Stanley S. Levinson

Towards a better understanding of heterophile (and the like) antibody interference with modern immunoassays.

BACKGROUND Heterophile antibodies interfere with immunoassays. Understanding the nature and characteristics of these antibodies provides a format for better identifying and removing them. Growing evidence suggests many of these antibodies are natural antibodies. Very large number of tests are being performed with automated analyzers and there has been a problem with misdiagnosis due to interference. New commercial agents for blocking heterophile antibodies have been developed. METHODS Review of the immunology and methodological literature with critical interpretation of the findings. CONCLUSIONS Heterophile antibodies consist of natural antibodies and autoantibodies. Both types are usually weak antibodies that interfere by noncompetitive mechanisms. Based on very strong circumstantial evidence, we propose that natural antibodies account for most interference with automated immunoassays. In terms of false positive results, the interference rate is very low, about 99.95% accuracy. Specific blocking agents have some theoretical advantage over nonspecific blocking agents, but in actual practice, the very low false positive frequency makes it difficult if not impossible to statistically compare blocking agents or other assay modifications with adequate statistical power. In the absence of a technique that lends itself to automation for removing all immunoglobulins, it appears that infrequent heterophile interference cannot be avoided.

Fibrinogen is an antioxidant that protects β-lipoproteins at physiological concentrations in a cell free system

Oxidation of beta-lipoproteins has been linked to the development of arteriosclerosis. Using a copper mediated cell free system to oxidize beta-lipoproteins, we found that beta -lipoproteins isolated from plasma were less susceptible to oxidation than lipoproteins from serum and that this was probably due to inhibition by fibrinogen, because removal of fibrinogen from plasma enhanced oxidation, while addition of fibrinogen restored inhibition. Fibrinogen inhibited conjugated diene formation and peroxide formation assayed by the xylenol orange assay (absorbance+/-confidence interval: 0.155+/-0.007 with fibrinogen vs 0.255+/-0.014 without) and retarded copper mediated oxidation of apolipoproteins in low density lipoproteins, reducing the distance of electrophoretic migration by 5 mm. The effect of fibrinogen was not due to chelation of copper, since it provided protection when hydrogen peroxide was substituted for copper as an oxidizing agent. At normal physiological concentration equivalents, fibrinogen showed superior antioxidant properties compared to albumin, melatonin, vitamin C and vitamin E and was superior to the vitamins when compared on an equimolar basis. Other studies have shown the fibrinogen to be more oxidizable than other major plasma proteins and to inhibit peroxide production. Because of its high mass concentration, we postulate fibrinogen is an important antioxidant protecting beta-lipoproteins in plasma and that it may be important in protecting lipoproteins in tissue spaces.

Serum free light chain analysis may miss monoclonal light chains that urine immunofixation electrophoreses would detect.

BACKGROUND A nephelometric assay for identifying free light chains (FLC) of immunoglobulin in serum is commercially available. Although this assay is not specific for monoclonal FLC, an abnormal kappa/lambda ratio is thought to presumptively identify monoclonal FLCs. It has been proposed that serum FLC assays can replace urine immunofixation electrophoresis (IFE) for routine detection. This proposal seems to be based upon testing of samples retrospectively drawn from patients mostly with myeloma or known primary amyloidosis-conditions that are less frequently observed in general laboratory practice. METHODS Prospective analysis of 5 patient samples by standard care techniques with serum/urine IFE compared with serum FLC nephelometric assay. Rather than classical myeloma, cases were selected on the basis of unusual or equivocal atypical serum protein electrophoresis patterns. RESULTS In all 5 cases, the nephelometric method for free light chains was negative by virtue of serum FLC kappa/lambda ratio being within the reference range, contrasting with monoclonal proteins or free light chains detected by serum/urine IFE. CONCLUSIONS Since peculiar or unusual patterns are more commonly observed in general practice, these cases illustrate the pitfalls of relying solely on the serum free light chain analysis method in order to establish the presence of monoclonal FLC.

Convenient and Effective Method for Removing Fibrinogen from Serum Specimens before Protein Electrophoresis

BACKGROUND Fibrinogen in serum specimens can be misinterpreted on protein electrophoresis as a monoclonal protein. We evaluated selective precipitation of fibrinogen with ethanol. METHODS Pooled human plasma was mixed with absolute ethanol or saline (final concentrations of 40, 80, 100, 120, and 160 mL/L) and incubated at 4 degrees C overnight or placed in an ice bath for 15 min. After centrifugation, the supernatants and resuspended pellets were used for protein electrophoresis and quantitative measurements of protein and fibrinogen. RESULTS The fibrinogen band was effectively eliminated from the electrophoretic pattern in the plasma samples treated with ethanol at 100 mL/L and incubated in an ice bath for 15 min without a significant change in immunoglobulin concentrations. The 100 mL/L ethanol did not noticeably change the electrophoretic pattern of monoclonal immunoglobulins. This approach allowed analysis of a sample collected from an arteriovenous shunt kept open with heparin. CONCLUSIONS Ethanol, 100 mL/L, can selectively precipitate fibrinogen without significantly interfering with the immunoglobulins. The precipitation process can be completed in 15 min at 0-4 degrees C and can avoid the need to obtain another blood sample.

Implications of reverse cholesterol transport: recent studies.

INTRODUCTION There is a strong epidemiological relationship between high density lipoproteins and atherosclerotic coronary vascular disease (ASCVD). The process of reverse cholesterol transport (RCT) has been hypothesized to help explain this relationship. The corollary that raising HDL should reduce ASCVD is also drawn from this relationship. In recent years, the metabolism of HDL has become better understood. A hypothetical process for explaining RCT has been superimposed on the currently understood HDL metabolic pathways. METHODS Outline of HDL metabolism and the superimposed RCT process. Literature review of studies of persons with genetic defects, HDL cholesterol raising clinical trials, Mendelian randomization studies and treatments with molecules that mimic HDL. CONCLUSIONS Mutation studies of ABCA1, LCAT and SR-B1 genes in humans showed expected variations in HDLC but little association with ASCVD and there was no significant association between HDLC and ASCVD in Mendelian randomization studies. Elevations in HDLC due to treatment with niacin and cholesteryl ester transport protein inhibitors in randomized trials raised HDLC but did not significantly reduce risk of ASCVD. Treatment with molecules that mimic HDL did not seem to reduce ASCVD. Thus, recent evidence does not seem to support RCT as currently proposed. This hypothesis seems to need substantial revision.

Comparison of apolipoprotein B and non-high-density lipoprotein cholesterol for identifying coronary artery disease risk based on receiver operating curve analysis.

Whether or non-high-density lipoprotein cholesterol is equivalent to apolipoprotein B (apo B) for screening remains controversial. One reason for continued controversy is that most studies express results as relative risk/hazard or odds ratios based on P values that reflect diagnostic values poorly. Apo B and lipoprotein lipids were compared in 437 men. The results were evaluated by multivariate techniques and by receiver operating characteristic (ROC) curves. When analyzed by ROC curves, the difference between Apo B and lipoprotein lipids proved to be less than would be anticipated from the odds ratios. Although, after adjustment, the difference was about 14% by odds ratios, ROC analysis showed only a small difference of about 1%. These data show that clinical studies should analyze the data using an absolute measure of risk such as ROC curves rather than just relative indexes. Such a small absolute difference may also explain discrepancies between studies.

7 – INTERFERENCES IN IMMUNOASSAYS

This chapter discusses the sources of interferences that are specific to immunoassays and their detection mechanisms. The interference may be positive or negative and may vary in magnitude depending on the concentration of the interfering substance in the sample. The sources of interferences include cross-reactivity, endogenous interfering antibodies, masked antigens, interferences with the indicator mechanism, and matrix effects. The examples will be predominantly for quantitative assays, but the same sources and mechanisms also apply to qualitative immunoassays. In addition, examples will include a variety of assay formats including two-site “sandwich” assays, competitive binding (CB) assays, and nephelometric assays, but the principles also apply to other types of immunoassays such as radial immunodiffusion and agglutination techniques. In recent years, there have been many methodological advances in immunoassays including monoclonal antibody technology, nonisotopic detection systems, and two-site sandwich assays. These advances have allowed great improvements in sensitivity, convenience in terms of automation and rapid turn-around times, and development of immunoassays for many “new” analytes. However, these improvements have been accompanied by some new types of interference.

An algorithmic approach using κ/λ ratios to improve the diagnostic accuracy of urine protein electrophoresis and to reduce the volume required for immunoelectrophoresis

Abstract The most sensitive routine method for identifying urinary monoclonal immunoglubulin κ and λ light chains, called Bence Jones proteins (BJPs), in clinical laboratories is immunofixation electrophoresis (IFE), but this procedure is time-consuming and expensive. As a result, may laboratories screen for paraproteins with urine protein electrophoresis (UPE), which is insensitive when low concentrations of BJP are present and is difficult to interpret with severe proteinuria. The purpose of this study was to determine whether κ/λ ratios can be used in conjunction with UPE to improve diagnostic reliability in identifying paraproteins, and decrease the need for IFE on all samples. Urine specimens from 243 patients were examined by UPE and κ/λ ratios, and compared with IFE. Due to poor analytical sensitivity, the urinary κ or λ concentrations could not be determined in many cases. As a result, many specimens showed κ/λ ratios that were indeterminate. Nevertheless, when both urinary κ and λ concentrations were undetectable, a BJP could be ruled out. A urinary κ/λ ratio between 0.75-3 also ruled out a BJP. The use of κ/λ ratios, in conjunction with UPE, resulted in a 52% decrease in the volume of IFE during the course of this study, with 100% sensitivity for detecting BJP.

Urine immunofixation electrophoresis remains important and is complementary to serum free light chain

Abstract Articles have debated whether or not urine analysis remains valuable for identifying monoclonal gammopathies. A general impression is that the newer serum free light chain (FLC) assay is more analytically sensitive, more quantitative and simpler to perform. Many laboratory directors may have seized on the idea of eliminating urine analysis because it is a tedious procedure and requires expert interpretation while most laboratories can perform automated serum FLC assay. Others have concluded that urine immunofixation electrophoresis (IFE) optimizes the diagnostic sensitivity and should be included when there is a clinical indication. Here, I show that papers faulting urine analysis often used inappropriate urine methodology and this helps explain why there was misinterpretation. Moreover, the literature, shows urine IFE is often more sensitive for identifying low-level monoclonal FLC than the serum assay because urine IFE is as sensitive when performed appropriately and generally more specific. Besides, the reference range for serum FLC assay is unclear which is a great problem in assessing response to treatment and in identifying diseases when there is low concentration monoclonal FLC. I conclude that urine IFE remains important and is complementary to serum FLC assay, although the best algorithms for use remains to be elucidated.

Mechanized lipoprotein(a) assay as a marker for coronary artery disease illustrates the usefulness of high lipoprotein(a) levels

Only a few simple lipoprotein(a) [Lp(a)] assays are available in kit form for use in clinical laboratories. The present study compares the analytical and clinical performance of a mechanized immunonephelometric method to enzyme-linked immunosorbent assay. Clinical performance was evaluated by measuring lipoprotein markers in 191 patients, with the extent of stenosis defined by angiography. Analytically, both methods showed little or no correlation with cholesterol, high density lipoprotein cholesterol, elevated triglycerides, apo A-I and apo B, while they showed good agreement with one another (r = 0.88). The methods showed comparable well known differences between black and white persons. Logistic regression indicated that Lp(a) was a weak but independent marker for coronary artery disease (CAD). Receiver operator characteristic curve analysis showed an association with CAD only at higher Lp(a) concentrations. We conclude that Lp(a) at higher concentrations may be a contributory marker for CAD and that mechanized nephelometric assays for it can be used in the clinical laboratory.