Evaluating automated titre score as an alternative to continuous flow analysis for the prediction of passive anti-D in pregnancy.

OBJECTIVES: To evaluate the potential of the automated titre score (TS) as an alternative method to continuous flow analysis (CFA) for the prediction of the nature of anti-D in pregnancy. BACKGROUND: The 2016 revised British Society for Haematology (BSH) antenatal guidelines recommended a measurement of anti-D concentration by CFA to ensure the detection of potential immune anti-D. Due to high referral costs and resource pressures, uptake has been challenging for hospital laboratories. Serious Hazards of transfusion (SHOT) data have previously shown that this has contributed to missed antenatal follow ups for women with immune anti-D and neonates affected by haemolytic disease of the fetus/newborn. METHODS/MATERIALS: In this multicentre comparative study, samples referred for CFA quantification were also tested by an ORTHO VISION automated anti-D indirect antiglobulin test (IAT) serial dilution and then converted to TS. CFA results and history of anti-D prophylaxis were used to categorise samples as passive or immune, with the aim of determining a potential TS cut-off for CFA referral of at risk patients. RESULTS: Five UK National Health Service (NHS) trusts generated a total of 196 anti-D TS results, of which 128 were classified as passive and 68 as immune. Diagnostic testing of CFA and TS values indicated a TS cut-off of 35 to assist in distinguishing the nature of anti-D. Using this cut-off, 175 (89%) results were correctly assigned into the passive or immune range, giving a specificity of 92.19% and a negative predictive value of 91.47%. CONCLUSION: TS in conjunction with clinical and anti-D prophylaxis history can be used as a viable and cost-effective alternative to CFA in a hospital laboratory setting.

Avaliação da validação da pesquisa de anticorpos irregulares no Hemocentro Regional de Montes Claros / Validation assessment of irregular antibodies investigation at the Regional Blood Center in Montes Claros City / Evaluación de la validación de la búsqueda de anticuerpos irregulares em la Región Hemocentro de Montes Claros

Objetivo: Analizar lavalidación de la detección de anticuerpos irregulares (PAI) mediante el uso del reactivo de control de Coombs en muestras de sangre tomadas de Febrero 2015 a Agosto 2016. Métodos: Estudio de naturaleza observacional, retrospectivo y prospectivo, con los procedimientos técnicos de carácter documental, que se sucederá em Laboratorio de Inmunohematología del Hemocentro Regional de Montes Claros - MG. Resultados: Se observó durante la encuesta que después de la no validación de algunos testes y sucedida la repetición del mismos individuos, no se ha encontrado la validación, por lo tanto requeiendo otra repetición hasta que la validación de la muestra. Esto plantea la posibilidad de interferencia que no sea el conocido y discutido, ya que la repetición se realiza aisladamente el análisis crítico de todos los pasos del proceso. Conclusión: El bajo porcentaje de resultados no validados ratifica la prueba de validación antiglobulínico es un buen método para confirmar el resultado de la búsqueda de anticuerpos irregulares

Objetivo: Analisar a validação da pesquisa de anticorpos irregulares (PAI) através da utilização do reagente Controle de Coombs em amostras sanguíneas coletadas de Fevereiro de 2015 à Agosto de 2016. Métodos: Estudo de natureza observacional, retrospectiva e prospectiva, apresentando procedimentos técnicos de caráter documental, a ser realizado no Laboratório de Imunohematologia do Hemocentro Regional de Montes Claros - MG. Resultados: Foi observado durante a pesquisa que após a não validação de alguns testes e realizada a repetição dos mesmos isoladamente, não foi constatado a validação sendo necessário outra repetição até que essa amostra validasse. Esse fato levanta a possibilidade de outras interferências além das conhecidas e discutidas, uma vez que a repetição foi realizada isoladamente analisando criticamente todas as etapas do processo. Conclusão: O baixo percentual de resultados não validados ratifica que o teste de validação antiglobulínico é um bom método para confirmar o resultado da pesquisa de anticorpos irregulares

Objective: The study's purpose has been to assess the validation of irregular antibodies investigation using the Coombs control reagent in blood samples collected over the period from February 2015 to August 2016. Methods: It is a observational, retrospective and prospective study, which presents technical procedures bearing a documentary character, and that was performed at the Laboratory of Immunohematology from the Regional Blood Center in Montes Claros-MG. Results: During the research, it was observed that after the non-validation of some tests and its repetition was then performed alone; the validation was not verified and once again a repetition was necessary until this sample was defined as validated. This fact raises the possibility of other interferences beyond those both known and discussed; bearing in mind that the repetition was carried out in isolation and also all stages of the process were performed under scrutiny. Conclusion: The low percentage of non-validated results ratifies that the antiglobulin validation test is a good method to confirm the result of the search for irregular antibodies

Paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.

A rapid and simple paper-based elution assay for red blood cell antigen typing by the indirect antiglobulin test (IAT) was established. This allows to type blood using IgG antibodies for the important blood groups in which IgM antibodies do not exist. Red blood cells incubated with IgG anti-D were washed with saline and spotted onto the paper assay pre-treated with anti-IgG. The blood spot was eluted with an elution buffer solution in a chromatography tank. Positive samples were identified by the agglutinated and fixed red blood cells on the original spotting area, while red blood cells from negative samples completely eluted away from the spot of origin. Optimum concentrations for both anti-IgG and anti-D were identified to eliminate the washing step after the incubation phase. Based on the no-washing procedure, the critical variables were investigated to establish the optimal conditions for the paper-based assay. Two hundred ten donor blood samples were tested in optimal conditions for the paper test with anti-D and anti-Kell. Positive and negative samples were clearly distinguished. This assay opens up new applications of the IAT on paper including antibody detection and blood donor-recipient crossmatching and extends its uses into non-blood typing applications with IgG antibody-based diagnostics. Graphical abstract A rapid and simple paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.

Enfermedad hemolítica perinatal causada por anticuerpos anti-M y tratada con inmunoglobulinas intravenosas fetales / Hemolytic disease of the fetus due to anti-M antibodies treated with fetal intravenous immunoglobulin therapy

Presentamos el caso de una mujer de 28 años, con 2 abortos tardíos previos causados por anticuerpos anti-M. En la actual gestación es tratada desde la semana 23 hasta la semana 34 con inmunoglobulinas intravenosas fetales, con resultado satisfactorio. Aunque no hay estudios randomizados y controlados que indiquen que las inmunoglobulinas fetales son efectivas en el manejo de la isoinmunización, pequeñas series de casos sugieren resultados prometedores (AU)

We present the case of a 28-year-old woman with two prior late miscarriages caused by anti-M antibodies, leading to alloimmunization of her previous pregnancies. During this pregnancy, she was successfully treated with intravenous immunoglobulins administered from the 23th to the 34th week of pregnancy. There are no randomized trials to indicate whether the antenatal use of intravenous immunoglobulin is effective in the management of fetal red blood cell alloimmunization. Several case series suggest a beneficial role in preventing severe fetal anemia (AU)

A multicenter study on the performance of a fully automated, walk-away high-throughput analyzer for pretransfusion testing in the US population.

BACKGROUND: Moving to automation is a major focus of transfusion centers. Erytra (Grifols) is a walk-away analyzer with high-performance and -throughput capacity for pretransfusion testing. Efficiency and performance of Erytra with its cards and reagents were evaluated in comparison to Food and Drug Administration (FDA)-approved reference methods. STUDY DESIGN AND METHODS: A total of 5279 blood samples (46% patients; 54% donors) were obtained from US blood establishment facilities. Samples were analyzed with Erytra and results were compared with the routine FDA-licensed automated platforms used by the clinical study sites. A total of 25,217 tests were performed (15,322 ABO/D/reverse typing; 4916 Rh phenotypes, 669 K typing, 838 antibody screens, 759 antibody identifications, 250 cross-matches, 244 ABO compatibilities by immediate-spin cross-match, and 219 direct antiglobulin tests [DATs]). RESULTS: Global agreement between Erytra and the comparison platforms was 99.66%, with 99.82% positive percent agreement (95% lower confidence bound [LCB], 99.75%) and 99.50% negative percent agreement (95% LCB, 99.37%). There were 85 discrepancies (0.34%), including cross-matches (n = 13), antibody screens (n = 10), antibody identifications (n = 21), and DATs (n = 5), whereas an excellent concordance was obtained in blood grouping determinations (ABO/D/C/E/c/e/K, 0.04%-0.22% discrepancies). Analysis of the discrepancies showed that Erytra provided the correct result in 51 of them (60%), with only five false negatives (one O patient transplanted with A, one mixed-field reaction in a very weak D, one anti-Vel, two A2rr). Erytra results were 100% reproducible in a series of 3760 repetition tests. CONCLUSION: Grifols' Erytra analyzer showed reliable efficacy compared with equivalent FDA-licensed reagents and FDA-cleared instruments.

Should a positive direct antiglobulin test be considered a prognostic predictor in chronic lymphocytic leukemia?

BACKGROUND: The clinical course of patients with B-cell CLL is often complicated by autoimmune phenomena. The DAT might be positive at some time during the course of the disease in up to 35% of cases. The aim of this retrospective study was to investigate the relationship between the occurrence of a positive DAT and biological features of CLL patients. PATIENTS AND METHODS: In our institution, 146 untreated patients with CLL were studied using the DAT. RESULTS: According to the statistical analysis, a high level of ß2-microglobulin and unmutated IgHV emerged as factors significantly related to the presence of DAT positivity. Time to first TFS was significantly shorter in DAT-positive patients. The adverse effect of a DAT positive result was maintained in terms of TFS when patients with mutated IgHV status were excluded from statistical analysis. CONCLUSION: These results suggest that the DAT might provide additional prognostic information regarding patients with IgHV unmutated status.

Multi-centre evaluation of pre-transfusional routine tests using 8-column format gel cards (DG Gel®).

BACKGROUND: Advances in immunohaematology laboratory practice to improve performance, cost-effectiveness and patient safety are desirable. OBJECTIVES: To perform a multi-centre evaluation of the 8-column Grifols DG Gel(®) cards and reagent system to assess its performance, suitability and adaptability to the daily blood transfusion laboratory routine in the United Kingdom. METHODS/MATERIALS: A total of 4281 immunohematological analyses {1825 ABO/D grouping, 1921 antibody screening, 75 Rh phenotyping and K antigen determination, 361 antibody identification and 99 neonates [ABO/D and DAT (direct anti-globulin test)]} were performed on 2255 specimens. All cases were run in parallel with the reference method of each laboratory (DiaMed-ID(®) cards or conventional tube technique in some cases). RESULTS: Concordant results between Grifols DG Gel(®) system and the reference method were obtained in 97·7% of tests. For ABO grouping by the Grifols DG Gel(®) system, sensitivity was 99·95%, specificity was 99·96%, predictive positive value (PPV) was 99·89% and predictive negative value (PNV) was 99·98%. For D grouping, sensitivity was 99·78%, specificity was 100%, PPV was 100% and PNV was 99·78%. For antibody screening, sensitivity was 90·63%, specificity was 99·94%, PPV was 99·32% and PNV was 99·15%. Of the Rh subgroups and K types, results were 100% concordant. For antibody specificity detection, accuracy was 96·95% for Grifols DG Gel(®) system and 95·29% for DiaMed-ID(®) system. For the newborn tests, concordant results were obtained in 100% of ABO/D grouping and in 89·9% of DAT. CONCLUSION: The Grifols DG Gel(®) 8-column system is reliable and safe for routine tests performed in the immunohaematology laboratory.

Antibody identification using both automated solid-phase red cell adherence assay and a tube polyethylene glycol antiglobulin method.

BACKGROUND: Because antibody identification is labor-intensive, facilities with high volume and/or marginal technical support could benefit from partial automation. STUDY DESIGN AND METHODS: After a validation study of automated solid-phase red cell (RBC) adherence assay (SPRCA; Galileo, Immucor) compared to tube polyethylene glycol antiglobulin antibody identification method (t-PEG), we evaluated Galileo followed by select RBC panels by t-PEG. Of 298 consecutive samples in which antibody identifications were performed in a 2-month period, 160 samples were examined by both Galileo and t-PEG. RESULTS: There were concordant results between Galileo and t-PEG in 120 of 160 (75%) samples including cases with identical alloantibody identification (n = 99), panagglutinin (n = 9), and negative results (n = 12). Of the samples in which alloantibodies were identified, 99 of 108 (91.7%) were identical. In 9 samples with discrepant antibody identifications, 2 samples showed alloantibody specificity by Galileo (possible anti-K and anti-Jk(b)) but were negative by t-PEG. These antibodies were identifiable by t-PEG in subsequent samples. One sample showed anti-E by Galileo, while t-PEG revealed anti-Fy(a) and -E. Five samples showed alloantibody specificity by t-PEG and nonspecific reactivity or panagglutinin by Galileo. These included samples with anti-C (n = 2), anti-E (n = 2), and anti-Fy(a) (n = 1). One sample showed anti-E by t-PEG but was negative by Galileo. Galileo found a panagglutinin in 23 samples and nonspecific reactivity in 22 samples, whereas t-PEG found a panagglutinin in 12 samples but no nonspecific reactivity. CONCLUSIONS: Automated Galileo solid-phase red cell adherence assay can be a useful adjunct for antibody identification, although it detects more nonspecific reactivity than does t-PEG.

Antibody screening in repeatedly transfused patients.

The purpose of pretransfusion compatibility testing is to prevent immune mediated hemolytic transfusion reactions. Our study aimed to evaluate the gel test for detection of clinically significant antibodies in repeatedly transfused patients. We investigated 200 thalassemic patients in whom, blood group, Rh-D, Rh phenotype determination, antibody screening and identification were done using an ID Microtyping System. Red cell alloantibodies were detected in 21 patients (10.5%). Among these patients, Anti-E was detected in 5 (23.8%), anti-D in 4 (19%), anti-K in 4 (19%), anti-e in 3 (14.3%) and each of anti-Fy(a), anti-Js(a), anti-Lu(a), anti-N and anti-s in one patient (4.8%). Anti-E showed the highest frequency in the 21 positive cases that developed clinically significant antibodies. The study revealed statistically significant correlation between development of transfusion reactions, frequency of blood transfusion and the duration of blood transfusion with the incidence of development of clinically significant alloantibodies. It is concluded that the gel test is an easy, quick and reliable method for detecting clinically significant antibodies. Antibody screening and identification is recommended prior to transfusion to detect if there is blood group incompatibility other than the ABO and Rh.

A comparison of conventional tube test and gel technique in evaluation of direct antiglobulin test.

In vivo coating of red cells by antibody and/or complement is detected using various sensitive techniques, however most hospitals even today rely on the conventional tube technique (CTT). We compared the performance of the CTT and recently introduced gel test (GT) in the evaluation of direct antiglobulin test (DAT). The CTT and GT were first compared using in-house prepared control cells. The polyspecific DATs were performed simultaneously by CTT and GT on 170 consecutive blood samples. Positive samples were further tested for monospecific IgG and C3d by both techniques. GT demonstrated stronger agglutination scores (60 vs. 43) compared to CTT using control cells. The sensitivity and specificity of the GT was 98.4 and 95.2%, respectively as compared to CTT for polyspecific DAT. Discordance between the two test systems was seen in 6/170 patients. Of these, 5 were missed by CTT while GT failed to detect in vivo coating in only 1 case. The agreement between two methods of DAT was 96.4% (kappa = 0.926) using polyspecific AHG and 95.7% (kappa = 0.379) with monospecific anti-IgG. We conclude that GT is a better alternative to CTT for detecting red cell bound antibodies in various clinical conditions.

Use of gel microcolumn assay for the detection of drug-induced positive direct antiglobulin tests.

Drugs can result in broad variety of hematologic abnormalities including positive direct antiglobulin test. In this study, we evaluated gel microcolumn assay for the detection of drug-induced antibodies. Direct antiglobulin test was performed by conventional tube and by gel microcolumn assay in 139 hospitalized patients. Drug in vitro studies were done in 34 patients with positive direct antiglobulin test by tube test and gel microcolumn assay using serum and eluate. None of them had signs of hemolytic anemia. A total of 1,000 blood samples from donors were used as control group. Gel microcolumn assay was more sensitive than in tube test for direct antiglobulin test (P<0.01). Positive direct antiglobulin test was more frequent in patients than in donors (P<0.01). Drug in vitro studies were positive with at least one drug in 76.5% of patients with positive direct antiglobulin test by immune complex and/or adsorption mechanisms. We found a high incidence of positive drug in vitro tests in positive direct antiglobulin test patients. Gel microcolumn assay showed appropriate results for drug in vitro studies. The combination of tube and gel microcolumn assay can improve detection of drug-induced positive direct antiglobulin tests.

Gel card in the diagnosis of autoimmune haemolytic anemia.

The diagnosis of autoimmune haemolytic anaemia (AIHA) requires the establishment of haemolysis and demonstration of autoantibodies against red cells. Most laboratories use the conventional Coomb's test for the demonstration of the autoantibodies. However, in approximately 2-6% of the patients who present with the clinical and haematological features of AIHA, the direct agglutination test is negative on repeated testing. Attempts are therefore being made to identify a test which could be more sensitive than the conventional test, yet retaining the simplicity and cost effectiveness of the test. In the present study, the efficacy of the newly developed gel card test has been compared with the conventional Coomb's test for detection of autoantibodies in 50 cases clinically suspected to have haemolytic anemia. The gel card picked up the antibodies in all the cases detected to be positive by the conventional test. In addition, the gel card also picked up 5 tests which were negative by the conventional method. The sensitivity and specificity of the gel card Direct Coomb's test (DCT) as compared to the conventional tube test for DCT was found to be 100% and 95.1% respectively. The Indirect Coomb's test (ICT) was 100% sensitive and 92.5% specific. In view of the high sensitivity and specificity and the simplicity of the procedure, this test may be effectively used for diagnosis of AIHA.

Evaluation of 2-column agglutination versus conventional tube technique for antibody screening.

The study aimed to determine the specificity and sensitivity of the Ortho BioVue two-column agglutination system for the detection of low concentrations of clinically significant antibodies in serum. The BioVue system was compared with the conventional tube technique (LISS-Coombs indirect antiglobulin test), and the two-stage Papenzyme test was used to resolve discrepancies between the two methods. We tested 3000 serum samples from randomly selected patients at King Hussein Medical Centre. Both the antibody screening and identification gave negative results in 2952 patients and positive results in 48 patients. We found the BioVue system to be the more sensitive technique. However, if papain enzyme-treated cells were included in the conventional tube technique when applied to antibody screening and identification, both methods would be of comparable sensitivity.

Comparison of low ionic diluents for use with the Diamed antiglobulin gel test.

Microtube column systems, although widely used in transfusion serology for the detection of red cell antibodies, may not detect weak Fy(a), Jk(a), S and K antibodies. A number of low ionic diluents are used to shorten the incubation time required for red cell antibody detection in the antiglobulin test. However, there are no published reports to show whether these low ionic diluents vary in their ability to detect red cell antibodies using microcolumn detection systems. Three low ionic diluents, Diamed ID-CellStab, Diamed ID-Diluent2 and an in-house produced low ionic strength solution (LISS), were assessed using the Diamed-ID LISS/Coombs microtube column system (in accordance with the manufacturer's instructions), to ascertain whether the choice of diluent influences red cell antibody detection. Two hundred and seventy patient samples were screened for red cell antibodies. The reaction strength was increased in 50% of the samples with detectable red cell antibodies using LISS as the diluent compared with ID-CellStab. Of the 51 red cell antibodies directed against Rhesus, Duffy, Kidd or Kell antigens, 21% reacted more strongly in LISS compared with Diamed ID-CellStab with a difference in grading of > or =1. Minimal disparity was found between ID-Diluent2 and LISS. Biochemical analysis of pH, osmolality, sodium, potassium and phosphate were comparable for ID-CellStab, ID-Diluent2 and LISS. Measurement of conductivity in each low ionic diluent was performed as a measure of ionic strength in the final reactant mix, as the same amount of low ionic diluent was used for each test. The conductivity was 3 x 5 mS cm for LISS and ID-Diluent2, and 5 x 8 mS cm for ID-CellStab; the acceptable range being 3 x 7 +/- 0 x 3 mS cm as cited in the Guidelines for the Blood Transfusion Services in the United Kingdom. This evaluation suggests that ID-CellStab is a suboptimal low ionic diluent for red cell antibody detection using Diamed-ID LISS/Coombs gel cards. The poorer performance of ID-CellStab compared with LISS may be explained by its higher ionic strength.