion and conclusionsAnti-recipient isohemagglutinins occur at low rates in ABO incompatible HSCT and are significantly more common in minor ABO incompatible transplant compared to bidirectional transplants. Larger cohort studies are needed to better understand the relationship between anti-recipient isohemagglutinins and HSCT outcomes.     

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.     

用分子生物学技术鉴定新生儿脐血ABO疑难血型

目的用分子生物学技术对新生儿脐血ABO疑难血型鉴定。方法新生儿产出后断脐带时留取脐血血样,先用传统的试管法对脐血标本作ABO血型鉴定,不能定出血型的标本用微柱凝胶技术鉴定,仍不能定出血型者再用分子生物学方法(聚合酶链反应-序列特异性引物分析PCR-SSP)作进一步鉴定。结果传统的试管法仅能对83.98%(4561/5431)的脐血血样正确定出ABO血型,用微柱凝胶技术仍有1.77%(96/5431)的标本不能定出ABO血型,对以上疑难标本用分子生物学技术容易地定出了ABO血型。结论微柱凝胶技术敏感,应取代试管法ABO血型定型方法,分子生物学技术鉴定新生儿脐血ABO疑难血型是一种有效的手段。     

Red Blood Cell Antigen Genotyping for Sickle Cell Disease,Thalassemia, and Other Transfusion Complications

Since the discovery of the ABO blood group in the early 20th century, more than 300 blood group antigens have been categorized among 35 blood group systems. The molecular basis for most blood group antigens has been determined and demonstrates tremendous genetic diversity, particularly in the ABO and Rh systems. Several blood group genotyping assays have been developed, and 1 platform has been approved by the Food and Drug Administration as a “test of record,” such that no phenotype confirmation with antisera is required. DNA-based red blood cell (RBC) phenotyping can overcome certain limitations of hemagglutination assays and is beneficial in many transfusion settings. Genotyping can be used to determine RBC antigen phenotypes in patients recently transfused or with interfering allo- or autoantibodies, to resolve discrepant serologic typing, and/or when typing antisera are not readily available. Molecular RBC antigen typing can facilitate complex antibody evaluations and guide RBC selection for patients with sickle cell disease (SCD), thalassemia, and autoimmune hemolytic anemia. High-resolution RH genotyping can identify variant RHD and RHCE in patients with SCD, which have been associated with alloimmunization. In the future, broader access to cost-efficient, high-resolution RBC genotyping technology for both patient and donor populations may be transformative for the field of transfusion medicine.     

Genotyping of samples lacking expected antibodies in ABO blood group

We report nine donations with ABO inconsistency in reverse typing caused by partly or entirely missing antibodies. A and B antigens and antibodies were examined by serological blood typing, and ABO deoxyribonucleic acid (DNA) analyses were performed by sequence-specific priming and sequencing. A B101 allele was demonstrable in a case with O phenotype. The molecular mechanisms in deficiency of natural ABO antibody could be partly clarified. The ABO genotyping technique is an accurate method for determining the blood samples involved in ABO grouping discrepancies and is a valuable complement to serology for correct determination of donor blood status. The mechanisms involved in the absence of potent natural antibodies directed against A and B antigen lacking on an individual's own red cell membranes remain to be further investigated.     

2种微柱凝胶卡鉴定3岁以下婴幼儿ABO及血型对比研究

目的比较国产微柱凝胶血型鉴定卡（甲卡）与进口微柱凝胶血型鉴定卡（乙卡）鉴定3岁以下婴幼儿ABO及RhD血型的效果。方法首先采用甲卡对3岁以下婴幼儿EDTA抗凝血标本进行A抗原、B抗原及D抗原初步鉴定,再采用乙卡对同一批婴幼儿不抗凝血标本的A抗原、B抗原、D抗原进行复核,对比两者血型鉴定结果符合程度及凝集强度。结果共检测6月龄以下患儿3 938例,其中早产儿96例,足月新生儿355例,血液病患儿570例,其他疾病患儿2 917例。A抗原、B抗原、D抗原初检及复检两种卡符合率100%;早产儿A、B抗原凝集强度3＋以上者两种卡均达83%左右,两者相比差异无统计学意义（P〉0.05）。检测7月龄至3岁患儿12 500例,A抗原、B抗原、D抗原初检及复检符合率100%。检出A亚型、B亚型各2例。结论甲卡在鉴定3岁以下婴幼儿A抗原、B抗原、D抗原的能力方面与乙卡没有差别,两者均能够保证患儿血型鉴定及安全输血的需求。     

Evaluation of immunohematologic routine methods using the new erythrocyte-magnetized technology on the QWALYS 2 system

BACKGROUND: QWALYS 2 is a fully automated system for ABO/D grouping, Rh phenotyping, K typing, and antibody screening (ABS). Its new erythrocyte-magnetized technology (EMT) is based on the use of magnetic nanoparticles and avoids centrifugation and washing steps.
STUDY DESIGN AND METHODS: Overall 499 blood samples were tested with our routine blood bank methods for ABO/D grouping, 313 samples for Rh phenotyping and K typing (microtiter plates; Olympus PK 7200), and 478 samples for ABS (gel centrifugation technique, DiaMed). All samples were tested in parallel with the EMT.
RESULTS: In 496 of 499 samples (99.4%), a complete concordance between the observed (QWALYS 2) and the expected results for ABO/D grouping was found. One sample with a weak A in an AB blood group and 2 samples with a weak D were not detected by the QWALYS system. Rh phenotyping and K tests revealed a 100% concordance. In the two ABS techniques, 427 samples were negative in both and 15 samples showed the same antibody specificity in both. Three immunoglobulin M antibodies were as expected negative in EMT and positive by DiaMed. In 32 cases (6.7%), false-positive reactions were observed by EMT due to 22 unspecific reactions (4.6%) and 10 lipemic or fibrinic plasmas (2.1%). One autoantibody was found by EMT only.
CONCLUSION: The EMT is reliably suited to ABO/D grouping, Rh phenotyping, and K testing and is suitable to detect immunoglobulin G red blood cell alloantibodies as well. The rate of false-positive reactions in ABS due to lipemic and fibrinic samples needs to be reduced.     

Evaluation of polyethylene terephthalate for ABO and Rh typing and alloantibody screening

BACKGROUND: For many years, hospitals and laboratories have used evacuated glass tubes for blood collection. To improve the safety of blood collection, plastic polyethylene terephthalate (PET) tubes (Vacutainer PLUS, Becton Dickinson) have been developed. The objectives of this study were to compare the accuracy of ABO grouping, Rh typing, and antibody screening of blood samples collected in plastic tubes with that in glass tubes and to determine if refrigerated blood samples collected in plastic tubes remained stable over a 28-day period. STUDY DESIGN AND METHODS: Samples were collected from 121 volunteers, at least 30 from each of the A, B, O, and AB blood groups, in four types of Vacutainer tubes: silica-coated plastic, K(2) EDTA plastic, uncoated glass, and K(2) EDTA glass. Samples from each tube were tested for ABO group and Rh type by use of the microtyping gel identification card system and the tube method. A three-cell antibody screen was performed by the microtyping gel card technique with a monospecific IgG reagent. Initial samples were tested within 3 hours of collection. Refrigerated samples were retested for ABO and Rh type and antibody screening 1, 2, 21, and 28 days later. Agreement between test results was determined by using Cohen's Kappa statistic. RESULTS: Complete agreement was observed between the ABO and Rh typing results in samples drawn into glass and plastic tubes of both the EDTA and nonanticoagulated type (kappa = 1.0). In retesting, there were no examples of a change in ABO or Rh type over the 28-day study period. Only two alloantibodies (1.7%) were identified in the 121 samples, and no difference was observed in alloantibody expression in either plastic or glass Vacutainer tubes over the 28-day study period. CONCLUSION: Samples collected into the PET serum or EDTA tubes provided accurate ABO and Rh typing results that remained consistent over a 28-day period. Samples collected in these tubes also appeared to enable accurate alloantibody identification. However, the number of alloantibodies identified in this study was small, and this result should be confirmed in a larger series.     

A novel microplate agglutination method for blood grouping and reverse typing without the need for centrifugation

BACKGROUND: Current agglutination tests and solid-phase adherence methods, employed as the techniques for RBC typing and antibody screening, require centrifugation and washing steps. This report describes a novel agglutination method for forward and reverse grouping that is based on the formation of an RBC monolayer on a microplate without the need for centrifugation and washing. STUDY DESIGN AND METHODS: In a comparative study, 2225 samples from healthy regular blood donors were tested for ABO, Rh (D, C, c, E, and e), K, and reverse grouping, in parallel, by the new microplate agglutination method and a commercially available blood testing system, which served as a reference method. RESULTS: In the case of forward grouping, 0.37 percent of samples tested were false negative in the new method and 1.35 percent tested false negative in the reference blood testing system. In addition, the reverse grouping reference method showed 0.4 percent false-positive and 2.6 percent false-negative results. In contrast, the new method gave false-positive results in only 0.09 percent and false-negative results in 0.67 percent of the cases tested. CONCLUSION: These results, as well as the possibility of adapting this method to a fully automated system, suggest that our novel agglutination method could be an important contribution to the field of immunohematology.     

Tetragametic chimerism detected in a healthy woman with mixed-field agglutination reactions in ABO blood grouping

BACKGROUND: The case of a healthy woman with serologic blood group AB and her biologic father showing blood group O was investigated. Further analysis, including blood, buccal swabs, and nail clippings, revealed a tetragametic chimerism. STUDY DESIGN AND METHODS: Blood grouping was performed with standard gel centrifugation test cards, ABO genotyping by sequence-specific primers (SSPs) and sequence-based typing, and HLA Class I and II typing by standard NIH cytotoxicity testing and SSP. Additionally, short-tandem-repeat (STR) and variable-number tandem-repeat (VNTR) typing was performed on blood, nail clippings, and buccal swab samples. The karyotype was analyzed by G-banded chromosomes. RESULTS: The proposita's RBCs were typed AB with a mixed-field agglutination whereas in molecular typing A, B, and O alleles were found. One paternal and two maternal haplotypes were determined by use of HLA typing. Interestingly, both paternal alleles were detected in 4 of 23 tested STR and VNTR loci only, with whole blood, nail clippings, and buccal swabs. The karyotype was identified as 46XX. The family members including the proposita's healthy twin children displayed no abnormal findings in tests performed. CONCLUSION: By investigation of DNA polymorphisms, it was possible to determine a rare case of tetragametic chimerism being the result of double parental contribution of nuclei.     

抗A和抗B血型定型试剂稳定性观察及对亚型检测的影响

目的检测国产单克隆抗A(B)试剂质量,观察对ABO亚型检测的影响。方法测定四个厂家"单抗A、抗B"试剂效价、亲和力等,并检测稀有的ABO亚型红细胞与单克隆试剂反应情况,同时用人源抗A、抗B试剂作对照。结果所有厂商的试剂均符合国家标准,单克隆抗体仍有漏检ABO亚型现象。结论目前市场销售的单克隆抗A、抗B血型定型试剂,质量基本可靠,但应定期测定其效价、亲和力,受检者ABO血型检测必须用ABO反定型试验复核。     

梯形微板法与手工试管法检测ABO血型比较

目的比较两种方法检测ABO血型的准确度以及对AB0亚型、不规则抗体的检出能力。方法通过实验结果的统计，分析POSEIDON全自动数字血型仪梯形微板法与手工试管离心法的差异。结果两种方法对血型判读准确率均为100％，梯形微板法对亚型的检出率为0．022％（3／13555），对不规则抗体的检出率为0．037％（5／13555）；手工试管离心法对亚型的检出率为0．015％（2／13555），对不规则抗体的检出率为0．007（2／13555）。结论两种方法对ABO血型判读准确率均为100％，但微板法对亚型和不规则抗体的检出率高于手工试管法。     

Exchange transfusion the hard way: massive hemolysis following transplantation of bone marrow with minor ABO incompatibility

BACKGROUND: Bone marrow transplantation using donors with minor ABO incompatibility may result in the rapid production of donor-derived red cell isohemagglutinins, causing hemolysis of recipient red cells. CASE REPORT: The transplant of sibling-donor marrow with minor ABO incompatibility (group O donor marrow to group A recipient), using FK- 506 as an immunosuppressant to prevent graft-versus-host disease, is reported. Following early myeloid engraftment, the recipient developed hemolysis of her entire A red cell population between Day 8 and Day 11. This brisk hemolytic anemia was due to rapid donor lymphoid engraftment that resulted in the explosive production of donor-derived anti-A. CONCLUSION: Patients undergoing the transplantation of marrow from donors with minor ABO incompatibility in which the donor cells can produce isohemagglutinins against the recipient's red cells must be kept under vigilant observation for the possible development of severe hemolysis, particularly in the setting of profound T-cell suppression without B-cell suppression.