Gel Test Application for IgG Subclass Detection in Auto-Immune Haemolytic Anaemia

Background and objectives: Commercially available gel test microtubes are not available with antisera for the determination of IgG subclasses. The aim of this study was to adapt the gel technique for this purpose and apply it to the investigation of patients with autoimmune haemolytic anaemia (AIHA). Materials and methods: We studied 66 red cell samples from 49 patients with AIHA of the warm-active IgG type. Standard serologic and haematologic methods were used. We adapted the DiaMed gel test by using IgG-subclass antisera. Results: We found the adapted test useful in determining the subclass of autoantibodies in eluates. We could identify the IgG subclass in all the AIHA patients, even those that were ‘Coombs-negative’, with less than 200 IgG molecules bound in vivo per red cell. Comparison of the gel test with the standard spin tube test and the microtiter plate test showed the superiority of the gel test, i.e., detection of IgG subclasses was much better than with the tube test and the results were more clear-cut than those of the microplate test. The gel test is also the simplest and least time-consuming and permits a later reading of results. Application of the test in our 66 samples confirmed that IgG1 was the most frequent (96%). In 59% of the cases it was accompanied by IgG of other subclasses. Multiple subclasses were most common in the cases with stronger in vivo IgG red cell sensitization and severe haemolysis. Accompanying IgG3 was detected only in patients with obvious haemolysis. Conclusion: The gel test is more sensitive than other procedures for the determination of IgG subclass and has the advantages of simplicity, rapidity, low cost, and stability of the agglutinates.     

A Quantitative Assay for Subclassing IgG Alloantibodies Implicated in Hemolytic Disease of the Newborn

Traditionally, IgG subclassing has been performed using qualitative assays. Quantitation of IgG subclasses may have prognostic value in evaluating alloimmunized pregnancies. A quantitative enzyme-linked immunosorbent assay (ELISA) was implemented for measuring IgG subclasses of red blood cell (RBC) antibodies (AB) isolated by adsorption/elution from the sera of alloimmunized pregnant women. The assay is a sandwich enzyme immunoassay using monoclonal antibodies specific for the relevant IgG subclasses and anti-human IgG peroxidase conjugate to quantitate the amount of bound IgG. The sensitivities of the assay for IgG1, 2, 3, and 4, respectively, were 4,23,4 and 2 μg/l. The results for each subclass for a given AB were expressed as a percentage of the total. In a series of pregnant mothers with ABs: E (4), Fy^a (2), Jk^a (1) and S (1), the mean percentage ± 1SD of each subclass was: IgG1 61±34; IgG2 14±22; IgG3 18±28 and IgG4 4±17. IgG1 or IgG3 accounted for greater than 50% of the AB subclass distribution in 5 cases that resulted in hemolytic disease of the newborn (HDN). Although only a small number of samples was studied, changes in the concentrations of IgG1 or IgG3 during gestation suggest a correlation with the presence or absence of HDN. The ELISA may be used to quantitate the IgG subclasses of RBC ABs and may be valuable in predicting the severity of HDN in alloimmunized pregnancies.     

Platelet-Associated IgG in Thrombocytopenia: A Comparison of Two Techniques

The results obtained in the analysis of 130 thrombocytopenic patients with a radioimmunoassay (RIA) for platelet-associated IgG (PA-IgG) and the platelet suspension immunofluorescence test (PIFT) were compared. The RIA was positive in 33 of 41 (82.9%) patients with idiopathic thrombocytopenia (ITP) and in 51 of 79 (64.4%) patients with secondary thrombocytopenia (STP). The PIFT was positive in 37 of the 41 (90.2%) ITP patients and in 57 of the 79 (72.2%) STP patients. Sensitivity and specificity for the diagnosis of ITP of both tests were comparable: 82.9 and 40.9% for the PA-IgG(RIA) and 90.2 and 36.7% for the PIFT. A significant positive correlation was observed between the mean amount of PA-IgG measured and the height of PIFT scores with anti-IgG. Of 38 discrepancies between PA-IgG(RIA) and PIFT with anti-IgG, 15 were due to borderline results, 17 were associated with abnormal platelet-size distribution and 20 were associated with occurrence of IgM antibodies. These results suggest influences of platelet fragments and/or aggregates on accurate measurement of PA-IgG. Both fragments and aggregates escape from accurate platelet counting, while their contribution to the total IgG content remains. Therefore, a falsely elevated PA-IgG (RIA) may be measured.     

Haematopoiesis in Busulphan-Treated Mice: A Comparison between Two Different Stem Cell Assays

The effect of busulphan on haematopoiesis was investigated in mice. Bone marrow and blood cells were sampled 2 h to 55 d after a single treatment with busulphan. The effect on progenitor cells was examined with the diffusion chamber (DC) technique and the spleen colony assay. Busulphan had a severe depressive effect on progenitor cells in bone marrow, and their number was reduced to 0.01–0.1 % of the control value on day 5–6. During the first 2 d the CFUs were reduced more than granulocyte progenitors as determined in the DC system. Histological examination of spleen colonies showed that in the same interval erythroid colony number was more depressed than granuloid colony number. This situation reversed after 4–6 d. Our interpretation is that busulphan affects all types of progenitor cells irrespective of their proliferative state. However, multipotent CFUs in G_o-phase are more depressed than granulocyte progenitor cells (DC), which have a higher proliferative rate. The selective effect on erythroid colonies during the initial period might indicate that busulphan impairs the ability of multipotent CFUs to erythroid differentiation. This damage was repaired within a few days. An abortive regeneration of bone marrow cells was observed on day 7–10. This can best be explained by an inflow from a cell pool intermediate between stem cells and early precursor cells, with some degree of ‘stem-cellness' (self-sustaining capacity).     

A Two-Stage Agglutination Test for the Detection of Antithrombocyte Antibodies

Abstract. A two-stage agglutination test is described, in which the synergistie action of autoantibodies and heteroantibodies in subagglutinating doses is utilized for the detection of thrombocyte autoantibodies. The test is more sensitive than the direct agglutination test for detecting thrombocyte agglutinins. It also detects 'incomplete' antithrombocyte antibodies with high reproducibility. The two-stage agglutination test gave positive results in 11 of 21 cases of ITP while the direct agglutination test was positive in only 3 of these cases. It is simple and easy to perform, as it utilizes very small quantities of thrombocytes and requires no washings of the thrombocytes. The direct variation of this test is discussed.     

Erythrocyte Antibody Screening in Solid-Phase: A Comparison of Two Solid-Phase Microplate Assays with the Indirect Antiglobulin Test in Polyethylene Glycol for the Detection of Irregular Erythrocyte Antibodies

The screening of a serum for irregular erythrocyte antibodies in the indirect antiglobulin test is a well-established technique. We compared the test results of two different solid-phase microplate indirect antiglobulin tests with a liquid-phase indirect antiglobulin test in tubes. Antibody screening with both solid-phase microplate techniques proved to be more sensitive than the liquid-phase indirect antiglobulin test. In addition, a difference in sensitivity between the two solid-phase techniques was observed: prior immobilization of test erythrocytes on the microplate followed by incubation with a serum and detection of sensitization with antihuman IgG-coated detector cells gave better test results than secondary immobilization on the microplate of test erythrocytes sensitized with antibodies and an antihuman globulin serum.     

Evaluation of Polysaccharide-Based Latex Agglutination Assays for the Rapid Detection of Antibodies to Burkholderia pseudomallei

Melioidosis is a severe disease caused by the Gram-negative bacterium Burkholderia pseudomallei. Diagnosis of melioidosis currently relies on the isolation of B. pseudomallei from clinical samples, which can take several days. An indirect hemagglutination assay (IHA) is widely used for serodiagnosis, but it has a short shelf life, is poorly standardized, and requires a viable bacteria culture performed in a biosafety level 3 (BSL-3) laboratory. To improve the diagnostic methods, we have developed two rapid latex agglutination tests based on purified B. pseudomallei O-polysaccharide (OPS) and capsular polysaccharide (CPS) antigens. The immunodiagnostic potential of these tests was evaluated using serum from culture-confirmed melioidosis patients (N = 143) and healthy donors from either endemic (N = 199) or non-endemic areas (N = 90). The sensitivity of the OPS-based latex agglutination assay (OPS-latex; 84.4%) was significantly higher than both the CPS-latex (69.5%) (P < 0.001) and IHA (69.5%) (P = 0.001). When evaluated with Thai donor serum, the OPS-latex had comparable specificity (56.9%) to the CPS-latex (63.8%) (P = 0.053), but was significantly lower than the IHA (67.6%) (P = 0.002). In contrast, all tests with U.S. donor serum were highly specific (≥ 97.8%). These results suggest that polysaccharide-based latex agglutination assays may be useful for serodiagnosis of melioidosis in non-endemic areas.     

Qualitative and Semi-Quantitative Comparison of an Rk39 Strip Test and Direct Agglutination Test for Detection of Anti-Leishmania Donovani Antibodies in the Sudan

Background: Until now, the comparison of the rK39 strip test (RKT) and direct ag-glutination test (DAT) for detection of visceral leishmaniasis (VL) is exclusively based on either positive or negative qualification of the reaction outcome. Objective: In this study, we compared the diagnostic performance of RKT and DAT for VL both qualitatively and semi-quantitatively. Methods: For comparison based on semi-quantitative grounds, the execution of RKT and DAT was according to the standard procedures. For comparison on semi-qualitative grounds with DAT, the RKT was ap-plied to aliquots from positive samples that were two-fold serially diluted in saline to determine, as for the DAT, the end-point reaction in RKT. Results: While qualita-tively both RKT and DAT demonstrated comparable reliability for VL detection (sen-sitivity = 96% and specificity = 98.7% or 99.3%), no significant correlation (r = 0.13) could be established between intensities of their positive reactions in 25 cases studied. A negative correlation was further determined in those 25 VL cases between the posi-tive intensities of the RKT and antibody levels measured semi-quantitatively with the same procedure (r = -0.36) or the DAT (r = -0.30). Irrespective of the low, moderate or high antibody levels measured with RKT (<1:8 and 1:16-1:32 >1:256) or DAT (< 1:25,600 and 1:51,200- 1:409,600 > 1:3,276,800) in patients with confirmed or uncon-firmed VL infection, exclusively strong positive intensities were obtained with RKT. Conclusion: For further optimizing diagnosis and simultaneously assessing magni-tude of immune response to L. donovani infection in Sudanese patients, the combined application of RKT and DAT is recommended.     

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

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

Two decades of haemophilia treatment in the Netherlands, 1972–92

Summary Four questionnaire surveys were conducted over a period of 20 years to evaluate long-term effects of haemophilia treatment in the Netherlands. The response to the prestructured questionnaires in 1972, 1978, 1985 and 1992 varied between 70% and 84%. Data concerned treatment modalities, bleeding episodes, hospitalization, absenteeism, joint impairment and employment. Results over the period 1972–92 for patients with severe and moderately severe haemophilia showed that the use of prophylaxis had sharply increased (from 21% to 45%), as was the case for home treatment (from 4% to 62%). Consequently, the annual mean number of bleeds diminished from 19 to 13. Absence from school was markedly reduced (from 32 to 5 days), and sick leave in employed patients had also diminished (from 26 to 22 days). Furthermore, the use of inpatient hospital facilities, as well as employment in haemophilia patients, had nearly equalled that of the general Dutch male population. The self-reported degree of joint impairment showed no overall improvement, but in patients aged under 35 years there seemed to be a slight reduction in severe impairment. Patients aged under 15 years finally had no severe impairment at all. Social participation can only be further improved if arthropathy is prevented from an early age. Therefore adequate prophylactic regimens and close monitoring of joint impairment in young adults are needed.     

Determination of heparin–platelet factor 4–IgG antibodies improves diagnosis of heparin-induced thrombocytopenia

Only a few patients with heparin-induced antibodies develop heparin-induced thrombocytopenia (HIT). In this study, we investigated whether different immunglobulin classes can be used to differentiate between antibody-positive patients with and without HIT. Four different patient populations were investigated: 32 patients with the immune type of HIT with thromboembolic complications, 13 patients with HIT without thromboembolism, 24 patients with heparin-platelet factor 4 (PF4) antibodies without clinical symptoms of HIT, and 20 heparin-treated patients with thrombocytopenia caused by other reasons. In all patients the immunglobulin mixture of IgG, IgM and IgA, and the single immunglobulin classes of heparin-PF4 antibodies, were investigated. No significant differences between HIT patients with thromboembolic complications and patients with isolated HIT were found concerning the different immunglobulin classes. Antibody-positive patients with HIT had significantly higher levels of IgG antibodies than those without HIT (P < 0.05), while they did not differ concerning IgM and IgA antibodies. By determining IgG antibodies, the specificity of the enzyme-linked immunosorbent assay (ELISA) system was increased without loss of sensitivity. Heparin-PF4-IgG antibodies can identify patients at risk of developing life-threatening HIT. Future ELISAs should only include this immunglobulin class, as the determination of the antibody mixture may lead to overestimation of HIT.     

A Test in Context: Interpretation of High-Sensitivity Cardiac Troponin Assays in Different Clinical Settings

High-sensitivity cardiac troponin (hs-cTn) assays have the ability to detect minute troponin concentrations and resolve minor changes in biomarker concentrations. Clinically, this allows for the ability to rapidly identify or exclude acute myocardial injury in the setting of acute chest discomfort—thus providing more rapid evaluation for acute myocardial infarction—but the improvements in troponin assays also create avenues for other applications where troponin release from the cardiomyocyte might confer prognostic information. These situations include cardiovascular risk assessment across a wide range of clinical circumstances, including apparently-well individuals, those at risk for heart disease, and those with prevalent cardiovascular disorders. The optimal hs-cTn threshold for each circumstance varies by the assay used and by the population assessed. This review will provide context for how hs-cTn assays might be interpreted depending on the application sought, reviewing results from studies leveraging hs-cTn for applications beyond “acute myocardial infarction diagnostic evaluation.”     

Comparison of the effects of ANF 99–126 and ANF 103–126 on captopril-induced renin release in man

OBJECTIVE: The aim was to examine the effects of atrial natriuretic factor (ANF) 99-126 and ANF 103-126, an N-terminal shortened analogue of the peptide, on the plasma renin activity response to captopril, an inhibitor of angiotensin-converting enzyme. DESIGN: Two protocols were performed. In the first protocol, subjects were studied on three occasions. Captopril 25 mg was given and a 60 minute infusion of 5% D-glucose (placebo), or ANF 99-126 3 or 10 pmol/kg/min, was administered in a single blind randomized manner. The second protocol was divided in two parallel phases comparing ANF 103-126 either 3 or 10 pmol/kg/min to placebo. SUBJECTS: Thirty-three salt-replete healthy male volunteers aged 21-39 years were studied in the supine position. MEASUREMENTS: Plasma renin activity, plasma ANF 99-126 and ANF 103-126 levels, heart rate and blood pressure were measured. RESULTS: Compared to placebo infusion, the rise in plasma renin activity after captopril was attenuated by ANF 99-126 infusion (from 755% of baseline to 294% by ANF 99-126 3 pmol/kg/min and from 755 to 202% by 10 pmol/kg/min; P less than 0.03 and P less than 0.01 respectively). The comparable findings with ANF 103-126 were 492 to 218% (3 pmol/kg/min) and 645 to 364% (10 pmol/kg/min) (P less than 0.01 and P less than 0.01 respectively). CONCLUSIONS: The results, taken in conjunction with previous findings, suggest that atrial natriuretic factor inhibits in a non-selective manner the renin response to all secretagogues so far tested in man. The current results also suggest that the anti-renin action of atrial natriuretic factor does not depend on the first four N-terminal amino acids of the native peptide.     

Comparison of Commonly Used Assays for the Detection of Microalbuminuria

There are a variety of methods for assessing urinary albumin excretion, extending from the very low-range microalbuminuria to higher ranges extending into macroalbuminuria or proteinuria. The recommendation for the initial screening of a new patient is to use a urine dipstick to assess for microalbuminuria. If positive, a spot urine for albumin:creatinine should be measured and reassessed annually. All patients with kidney disease, diabetes, or hypertension and metabolic syndrome should be screened for albuminuria. New methodologies using high-performance liquid chromatography are much more sensitive and specific when compared with older methods of detection and may prove very useful for earlier identification of high-risk patients. This is important since studies have shown that albuminuria levels below the microalbuminuria range, determined by conventional methodologies in uncomplicated essential hypertensive men, are associated with an adverse cardiovascular and metabolic risk profile. High performance liquid chromatography methodology, in contrast to older studies, detects all intact albumin and enables clinicians to assess disease severity and monitor therapeutic effectiveness with confidence in the accuracy of the microalbuminuria data reported to them.     

Performance Evaluation of a Particle Agglutination Test for Antibody to Human Immunodeficiency Virus 1: Comparison with Enzyme Immunoassay

A performance evaluation of a particle agglutination test (PAT), manufactured by Fujirebio Inc., Japan (Serodia-HIV), for antibody to human immunodeficiency virus type 1 (anti-HIV-1) was carried out and compared with a currently available enzyme immunoassay (EIA), manufactured by Genetic Systems Corp., USA, (HIV-1/HIV-2 EIA). Testing 2,878 Indian donor and patient samples, both tests showed 100% sensitivity and comparable specificity (PAT: 99.8%; EIA: 99.7% among donor samples). We conclude that PAT is a specific and sensitive test for anti-HIV-1; it is simple to perform and does not require sophisticated equipment. Hence it is suitable for mass screening of blood donors in a developing country like India, especially in rural areas where presently no HIV-testing facilities are available.     

Comparison of Serological Assays for the Detection of SARS-CoV-2 Antibodies

SARS-CoV-2 virus was first detected in late 2019 and circulated globally, causing COVID-19, which is characterised by sub-clinical to severe disease in humans. Here, we investigate the serological antibody responses to SARS-CoV-2 infection during acute and convalescent infection using a cohort of (i) COVID-19 patients admitted to hospital, (ii) healthy individuals who had experienced ‘COVID-19 like-illness’, and (iii) a cohort of healthy individuals prior to the emergence of SARS-CoV-2. We compare SARS-CoV-2 specific antibody detection rates from four different serological methods, virus neutralisation test (VNT), ID Screen^® SARS-CoV-2-N IgG ELISA, Whole Antigen ELISA, and lentivirus-based SARS-CoV-2 pseudotype virus neutralisation tests (pVNT). All methods were able to detect prior infection with COVID-19, albeit with different relative sensitivities. The VNT and SARS-CoV-2-N ELISA methods showed a strong correlation yet provided increased detection rates when used in combination. A pVNT correlated strongly with SARS-CoV-2 VNT and was able to effectively discriminate SARS-CoV-2 antibody positive and negative serum with the same efficiency as the VNT. Moreover, the pVNT was performed with the same level of discrimination across multiple separate institutions. Therefore, the pVNT is a sensitive, specific, and reproducible lower biosafety level alternative to VNT for detecting SARS-CoV-2 antibodies for diagnostic and research applications. Our data illustrate the potential utility of applying VNT or pVNT and ELISA antibody tests in parallel to enhance the sensitivity of exposure to infection.