La Assay Research Articles

Utilization of an Automated Latex Agglutination Turbidity Assay for Assessing Gastric Mucosal Alteration during Helicobacter pylori Infection.

: A latex agglutination turbidity (LA) assay to test for serum antibodies has been approved in Japan and Korea for mass screening of Helicobacter pylori infection. In this study, we evaluated the LA assay for diagnosing H. pylori infection and predicting gastric mucosal changes in a Mongolian population. : In total, 484 individuals were classified into H. pylori-positive (n=356) and H. pylori-negative (n=128) groups, as determined by histology and H. pylori culture. : The best cutoff, sensitivity, and specificity values for the LA assay were 18.35 U/mL, 74.2%, and 65.6%, respectively. The LA values in the atrophic gastritis group were statistically higher than those in the other groups (healthy, chronic gastritis, intestinal metaplasia, and gastric cancer, p<0.0001). The cutoff value to distinguish the atrophic gastritis group from the other four groups was 32.0 U/mL, and its area under the curve was 0.673, which was the highest among the E-plate, pepsinogen (PG) I, PG II, and PG I/II ratio tests in our data. The odds ratios for atrophic gastritis determined by the LA assay and PG I test in multiple logistic regression were 2.5 and 1.9, respectively, which were significantly higher than for the other tests. : The LA assay can determine the risk of atrophic gastritis, which in turn is a considerable risk factor for gastric cancer. We propose using this assay in combination with the PG I/II ratio to avoid missing gastric cancer patients who have a low LA value (less than 32.0 U/mL).

Direct oral anticoagulant adsorption and laboratory detection of lupus anticoagulant.

In this study, the effect of activated charcoal (DOAC-Stop, DS) as a DOAC-adsorbent was investigated on samples from DOACs treated and untreated patients. 165 plasma samples with a LA request were collected in three laboratories: 105 were from patients receiving DOACs and 60 were from nonanticoagulated patients with 30 LA negative and 30 LA positive. All coagulation screening assays and LA assays were evaluated before and after DS treatment. The adsorption technique reduced DOACs concentration below the Lower Limit of Quantification. For nonanticoagulated patients: no significant difference in ratio results of coagulation screening (prothrombin time, activated partial thromboplastin time and thrombin time) and LA tests were observed before and after addition of DS in LA positive and negative patients. Every LA was correctly classified. For anticoagulated patients: a statistically significant difference was found for coagulation screening assays and LA assays. Final LA conclusions changed after DS addition from positive to negative in 58.9% of patients (more frequently with Rivaroxaban) and from negative to positive in 8% of patients (more frequently with Apixaban). Our study suggests that DOAC-Stop can be used in daily laboratory practice to remove DOACs interference for a more accurate assessment of LA that is essential for diagnosis and management of APS patients.

Resolving DOAC interference on antithrombin activity testing on a FXa based method by the use of activated carbon

BackgroundDirect oral anticoagulants (DOACs) may cause falsely increased levels of antithrombin (AT) activity depending on the AT activity method and the specific target of the DOAC. Activated carbon (AC) has proven to remove DOAC interference on PT, aPTT and LA assays. We evaluate whether AC could be useful to resolve DOAC interference on AT assays. MethodsNormal pooled plasma (NPP) was diluted to obtain AT activity of 25 %, 50 % and 75 % respectively. The diluted NPPs were spiked with DOACs (apixaban, edoxaban, dabigatran and rivaroxaban) in concentrations of respectively 100, 250 and 500 ng/ml. DOAC concentrations and AT activity were tested at baseline and after treatment with 20 mg/ml AC. AT activity was measured with a FXa-based method (HemosIL Liquid Antithrombin®, Werfen). ResultsAll DOAC concentrations were below the limit of quantification (LoQ) after addition of AC. DOAC interference on AT activity testing was removed by adding AC, resulting in correctly diagnosing low levels of AT for all dilutions. The influence of DOACs on AT activity was directly correlated to the concentration of the DOAC. As expected, only the anti-FXa DOACs influenced the used assay. ConclusionsAC effectively removes anti-FXa DOAC interference on FXa-based AT assays.

Practical Considerations and Testing Nuances for the Detection of Lupus Anticoagulant: Do Low Phospholipid Screen Results, Assay Type, and Test Ratio Matter?

Lupus anticoagulant (LA) detection requires (1) prolongation of a phospholipid (PL)-dependent clot-based screening assay, (2) noncorrection upon adding normal pooled plasma, and (3) a confirmatory PL dependency test. Paired LA assays run screening and confirmatory tests simultaneously, with their test ratio (TR) or differences used to evaluate test results. We evaluated patients whose paired testing demonstrated PL dependence suggestive of LA, yet the low PL screen was not prolonged. Clinical and laboratory parameters are compared across (1) true positive (screen prolonged, TR positive) vs borderline (screen not prolonged, TR positive); (2) low-, moderate-, and high-TR subgroups; and (3) dilute Russell viper venom time (dRVVT) vs silica clotting time (SCT). Borderline samples are not statistically different from true positives in their rate of repeat LA positivity or association with other anti-PL antibodies. Compared with true positives, borderline dRVVT is more frequent in pregnancy, women, and younger age. Elevated activated partial thromboplastin time is more frequent in true-positive dRVVT and SCT vs borderline and with an increasing dRVVT TR. LA persistence is more frequent with an increasing SCT TR. In addition, dRVVT true positivity is more frequent with thromboembolic events, while SCT is more frequent with autoimmunity and pregnancy complications. Negative low-PL screens may not necessarily lack LA. A reevaluation of the laboratory criteria for LA detection may be needed.

Synthesis of Guaianolide Analogues with a Tunable α-Methylene-γ-lactam Electrophile and Correlating Bioactivity with Thiol Reactivity.

α-Methylene-γ-lactones are present in ∼3% of known natural products, and compounds comprising this motif display a range of biological activities. However, this reactive lactone limits informed structure-activity relationships for these bioactive molecules. Herein, we describe chemically tuning the electrophilicity of the α-methylene-γ-lactone by replacement with an α-methylene-γ-lactam. Guaianolide analogues having α-methylene-γ-lactams are synthesized using the allenic Pauson-Khand reaction. Substitution of the lactam nitrogen with electronically different groups affords diverse thiol reactivity. Cellular NF-κB inhibition assays for these lactams were benchmarked against parthenolide and a synthetic α-methylene-γ-lactone showing a positive correlation between thiol reactivity and bioactivity. Cytotoxicity assays show good correlation at the outer limits of thiol reactivity but less so for compounds with intermediate reactivity. A La assay to detect reactive molecules by nuclear magnetic resonance and mass spectrometry peptide sequencing assays with the La antigen protein demonstrate that lactam analogues with muted nonspecific thiol reactivities constitute a better electrophile for rational chemical probe and therapeutic molecule design.

Resolving DOAC interference on aPTT, PT, and lupus anticoagulant testing by the use of activated carbon

BackgroundDirect oral anticoagulants (DOACs) affect laboratory coagulations tests. Activated carbon (AC) can be used for adsorption of DOACs during acute human intoxications. ObjectivesThis study evaluates whether AC can also be used to resolve DOAC interference on in vitro clotting tests (prothrombin time [PT], activated partial thromboplastin time [aPTT], and lupus anticoagulant [LA] assays). Patients/MethodsInterference on PT, aPTT, Liquid anti‐FXa, DTI, and LA screening/confirmation (SCT and dRVVT) was determined by spiking citrated plasma from 5 adult controls with 0, 20, 40, 80, 120, or 160 mg/mL AC. DOAC concentrations, PT, and aPTT were compared before and after AC addition to citrated plasma from patients receiving DOACs (n = 29), low molecular weight heparin (n = 10), and coumarin (n = 10) therapy. Samples from 69 LA screened patients were compared before and after AC addition. ResultsA concentration of 20 mg/mL AC had the lowest interference and was selected for further experiments. After AC addition, all DOAC concentrations were below the limit of quantification in the 29 treated patients, except for 2 apixaban samples. AC removed DOAC interference on PT and aPTT but had no impact on results obtained during coumarin or low molecular weight heparin therapy. Of 15 LA samples with interference resulting from DOAC therapy, 14 samples became negative and 1 positive after AC addition. Interference from coumarin therapy was not resolved. All 19 LA negative samples remained negative. AC treatment of the negative pooled plasma was required to avoid false‐negative LA results in 21 known LA‐positive samples. ConclusionsAC selectively removes DOAC interference on PT, aPTT, and LA assays.

The Effect of 3.2% and 3.8% Sodium Citrate on Specialized Coagulation Tests.

- Coagulation testing is challenging and depends on preanalytic factors, including the citrate buffer concentration used. - To better estimate preanalytic effects of the citrate buffer concentration in use, the difference between results obtained by samples with 3.2% and 3.8% citrate was evaluated. - In a prospective observational study with 76 volunteers, differences related to the citrate concentration were evaluated. For both buffer concentrations, reference range intervals were established according to the recommendations of the C28-A3 guideline published by the Clinical and Laboratory Standards Institute. - In our reagent-analyzer settings, most parameters evaluated presented good comparability between citrated samples taken with 3.2% and 3.8% trisodium buffer. The ellagic acid containing activated partial thromboplastin time reagent (aPTT-FS) indicated a systemic and proportional difference between both buffer concentrations, leading to an alteration in its reference ranges. Further, a confirmation test for lupus anticoagulant assessment (Staclot LA) showed only a moderate correlation ( rρ = 0.511) with a proportional deviation between both citrate concentrations. Further, a statistically significant difference was found in the diluted Russell viper venom time confirmation testing, coagulation factors V and VIII, and the protein C activity, which was found to be of minor clinical relevance. - With caution regarding the potential impact of the reagent-analyzer combination, our findings demonstrate the comparability of data assessed with 3.2% and 3.8% buffered citrated plasma. As an exception, the aPTT-FS and the Staclot LA assay were considerably affected by the citrate concentration used. Further studies are required to confirm our finding using different reagent-analyzer combinations.

ALARM NMR for HTS triage and chemical probe validation.

Nonspecific target engagement by test compounds and purported chemical probes is a significant source of assay interference and promiscuous bioactivity in high-throughput screening (HTS) and chemical biology. Most counter-screens for thiol-reactive compounds utilize mass spectrometry or fluorescence detection, and non-proteinaceous reporters like glutathione that may not always approximate the reactivity of protein side-chains. By contrast, a La assay to detect reactive molecules by nuclear magnetic resonance (ALARM NMR) is an industry-developed protein-based [1H-13C]-heteronuclear multiple quantum coherence (HMQC) NMR counter-screen to identify nonspecific protein interactions by test compounds by reporting their tendencies to modulate the human La antigen conformation. This Current Protocol is a users-guide to the production of the 13C-labeled La antigen reporter protein, the reaction of test compounds with this reporter protein, as well as the collection and analysis of characteristic NMR spectra. Combined with other assay interference counter-screens, this assay will enhance chemical biology by helping researchers better prioritize chemical matter and which will increase the number of tractable HTS screening actives and aid in the development of better chemical probes.

Comparison of the clinical performance of restriction fragment mass polymorphism (RFMP) and Roche linear array HPV test assays for HPV detection and genotyping

BackgroundThe need for accurate genotyping of human papillomavirus (HPV) infections is becoming increasingly important as HPV is the primary cause of cervical cancer worldwide. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry-based restriction fragment mass polymorphism (RFMP) assay provides accurate, broad-spectrum, high-throughput genotyping of HPV. ObjectivesWe evaluated the clinical performance of the RFMP assay compared to a commercially available Roche linear array HPV genotyping test (LA) for detecting and genotyping of HPV. Study designThe RFMP assay and the LA were compared for detecting and genotyping HPV among a cohort of 244 liquid-based cytology samples. ResultsOverall, 216 specimens (93.1%, κ=0.86) generated concordant results for the presence or absence of high-risk HPV (HR-HPV) by the two assays. The RFMP assay and the LA assay generated concordant, compatible, and discordant genotyping results for 79.3, 9.9, and 10.8%, respectively. The diagnostic sensitivity and specificity of RFMP and LA for the cervical lesions of squamous cell carcinoma (SCC) were similar, at 92.9 and 85.0% (RFMP) and 92.9 and 83.8% (LA), respectively. In addition, the odds ratio for SCC with HR-HPV positivity estimated by the RFMP assay (73.7, 95% CI: 8.9–3173.3) was higher than the LA assay (67.0, 95% CI: 8.2–2887.0). ConclusionsThe RFMP and the LA assays were highly comparable with regard to detection and genotyping analysis of HPV. The sensitivity and specificity of RFMP assay for the detection of HR-HPV in various levels of cervical lesions seems to be valuable in the monitoring of HPV-associated cervical cancer.

Comparison of the clinical performance of an HPV mRNA test and an HPV DNA test in triage of atypical squamous cells of undetermined significance (ASC-US)

The effect of triaging women with atypical squamous cells of undetermined significance (ASC-US) with human papillomavirus (HPV) DNA testing has been well documented. New tests detecting HPV E6/E7 mRNA are emerging, claiming to be more specific for detecting high-grade disease. We evaluated the clinical performance of two HPV tests: the Linear Array HPV genotyping test (LA) detecting HPV DNA from 37 oncogenic and non-oncogenic HPV types and the Aptima HPV assay detecting E6/E7 mRNA from 14 oncogenic HPV types. We identified 369 consecutive PreservCyt samples diagnosed with ASC-US tested for HPV DNA using the LA test. The Aptima HPV test was performed on residual material in the same vial. Follow-up of 325 women was available. The gold standard used was histologically confirmed cervical intraepithelial neoplasia (CIN) grade 2+ or 3+. LA and Aptima HPV assays were positive in 44.3% and 31.7% of the cases, respectively. The concordance was 81.2%. The two tests had identical sensitivity for detecting CIN3+ [92.6% (95% CI, 75.7-99.1)] but the Aptima HPV assay showed a significantly better specificity of 73.8% (95% CI, 68.5-78.7) versus 60.1% (95% CI, 54.3-65.7) for LA for detecting CIN3+. When using CIN2+ as the gold standard the sensitivity for LA was higher than for the Aptima HPV assay [93.8% (95% CI, 82.8-98.7) versus 87.5% (95% CI, 74.8-95.3)], but the specificity was higher for the Aptima HPV assay: 78.0% (95% CI, 72.6-82.7) versus 64.3% (95% CI, 58.3-69.9). Both tests showed good and equal clinical sensitivities for detecting CIN3+, but the Aptima HPV assay had significantly higher specificity for detecting CIN2+ and CIN3+ in women aged 30 years or older with ASC-US.

Human papillomavirus detection and typing in thin prep cervical cytologic specimens comparing the Digene Hybrid Capture II Assay, the Roche Linear Array HPV Genotyping Assay, and the Kurabo GeneSquare Microarray Assay

Three methods for the detection of HPV DNA were compared in cervical cytologic specimens: the Digene Hybrid Capture II Assay (HC), the Roche Linear Array HPV Genotyping Assay (LA) and the Kurabo GeneSquare Microarray (GS). The main goals of the study were to correlate cytology with HPV detection and to determine agreement between assay pairs for HPV detection. Thin-prep Pap smears were performed and supernates were tested by HC, LA, and GS. For specimens reacting with the HPV 52/33/35/58 probe in the LA assay, type-specific PCR was performed for HPV types 52, 33, 35, or 58. Binomial proportions and kappa coefficients were calculated for agreement between assays. Cytology results and supernatant were available for 202 subjects. HPV detection increased with worsening cytologic abnormality in all three assays. For all cytologic groups, LA and GS detected more HPV (all and oncogenic) than HC. However, for detection of oncogenic HPV types represented in all three assays, differences between assays were less pronounced. The highest agreement was between LA and GS. In four of 12 specimens reacting with the HPV 52/33/35/58 probe in the LA assay but deemed HPV 52-LA-negative using an algorithm provided by the manufacturer, the presence of HPV 52 was confirmed using type-specific HPV 52 PCR. All four of these specimens were also GS-positive for HPV 52.

Pathophysiology of Anti-PL Syndrome in Thrombosis.

Abstract SCI-45The antiphospholipid (aPL) syndrome (APS) is an enigmatic autoimmune thrombotic disorder that was initially identified through astute clinical observations and the development of a quantitative test for aPL antibodies. The condition is marked by 2 types of assays: 1) immunoassays that were derived from the “biologic false positive syphilis” test and 2) coagulation assays that detect “lupus anticoagulants” (LAs), which are inhibitors of phospholipid-dependent coagulation reactions. The most recent consensus investigational criteria for diagnosing APS require that patients have evidence for thrombosis and/or pregnancy complications attributable to placental insufficiency and also laboratory evidence for persistent (aPL) antibodies, detected by high levels of IgG or IgM antibodies against cardiolipin and β2-glycoprotein I (β2GPI) and/or through abnormal LA assays. The thrombotic disorder requires long term anticoagulant treatment, which is accompanied by the risk of bleeding complications. Several mechanisms have been postulated and can be classified as involving: 1) aPL antibody-mediated inhibition of endogenous anticoagulant mechanisms; 2) aPL antibody-triggered signaling events on target cells (vascular endothelial cells, monocytes, platelets and trophoblasts) that promote proadhesive and prothrombotic phenotypes; and 3) aPL antibody-mediated complement activation. At present, it appears that the APS diagnostic entity actually includes several distinct subsets that reflect the actions of heterogeneous antibodies directed against different epitopes of phospholipid-binding proteins which then may yield different clinical sequelae. We have accumulated significant data indicating that a major one of these mechanisms involves aPL antibody-mediated disruption of annexin A5 (AnxA5) activity. AnxA5 is a potent anticoagulant protein whose anticoagulant properties are a consequence of its high affinity for anionic phospholipid. The protein forms 2-dimensional crystals on phospholipid surfaces that shield the phospholipids from availability for coagulation reactions. AnxA5 appears to play a thrombomodulatory role on the surfaces of cells lining the placental and systemic vasculatures. It is highly expressed on the apical membranes of placental syncytiotrophoblasts, the location where maternal blood interfaces with fetal cells. aPL antibody-antigen complexes disrupt the ordered crystallization of AnxA5, displace the protein from phospholipid membrane surfaces and thereby accelerate coagulation reactions. This effect of the antibodies has been demonstrated on artificial bilayers, on cultured trophoblasts and on endothelial cells and platelets. This disruption has been appears to be a consequence of aPL antibodies that recognize a specific epitope within domain I of β2GPI, the key antigen recognized by aPL antibodies. Based upon these data, we developed a novel clinical assay “the AnxA5 resistance (A5R) test” to identify patients who have antibodies that interfere with the anticoagulant activity of AnxA5. Initial studies indicate that a large proportion of APS patients have evidence for A5R. It therefore appears possible that measurement for A5R may provide a mechanistic assay for APS. We are also developing treatments to target this mechanism and protect AnxA5 from antibody-mediated disruption which may open novel nonanticoagulant approaches to treating APS. DisclosuresOff Label Use: The presentation will include research on in vitro effects of hydroxychloroquine; the drug is not FDA approved for the treatment of patients with antiphospholipid syndrome who do not also have concurrent systemic lupus erythematosus or rheumatoid arthritis.

Toxicological Evaluation of Thiol-Reactive Compounds Identified Using a La Assay To Detect Reactive Molecules by Nuclear Magnetic Resonance

We have recently reported on the development of a La assay to detect reactive molecules by nuclear magnetic resonance (ALARM NMR) to detect reactive false positive hits from high-throughput screening, in which we observed a surprisingly large number of compounds that can oxidize or form covalent adducts with protein thiols groups. In the vast majority of these cases, the covalent interactions are largely nonspecific (e.g., affect many protein targets) and therefore unsuitable for drug development. However, certain thiol-reactive species do appear to inhibit the target of interest in a specific manner. The question then arises as to the potential toxicology risks of developing a drug that can react with protein thiol groups. Here, we report on the evaluation of a large set of ALARM-reactive and -nonreactive compounds against a panel of additional proteins (aldehyde dehydrogenase, superoxide dismutase, and three cytochrome P450 enzymes). It was observed that ALARM-reactive compounds have significantly increased risks of interacting with one or more of these enzymes in vitro. Thus, ALARM NMR seems to be a sensitive tool to rapidly identify compounds with an enhanced risk of producing side effects in humans, including alcohol intolerance, the formation of reactive oxygen species, and drug-drug interactions. In conjunction with other toxicology assays, ALARM NMR should be a valuable tool for prioritizing compounds for lead optimization and animal testing.

Elevated activated partial thromboplastin time during administration of first-generation adenoviral vectors for gene therapy for prostate cancer: Identification of lupus anticoagulants

Objectives To evaluate the cause and significance of elevated activated partial thromboplastin time (aPTT) in a group of patients who received a first-generation adenoviral vector (Ad-OC-TK) delivering a toxic gene to prostate cancer cells as part of a Phase I clinical trial at the University of Virginia. Methods Eleven subjects were injected intratumorally to metastatic lesions of prostate cancer in the prostatic fossa, retroperitoneal lymph nodes, or bone (iliac, ischium, or vertebrae). After the initial laboratory evaluation, patients with elevated aPTT underwent a series of additional tests, including mixing studies, coagulation factor, prekallikrein, and high-molecular-weight kininogen, and lupus anticoagulant studies (modified Russell viper venom time) with phospholipid correction, and a Staclot LA assay. Results Of the 11 subjects who were enrolled in the trial, 6 had elevated aPTT values. Of the 6 patients, 3 had aPTT elevation of more than 10 seconds above normal. Two of the subjects with higher values demonstrated an inhibitory pattern with the factor VIII and XI assays, and the lupus anticoagulant studies were positive. No clinical sequelae to the elevated aPTT values were observed. Conclusions This is, to our knowledge, the first formal report of a first-generation adenoviral vector causing a slight transient elevation of the aPTT through the induction of an antiphospholipid antibody. No clinical sequelae related to elevated aPTT values were observed. The adenoviral protocol was safe; similar protocols should be aware of this phenomenon.

ALARM NMR: A Rapid and Robust Experimental Method To Detect Reactive False Positives in Biochemical Screens

High-throughput screening (HTS) of large compound collections typically results in numerous small molecule hits that must be carefully evaluated to identify valid drug leads. Although several filtering mechanisms and other tools exist that can assist the chemist in this process, it is often the case that costly synthetic resources are expended in pursuing false positives. We report here a rapid and reliable NMR-based method for identifying reactive false positives including those that oxidize or alkylate a protein target. Importantly, the reactive species need not be the parent compound, as both reactive impurities and breakdown products can be detected. The assay is called ALARM NMR (a La assay to detect reactive molecules by nuclear magnetic resonance) and is based on monitoring DTT-dependent (13)C chemical shift changes of the human La antigen in the presence of a test compound or mixture. Extensive validation has been performed to demonstrate the reliability and utility of using ALARM NMR to assess thiol reactivity. This included comparing ALARM NMR to a glutathione-based fluorescence assay, as well as testing a collection of more than 3500 compounds containing HTS hits from 23 drug targets. The data show that current in silico filtering tools fail to identify more than half of the compounds that can act via reactive mechanisms. Significantly, we show how ALARM NMR data has been critical in identifying reactive compounds that would otherwise have been prioritized for lead optimization. In addition, a new filtering tool has been developed on the basis of the ALARM NMR data that can augment current in silico programs for identifying nuisance compounds and improving the process of hit triage.

Thrombogenic Mechanisms of Antiphospholipid Antibodies

These studies indicate how immunoglobulin populations that react with phospholipid surfaces in the absence of other cofactor molecules can selectively inhibit anticoagulant pathways and lead to a prothrombotic state. These studies combined with those of others indicating the presence of a-PE antibodies (often in isolation) in thrombotic patients illustrate the need to better define the assays to determine patients at risk. Neither the LA assays nor the anti-cardiolipin assays presently in use may be testing for the population(s) of clinical importance. A better understanding of the biochemical requirements of the various reactions involved should help the rational design of such assays. The preliminary studies of Salmon, et al, also show that the genetic context of the patient may contribute to the thrombotic mechanism of any APA present. It is unlikely any single mechanism is responsible for the thrombogenic activity of all APAs associated with thrombosis and this will be a fertile field of investigation for a significant time to come.

Antiphospholipid Syndromes with Anti-Human β2-Glycoprotein I Antibodies despite Negative Reactivity in Conventional aPL and LA Assays

Antiphospholipid Syndromes with Anti-Human β2-Glycoprotein I Antibodies despite Negative Reactivity in Conventional aPL and LA Assays

Soluble transferrin receptor-transferrin complex in serum: measurement by latex agglutination nephelometric immunoassay

The transmembrane protein, transferrin receptor (TfR), exists in serum as a soluble form that lacks cytoplasmic and transmembrane domains (residues 1–100). The level of soluble TfR in serum is a sensitive indicator of total erythropoiesis and iron deficiency. This study revealed that the major part of soluble TfR was saturated by transferrin (To in serum, forming a stable complex which was more immunoreactive than intact TfR. Thus, we proposed that serum soluble TfR should be measured as the TfR-Tf complex (TRC), using prepared TRC for assay standardization. We developed a new assay for TRC, employing antibody-coated latex agglutination nephelometry (LA). Rapid and reproducible measurements were achieved using an automated analyzer. The values obtained by this LA assay were closely correlated with those obtained by conventional enzyme immunoassay ( r = 0.967). The mean level of TRC in 179 adult healthy subjects was 1.62 mg/1. Patients with iron-deficient anemia showed significantly higher TRC levels than the healthy subjects.

New diagnostic strategies for lupus anticoagulants and antiphospholipid antibodies.

Laboratories are currently challenged with the problem of ruling out APA in a patient plasmas. In order to optimize the identification of these antibodies, it is necessary to institute a well-planned approach. Both coagulation assays for the identification of LA and solid phase ELISA assays for the identification of ACA are indicated. In the case of LA, it is often necessary to screen the patient plasma with more than one test system. If two screening tests are negative, no further evaluation for the presence of LA is indicated. However, it is imperative to also perform ACA testing. The literature describing LA and ACA testing is growing exponentially. Several new integrated test systems which seek to simplify the approach to the diagnosis of LA have been introduced. These systems employ one test performed under two different conditions (low and high PL concentration). This approach may prove to be the most cost-efficient way of establishing the diagnosis of LA.