Mariacristina Varagnolo

Quantitative automated particle-enhanced immunonephelometric assay for the routinary measurement of human cystatin C

Abstract Human cystatin C is a low molecular mass protein of 13359 Dalton recently proposed as a new very sensitive marker of changes in glomerular filtration rate. Serum cystatin C concentration correlates negatively with glomerular filtration rate as well as or better than creatinine. We evaluated a recently introduced automated nephelometric immunoassay for cystatin C in serum or EDTA-plasma samples on the Behring Nephelometer System. The assay consists of incubating the 100-fold diluted sample for 6 minutes with latex particles covalently coated with anti-human cystatin C antibodies, and then quantifying the change of light-scatter produced. Method reproducibility is satisfactory, the intra-and inter-assay coefficients of variation ranging from 1.58 % to 3.77 % and from 5.6 % to 11.47 % respectively. Rheumatoid factor (≤ 1116 IU/ml), bilirubin (≤ 418 μmmol/l), triglycerides (10.47 mmol/l), and haemoglobin (12 g/l) do not significantly interfere in the assay. No significant difference was found in cystatin C concentration between serum and EDTA-plasma samples. Cystatin C is stable in serum samples stored under different conditions up to one month. This method correlates well (mean difference = −0.536 ± 0.307 mg/l) with another commercially available particle-enhanced turbidimetric immunoassay. Cystatin C offers better clinical sensitivity than creatinine for discriminating patients with normal renal function and those with mild-to-moderate reduction in renal function. This method is suitable for routine cystatin C measurement, including emergencies.

Heterophilic antibody interference in a non-endogenous molecule assay: an apparent elevation in the tacrolimus concentration.

BACKGROUND In drug monitoring assays the most common interferences are due to hematocrit, other drugs or their metabolites, while the interference by heterophilic antibodies has been reported only when measuring endogenous molecules. In the present paper a heterophilic antibody interference in the tacrolimus measurement is described. METHODS Samples from a patient treated with tacrolimus were analyzed on RxL Dimension analyzer. Ranging drug concentrations from 49 to 12.5 microg/L, even after the interruption of the treatment, confirmation analysis were performed using heterophilic blocking tubes before tacrolimus measurement on the same analyzer, then testing the samples on V-Twin System, finally incubating the samples with chlorophenol red beta-d-galactopyranoside, beta-galactosidase, polyclonal mouse IgG, protein A and Protein G resin. RESULTS The elevated tacrolimus concentrations were due to the presence of an interference attributable to heterophilic antibodies, as confirmed by treating the samples with heterophilic blocking tubes and protein G resin. CONCLUSIONS a) The interference caused by heterophilic antibodies can be found not only in immunoassays measuring endogenous molecules, but also in those for exogenous molecules; b) the pre-treatment sample procedure, which represent the main difference between the methods affected and unaffected by the interference, is a fundamental step in removing the antibodies responsible of the interference.

Caffeine as indicator of metabolic functions of microsomal liver enzymes.

Many studies have investigated the clinical utilization of methylxanthines as in vivo probes for the assessment of liver dug-meta~~g enzyme function. These investigations demonstrated that the demethylation reaction largely reflects hepatic cytochrome Pi-450 activity. However, the dominant pathway is the demethylation of the 3-position [l]. The clearance of exogenous compounds is clinically relevant in pharmacokinetic studies for evaluation of hepatic function. Such tests can dissect the liver cell volume with unimpaired metabolic functions, and can provide an estimate of functional reserve capacity. Caffeine appeared an ideal ‘model compound’ for this investigation, since its administration during clearance tests demonstrated complete absence of toxicity in men and women [2]. In addition, caffeine presents homoge- neous volume of distribution, low protein binding, low extraction and complete hepatic metabo~sm by oxidative demethylation. Our purpose was to evaluate an enzyme multiplied i~~oassay test (EMIT) for plasma caffeine measurement. We also evaluated the results of serum caffeine and its clearance in healthy people and patients with liver disease.

Serum free light chain reference values: a critical approach.

OBJECTIVES The clinical usefulness of serum free light chain (FLC) measurement in the management of patients with plasma cell proliferative disorders has been reported in several papers, and most clinical studies use the reference ranges declared by the manufacturer. The aim of the present study was to evaluate the reproducibility of FLCs immunoassay and to validate the reference range, before introducing it in routine setting. DESIGN AND METHODS Internal quality control materials and a pool of fresh serum samples were used to evaluate imprecision; 162 fresh sera from healthy blood donors were analyzed to evaluate the reference range for FLCs. In order to verify the κ/λ FLC ratio, 43 sera from patients with polyclonal hypergammaglobulinemia were tested. The FLC immunoassay was performed using a nephelometer with the Freelite reagents. RESULTS The imprecision studies performed using a serum pool tested with two different lots of reagents showed a mean CV of 16.09% for κFLC and of 16.72% for λFLC. Lower CV%s and different mean values were found by calculating the results from each specific lot separately, while different results were obtained using the control materials provided by the manufacturer. In reference subjects, the 2.5-97.5th percentiles were found to be 4.52-22.33 and 4.84-21.88mg/L for κFLC and λFLC, respectively. The range for κ/λ ratio (0.65-2.36) was validated with the values obtained from subjects with polyclonal hypergammaglobulinemia. In retesting 15 samples from blood donor subjects with a different lot of reagents, mean bias percentages of 17.60 for κFLC and 15.26 for λFLC were obtained. CONCLUSIONS These findings confirm the lot-to-lot variability of the FLC assays also in the measurement of polyclonal light chains, as well as the need to carefully validate the reference values.

Identification and quantification of hemoglobins in whole blood: the analytical and organizational aspects of Capillarys 2 Flex Piercing compared with agarose electrophoresis and HPLC methods.

Abstract Background: The present study was conducted to evaluate the analytical performance and the organizational aspects of Capillarys 2 Flex Piercing system (CFP) respect to agarose electrophoresis and HPLC methods in hemoglobinopathies screening. Methods: The measurement of imprecision in HbA2 and HbF quantification was verified on HbA2 CFP control and on three samples; 74 whole blood samples were used to evaluate migration time imprecision of hemoglobin variants S, C and E (HbS, HbC, and HbE); to compare methods, 451 samples were tested on CFP and HPLC; reference values were verified as value distribution in 160 blood donors and at ROC curve analysis on 449 samples from routine analysis. Results: Imprecision: the analytical CV%s ranged from 1.25 to 3.9 at HbA2 quantification, the CV% was 3.78 at HbF quantification; the running time imprecision for HbS and HbC and HbE ranged from 0.20 to 0.69%. Method comparison: at regression analysis findings were HbA2: CFP=1.21×HPLC–0.64, HbF: CFP=1.31×HPLC-0.75, HbS: CFP=1.10×HPLC-3.24. Reference values: the HbA2 95th percentile range was 2.5–2.8; HbF was undetectable in 154 out 160 samples tested; at ROC curve analysis the best combination of sensitivity and diagnostic efficiency was obtained using 2.2 and 3.0, as reference values, for HbA2 and 1.1 as the upper reference limit for HbF. Organizational aspects: with respect to the procedures currently implemented in our laboratory CFP requires 2 h less time and obviates the need for some manual steps. Conclusions: The quantification, reproducibility and diagnostic efficiency provided by CFP in identification and quantification of hemoglobins appear accurate. In addition, the use of primary tubes allows improved safety, and the avoidance of some manual steps, that prolong working time and are a source of possible errors.

Performance characteristics of laboratory testing and clinical outcomes.

In order to demonstrate the relationship between performance characteristics of laboratory tests and clinical outcomes, diabetes seems to represent a paradigmatic disease: diagnosis, monitoring of therapeutic efficacy and prognosis are adequately achieved by means of laboratory testing. Starting from a simple molecule, glucose, used for the diagnosis of diabetes, continuing with creatinine, used for monitoring renal function in diabetic patients and concluding with cardiac troponins, a recognised gold standard for the diagnosis and risk stratification of cardiovascular diseases, several criticisms may be stressed considering the current methodological state-of-the art. Finally, an often overlooked aspect of performance, the analytical interferences, being responsible of unexpected results, that in turn depend from unknown or undisclosed factors will be discussed, concerning in particular, in our paper, the macroprolactin and the heterophilic antibodies aspects.

An IgE multiple myeloma: contradictory findings in clinical laboratory testing.

BACKGROUND IgE multiple myeloma is a rare kind of plasma cell disorder, characterized by an aggressive clinical course, where laboratory testing plays a fundamental role for the correct diagnosis in order to start a targeted therapy. In the present paper it is described a case of IgE myeloma where contradictory findings between immunometric and separative techniques were found. MATERIALS AND METHODS Serum and 24h urine samples were tested using electrophoresis and immunofixation electrophoresis employing IgE antiserum and IgE were quantified using an immunometric method. RESULTS Serum immunofixation evidenced a monoclonal band ascribable to IgE lambda and IgE serum concentration was 1,364,00 kU/L. Urine electrophoresis evidenced a band compatible with IgE, and urine concentration was 2715 kU/L. On the contrary in the immunofixation of the urine sample no band reacting with IgE antiserum was found. CONCLUSIONS Considering that the immunometric method measured IgE in urine sample and the electrophoresis of urine sample evidenced a band compatible with IgE, the explanation could be that during renal filtration or because of some characteristics related to urine matrix, the immunoglobulin IgE could had been modified in the site recognized by the antiserum used in immunofixation, and not in the one used in the immunometric method.

Biological variation of free light chains in serum

We read with interest the paper by Braga F. and colleagues, recently published in the journal [1], concerning the biological variation of free light chains in serum, a well-known and clinically useful test, particularly in the monitoring of patients suffering from multiple myeloma and related plasma cell disorders [2]. The biological variation study represents a fundamental issue, and the Reference Change Value (RCV) is an important parameter to objectively evaluate the significance of changes in concentrations observed in serial samples for patient monitoring. However, the data reported in the paper, in particular the analytical coefficients of variation utilized in the calculation of RCVs (CVa=kFLC 1.2%, λFLC 0.9%, FLC ratio 1.7%) present, in our opinion, some criticisms. According to the theory of the biological variation study, in order to maximize the information on the biological components of the variation, is mandatory to minimize the pre-analytical as well as the analytical sources of variability [3]. For this reason, the experimental design calls for samples analyzed in a single run in duplicate and in random order and the estimation of the analytical variance from the duplicate results for each specimen. However, in the calculation of the RCV, the analytical imprecision obtained from the internal quality control data should be considered [4]. For example, in our routine setting (Freelight reagents, Binding Site, automatized on BN II nephelometer, Siemens), the within-run imprecision (CV%) resulted around 6% for kappa and 7.5% for lambda light chains. Therefore, the analytical performances seem to be quite different from those considered in the paper for RCV calculation, making the obtained value not suitable for the application

Anti-heparan sulphate antibodies and homocysteine in dementia: markers of vascular pathology?

Increasing evidence supports a pathogenic role of heparan sulphate (HS) in the development of dementia. Since HS proteoglycans are present in the endothelial cells and perivascular basement membrane, we wanted to assess blood titres of HS antibodies (Abs) in patients with vascular dementia (VD) and in patients with Alzheimers disease (AD) with cerebrovascular disease (CVD) [mixed dementia (MixD)]. Moreover, plasma levels of homocysteine, an independent risk factor for the development of dementia as well as for CVD, were also determined. High HS Abs titres were present in one patient with VD and in two patients with mixed dementia, as well as in two neurological control patients (stroke and epilepsy). Increased homocysteine levels were found in 62.5% of patients with mixed dementia, in 22.2% of the VD subjects, in 54.2% of patients with CVD, and in 41.2% of patients with other neurological diseases. The present findings suggest that neither elevated HS Abs titres nor increased homocysteinemia may represent a useful biochemical marker for the diagnosis of VD.

Free light chains and heavy/light chains in monitoring POEMS patients.

Abstract Background: POEMS syndrome is defined by Polyneuropathy, Organomegaly, Endocrinopathy, Monoclonal gammopathy and Skin changes. The vascular endothelial growth factor (VEGF) appears to play a key role in the pathogenesis of the syndrome, and its concentrations are deemed to correlate to disease activity. The aim of the present study was to verify whether other biochemical markers including serum free light chains (FLC) and heavy/light chains (HLC) would be of value in monitoring POEMS patients. Methods: Fifty-three serum samples were collected from seven POEMS patients at diagnosis and during a follow-up period (range 14–56 months). VEGF was measured using an ELISA method, while FLC and HLC concentrations were measured using Binding Site reagents on a BNII (Siemens) nephelometer. Results: At diagnosis all patients presented high VEGF concentrations, while the κ/λFLC ratio (FLCr) was within the reference range. Four patients had abnormal HLC, HLCκ/HLCλ (HLCr) and FLC values. The relationship between the trend of VEGF and both HLC and FLC during the follow-up was analysed by means of Cohen’s κ coefficient. VEGF and HLC values displayed a significant κ-Cohen (0.537, p=0.002) in all chemotherapy-responder patients while in non-responders it did not. Conversely, in both responders and non-responders, VEGF and FLC values did not attain a significance on κ-Cohen analysis. In three out of four responders HLCr values increased, thus reflecting an improved clinical condition. Conclusions: The findings made in the present study indicate that HLC, either as intact immunoglobulin or as HLCr, may provide useful information, particularly in identifying responders and confirm that the role of FLC is unreliable in monitoring patients with POEMS syndrome.