Enzyme Immunoassay Optical Density Research Articles

Quantitative interpretation of PF4/heparin‐EIA optical densities in predicting platelet‐activating VITT antibodies

BackgroundVaccine‐induced immune thrombotic thrombocytopenia (VITT) is a prothrombotic, heparin‐induced thrombocytopenia (HIT)‐mimicking, adverse reaction caused by platelet‐activating anti‐platelet factor 4 (PF4) antibodies that occurs rarely after adenovirus vector‐based COVID‐19 vaccination. Strength of PF4‐dependent enzyme immunoassay (EIA) reactivity—judged by optical density (OD) measurements—strongly predicts platelet‐activating properties of HIT antibodies in a functional test. Whether a similar relationship holds for VITT antibodies is unknown. ObjectivesTo evaluate probability for positive platelet activation testing for VITT antibodies based upon EIA OD reactivity; and to investigate simple approaches to minimize false‐negative platelet activation testing for VITT. MethodsAll samples referred for VITT testing were systematically evaluated by semiquantitative in‐house PF4/heparin‐EIA (OD readings) and PF4‐induced platelet activation (PIPA) testing within a cohort study. EIA‐positive sera testing PIPA‐negative were retested following 1/4 to 1/10 dilution. Logistic regression was performed to predict the probability of a positive PIPA per magnitude of EIA reactivity. ResultsGreater EIA ODs in sera from patients with suspected VITT correlated strongly with greater likelihood of PIPA reactivity. Of 61 sera (with OD values >1.0) testing negative in the PIPA, a high proportion (27/61, 44.3%) became PIPA positive when tested at 1/4 to 1/10 dilution. ConclusionsVITT serology resembles HIT in that greater EIA OD reactivity predicts higher probability of positive testing for platelet‐activating antibodies. Unlike the situation with HIT antibodies, however, diluting putative VITT serum increases probability of a positive platelet activation assay, suggesting that optimal complex formation depends on the stoichiometric ratio of PF4 and anti‐PF4 VITT antibodies.

Most anti-PF4 antibodies in vaccine-induced immune thrombotic thrombocytopenia are transient

Vaccine-induced thrombotic thrombocytopenia (VITT) is triggered by vaccination against COVID-19 with adenovirus vector vaccines (ChAdOx1 nCoV-19; Ad26.COV2-S). In this observational study, we followed VITT patients for changes in their reactivity of platelet-activating antiplatelet factor 4 (PF4) immunoglobulin G (IgG) antibodies by an anti-PF4/heparin IgG enzyme immunoassay (EIA) and a functional test for PF4-dependent, platelet-activating antibodies, and new thrombotic complications. Sixty-five VITT patients (41 females; median, 51 years; range, 18-80 years) were followed for a median of 25 weeks (range, 3-36 weeks). In 48/65 patients (73.8%; CI, 62.0% to 83.0%) the functional assay became negative. The median time to negative functional test result was 15.5 weeks (range, 5-28 weeks). In parallel, EIA optical density (OD) values decreased from median 3.12 to 1.52 (P < .0001), but seroreversion to a negative result was seen in only 14 (21.5%) patients. Five (7.5%) patients showed persistent platelet-activating antibodies and high EIA ODs for >11 weeks. None of the 29 VITT patients who received a second vaccination dose with an mRNA COVID-19 vaccine developed new thromboses or relevant increase in anti-PF4/heparin IgG EIA OD, regardless of whether PF4-dependent platelet-activating antibodies were still present. PF4-dependent platelet-activating antibodies are transient in most patients with VITT. VITT patients can safely receive a second COVID-19 mRNA-vaccine shot.

Comparative evaluation of commercially available manual and automated nucleic acid extraction methods for rotavirus RNA detection in stools

Rotaviruses are a major cause of viral gastroenteritis in children. For accurate and sensitive detection of rotavirus RNA from stool samples by reverse transcription-polymerase chain reaction (RT-PCR), the extraction process must be robust. However, some extraction methods may not remove the strong RT-PCR inhibitors known to be present in stool samples. The objective of this study was to evaluate and compare the performance of six extraction methods used commonly for extraction of rotavirus RNA from stool, which have never been formally evaluated: the MagNA Pure Compact, KingFisher Flex and NucliSENS® easyMAG® instruments, the NucliSENS® miniMAG® semi-automated system, and two manual purification kits, the QIAamp Viral RNA kit and a modified RNaid® kit. Using each method, total nucleic acid or RNA was extracted from eight rotavirus-positive stool samples with enzyme immunoassay optical density (EIA OD) values ranging from 0.176 to 3.098. Extracts prepared using the MagNA Pure Compact instrument yielded the most consistent results by qRT-PCR and conventional RT-PCR. When extracts prepared from a dilution series were extracted by the 6 methods and tested, rotavirus RNA was detected in all samples by qRT-PCR but by conventional RT-PCR testing, only the MagNA Pure Compact and KingFisher Flex extracts were positive in all cases. RT-PCR inhibitors were detected in extracts produced with the QIAamp Viral RNA Mini kit. The findings of this study should prove useful for selection of extraction methods to be incorporated into future rotavirus detection and genotyping protocols.

Temporal and spatial patterns of serologic responses to Plasmodium falciparum antigens in a region of declining malaria transmission in southern Zambia

BackgroundCritical to sustaining progress in malaria control is comprehensive surveillance to identify outbreaks and prevent resurgence. Serologic responses to Plasmodium falciparum antigens can serve as a marker of recent transmission and serosurveillance may be feasible on a large scale.MethodsSatellite images were used to construct a sampling frame for the random selection of households enrolled in prospective longitudinal and cross-sectional surveys in two study areas in Southern Province, Zambia, one in 2007 and the other in 2008 and 2009. Blood was collected and stored as dried spots from participating household members. A malaria rapid diagnostic test (RDT) was used to diagnose malaria. An enzyme immunoassay (EIA) was used to detect IgG antibodies to asexual stage P. falciparum whole parasite lysate using serum eluted from dried blood spots. The expected mean annual increase in optical density (OD) value for individuals with a documented prior history of recent malaria was determined using mixed models. SatScan was used to determine the spatial clustering of households with individuals with serological evidence of recent malaria, and these households were plotted on a malaria risk map.ResultsRDT positivity differed markedly between the study areas and years: 28% of participants for whom serologic data were available were RDT positive in the 2007 study area, compared to 8.1% and 1.4% in the 2008 and 2009 study area, respectively. Baseline antibody levels were measured in 234 participants between April and July 2007, 435 participants between February and December 2008, and 855 participants between January and December 2009. As expected, the proportion of seropositive individuals increased with age in each year. In a subset of participants followed longitudinally, RDT positivity at the prior visit was positively correlated with an increase in EIA OD values after adjusting for age in 2007 (0.261, p = 0.003) and in 2008 (0.116, p = 0.03). RDT positivity at the concurrent visit also was associated with an increase in EIA OD value in 2007 (mean increase 0.177, p = 0.002) but not in 2008 (−0.063, p =0.50). Households comprised of individuals with serologic evidence of recent malaria overlapped areas of high malaria risk for serologic data from 2009, when parasite prevalence was lowest.ConclusionsSerological surveys to whole asexual P. falciparum antigens using blood collected as dried blood spots can be used to detect temporal and spatial patterns of malaria transmission in a region of declining malaria burden, and have the potential to identify focal areas of recent transmission.

Incidence of Direct Thrombin Inhibitor Therapy for Presumed Heparin Induced Thrombocytopenia Before and After Implementation of Heparin Induced Thrombocytopenia IgG Subtype Antibody Test

AbstractAbstract 3336 Introduction:Heparin Induced thrombocytopenia (HIT) is a clinicopathological entity in which thrombocytopenia occurs in temporal relation to the initiation of heparin therapy. The diagnosis is made using clinical criteria and a scoring system (4T) can be applied to assess the probability of the diagnosis. A variety of sensitive tests are available but the most widely applied is an enzyme immunoassay (EIA), which detects and semi-quantifies antibodies of any class to the heparin-PF4 complex. The role of testing is largely supplementary, but recently focus has been placed on the epidemic of HIT overdiagnosis due to overreliance on laboratory tests irrespective of the clinical scenario. A newer EIA can measure the pathogenic IgG antibody only. This study examines the number of patients started on direct thrombin inhibitor (DTI) therapy for a presumptive diagnosis of HIT prior to the implementation of the IgG antibody and after its introduction at one large academic tertiary care center. Methods:All patients placed on the DTIs argatroban and lepirudin between October 2006-May 2009 in the pre IgG subtype antibody period and May 2009-February 2012 in the post IgG subtype period for a presumptive diagnosis of HIT were identified. Demographic data including age, sex, and primary service were recorded. All HIT antibody (Ab) immunoglobulin class EIA and IgG specific EIA optical density were recorded, as available. 4T criteria and rates of thrombosis were assessed retrospectively. Incidence of WHO Grade III (requiring transfusion) or IV (life threatening) bleeding was documented. Chi Square, Mann Whitney U, and Fisher's exact tests were used as appropriate for statistical analysis. Results:89 patients pre availability of the IgG EIA assay and 100 patients post availability of the IgG EIA assay met inclusion criteria and were evaluated. Groups were matched in terms of sex and 4T pretest probability distribution as well as rates of thrombosis. Patients post IgG EIA availability were older and had lower mean total HIT Ab optical densities. Post IgG EIA patients were treated with DTI therapy 5 days less, which was statistically significant. The incidence of Grade III and IV bleeding episodes, however, was statistically similar between groups. In the pre IgG EIA group, 7 bleeds (4 Grade III, 3 Grade IV) occurred in patients with low pretest probability for HIT based on 4T criteria. In the post IgG EIA group, 8 bleeds (5 Grade III, 3 Grade IV) occurred in patients with low pretest probability for HIT based on 4T criteria.Table 1Comparison between demographic and clinical outcome data derived from patients treated with DTI therapy for presumed HIT prior to IgG EIA availability and post IgG EIA availabilityPre IgG EIA (N=89)Post IgG EIA (N=100)P valueMedian Age (Range)60.5 (18–91)67 (25–98)0.03M:F46:4354:460.754T Pretest ProbabilityLow34460.28Intermediate44400.19High11140.74Median Total Antibody Optical Density0.620.490.02Median Days on Direct Thrombin Inhibitors (DTIs)83<0.0001Grade III/IV Episodes of Bleeding20220.92Grade III12170.50Grade IV850.28Episodes of Thrombosis16171 Conclusion:Implementation of the IgG HIT EIA resulted in a decrease in the number of days on a DTI without an observed change in serious bleeding events, indicating that such bleeding occurs early after initiation of DTI therapy. Further, patients pre and post availability of the IgG EIA were started on DTI therapy despite a low pretest clinical risk for HIT. The use of a test with a shorter turn-around time to exclude HIT or more rigorous enforcement of clinical guidelines will be required to minimize misdiagnosis and avoid therapy related morbidity. Disclosures:No relevant conflicts of interest to declare.

Galactomannan Antigen Testing for Diagnosis of Invasive Aspergillosis in Pediatric Hematology Patients

Invasive aspergillosis (IA) can cause significant morbidity and mortality in immunocompromised children. The galactomannan (GM) enzyme immunoassay (EIA) has been shown in adult studies to be a useful adjunct in diagnosing IA. Data on this assay in children are limited by small sample sizes and conflicting results; false-positive assays were a concern in historical studies. We sought to evaluate the GM EIA in a large cohort of children who received intensive chemotherapy and/or hematopoietic stem cell transplant. A focus was placed on evaluating the assay specificity, and the potential of measuring GM antigen in urine. A multicenter prospective observational study in children with anticipated prolonged neutropenia was performed. Serum specimens were collected twice weekly, and urine was collected once weekly during neutropenic periods. Operating characteristics were calculated using the GM EIA optical density index cutoffs of 0.5 and 1.0 for both serum and urine specimens. At least one serum or urine specimen was tested from 198 patients. Ten patients had one or more repeatedly positive serum specimens, while 37 patients had one or more repeatedly positive urine specimens. The specificity of serum and urine testing was 95% and 80%, respectively. Although the urine test resulted in a higher false positivity rate, it successfully identified the only case of probable IA. Data suggest that the serum GM EIA does not provide frequent false-positive results as previously reported. Screening for galactomannan, or a related antigen in urine, needs to be further evaluated as it may be amenable to development of surveillance strategies.

The temporal profile of the anti-PF4/heparin immune response

The immune response in heparin-induced thrombocytopenia (HIT) is puzzling: heparin-naive patients can develop IgG antibodies and clinical HIT as early as day 5, and evidence for an anamnestic response on heparin reexposure is lacking. We assessed daily serum samples by anti-PF4/heparin enzyme-immunoassay (EIA) in patients receiving heparin thromboprophylaxis. Of 435 patients, 56.1% showed an increase in EIA optical density (OD) of more than or equal to 15%, with more than 90% starting between days 4 and 14. After reaching maximum reactivity by days 10 to 12, ODs declined despite heparin continuation, including in 2 patients with clinical HIT. Individual IgG/A/M classes showed identical time of onset (median, day 6). Most (58.7%) antibody-positive patients developed all 3 Ig classes; only 11.3% lacked IgG response. IgG/A/M increase usually occurred simultaneously (+/- 1 day) with no general tendency for IgM precedence. Consistent with the transient immune response, none of the IgG-EIA-positive (OD > 0.5) patients at discharge developed clinically evident thrombosis during extended low-molecular-weight heparin thromboprophylaxis. The rapid onset of the anti-PF4/heparin immune response, its transience, and the simultaneous appearance of antibodies of different classes with no IgM precedence suggest short-term activation of B cells that have previously undergone Ig-class switching even without previous pharmacologic heparin exposure.

Evidence for anti-Plasmodium falciparum antibodies that cross-react with human T-lymphotropic virus type I proteins in a population in Irian Jaya, Indonesia.

This study was performed to demonstrate the presence of anti-Plasmodium falciparum antibodies in a population living in Irian Jaya, Indonesia that cross-react with human T-lymphotropic virus type I (HTLV-I) proteins. Serum samples from 63 volunteers living in Oksibil, a secluded highland valley in Irian Jaya, were tested for anti-P. falciparum antibodies by an immunofluorescence assay and for anti-HTLV-I antibodies by an enzyme immunoassay (EIA). All samples were positive for anti-P. falciparum antibodies at titers of > or = 1:256. Twenty-four samples were reactive by EIA for HTLV-I, and of these, 23 were tested by western blotting (immunoblotting). Five of the 23 samples were classified as western blot positive and 18 were classified as western blot indeterminate. In competitive blocking assays with malaria proteins, western blot immunoreactivity to all HTLV-I Gag proteins was either reduced or eliminated. Significant reductions in the HTLV-I EIA optical density values of the Oksibil sera occurred when the sera were competitively blocked with the malaria antigens. The optical density values of HTLV-I-positive control sera showed no significant change. Competitive blocking with HTLV-I antigens produced reductions in the optical density values of both the Oksibil sera and the HTLV-I-positive control sera. These data suggest that in this population, anti-P. falciparum antibodies are cross-reactive with HTLV-I proteins in the western blot and EIA tests.

Confirmation of second generation anti-hepatitis C virus enzyme immunoassays by antigenic cross-validation.

To determine if a scheme for validating enzyme immunoassay (EIA) results could be devised that did not require costly and methodically elaborate supplemental assays. Samples (n = 525) from patients with haemophilia A, leukaemia, and chronic liver disease and at increased risk of hepatitis C virus infection were tested by EIA-1 (Ortho Diagnostics), an assay which uses recombinant HCV fusion proteins as antigens, and by EIA-2 (United Biomedical), an assay based on synthetic HCV oligopeptide antigens. Samples (n = 193) were repeatedly reactive in both EIAs. Of these, 190 (98%) yielded reactivities in both of two supplemental assays used, one an immunoblot assay (RIBA) using recombinant HCV polypeptides similar to EIA-1 antigens, and the other a neutralisation EIA (EIA-2N) based on antigenic competition with HCV peptides similar to EIA-2 antigens. The three samples not reactive in supplemental tests exhibited low EIA optical density (OD) values (signal/cutoff ratios of less than 3). Hence, all specimens reactive and yielding high OD values in both EIAs were also reactive in supplemental assays. Twenty four samples were reactive in EIA-1 only and nine (38%) of these were reactive in RIBA. Fourteen of the 15 (93%) specimens reactive in EIA-1 but not RIBA were derived from patients with chronic liver dysfunction. Two samples were reactive in EIA-2 only, of which one was reactive in EIA-2N and none in RIBA. Compared with EIA-2, EIA-1 yielded more validated reactive samples and resulted in more non-validated reactivities. It is therefore suggested that for clinical diagnosis: (i) EIA-1 be used for anti-HCV testing and EIA-2 for validation of EIA-1 reactivities; (ii) samples concordantly reactive in EIA-1 and EIA-2 and displaying high OD readings be considered HCV antibody positive without supplemental testing; (iii) supplemental testing by RIBA be limited to samples reactive in EIA-1 but equivocal or unreactive in EIA-2 and those concordantly reactive but exhibiting low absorbance readings.

Use of high-speed rolling to detect respiratory syncytial virus in cell culture.

We examined the effect of motion on respiratory syncytial virus (RSV) growth in cell culture. Infected cultures were incubated stationary, rolling, or on an orbital shaker. Enzyme immunoassay (EIA) results for cultures infected with high concentrations of a laboratory strain of RSV were similar for all incubation conditions. However, cultures infected with low concentrations of virus and rolled at 96 rpm had a significantly greater mean EIA optical density (1.78 +/- 0.22) than cultures rolled at 2 rpm (1.42 +/- 0.08) (P less than 0.05). The mean EIA optical density of high-speed-rolled cultures was also significantly greater than for cultures on an orbital shaker (1.25 +/- 0.08) or for stationary cultures (0.21 +/- 0.17) (P less than 0.01). The amount of virus measured by EIA from cultures infected with clinical specimens was also found to be significantly greater at 96 rpm than for stationary cultures. Cultures infected with cell culture isolates were detected significantly earlier at 96 rpm than when stationary. We suggest that high-speed-rolling can be used to enhance the detection of RSV in clinical specimens, especially if the virus is present in low concentrations.

QUANTITATIVE EVALUATION OF NEURITE OUTGROWTH AND NEURONAL SURVIVAL OF CULTURED NERVE CELLS BY ENZYME IMMUNOASSAY AND ITS APPLICATION

Neuronal survival and neurite elongation in tissue culture are usually evaluated by counting the number of nerve cells and measuring neurite length on photographs, respectively. Because this is very time-consuming and not free of some artificial aberration, more reliable and simpler methods are needed. In this report, enzyme immunoassay (EIA) for quantitation of neurofilament protein is demonstrated to be an excellent method for evaluating neurite outgrowth and the number of surviving neurons in tissue culture. EIA is applied to the evaluation of nerve growth factor (NGF) -dependent neurite regeneration capacity in aged animals and to a study of the cytotoxic effects of dopa and dopamine on cultured nerve cells.Dorsal root ganglion (DRG) neurons were obtained from 10-week-old (Group Y), 72-week-old (Group M) and 114-week-old (Group O) male mice. Dissociated neuronal cells from each group were cultured for 4 days in chemically defined serum-free medium including various levels of NGF up to 160 ng/ml. Then EIA was performed using rabbit anti-neurofilament protein (NF) serum as the first antibody. NF content was expressed as optical density.EIA revealed less neurite outgrowth in Group M than in Group Y, but acceleration after the addition of a large amount of NGF. Neurite elongation capacity in Group O was moderately reduced at all levels of NGF when compared with the other groups. At the same time, however, significant NGF-dependent neurite regeneration capacity was preserved in aged animals. In Group Y and M, maximum length (ML : length of the longest neurite of an individual neuron) and total length (TL : sum of the length of all neurites of the neuron) were also quantified by tracing the distance on photographs. Optical densities were well correlated with ML in both groups and TL in Group Y. There was no correlation between optical densities and TL in Group M because of considerable fluctuation in TL.EIA optical density, i.e., NF content, was determined to be a reliable index for evaluating the number of surviving neurons in tissue culture. DRG neurons were obtained from 5-week-old mice and were seeded at various densities. The number of neurons counted and EIA optical density were significantly correlated. Using this method the effects of dopa and dopamine on the DRG neurons were tested. Neurons were destroyed by exposure to 0.5 mM dopa or 1.0 mM dopamine with or without superoxide dismutase and catalase, but they were spared by pretreatment with 1.0 mM deferoxamine mesylate. These results indicate that dopa and dopamine cause neuronal cell death in the presence of a small amount of iron in the culture with little or no participation by reactive oxygen species. As reported elsewhere, formation of malondialdehyde is also observed in the reaction between dopa and dissociated cerebral cortical neurons only when iron is present, which suggests that the complex of dopa (or dopamine) and iron initiates cell membrane lipid peroxidation leading to the neuronal destruction. In Parkinson's disease the mechanism of cytotoxicity may involve neuronal degeneration in the substantia nigra, which has an abundance of both iron and dopaminergic neurons. Further study is required to elucidate what triggers iron and dopa (dopamine) -mediated lipoxigenation of cell membranes in human brain in the presence of protective factors such as iron-transporters, tocopherols, phosphates, etc.

Antigens recognised by the human immune response to infection with Leptospira interrogans serovar hardjo.

Serum samples from patients infected with Leptospira interrogans serovar hardjo were tested by the microscopic agglutination test (MAT), enzyme immunoassay (EIA) and immunoblotting. There was no apparent correlation between MAT titre and EIA optical density (OD) for individual serum samples, but sequential serum samples produced similar profiles in both tests during the course of an infection. Immunoblotting of hardjo sonicate with patients' sera revealed reactions with a number of bands, in the mol. wt (10(3] range 14.4-95. However, all serum samples reacted with the major 28 x 10(3)-mol. wt sub-unit of hardjo lipopolysaccharide (LPS) and most reacted with a (34.5-35) x 10(3)-mol. wt flagella doublet. Examination of sequential serum samples obtained over a period of about 3 months after infection revealed little change in the antigens detected after the second to third week of infection. Absorption of patients' sera with whole viable leptospires revealed that antibodies to several exposed antigens, including LPS, were produced. Sera which reacted with hardjo flagella also reacted with bands of similar mol. wts in preparations from other serovars.