The harmonization issue in laboratory medicine: the commitment of CCLM.

The analytical quality of the clinical laboratory results has shown a significant improvement over the past decades, thanks to the joint efforts of different stakeholders, while the comparability among the results produced by different laboratories and methods still presents some critical issues. During these years, Clinical Chemistry and Laboratory Medicine (CCLM) published several papers on the harmonization issue over all steps in the Total Testing Process, training an important number of laboratory professionals in evaluating and monitoring all the criticisms inherent to the pre-analytical, as well as analytical and post analytical phases: from the consensus statement on the most informative testing in emergency setting, to the prevention and detection of hemolysis or to patients identification and tube labeling procedures, as far as to different approaches to harmonize hormones measurements or to describe new reference methods or to harmonize the laboratory report. During these years the commitment of the journal, devoted to the harmonization processes has allowed to improve the awareness on the topic and to provide specific instruments to monitor the rate of errors and to improve patients safety.

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.

ANMCO/ISS/AMD/ANCE/ARCA/FADOI/GICR-IACPR/SICI-GISE/SIBioC/SIC/SICOA/SID/SIF/SIMEU/SIMG/SIMI/SISA Joint Consensus Document on cholesterol and cardiovascular risk: diagnostic-therapeutic pathway in Italy.

Atherosclerotic cardiovascular disease still represents the leading cause of death in Western countries. A wealth of scientific evidence demonstrates that increased blood cholesterol levels have a major impact on the outbreak and progression of atherosclerotic plaques. Moreover, several cholesterol-lowering pharmacological agents, including statins and ezetimibe, have proved effective in improving clinical outcomes. This document focuses on the clinical management of hypercholesterolaemia and has been conceived by 16 Italian medical associations with the support of the Italian National Institute of Health. The authors discuss in detail the role of hypercholesterolaemia in the genesis of atherosclerotic cardiovascular disease. In addition, the implications for high cholesterol levels in the definition of the individual cardiovascular risk profile have been carefully analysed, while all available therapeutic options for blood cholesterol reduction and cardiovascular risk mitigation have been explored. Finally, this document outlines the diagnostic and therapeutic pathways for the clinical management of patients with hypercholesterolaemia.

Measurement of free light chains - pros and cons of current methods.

The measurement of the serum free light chains (FLC) is of paramount importance in the management of patients with plasma cell dyscrasias (PSD). The immunoassays for FLC measurement require adequate precision, accuracy, specificity and reproducibility between batches to prevent under or over estimation of FLC concentration and for an adequate patient monitoring. Considering the peculiarity of the measurand (monoclonal proteins), the optimization of any analytical aspect is difficult to achieve. Three methods are currently available for the assay. The first one has been on the market for over 15 years, and it is based on polyclonal antibodies. The vast majority of the clinical studies demonstrating the utility of the serum FLC measurement have been performed using this assay. A second method based on monoclonal antibodies (mAbs) was marketed in 2011; a third one, also employing mAbs and allowing the simultaneous measurement of κ and λ FLC is in the process of publication. These methods show relevant differences in the type of antibodies used and in the assay design and it is not possible to identify an immunoassay that is superior to the others in any analytical aspect. The comparison studies show that the three methods differ significantly in terms of quantitative values, especially when samples containing monoclonal proteins are compared. Hence the methods cannot be used interchangeably, in particular when the assay is used to monitor the patient response to therapy. In the absence of an international standard for FLC measurement, it is impossible, at this stage to establish, which method shows the best accuracy.

Comparison of serum creatinine and cystatin C for early diagnosis of contrast-induced nephropathy after coronary angiography and interventions.

BACKGROUND: The diagnostic accuracy of serum creatinine and cystatin C (Cys) as early predictors of contrast-induced nephropathy (CIN) has been debated. We investigated the diagnostic sensitivities, diagnostic specificities, and variations from baseline for serum creatinine and Cys in CIN. METHODS: We prospectively evaluated 166 patients at risk for CIN at baseline, and at 12, 24, and 48 h after exposure to contrast media. CIN occurred in 30 patients (18%). Changes (Δ) compared to baseline in serum creatinine and Cys were evaluated at the predefined time points. ROC curve analysis was performed for the Δ 12-h basal serum creatinine and Cys. RESULTS: The Δ serum creatinine at 12 h from baseline was the earliest predictor of CIN [area under the ROC curve (AUC) = 0.80; P < 0.001]. The Δ serum creatinine 15% variation [0.15 mg/dL (13.2 µmol/L)] yielded 43% diagnostic sensitivity and 93% diagnostic specificity. The ΔCys at 12 h from baseline performed significantly worse than serum creatinine (AUC = 0.48; P = 0.74). CONCLUSIONS: Variations from the serum creatinine baseline offer better diagnostic accuracy for predicting CIN at an earlier stage than similar variations in Cys. An additional diagnostic value of Cys over the determination of serum creatinine in the setting of CIN was not observed.

Serum thyroglobulin reference values according to NACB criteria in healthy subjects with normal thyroid ultrasound.

BACKGROUND: The present study was undertaken to establish serum thyroglobulin (Tg) normal reference values in a large group of healthy subjects. METHODS: Four hundred and thirty-eight non-smoking healthy subjects were selected to assess the Tg reference values (209 males, 229 non-pregnant females, age 34.7±13.1 years). Inclusion criteria were: no personal or familial history of thyroid disease, thyrotropin levels from 0.5 to 2.00 mUI/L, negative thyroperoxidase and thyroglobulin antibodies. In addition, the patients had a normal size thyroid (females ≤18 mL, males ≤25 mL) without nodules on the thyroid ultrasound (TUS). According to National Academy of Clinical Biochemistry (NACB) criteria the Tg results were transformed to a logarithmic scale and reference ranges were calculated as mean±2 SD. RESULTS: Serum Tg was measured on the Beckman Coulter UniCel DxI 800 automated platform by the simultaneous 1-step immunoenzymatic Access Thyroglobulin assay (Beckmann-Coulter SA, Nyon, Switzerland). Serum Tg levels were higher in females than in males (p=0.0022). Accordingly, gender-specific reference values were calculated (i.e., males: 1.40-29.2 ng/mL; females: 1.50-38.5 ng/mL). CONCLUSIONS: To the best of the authors' knowledge, the first reference interval study for Tg that integrates NACB criteria and TUS assessment for the selection of the reference population is provided here.

Age dependence of within-subject biological variation of nine common clinical chemistry analytes.

BACKGROUND: The knowledge of biological variation (BV) data is important for clinical decisions and as a basis for defining analytical quality specifications. However, in generating reliable data of biological variation there are still some unsolved problems, such as age dependence. The aim of our work is to verify this aspect. METHODS: Twenty-six subjects divided into three groups by age were studied. Blood samples were collected in lithium heparin tubes for four weeks at one week intervals, on the same day of the week (Tuesday) and at the same time of day (8-9 a.m.) by the same phlebotomist. They were analysed in duplicate for creatinine, urate, calcium, albumin, total cholesterol, high density lipoprotein (HDL) and low density lipoprotein (LDL)-cholesterol, triglycerides and iron. After outlier exclusion by Cochran's test, components of biological variation were calculated by ANOVA. The significance of the differences between results of the classes was also calculated with the Student's test (t-test) and the Fisher's test (F-test). RESULTS: Excluding albumin, the group 3 results (age range from 78 to 98 years) showed significantly lower CV within subjects (CV(W)) than the other two groups. CONCLUSIONS: Our data seem to highlight the relevance of the age when choosing the reference subjects for biological variation studies. The level of within-subject biological variation of the elderly group may have been further reduced by the homogeneity of the group constituted by individuals living together in the same nursing home.

Thyroid volume influences serum calcitonin levels in a thyroid-healthy population: results of a 3-assay, 519 subjects study.

BACKGROUND: We aimed to assess determinants for serum calcitonin (CT) levels and to define reference ranges for different CT immunoassays integrating thyroid ultrasonography (TUS) and conventional clinical and biochemical criteria to select the reference population. METHODS: Five hundred and nineteen thyroid-healthy subjects were included in this prospective cross-sectional population study. Thyroid volume was measured by TUS, while serum CT levels were measured by three different immunoassays. RESULTS: Significant interassay differences were found and the agreement between assays was poor. CT levels were higher in males than in females in all immunoassays. Using the first two assays, both gender and thyroid volume were independent determinants for CT levels. While using the third assay, one thyroid volume was the only determinant for CT levels. CONCLUSIONS: Thyroid volume is a relevant determinant for CT levels. In the clinical practice, the difference of the thyroid sizes in males and females warrants gender-specific reference ranges.

Identification of amyloidogenic light chains requires the combination of serum-free light chain assay with immunofixation of serum and urine.

BACKGROUND: The diagnosis of systemic immunoglobulin light-chain (AL) amyloidosis requires demonstration of amyloid deposits in a tissue biopsy and amyloidogenic monoclonal light chains. The optimal strategy to identify the amyloidogenic clone has not been established. We prospectively assessed the diagnostic sensitivity of the serum free light chain (FLC) kappa/lambda ratio, a commercial serum and urine agarose gel electrophoresis immunofixation (IFE), and the high-resolution agarose gel electrophoresis immunofixation (HR-IFE) developed at our referral center in patients with AL amyloidosis, in whom the amyloidogenic light chain was unequivocally identified in the amyloid deposits. METHODS: The amyloidogenic light chain was identified in 121 consecutive patients with AL amyloidosis by immunoelectron microscopy analysis of abdominal fat aspirates and/or organ biopsies. We characterized the monoclonal light chain by using IFE and HR-IFE in serum and urine and the FLC kappa/lambda ratio in serum. We then compared the diagnostic sensitivities of the 3 assays. RESULTS: The HR-IFE of serum and urine identified the amyloidogenic light chain in all 115 patients with a monoclonal gammopathy. Six patients with a biclonal gammopathy were omitted from the statistical analysis. The diagnostic sensitivity of commercial serum and urine IFE was greater than that of the FLC kappa/lambda ratio (96% vs 76%). The combination of serum IFE and the FLC assay detected the amyloidogenic light chain in 96% of patients. The combination of IFE of both serum and urine with the FLC kappa/lambda ratio had a 100% sensitivity. CONCLUSIONS: The identification of amyloidogenic light chains cannot rely on a single test and requires the combination of a commercially available FLC assay with immunofixation of both serum and urine.

Diagnostic accuracy of a reagent strip for assessing urinary albumin excretion in the general population.

BACKGROUND: Albuminuria is a sensitive marker of renal derangement and has been included in a number of studies investigating chronic kidney diseases (CKDs). This study is aimed to evaluate the diagnostic performances of a strip for measuring the albumin/creatinine ratio (ACR) in the general population and to compare it with those found in a diabetic population. METHODS: Urine samples were obtained from 201 consecutive subjects enrolled in an epidemiological study and from 259 type 2 diabetic patients. Urine was tested for albumin and creatinine using the strip (Clinitek Microalbumin) and laboratory methods. A hundred samples were stored under various conditions to assess analyte stability. RESULTS: In the general population, the strip test reached a 90% sensitivity and 91% specificity, considering the laboratory method as the 'gold standard', sparing >80% of subjects the laboratory tests at the expense of a 1% false negative rate and an 8% false positive rate. Regarding sensitivity and specificity, the ACR test performs very similarly in the general population and in the diabetics. The stability study showed that storage at -20 degrees C induced a significant decrease in the albumin concentration with both methods, such that 5% of the samples were re-classified in the lower ACR class. Storage at -80 degrees C for up to 12 months did not affect the measurement with both methods. CONCLUSION: Clinitek Microalbumin strips can be used for screening purposes in the general population since they correctly classify a significant percentage of subjects, particularly those with a normal albuminuria. Storage at -80 degrees C does not affect strip results. Screening with the strip and confirming positive results with a wet chemistry method are an efficient strategy for detecting albuminuria in the general population.

A Summary of Worldwide National Activities in Chronic Kidney Disease (CKD) Testing.

Chronic kidney disease (CKD) is a major public health issue worldwide and is associated with adverse health outcomes, especially in low- and middle-income countries. In a cash limited healthcare system, guidelines that improve the efficiency of health care free up resources needed for other healthcare services. This short review presents some examples from national acitivities in CKD testing, including countries throughout the globe: Mexico in North America, Uruguay in South America, Italy in Europe, Nigeria in Africa and India in Asia. Considering the fact that treatment of CKD is cost-effective and improves outcomes, this observation argue in favor of including CKD in national guidelines and noncommunicable chronic disease (NCD) programs. This diverse example of national activities fullfil the very first step in achieving this goal.

[ANMCO/ISS/AMD/ANCE/ARCA/FADOI/GICR-IACPR/SICI-GISE/SIBioC/SIC/SICOA/SID/SIF/SIMEU/SIMG/SIMI/SISA Consensus document. Hypercholesterolemia and cardiovascular risk: diagnostic and therapeutic pathways in Italy]. / Documento di consenso intersocietario ANMCO/ISS/AMD/ANCE/ARCA/FADOI/ GICR-IACPR/SICI-GISE/SIBioC/SIC/SICOA/ SID/SIF/SIMEU/SIMG/SIMI/SISA Colesterolo e rischio cardiovascolare: percorso diagnostico-terapeutico in Italia.

Atherosclerotic cardiovascular disease still represents the leading cause of death in western countries. A wealth of scientific evidence demonstrates that increased blood cholesterol levels have a major impact on the outbreak and progression of atherosclerotic plaques. Moreover, several cholesterol-lowering pharmacological agents, including statins and ezetimibe, have proven effective in improving clinical outcomes. This document is focused on the clinical management of hypercholesterolemia and has been conceived by 16 Italian medical associations with the support of the Italian National Institute of Health. The authors have considered with particular attention the role of hypercholesterolemia in the genesis of atherosclerotic cardiovascular disease. Besides, the implications of high cholesterol levels in the definition of the individual cardiovascular risk profile have been carefully analyzed, while all available therapeutic options for blood cholesterol reduction and cardiovascular risk mitigation have been considered. Finally, this document outlines the diagnostic and therapeutic pathways for the clinical management of patients with hypercholesterolemia.

Serum free light chain analysis in the diagnosis and management of multiple myeloma and related conditions.

The serum free light chain (FLC) assay is an important tool in the management of patients with monoclonal gammopathies. MEDLINE, the Cochrane Central Register of Controlled Trials and the Cochrane Database of Systematic Reviews from January 2000 through July 2013, were used as data sources. The available evidence is rather weak. For screening of multiple myeloma and related conditions, the association of the FLC assay with the traditional serum tests avoids urine study. Screening for immunoglobulin light-chain (AL) amyloidosis or other rare syndromes requires the urine examination. FLC measurement is used in the assessment of the risk of progression of precursor diseases to overt myeloma, and for risk stratification in solitary plasmacytoma, multiple myeloma and AL amyloidosis. In patients with oligosecretory myeloma and AL amyloidosis, the quantification of FLC is essential for monitoring and categorization of response to therapy. Further studies with improved design are warranted to strengthen the available evidence.