[The ways of harmonization of clinical laboratory measurement techniques].

The results of implementation of different clinical laboratory techniques are to be equal in clinically significant limits to be optimally applied in diagnostics of diseases and treatment of patients. When the results of laboratory tests are not standardized and harmonized for the very same clinical assay the results can be expressed by unmatched numbers. Unfortunately, in some handbooks the values are presented based on the results of application of specific laboratory techniques without considering possibility or likelihood of differences between various techniques. When this is a case, accumulation of data of diferent clinical research studies and working out of clinical handbooks on this basis will be inconsistent. Inadequate understanding of issue that the results of laboratory tests are not standardized and harmonized can lead to incorrect clinical, financial, managerial or technical decisions. The standardization of clinical laboratory techniques was applied to many measurands related to primary referent techniques (standard specimen of pure substance) or/and developed referent measurement techniques. However, harmonization of clinical laboratory techniques for those measurands which are not related any developed measurement techniques is quite problematic due to inadequate determination of measurand, its inadequate analytical specificity, insufficient attention to commutability of referent materials and poor systematic approach to harmonization. To overcome these issues an infrastructure is to be developed to support systematic approach to identification and prioritization of measurands which are to be harmonized on the basis of clinical importance and technical applicability. The management of technical implementation harmonization process for specific measurands.

Diagnostic terminology for reporting thyroid fine needle aspiration cytology: European Federation of Cytology Societies thyroid working party symposium, Lisbon 2009.

A European Federation of Cytology Societies (EFCS) working party of 28 members from 14 European countries met at the European Congress of Cytology in Lisbon in September 2009, with two observers from the USA, to discuss the need for standardising thyroid FNA nomenclature in the light of the National Institute of Cancer (NCI) recommendations resulting from the State of the Science conference in Bethesda in 2007. The data were obtained through two questionnaires sent by email and a transcript of the live discussion at the congress, which is presented in full. The surveys and discussion showed that there were currently no national terminologies for reporting thyroid FNA in the different European countries except in Italy and the UK. Personal, 'local', surgical pathology and descriptive terminologies were in use. All but one of the working party members agreed that thyroid FNA reporting should be standardised. Whilst almost a third would adopt the NCI Bethesda terminology, which offers the advantages of a 'risk of cancer' correlation and is linked to clinical recommendations, more than half favoured a translation of local terminology as the first step towards a unified nomenclature, as has been done recently in the UK. There was some disagreement about the use of: a) the six-tiered as opposed to four or five-tiered systems, b) the use of an indeterminate category and c) the 'follicular neoplasm' category, which was felt by some participants not to be different from the 'suspicious of malignancy' category. The conclusions will be passed to the different national societies of cytology for discussion, who will be asked to map their local terminologies to the Bethesda classification, observe its acceptance by clinicians and audit its correlation with outcome.

Belgian consensus guidelines for follow-up of women with cervical cytological abnormalities.

In medical care cervical cancer screening is important because it enables the detection of precancer and cancer at an early stage. By adequate treatment after a screening-detected lesion it helps to reduce the mortality related to cervical cancer. Worldwide, many millions of women have smears taken at a more or less regular base and of these, approximately 7% are abnormal, and follow-up is thus required.As this represents an important cost in medical health care and has serious consequences for the affected women, it is important to have uniform and clear guidelines to allow an optimal follow-up and clinical management. A system for the uniform reporting of cervical cytology has been designed by the National Cancer Institute (U.S.A.) and resulted in the Bethesda System 1991. The present paper and the terminology used are based on the Bethesda System revised in 2001. It explains the guidelines, based on the 2001 Bethesda System and the 2004 consensus guidelines for the management of women with cervical cytological abnormalities, as developed by the members of the Board of the Belgian Society of Clinical Cytology, and adapted to the Belgian situation.

Validation of 5 routine assays for serum free testosterone with a candidate reference measurement procedure based on ultrafiltration and isotope dilution-gas chromatography-mass spectrometry.

BACKGROUND/METHOD: The analytical validity of free testosterone (FTe) analog immunoassays is subject to much controversy. We revisited the validation of 4 analog assays and 1 FTe calculation procedure with a metrologically traceable reference measurement procedure (RMP) based on ultrafiltration and isotope dilution-mass spectrometry for direct measurement of Te in the ultrafiltrate. To this end, we performed split-sample measurements of 40 male sera. RESULTS: Deming regression showed that 3 of the immunoassays had moderate to good correlation (0.8474 < or = r < or = 0.9241) with the RMP; however, the slope was markedly below 1. The FTe calculation procedure was in good agreement with this result. The Sy/x values for all assays were higher than the combined imprecision values, which indicate their susceptibility to matrix-related effects. CONCLUSIONS: The study demonstrated substantial differences in analytical quality of FTe assays; however, the results suggested that after extending the validation with a larger variety of samples, recalibration of some analog assays might be worth further investigation.

Variant t(2;11)(p11;q13) associated with the IgK-CCND1 rearrangement is a recurrent translocation in leukemic small-cell B-non-Hodgkin lymphoma.

Classical t(11;14)(q13;q32) involving IGH-CCND1 is typically associated with aggressive CD5-positive mantle cell lymphoma (MCL). Recently, we identified the IGK variant of this translocation, t(2;11)(p11;q13), in three patients with a leukemic small-cell B-non-Hodgkin lymphoma. In all cases, rearrangements of the IGK and CCND1 genes were demonstrated by fluorescence in situ hybridization. Moreover, we mapped the 11q13 breakpoint of this variant translocation in the 3' region of CCND1 which contrasts with the 5' breakpoints in a standard t(11;14)(q13;q32). Expression of cyclin D1 was shown in two cases analyzed either at diagnosis or during disease progression. All three patients were asymptomatic at presentation and no initial therapy was required. One patient died of a progressive disease 58 months from diagnosis, and two patients showed stable disease after 12 months of follow-up. In two analyzed cases, mutated IGVH genes were identified. Our findings indicate that variant t(2;11)(p11;q13) does not typify a classical MCL but possibly a more indolent leukemic lymphoma originating from an antigen experienced (mutated) B cell.

Preoperative breast MRI in patients with invasive lobular breast cancer.

To investigate the use of MRI in preoperative characterization of invasive lobular breast cancer (ILC) and in detection of multifocal/multicentric disease. We retrospectively reviewed T1-weighted FLASH 3D precontrast and postcontrast MR images together with subtraction images of 26 women with histopathologically proven invasive lobular cancer. Two experienced radiologists described tumor patterns of ILC independently. MR findings of unifocal, multifocal, single quadrant and multiquadrant disease were correlated with results of other imaging techniques and compared with histopathological findings as gold standard. Most ILC presented on MRI as a single spiculated/irregular, inhomogeneous mass (pattern 1, n=12) or as a dominant lesion surrounded by multiple small enhancing foci (pattern 2, n=8). Multiple small enhancing foci with interconnecting enhancing strands (pattern 3) and an architectural distortion (pattern 4) were both described in three cases. There was one case of a focal area of inhomogeneous enhancement (pattern 5) and one normal MR examination (pattern 6). Unifocal and multifocal lesions were identified on MRI in four patients with normal conventional imaging. In nine women, multiple additional lesions or more extensive multiquadrant disease were correctly identified only on MRI. MRI may play an important role in the evaluation of patients with ILC, which is often difficult to diagnose on clinical examination and conventional imaging and more likely occur in multiple sites and in both breasts. However, false-negative MR findings do occur in a small percentage of ILC.

Thyroid fine needle aspiration: the morphological features on ThinPrep slide preparations. Eighty cases with histological control.

This study had several purposes: to define cytomorphological features of thyroid cells that might be modified by alcohol fixation; to optimize May-Grünwald-Giemsa (MGG) staining on ThinPrep(R) (TP; Cytyc Inc., Bexborough, MA, USA) slides and to compare the diagnostic accuracy of slides prepared by a liquid-based method with those obtained by conventional technique. This study included 120 cases of ultrasound-guided fine needle aspiration (FNA) of the thyroid and 55 FNAs performed on surgically resected thyroid specimens. Histological control was available in 80 cases. In the first group of 120 FNAs, a split-sample technique was used for the TP. Three screenings were performed: first, an individual screening of the conventional smears (CS) and of the TP, a second screening to compare cells observed on the TP with the histological control and a third screening to assess the previously defined diagnostic criteria. Twenty-seven TP cases (22%) were considered unsatisfactory for diagnosis compared with 10 in CS (8%). The high rate of unsatisfactory cases with TP is likely to be due to the use of the split-sample technique. The sensitivity was 94% for CS and 81% for TP. The specificity was 67% and 60% for CS and TP, respectively. Two occult papillary carcinomas were missed by both methods. As for the MGG staining, the modified technique used for TP resulted in the same quality as the standard procedure. Conversely, TP did however induce uncommon morphological features. In this study, sensitivity and specificity levels are higher for CS than for TP; the difference may be explained by the fact that the methanol fixative used for TP induces some cytological alterations, especially in oncocytic tumours and lymphocytic thyroïditis.

Breast MR imaging in a patient with unilateral axillary lymphadenopathy and unknown primary malignancy.

Bilateral invasive breast cancers were detected on MR imaging in a patient with left unilateral axillary lymphadenopathy and normal findings at physical examination, mammography, and ultrasound of both breasts. One spiculated and a second ill-defined enhancing lesion in the left breast as well as a superficial circumscribed lesion in the right breast were proven to be invasive malignancies. In patients with isolated axillary lymph nodes and occult primary malignancy, breast MRI can identify or exclude the breast as primary site, which proves the superiority and diagnostic benefit of this imaging method and also its contribution to the therapeutic approach.

Trueness verification in European external quality assessment schemes: time to care about the quality of the samples.

An external quality assessment (EQA) survey on 14 fresh-frozen, single-donation sera assigned with reference measurement procedure (RMP) values revealed a mean bias of + 5.2% and + 3.7% for the cholesterol oxidase and the photometric glucose oxidase procedure groups, respectively. Conversely, on lyophilized sera, the same procedure groups showed almost bias-free results, the differences from the RMP values being only -0.8% for cholesterol and + 0.7% for glucose. These data, which are in fairly good agreement with the literature, suggest the existence of artificial matrix effects in processed materials. Therefore they indicate that, currently, assessment of trueness is hampered in many European EQA schemes, as most of them use lyophilized sera. This approach may give a false impression about the trueness of laboratory results as well as carrying the risk that laboratories calibrated on the RMP values of the survey samples could make errors in patient testing. Consequently, if European EQA is willing to fulfil a post-market vigilance function of the performance of in vitro diagnostic medical devices, then the time has come to tackle the problem of the quality of the survey samples. EQA organizers urgently need to make an effort to seek out materials that analytically behave like authentic clinical specimens. In the meantime, alternative approaches should be used. Although not ideal, the special survey described in this article is one of the possibilities. Naturally, it implies logistic problems and increased costs for the individual EQA schemes. However, both can be overcome with the cooperation of the predominantly nationally organized schemes.

The ionized fraction of serum total magnesium in hemodialysis patients: is it really lower than in healthy subjects?

AIM OF THE STUDY: Based on data in the literature, it remains unclear whether the ionized fraction of serum total magnesium (Mg) is lower in chronic hemodialysis (HD) patients compared to healthy subjects. PATIENTS AND METHODS: The ionized fraction of serum total Mg was investigated in 49 HD patients, pre- and post-dialysis, and compared to 30 healthy controls. The quality of the analytical performance of the Mg measurements has been emphasized by applying a reference method and/or rigorous internal quality control (IQC). In addition, the ionized fraction of serum total calcium (Ca) was measured in both populations, because the results for Mg should be related to those of Ca. RESULTS: In HD patients, the ionized fraction of serum total Mg was on average 65% (pre-dialysis 64.2% and post-dialysis 66.2%). In healthy controls, the ionized fraction was 64.9%. When the analytical variability was taken into account, no significant differences (p > 0.05) were observed between pre- and post-dialysis samples and controls. For Ca, an ionized fraction of 55.3% was found in HD patients, which was not significantly different from the fraction obtained in the control group (55.7%). CONCLUSION: The present study demonstrates that, compared to healthy controls, the ionized fraction of serum total Mg is not different in hemodialysis patients.

Strategies for determination of insulin with tandem electrospray mass spectrometry: implications for other analyte proteins?

Using human insulin (MW 5808 Da) as a model compound, the possible strategies towards optimization of sensitivity and selectivity of measurement by electrospray ionization with a standard triple quadrupole mass spectrometer were investigated. For measurement in selected ion-monitoring (SIM) mode, these strategies involved systematic variation of instrumental parameters and spray pH. In this investigation four different operating modes were used corresponding to positive/negative ionization modes with acidic/basic sprays and pH reversed (hereafter termed 'wrong-way-round' operation); the cone voltage was optimized for each mode of operation. When collision-activated dissociation (CAD) is employed, two additional operation modes are possible: namely, low collision energies (10-35 eV, CAD-l) for the generation of sequence-specific fragments and high collision energies (>80 eV, CAD-h) for the generation of nonspecific fragments. Overall, this results in twelve different modes of operation. Loop-injection of aqueous insulin standards were run for each of the twelve operating modes and measurements made for five different charge states (n = 2-6) observable with our instrument that has an upper mass limit of m/z 4000. The signal/noise (S/N) ratio was optimized for each charge state, resulting in 60 measurements. The best S/N ratios (20 000) were achieved under positive SIM conditions with charge state 6 (m/z 969) and under 'wrong-way-round' negative SIM conditions with charge state 3 (m/z 1935). Lower S/N ratios were observed under positive CAD-h conditions with charge state 5 (m/z 1163, S/N 15 000) and positive CAD-l conditions with charge state 6 (m/z 969, S/N 10 000). All other operating modes gave maximum S/N ratios of 4000. For measurement of insulin standards, the results obtained show SIM to give the best S/N ratio. However, for samples in complex matrices, our general experience suggests CAD to be the preferable operating mode. Consequently, for the development of a quantitative method for proteins in general, it might be advocated that all of the twelve operating modes and all relevant charge states be investigated to find the optimum S/N ratio.

Models for combining random and systematic errors. assumptions and consequences for different models.

A series of models for handling and combining systematic and random variations/errors are investigated in order to characterize the different models according to their purpose, their application, and discuss their flaws with regard to their assumptions. The following models are considered 1. linear model, where the random and systematic elements are combined according to a linear concept (TE = absolute value(bias) + z x sigma), where TE is total error, bias is the systematic error component, sigma is the random error component (standard deviation or coefficient of variation) and z is the probability factor; 2. squared model with two sub-models of which one is the classical statistical variance model and the other is the GUM (Guide to Uncertainty in Measurements) model for estimating uncertainty of a measurement; 3. combined model developed for the estimation of analytical quality specifications according to the clinical consequences (clinical outcome) of errors. The consequences of these models are investigated by calculation of the functions of transformation of bias into imprecision according to the assumptions and model calculations. As expected, the functions turn out to be rather different with considerable consequences for these types of transformations. It is concluded that there are at least three models for combining systematic and random variation/errors, each created for its own specific purpose, with its own assumptions and resulting in considerably different results. These models should be used according to their purposes.

Quantitative analysis of urinary C-peptide by liquid chromatography-tandem mass spectrometry with a stable isotopically labelled internal standard.

We describe the first results of a quantitative LC-tandem mass spectrometry method for urinary C-peptide with the use of [2H14]C-peptide as internal standard. LC was based on gradient elution of a Hypersil PEP C18 column. Mass spectrometry was performed in the negative electrospray ionization mode and by monitoring of the transitions at m/z 1514/1334 ([2H14]C-peptide) and 1507/1320 (C-peptide). For sample preparation, we applied ultrafiltration. The analytical performance of the method in terms of measurement precision gave an RSD of <2% (n=10). The overall imprecision was investigated from independent analysis of two urine samples in six-fold and resulted in an RSD<5%. The limit of detection, expressed as signal-to-noise ratio 3, was approximately 0.15 ng C-peptide injected. Analysis of 10 random urine samples from laboratory volunteers showed interference-free ion chromatograms at a signal-to-noise ratio of approximately 75 on average. The C-peptide concentrations calculated from quantification by the bracketing calibration technique ranged from 32 to 165 ng/ml.