Drug-induced immune hemolytic anemia investigation: Comparison between tube test and microcolumn agglutination (gel test) for the detection of drug-dependent antibodies in the presence of soluble drug.

OBJECTIVES: Testing for drug-dependent antibodies is traditionally performed with the tube method either with drug-treated red blood cells or with untreated red blood cells in the presence of soluble drug. Gel microcolumn agglutination method was compared to tube testing for the demonstration of drug-dependent antibodies in the presence of soluble drug. MATERIALS AND METHODS: Patient's samples were tested in parallel by tube and gel microcolumn agglutination method with untreated and/or enzyme-treated red blood cells in the presence of soluble drug. RESULTS: Twenty six different patient's samples were studied and thirty nine tests performed to investigate antibodies directed against fifteen different drugs. There was a good correlation between the results obtained by tube and gel method in terms of analytical sensitivity and specificity. Reactions appeared to be stronger with the gel test than seen with the conventional tube method for most of the drug antibodies investigated. Enzyme-treated cells should be used in addition to untreated cells to improve the sensitivity of the method for detecting drug-dependent antibodies especially those directed against drugs that do not bind firmly to red blood cells. CONCLUSIONS: Gel method appeared to be sensitive, reliable, reproducible, and comparable to the conventional tube method for the detection of all the drug-dependent antibodies investigated in this study. Further studies need to be performed to evaluate gel testing for the detection of drug-dependent antibodies that only react with drug-treated red blood cells.

Evaluation of the Ph. Eur. anti-A and anti-B assay on a pipetting robot in combination with a camera guided reading mode.

The main objective of this study was the standardization of the direct anti-A, anti-B haemagglutination assay for immunoglobulin products in microtitre plates and gelcards by automation on a liquid handling robot. In addition, the evaluation of the pipetted microtitre plates with a computer-controlled camera was investigated and these results were related to titres from visual live reading. The titres obtained with the automated and the manual assay in microtitre plates and gelcards were compared. They were in excellent agreement: the titres of samples processed with the automated method varied maximally one titre step relative to the median titres of the same sample analysed with the manual method. The implementation of a camera guided test plate reading further increased the repeatability of the reported titres. In summary, the automated haemagglutination assay combined with a camera improved the consistency plus the traceability of the results and reduced the required hands-on time.

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.

[Sensitivity and specificity of new commercial tests for the detection of specific Echinococcus antibodies]. / Sensitivität und Spezifität neuer kommerziell erhältlicher Tests zum Nachweis von Echinococcus-Antikörpern.

Cystic echinococcosis (CE), caused by Echinococcus granulosus, and alveolar echinococcosis (AE), caused by E. multilocularis belong to the most serious parasitic diseases. Both forms of echinococcosis occur in Austria; in addition, imported cases are diagnosed and treated regularly in Austria. Diagnosis of echinococcosis is based on clinical symptoms, imaging techniques and particularly on the detection of specific antibodies in serum specimens of patients. For decades several companies have been providing commercial Echinococcus antigens and echinococcosis tests based on different methods, i.e. complement fixation test (CFT) and electrophoretic methods (CIEP, IEP) in the past and enzyme-linked immunosorbent assays (ELISA), western blot assays (WB) and indirect haemagglutination assays (IHA) in recent years. During the last years two studies have been carried out in our laboratory in order to evaluate the sensitivity and specificity of two commercial E. granulosus antigens (the synthetic p176 antigen, arc 5 antigen) and three commercial testkits (IHA from Dade Behring, IHA from Fumouze, ELISA from Novagnost-Dade Behring). Sera of patients with histologically and/or molecular biologically confirmed cystic or alveolar echinococcosis, of patients with other parasitic infections and of healthy people were tested comparatively for specific Echinococcus antibodies. The synthetic p176 antigen proved to be a highly specific but a insensitive antigen, whereas both the indirect haemagglutination assay as well as the Novagnost-ELISA showed a much higher sensitivity but only moderate specificity. Our studies demonstrated that neither the commercial antigens nor the test kits tested should be used as a primary test in a routine laboratory for the diagnosis of cystic echinococcosis or of alveolar echinococcosis.

An integrated fiberoptic-microfluidic device for agglutination detection and blood typing.

In this paper, an integrated fiberoptic-microfluidic device for the detection of agglutination for blood type cross-matching has been described. The device consists of a straight microfluidic channel through with a reacted RBC suspension is pumped with the help of a syringe pump. The flow intersects an optical path created by an emitter-received fiber optic pair integrated into the microfluidic device. A 650 nm laser diode is used as the light source and a silicon photodiode is used to detect the light intensity. The spacing between the tips of the two optic fibers can be adjusted. When fiber spacing is large and the concentration of the suspension is high, scattering phenomenon becomes the dominant mechanism for agglutination detection while at low concentrations and small spacing, optointerruption becomes the dominant mechanism. An agglutination strength factor (ASF) is calculated from the data. Studies with a variety of blood types indicate that the sensing method correctly identifies the agglutination reaction in all cases. A disposable integrated device can be designed for future implementation of the method for near-bedside pre-transfusion check.

Comparison of the performance of microtube column systems and solid-phase systems and the tube low-ionic-strength solution additive indirect antiglobulin test in the detection of red cell alloantibodies.

To compare the performance of seven currently available test systems in the detection of erythrocyte alloantibodies (ab), we tested in parallel 446 sera samples containing red cell ab [368 sera samples with ab that are assumed to be clinically significant (cs-ab) and 78 sera samples with ab that are assumed to be of minor clinical significance (ms-ab)] using the tube spin low-ionic-strength solution (addition method) indirect antiglobulin test (tube LISS-IAT), three microtube column agglutination techniques (DiaMed-ID, Ortho BioVue and Bio-Rad Scangel), one affinity adherence test system (CLB/Mast CellBind Screen) and two solid-phase tests [Biotest Solidscreen II and Immucor Capture-R Ready-Screen (4)]. To address the specificity of the three test systems under routine conditions, results of 4566 patient samples obtained using the tube LISS-IAT, results of 5205 patient samples obtained using the Scangel and results of 3560 samples obtained using the Capture-R were evaluated. The DiaMed-ID detected 344 cs-ab and 43 ms-ab, BioVue 333 cs-ab and 48 ms-ab, Scangel 348 cs-ab and 62 ms-ab, CellBind Screen 346 cs-ab and 47 ms-ab, Solidscreen 330 cs-ab and 38 ms-ab, Capture-R 358 cs-ab and 45 ms-ab and LISS-IAT 159 cs-ab and 12 ms-ab. In routine practice, erythrocyte cs-ab could be identified in 61 (67.8%) of 90 reactive sera (specificity: 98.6%) in the tube LISS-IAT, in 169 (58.7%) of 288 (94.4%) in Bio-Rad Scangel and in 101 (51.0%) of 198 reactive sera (94.3%) in Capture-R. We conclude that the sensitivity of the microcolumn, affinity adherence and solid-phase test systems in the detection of cs-ab was similar and was markedly superior to that of the conventional tube LISS-IAT. All high-sensitive test systems produced higher rates of false positives and ms-ab compared to the tube test. An individual cost-benefit analysis, considering the recent knowledge about the clinical significance of weak-reactive cs-ab, should be performed in every institution to decide whether and if so which high-sensitive screening system should be applied.

Detection of weak D with a fully automated solid-phase red cell adherence system.

BACKGROUND: Microplate agglutination methods (MAMs) and column agglutination technology are widely employed for red cell typing and can be automated. Some tests, however, such as detection of weak D, require manual testing. The possibility of detecting weak D by a solid-phase RBC adherence (SPRCA) test was studied in a fully automated system. STUDY DESIGN AND METHODS: The results of 2609 blood samples, characterized as being D- or with a incomplete agglutination reaction, were analyzed for the presence of the weak D phenotype. The 2609 samples were tested by a weak D tube test (antiglobulin method) and a weak D-test with the new SPRCA method. When weak D was detected, which D epitope was involved and whether it was associated with a partial D phenotype were determined. RESULTS: Weak D was detected in 60 (2.3%) of the 2609 samples. The 60 samples that were weak D by the tube test were also weak D+ with the new automated SPRCA test. The sensitivity and specificity for the new weak D typing method were 100 percent, when compared with the standard weak D manual test. CONCLUSIONS: These results support the possibility of performing weak D detection with a SPRCA fully automated system. These preliminary results are encouraging, showing good sensitivity and specificity of the test. Detection of weak D will permit full automation of blood typing.

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.

Detection and quantitation of ABO RBC chimerism by a modified coil planet centrifuge method.

BACKGROUND: Differential agglutination procedures and flow cytometric analysis have been used for detecting and quantitating mixed cell populations. For more than 20 years in our laboratory, a differential agglutination method using the coil planet centrifuge and polyclonal anti-A or anti-B has been used. However, it is now difficult to obtain polyclonal antisera, and it is unknown whether MoAbs can take the place of polyclonal antisera in the coil planet centrifuge method. STUDY DESIGN AND METHODS: Polyclonal antisera and MoAbs were filled into a coil first and then unsensitized packed RBCs from ABO variants, and from chimeras and patients after ABO-incompatible HPC trans- plantation (HPCT) were loaded. After centrifugation, agglutinated and nonagglutinated RBCs were collected, hemolyzed, and subjected to colorimetric analysis for quantitation. RESULTS: ABO chimerism was quantitatively estimated with detection as low as 0.1 percent. ABO variants showed different patterns of agglutination and nonagglutination. The reconstitution status of the erythroid lineage after ABO-mismatched HPCT was also quantitatively evaluated. CONCLUSION: A modified coil planet centrifuge method is established by which ABO chimerism could quantitatively be analyzed and ABO variants identified to the same degree of accuracy as the other differential methods and flow cytometry. The monitoring of ABO chimerism might also help the diagnosis of early relapse or rejection after ABO incompatible HPCT.

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.

Comparison of the performance of four microtube column agglutination systems in the detection of red cell alloantibodies.

BACKGROUND: The purpose of this study was to compare the performance of four currently available microtube column agglutination systems in the detection of red cell alloantibodies to that of the standard tube low-ionic-strength solution (LISS) indirect antiglobulin test (IAT) (tube LISS-IAT). STUDY DESIGN AND METHODS: In a comparative study, 172 sera, previously demonstrated to contain red cell alloantibodies, were tested in parallel by the tube LISS-IAT and three microtube column agglutination techniques (DiaMed-ID, Ortho BioVue, and Sanofi-Pasteur Scangel) and one affinity-adherence test system (Gamma ReACT). Tests were performed simultaneously by a single person on freshly thawed sera that had been frozen at -20 degrees C. RESULTS: The rate of detection of clinically significant alloantibodies (n = 154) in microtube column systems was very similar. One hundred forty-one sera (91.6%) reacted in the DiaMed-ID, 139 (90.3%) in the ReACT, 139 (90.3%) in the BioVue, and 142 (92.2%) in the Scangel. Only 117 (76.0%) of these sera reacted in the tube LISS-IAT. The detection rates for 18 antibodies of minor clinical significance (anti-M, -N, -P1, -Le(a), and -Le(b)) varied among the test systems: DiaMed-ID, 5 (28%); ReACT, 7 (39%); BioVue, 14 (78%); Scangel, 10 (56%); and tube LISS-IAT, 6 (33%). Antibody reactivity as determined by titer and score was very similar in all microtube column systems and higher in these systems than in the tube LISS-IAT. CONCLUSION: The sensitivity of all four microtube column systems in the detection of clinically significant red cell alloantibodies was similar and was markedly superior to that of the tube LISS-IAT. An individual cost-benefit analysis should be performed in every institution to decide whether a microtube column system should be applied. If so, the antibody screen in the microtube column agglutination system should ideally be performed in advance of the crossmatch to provide time to screen for compatible units.

[The characteristics of detecting the tick-borne encephalitis virus antigen in the ELISA and indirect hemagglutination reaction by means of scanning electron microscopy]. / Osobennosti vyiavleniia antigena virusa kleshchego éntsefalita v IFA i RNGA metodom skaniruiushchei élektronnoi mikroskopii.

The surface of polystyrene plates was studied at different stages of the enzyme immunoassay (EIA) and the passive hemagglutination (PHA) test by the method of scanning electron microscopy in the detection of tick-borne encephalitis (TBE) virus antigen. The study revealed that in the process of EIA larger antigens were washed away from the plate surface. The objects detected on the polystyrene surface were identified as conglomerations of the virions of TBE virus, but whole virions were shown to play no decisive role in EIA. The conclusion was made that, due to some specific features of this method, EIA was more sensitive in reaction with small antigens (individual glycoproteids, their small complexes). And, respectively, the PHA test was more sensitive in reaction with large antigenic complexes (whole virions, their conglomerations, immune complexes).

Comparison and simulation of different levels of erythrocyte aggregation with pig, horse, sheep, calf, and normal human blood.

Erythrocyte aggregation levels in pig, horse, sheep, and calf blood samples were investigated and compared to that of normal human blood. The aggregation kinetics and adhesive forces between red cells, and an index of structure of the aggregates were determined with an erythroaggregameter (Regulest, France) at constant hematocrit (0.40 l/l) and temperature (37 degrees C). The adhesive forces and the index of structure in pig blood were close to those of normal human blood. The results for horse blood showed a very high level of aggregation kinetics and adhesive forces between red cells. For sheep and calf blood, little erythrocyte aggregation was found. To simulate different levels of red cell hyperaggregation in humans, a volume of horse plasma was replaced by isotonic NaCl in different proportions (5 to 40% V/V). The kinetics of rouleaux formation and especially the adhesive forces between erythrocytes were systematically decreased, while the index of structure was raised with increasing concentrations of isotonic NaCl. By replacing the porcine plasma with isotonic NaCl, normal and hypoaggregating levels of human red cells were simulated. The aggregation kinetics and the adhesive forces were reduced and the index of structure was raised when the concentration of isotonic NaCl was increased. In summary, large differences in the aggregation parameters were found between mammals. This study also showed that different human erythrocyte aggregation levels can be simulated by diluting the concentration of plasma proteins in equine and porcine bloods.

[The microagglutination test--a simple and sensitive procedure for serodiagnosis of pertussis infections]. / Der Mikroagglutinationstest--ein einfaches und sensitives Verfahren zur Serodiagnostik von Pertussisinfektionen.

The microagglutination assay is a useful method for the diagnosis of B. pertussis infections. In a group of 30 patients with culture proven pertussis 27 (90%) had > or = fourfold increases in titers between acute and convalescent phase serum specimens. The microagglutination test offers several advantages over other more sophisticated B. pertussis antibody tests: only 50 microliters of serum is required, it is a standardized test, which doesn't require specialized technical expertise or equipment, it is easy to perform and good results are noted in a broad age range of patients. Disadvantages of the microagglutination test are: two separate serum specimens are necessary (acute and convalescent phase), the test does not differentiate IgA and IgG antibodies and the temporal association with recent immunization can lead to false positive results. In our opinion the microagglutination test is a useful method for the diagnosis of B. pertussis infections. This is especially true in cases where more sophisticated serologic tests such as ELISA can not be performed immediately but physicians and patients expect to get a result quickly.

Red cell antibody screening, red cell antibody identification and compatibility testing with the Column Agglutination Technology (CAT). The Bio Vue system.

A new system for irregular antibody screening was described in 1993 by Reis K.J. This test is performed in a microcolumn prefilled with glass microbeads in suspension in a neutral or Anti Human Globulin isotonic solution. When the red cells are sensitized they are trapped by the microbead suspension during column centrifugation. 21365 irregular antibody screenings were performed with this Column Agglutination Technology (CAT) and the results were compared to those obtained with conventional manual tests. The CAT was more efficient than the manual tests. The number of positive samples containing specific antibodies was higher with CAT tests (924 samples) than with manual tests (802 samples). The CAT is easy to perform and the elimination of washing steps decreases the overall test time. It allows the use of this test for pre-transfusion compatibility testing particularly in emergency transfusion cases. The red cell age and more generally the red cell storage conditions seem to be an influential parameter on the percentage of unspecific reactions. In this study the unspecific reaction rate was low but we used only reagent red cells prepared every day. This new technology may be considered as an alternative technology to the Gel Test in Blood Transfusion security.

Column agglutination technology: the antiglobulin test.

A new system for typing and screening blood, based on the sieving effect of glass bead microparticles, has been developed. The test is performed in a microcolumn in which the red cell agglutinates are trapped in the glass bead matrix during centrifugation, and unagglutinated cells form a pellet at the bottom of the column. Anti-human globulin reagents were incorporated in the diluent and the new test system, column agglutination technology, was compared to conventional tube tests and low-ionic-strength method. Sera and plasmas (228 samples) were screened for red cell antibodies with two anti-human globulin reagents: one containing only anti-IgG and the other containing both anti-IgG and anti-C3b, -C3d. After initial testing, there was 94-percent agreement between column agglutination technology and tube tests, and after repeat testing, there was 97-percent agreement. The column agglutination technology anti-human globulin test eliminates the need to wash red cells, which decreases the overall test time. The test is easy to perform, and the results are more objective than those with tube and microplate methods.

[Solid phase techniques in blood group serology]. / Solid-Phase-Techniken in der Blutgruppenserologie.

As alternatives to hemagglutination, solid-phase red blood cell adherence assays are of increasing importance. The adaptation of the new techniques to microplates offers several advantages over hemagglutination. Using microplates the assays may be processed semiautomatically, and the results can be read spectrophotometrically and interpreted by a personal computer. In this paper, different red blood cell adherence assays for AB0 grouping, Rh typing, Rh phenotyping, antibody screening and identification, as well as crossmatching will be described.

[Initial results of a prospective study with automated solid phase and column/agglutination techniques in screening for transfusion relevant IgG antibodies]. / Erste Ergebnisse einer prospektiven Studie mit automatisations-fähigen Festphasen- und Säulen-/Agglutinationstechniken beim Screening auf transfusions-relevante IgG-Antikörper.

We report the first results of a prospective study concerning antibody screening in transfusion recipients. We compared the standard tube test (Liss Coombs) with two commercial tests for the detection of red cell IgG antibodies: (1) a column/agglutination test and (2) a microplate/solid-phase system. The results of the study demonstrate several significant advantages of the two methods as compared with the standard tube test. The new methods, especially the microplate test, are superior in determining red cell antibodies which are relevant to transfusion. The two methods are more sensitive and, when automated, more efficient and safer as compared with the standard tube test.