A blood grouping and antibody screening system using image analysis

Summary: An effective, modular automated blood grouping system has been developed which performs more effectively than the AG16C autogroupers and which uses readily obtainable reagents and disposables. Operating and maintenance costs are low by comparison with currently available equipment of similar capacity. The 99·2% success rate for blood grouping using this image analysis-based system compares well with the 98·3% success rate on the AG16C autogroupers. Staffing requirements are reduced and the flexible modular approach allows this Newcastle system to be used for a range of serological functions, including grouping, antibody screening and screening for antigen donors. The ability to operate with small sample numbers without long start-up times and the relatively low capital cost makes the Newcastle system an option that could be considered by the larger hospitals as well as transfusion centres. The sensitivity of the system could be developed to include compatibility testing as a feature of the system.     

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.     

两株抗M和两株抗N单克隆抗体的血型血清学分析

用人O型红细胞免疫Balb/C小鼠,取其脾细胞与鼠骨髓瘤细胞系Ns-1以PEG为助融剂进行融合。用人的标准谱细胞筛选,一次融合得到2株抗M杂交瘤细胞株(2A1和3F6)及2株抗N杂交瘤细胞株(4E3和7G5),均能稳定地分泌IgM型抗体,与人血清试剂平行鉴定300份血样,测定结果完全一致。4E3单抗完全不被O型M红细胞所吸收,7G5能被吸收。     

不规则抗体导致ABO正反定型不符处理探讨

目的探讨不规则抗体导致ABO正反定型不符原因,并提出解决方法。方法总结分析12例不规则抗体导致ABO正反定型不符血型血清学试验结果,通过不规则抗体筛查、不规则抗体鉴定、相应血型检测明确不规则抗体导致ABO正反定型不符确切原因,用相应抗原阴性红细胞重新进行ABO血型反定型。结果 12例标本中抗-M 4例,抗-N 1例,抗-M合并抗-N 1例,抗-Lea1例,抗-P11例,抗-I 1例,抗-D 1例,抗-E 1例,抗-A11例。用相应抗原阴性红细胞重新进行ABO血型反定型,正反定型结果均相符。结论不规则抗体是导致ABO正反定型不符的常见原因,经过适当处理可以正确鉴定ABO血型。     

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.     

Evaluation of the gel system for ABO grouping and D typing

BACKGROUND: The gel agglutination assay has been approved by the Food and Drug Administration as an alternative to the tube assay for the detection of red cell antibodies. It has also been approved recently by the Food and Drug Administration for ABO blood grouping and D typing. STUDY DESIGN AND METHODS: Tube and gel agglutination assays were compared for ABO grouping and D typing of 100 donor and 100 patient specimens. ABO grouping of 14 specimens of known ABO groups and D typing of 10 specimens with weak D were also compared. When antigen typing or isohemagglutinin results differed, gel testing was repeated by the use of modified incubation times, reagent or specimen volumes, and red cell concentrations. RESULTS: ABO grouping and D typing in all patient and donor specimens concurred. B isohemagglutinins were not detected in seven group A specimens. Six of seven discrepancies were resolved when gel tests were incubated at room temperature with increased serum or plasma volume. Weak D was detected in all 10 specimens tested by both assays. When weak A and/or B were tested with monoclonal antibody reagents, the correct phenotypes were identified in 9 specimens by gel assay and in 10 by tube assay. Using human antisera, 6 specimens were correctly phenotyped by gel assay and 7 by tube assay. CONCLUSION: The gel assay performed as well as the tube assay in detection of A, B, and D, but the tube assay was slightly better at detecting B isohemagglutinins. The gel assay can be used in place of the tube assay for ABO blood grouping and D typing.     

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。     

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.     

A fully automated blood typing system for hospital transfusion services. ABS2000 Study Group

BACKGROUND: The results of routine blood bank testing by a fully automated blood typing system (ABS2000) were compared with those obtained by standard manual methods in six hospital transfusion services. STUDY DESIGN AND METHODS: The ABS2000 system uses microtiter plates for determining ABO and D types, solid-phase red cell adherence (SPRCA) assays for antibody detection, and modified SPRCA plates for IgG crossmatches. The transfusion services used their standard manual test tube methods. RESULTS: Of 3779 donors' samples tested for ABO types (red cell typings only), 3.0 percent could not be interpreted by the ABS2000 system's neural network, because of clots, hemolysis, or lipemic samples. The results for ABO types were concordant for 99.8 percent of the remaining samples. Of 3779 donors' samples tested for D types, the results were concordant for 98.7 percent. Of 7580 patients' samples tested for ABO types (red cell and plasma typings), 5.8 percent could not be interpreted by the ABS2000 system. There was 100-percent concordance of ABO typing results for the remaining 7140 samples. There was 99. 7-percent concordance of results for patients' D types. The results of 96.7 percent of antibody detection tests and 98.8 percent of crossmatches were concordant. Neither method failed to detect a serologically incompatible crossmatch that was associated with a specific, clinically significant alloantibody. The ABS2000 system performed 45 confirmatory donor ABO and D types in 115 minutes, 22 antibody detection tests in 116 minutes, 16 patients' ABO/D types in 149 minutes, and 40 crossmatches in 140 minutes. CONCLUSION: The ABS2000 blood typing system automates routine blood bank tests with accuracy comparable to that of hospital transfusion services' standard manual methods.     

Monoclonal anti-D specificity and Rh D structure: criteria for selection of monoclonal anti-D reagents for routine typing of patients and donors

SUMMARY. The Rh blood group system is the next most important to the ABO system in terms of its clinical significance in blood transfusion. It is vital to the safe, efficient practice of transfusion medicine that Rh D phenotyping tests are selected, executed and interpreted correctly. However, the Rh D blood group antigen has been shown to be subject to many phenotypic variations, and different reagents and typing techniques vary in their ability to detect these variants. The range of D-positive phenotypes are reviewed in terms of their reactivity with monoclonal antibody reagents and their clinical significance. In view of the available evidence, it is suggested that patient typing can be safely achieved by the duplicate use of one high-avidity or two very similar IgM monoclonal anti-D reagents that detect most variants except category D^VI in simple tube or microplate saline tests. Antiglobulin testing for weak D should not be carried out on patient samples. Donor typing can be safely achieved by the use of the same monoclonal, used in parallel with a polyclonal anti-D reagent that detects D^VI on sensitive automated equipment.     

Automated blood typing of patients

The 15-channel Technicon blood typing machine is shown to be equally useful for patient and donor blood typing. It provides increased accuracy as well as increased speed and economy. Test capabilities are broadened by permitting additional simultaneous determinations such as the automated reagin test for syphilis or tests for additional erythrocyte antigens. Automated antibody screening, performed on the blood typing machine in conjunction with patient typing, was found to be of limited use because it failed to detect as much as 60 per cent of some clinically significant antibodies.     

全自动血型分析仪应用于献血者血型筛查

目的探讨并评价全自动血型分析仪应用于献血者血型筛查和盐水不规则抗体检测。方法采用全自动血型分析仪(全自动法)对25 554例献血者标本作ABO及RhD血型鉴定、盐水不规则抗体初筛,并与加样仪加样手工比色法(半自动法)作比对实验。ABO正反定型不一致而无法定型、O细胞凝集、RhD阴性的标本送血型红细胞参比实验室鉴定。结果全自动法与半自动法比较,ABO、RhD阴性血型1次准确定型率:99.93%(25 535/25 554)vs99.95%(25 542/25 554)(P>0.05);O细胞凝集阳性率:0.18%(46/25 554)vs0.10%(26/25 554)(P<0.05),经参比实验室确认,盐水不规则抗体分别为43、24例(0.17%vs0.09%,P<0.05);正反定型不符:17例(0.06%)vs10例(0.04%),参比实验室确认,经参比实验室确认2种方法共同拥有亚型5例,亚型漏检各2例(0.01%),余为正常血型(10/17vs3/10,P>0.05)。结论全自动血型分析仪作ABO、RhD血型筛查的技术相关性好、重复性好;除了全自动化、操作规范化、标准化等优点外,全自动血型分析仪更易发现盐水不规则抗体。     

Automated Duffy Typing Using a Multichannel Blood Grouping Machine

Successful Fy^a blood typing has been achieved by using a multichannel automated blood grouping machine with a filter-paper readout. The Fy^a channel was modified by increasing the temperature of incubation and by using a selected Fy^a antibody in an alkaline polyvinylpyrrolidone-methyl cellulose reagent.     

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.     

Monoclonal antibodies in blood group serology

The state of the art of monoclonal antibody specifications and blending characteristics needed to produce high quality reagents are described for ABO, RhD and polyspecific anti-human globulin (AHG) reagents.Some interesting advances have been made. Thus some murine monoclonal anti-As (e.g. MH04) see Ax. Also, they may show feeble reactions with the traces of A on some group B cells (the B(A) phenomenon) and the quality control of these reagents is discussed. The use of anti-A,B reagents is now debatable as monoclonal anti-A/anti-B reagents are now more reliable than conventional reagents for detecting weak sub-groups of A and B.RhD typing by saline routine and rapid tests can now be performed with IgM monoclonal anti-D reagents (e.g. MAD-2), but the old problem of Du and D variants is discussed as IgM anti-D is not reliable for their detection.Monoclonal anti-C3c/C3d or just selected anti-C3d (BRIC-8) blended with rabbit anti-IgG make excellent potent clean AHG reagents. The essential false positive test of fresh serum incubated with CPD-A1 red cells from donor pack segment lines is essential for the adjustment and evaluation of AHG reagents.Selected monoclonal anti-M, anti-N, anti-Le^a and Le^b are established as excellent typing reagents and monoclonal antibodies have now been developed to e, E, K, k and other red cell antigens.     

Thimerosal dependent agglutination, a newly described blood bank problem

A number of ABO grouping, Rh typing, antibody screening, and antibody identification problems are associated with chemicals in blood bank reagents. We describe a newly discovered agglutination phenomenon due to a thimerosal (Merthiolate)-dependent agglutinin found in the serum of a normal blood donor. Thimerosal is used as a preservative in several low-ionic strength reagents. This agglutination phenomenon is detected only in test systems (low-ionic-strength, albumin, saline, ficin treated test cells) in which test cells are incubated in the presence of thimerosal. Agglutination does not occur in the absence of thimerosal. The thimerosal-dependent agglutinin behaves like an IgG IgG autoantibody. There is no evidence that the thimerosal-dependent agglutinin is responsible for increased red cell destruction.     

Correlation between serology and genetics of weak D types in Denmark

BACKGROUND: To date more than 100 variant D types have been reported and the frequencies vary among populations. Blood donor typing should reveal all donors expressing D antigens, while patient typing should prevent the development of anti-D in patients with a D- or variant D blood type. Serotyping is the standard method to assign transfusion strategies, whereas molecular classification offers a more specific grouping of weak and partial D. STUDY DESIGN AND METHODS: Blood donor and patient samples with discrepant results of D phenotyping were collected to investigate the frequency of weak D subtypes in Denmark and to evaluate currently used serologic methods. RESULTS: Nine different weak D types were identified among the 101 samples. Weak D Types 1, 2, and 3 constituted 80 percent of the analyzed samples and 10 percent of the samples identified as weak D from serology were actually partial D. CONCLUSION: The distribution of weak D types in Denmark was found to resemble the distribution in Northern Germany in respect to the three most prevalent weak D types. Correctly defining all samples that show weak reactions in D typing as weak D or partial D is not possible with serotyping alone; genotyping offers the only exact categorization. Serology is superior for routine blood typing, however.     

A microplate system for ABO and Rh(D) blood grouping

A microplate system for performing ABO and Rh(D) blood group determinations with a Kemble Kemtek 1000SP liquid handling processor, an Anthos 2001 microplate reader, an IBM Personal System 2 microcomputer, and Sanguin Forma software is described. The performance of this Kemble/Anthos/IBM/Sanguin microplate system for ABO and D grouping was evaluated by testing 10,042 routine blood donor samples in parallel with the forward-grouping channels of a Technicon AutoAnalyzer blood grouping system. Manual techniques were used to perform ABO reverse groupings and to resolve all ABO forward- and reverse-grouping discrepancies. D-negative test results were investigated and confirmed manually by the indirect antiglobulin test. Of the 10,042 samples tested, 97.3 percent were grouped correctly. There were 266 samples whose results were flagged as no type determined, of which 124 were ABO tests and 142 were Rh tests. In addition, 30 weak D samples missed by both automated systems were detected by manual indirect antiglobulin test. The system is flexible and easy to maintain and operate.     

PK7300全自动血型分析仪测定ABO血型抗体效价的初步研究

目的：探索使用PK7300全自动血型分析仪测定ABO血型抗体效价的方法,探讨献血者血型检测中与ABO血型抗体效价相关的影响因素及注意事项。方法仪器微板法测定抽样标本的ABO血型抗体效价;仪器微板法及手工试管法测定各血型混合血浆的ABO血型抗体效价;统计分析该中心2013年5～7月检测出的ABO血型抗体效价降低的情况。结果A型献血者与B型献血者的ABO血型抗体效价比较,差异无统计学意义（P＞0．05）,O型献血者的ABO血型抗体效价明显高于A型或B型献血者（P＜0．05）;手工试管法的灵敏度明显优于仪器微板法,而手工试管法二的灵敏度又明显优于手工试管法一（P＜0．05）;ABO血型抗体效价降低的A型献血者明显多于B型或O型（均P＜0．01）,ABO血型抗体效价降低的男性献血者明显多于女性（P＜0．01）。结论仪器微板法测定ABO血型抗体效价操作简便、结果判读客观、重复性好,1∶1～1∶80的稀释比例范围能满足绝大多数标本的测定需要,采用梯度稀释有助于减小测定误差;血型、性别、年龄、亚型等因素可能与献血者ABO血型抗体效价降低相关。     

Developing a databank for multiple transfusion patients: Rh antigen and phenotype distribution among 3000 regular blood donors in Iran

BackgroundRhesus (Rh) blood group system is clinically the most significant protein-based grouping system. The Rh system is of vital importance in blood transfusion, and incompatibility between the donor and recipient leads to alloimmunization. Alloimmunization is commonly seen in multiple-transfusion recipients (e.g. thalassemia patients). There are a few studies about the prevalence of Rh antigens, except for D, in Iran; and regarding the high prevalence of thalassemia in the country, in this study we have determined antigens and phenotypes of the Rh among population of regular blood donors with the aim of developing a detailed Rh databank.Materials and methodsThis cross-sectional study randomly enrolled 3000 regular blood donors from three provinces of Sistan-Balouchestan, Khuzestan and Gilan in Iran, from September 2018 to May 2019. A fully automated system, based on hemagglutination, was used to Rh typing of blood samples.ResultsThe prevalence of Rh antigens were as follows: D: 88.9 %; E: 30.9 %; C: 74.1 %; e: 96.2 %; and c: 72.8 %. The most common antigen and phenotype were "e" and R₁r (DCcee), respectively.ConclusionDue to the high rate of alloimmunization incidence against Rh blood group antigens among multiple transfusion recipients, development of regular blood donor's Rh databank can facilitate extensive matching for the Rh antigens and it likely reduces the risk of alloimmunization.