An international multi-center serum protein electrophoresis accuracy and M-protein isotyping study. Part II: limit of detection and follow-up of patients with small M-proteins.

Background Electrophoretic methods to detect, characterize and quantify M-proteins play an important role in the management of patients with monoclonal gammopathies (MGs). Significant uncertainty in the quantification and limit of detection (LOD) is documented when M-proteins are <10 g/L. Using spiked sera, we aimed to assess the variability in intact M-protein quantification and LOD across 16 laboratories. Methods Sera with normal, hypo- or hyper-gammaglobulinemia were spiked with daratumumab or elotuzumab, with concentrations from 0.125 to 10 g/L (n = 62) along with a beta-migrating sample (n = 9). Laboratories blindly analyzed samples according to their serum protein electrophoresis (SPEP)/isotyping standard operating procedures. LOD and intra-laboratory percent coefficient of variation (%CV) were calculated and further specified with regard to the method (gel/capillary electrophoresis [CZE]), gating strategy (perpendicular drop [PD]/tangent skimming [TS]), isotyping (immunofixation/immunosubtraction [ISUB]) and manufacturer (Helena/Sebia). Results All M-proteins ≥1 g/L were detected by SPEP. With isotyping the LOD was moderately more sensitive than with SPEP. The intensity of polyclonal background had the biggest negative impact on LOD. Independent of the method used, the intra-laboratory imprecision of M-protein quantification was small (mean CV = 5.0%). Low M-protein concentration and high polyclonal background had the strongest negative impact on intra-laboratory precision. All laboratories were able to follow trend of M-protein concentrations down to 1 g/L. Conclusions In this study, we describe a large variation in the reported LOD for both SPEP and isotyping; overall LOD is most affected by the polyclonal immunoglobulin background. Satisfactory intra-laboratory precision was demonstrated. This indicates that the quantification of small M-proteins to monitor patients over time is appropriate, when subsequent testing is performed within the same laboratory.

An international multi-center serum protein electrophoresis accuracy and M-protein isotyping study. Part I: factors impacting limit of quantitation of serum protein electrophoresis.

Background Serum protein electrophoresis (SPEP) is used to quantify the serum monoclonal component or M-protein, for diagnosis and monitoring of monoclonal gammopathies. Significant imprecision and inaccuracy pose challenges in reporting small M-proteins. Using therapeutic monoclonal antibody-spiked sera and a pooled beta-migrating M-protein, we aimed to assess SPEP limitations and variability across 16 laboratories in three continents. Methods Sera with normal, hypo- or hypergammaglobulinemia were spiked with daratumumab, Dara (cathodal migrating), or elotuzumab, Elo (central-gamma migrating), with concentrations from 0.125 to 10 g/L (n = 62) along with a beta-migrating sample (n = 9). Provided with total protein (reverse biuret, Siemens), laboratories blindly analyzed samples according to their SPEP and immunofixation (IFE) or immunosubtraction (ISUB) standard operating procedures. Sixteen laboratories reported the perpendicular drop (PD) method of gating the M-protein, while 10 used tangent skimming (TS). A mean percent recovery range of 80%-120% was set as acceptable. The inter-laboratory %CV was calculated. Results Gamma globulin background, migration pattern and concentration all affect the precision and accuracy of quantifying M-proteins by SPEP. As the background increases, imprecision increases and accuracy decreases leading to overestimation of M-protein quantitation especially evident in hypergamma samples, and more prominent with PD. Cathodal migrating M-proteins were associated with less imprecision and higher accuracy compared to central-gamma migrating M-proteins, which is attributed to the increased gamma background contribution in M-proteins migrating in the middle of the gamma fraction. There is greater imprecision and loss of accuracy at lower M-protein concentrations. Conclusions This study suggests that quantifying exceedingly low concentrations of M-proteins, although possible, may not yield adequate accuracy and precision between laboratories.

Preclinical evaluation of a semi-automated and rapid commercial electrophoresis assay for von Willebrand factor multimers.

BACKGROUND: The von Willebrand factor (VWF) multimer test is required to correctly subtype qualitative type 2 von Willebrand disease (VWD). The current VWF multimer assays are difficult, nonstandardized, and time-consuming. The purpose of this study was to evaluate the clinical utility of the commercial VWF multimer kit by Sebia (Lisses, France), an electrophoresis technique yielding same-day results. METHODS: Ten healthy volunteer plasma samples, in-house reference plasma (IRP) and commercial normal plasma (CNP) samples, 10 plasma samples from patients with a known VWD type, 1 hemophilia A plasma sample, and 7 external quality assurance (EQA) samples were analyzed using the commercial VWF multimer kit. Additional coagulation testing included measurements of VWF antigen (VWF:Ag), VWF activity (VWF:Ac), and FVIII activity (FVIII:C). RESULTS: The CNP results revealed a relative loss of the highest molecular weight multimers; therefore, IRP was preferred as the reference sample. The interpretations of 10 patients with a known VWD type could be successfully reproduced and agreed with previous VWF multimer results. In all EQA surveys, the multimer results and final VWD diagnosis agreed with expert opinion. CONCLUSIONS: The VWF multimer assay by Sebia is easy to perform and can be successfully implemented in any clinical laboratory for second-stage evaluation of VWD. The resolution power of multimer distribution is adequate to correctly classify VWD types 1, 2A, 2B, and 3.

Pulse wave velocity and its gender-related associations with cardiovascular risk factors in a high cardiovascular risk population.

INTRODUCTION: Associations found between pulse wave velocity (PWV) and cardiovascular risk factors (CVrF) are diverse. We aimed to evaluate whether differences in PWV and its associations with CVrF in a high cardiovascular risk population exist between genders and between the whole population (WHgr) and groups of apparently healthy (AHgr) and those of hypertensive, obese or diabetics (Rgr). MATERIAL AND METHODS: Pulse wave velocity measured by Arteriograph was investigated in 805 adults aged 20-65, randomly selected from the Tallinn Population Register. RESULTS: Pulse wave velocity was the highest in Rgr and no differences were found between genders of the same group. In women of WHgr and AHgr age and SBP with addition of BMI and apolipoprotein B (ApoB) were associated with 54% and 48%, and without ApoB in Rgr with only 30% of PWV values. In men aged ≥ 50 of WHgr with elevated SBP odds ratios for increased PWV were 25.3 and 3.5, in Rgr 21.2 and 2.2, in those aged ≥ 50 AHgr 28.4. In women aged ≥ 50 of WHgr with elevated SBP and diabetes odds ratios were 5.5, 4.9 and 4.0, in Rgr with elevated SBP and diabetes 3.6 and 3.7, in those aged ≥ 50 AHgr 29.3. CONCLUSIONS: The associations of ApoB and BMI with PWV and diabetes with elevated PWV indicative of increased aortic stiffness were unique for women. Aging and SBP were related to PWV even in AHgr, although age ≥ 50 years in Rgr women and normal SBP in AHgr were not associated with elevated PWV.

Prevalence of cardiovascular disease risk factors in Tallinn, Estonia.

BACKGROUND AND OBJECTIVE: Cardiovascular diseases are still a major public health concern in Estonia despite the decline in the mortality rate during the past decade. For better preventive strategies we aimed to investigate the prevalence of cardiovascular disease risk factors and their relations with age, gender and ethnicity. MATERIALS AND METHODS: The cross-sectional study was carried out in Tallinn, Estonia. Two hundred individuals from each of the sex and 10-year age group (range 20-65 years of age) were randomly selected and invited to participate. Final study sample consisted of 511 men and 600 women (mean age of 46 years). Physiological measurements were taken and blood samples were drawn for standard measurements of the following markers: total cholesterol, high- and low-density lipoprotein cholesterol, apolipoproteins, triglycerides, glucose and inflammatory markers. RESULTS: Overall, 31% of the study subjects had high blood pressure, 23% had metabolic syndrome, and 55% were overweight/obese. The prevalence of all risk factors increased with age amongst both genders. The proportion of individuals having increased cholesterol, apolipoprotein B-100, and homocysteine levels was very high amongst both genders (60-80%). More Russians and other ethnic minorities compared to ethnic Estonians had calculated 10-year CHD risk≥10%. CONCLUSIONS: The study established a high prevalence of cardiovascular disease risk factors in Estonian adults (20-65 years of age). Younger portion of the population and some extent ethnic considerations should be taken into account when designing future studies, health prevention activities and interventions.

Prevalence and determinants of hypertension in Estonian adults.

BACKGROUND: Although Eastern Europe, including Estonia, has one of the highest morbidity and mortality rates associated with hypertension, there is little information in the literature concerning the biochemical risk factor profile or its association with hypertension in Estonia. This study examined the cross-sectional gender-stratified association between biochemical risk markers and hypertension in a population-based sample of adults in Estonia. METHODS: The study was carried out in Tallinn, Estonia and consisted of 511 men and 600 women with a mean age of 46 years. Physiological measurements were taken and blood samples drawn to measure the following markers: cholesterol, high- and low-density lipoprotein cholesterol, apolipoproteins A-1 and B, lipoprotein(a), triglycerides, glucose, fibrinogen, high-sensitivity C-reactive protein and homocysteine. RESULTS: Overall, 36% of participants had hypertension, with approximately 80% being aware of their condition. A total of 40% of participants reported taking antihypertensive medication. Multivariate binary logistic regression analysis showed that a decrease in high-density lipoprotein cholesterol and increases in age, body mass index, apolipoprotein B, triglyceride and homocysteine levels were associated with an increased probability of hypertension. CONCLUSIONS: Elevations in biochemical markers and cardiovascular risk factors are associated with hypertension. Increasing body mass index, triglyceride, apolipoprotein B and homocysteine levels with decreasing high-density lipoprotein cholesterol level should be investigated and monitored in Estonian adults.

Cardiovascular disease risk factors in homeless people.

BACKGROUND: Cardiovascular diseases (CVD) are associated with significant morbidity and mortality, which is highest in Eastern Europe including Estonia. Accumulating evidence suggests that life-style is associated with the development of CVD. The aim of this study was to evaluate the informative power of common CVD-related markers under unhealthy conditions. SUBJECTS: Subjects (n = 51; mean age 45 years; 90% men) were recruited from a shelter for homeless people in Tallinn, Estonia, and consisted of persons who constantly used alcohol or surrogates, smoked, and were in a bad physical condition (amputated toes, necrotic ulcers, etc.). METHODS: Blood pressure, pulse rate, and waist circumference were measured, and body mass index (BMI) was calculated. The following markers were measured in blood serum: total cholesterol (TChol), high-density lipoprotein cholesterol (HDL-Chol), low-density lipoprotein cholesterol (LDL-Chol), plasma triglycerides (TG), apolipoproteins A-l (ApoA1) and B (ApoB), lipoprotein(a) (Lp(a)), glycated hemoglobin (HbA1c), glucose (Gluc), high-sensitivity C-reactive protein (hsCRP), serum carbohydrate-deficient transferrin (CDT), gamma-glutamyltransferase (GGT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Except smoking, the anamnestic information considering eating habits, declared alcohol consumption and medication intake were not included in the analysis due to the low credibility of self-reported data. RESULTS: More than half of the investigated patients had values of measured markers (hsCRP, TChol, LDL-Chol, TG, HbA1c, ApoA1, ApoB, Lp(a), Gluc) within normal range. Surprisingly, 100% of subjects had HDL-Chol within endemic norm. CONCLUSION: This study demonstrates that traditional markers, commonly used for prediction and diagnosis and treatment of CVD, are not always applicable to homeless people, apparently due to their aberrant life-style.