Volume 54, Issue 12

Considering the high incidence of cryoglobulins in hepatitis C virus (HCV) infection together with the high worldwide prevalence of HCV infection, identification of clinically apparent mixed cryoglobulinemia syndrome is increasingly important as most patients who are identified can now be successfully treated. Different approaches for the detection, analysis and reporting of cryoglobulins have been described and there is a wide variation in results reported, ranging from a qualitative “negative” or “positive”, to a quantitative report including cryoglobulin type and the total protein. Protein and immunofixation (IFE) electrophoresis are generally used to identify and characterize cryoglobulins, as these methods quantify and phenotype. Here, we present a brief review of the literature and demonstrate a case oriented approach for identifying mixed cryoglobulinemia from the preanalytical phase, leading up to and including the analytical phase with characterization by IFE. Most patients with mixed cryoglobulinemia can now be treated with success. Nevertheless, the high cost may limit treatment of those with symptoms unless there is laboratory evidence for mixed cryoglubulinemia. Low levels of cryoglobulins can be associated with severe symptoms; as a result, accurate identification of cryoglobulins may be of increasing importance since clear identification may be a good reason to initiate treatment.

Calcitonin (CT) is currently the most sensitive serological marker of C-cell disease [medullary thyroid carcinoma (MTC) and C-cell hyperplasia]. Starting with a report on a case that occurred at our institution, this review focuses on trying to explain the reasons behind the poor specificity and sensitivity of the various CT immunoassays. A 15-year-old patient was referred to our institution in May 2014 for moderately elevated CT levels. Thyroid ultrasonography (US) documented a colloidal goiter. Secondary causes of the hypercalcitoninemia (hyperCT) were ruled out. The mismatch between the clinical picture and the laboratory results prompted us to search for other reasons for the patient’s high CT levels, so we applied the heterophilic blocking tube (HBT) procedure to the patient’s sera before the CT assay. Using this pretreatment step, his serum CT concentration dropped to <1 ng/L, as measured at the same laboratory. Measuring plasma CT has an important role in screening for C-cell disease, but moderately elevated serum CT levels need to be placed in their clinical context, bearing in mind all the secondary causes of C-cell hyperplasia and the possibility of laboratory interference, before exposing patients to the risks and costs of further tests.

Novel and non-invasive biomarkers are urgently needed for early detection of cancer. Exosomes are nano-sized particles released by cells and contain various bioactive molecules including proteins, DNA, mRNAs, and non-coding RNAs. Increasing evidence suggests that exosomes play critical roles in tumorigenesis, tumor growth, metastasis, and therapy resistance. Exosomes could be readily accessible in nearly all the body fluids. The altered production of exosomes and aberrant expression of exosomal contents could reflect the pathological state of the body, indicating that exosomes and exosomal contents can be utilized as novel cancer biomarkers. Herein, we review the basic properties of exosomes, the functional roles of exosomes in cancer, and the methods of detecting exosomes and exosomal contents. In particular, we highlight the clinical values of exosomal non-coding RNAs in cancer diagnosis and prognosis.

A body of evidence collected in the last few decades demonstrates that the pre- and post-analytical phases of the testing cycle are more error-prone than the analytical phase. However, the paradigm of errors and quality in laboratory medicine has been questioned, analytical mistakes continuing to be a major cause of adverse clinical outcomes and patient harm. Although the brain-to-brain concept is widely recognized in the community of laboratory professionals, there is lack of clarity concerning the inter-relationship between the different phases of the cycle, interdependence between the pre-analytical phase and analytical quality, and the effect of the post-analytical steps on the quality of ultimate laboratory information. Analytical quality remains the “core business” of clinical laboratories, but laboratory professionals and clinicians alike should never lose sight of the fact that pre-analytical variables are often responsible for erroneous test results and that quality biospecimens are pre-requisites for a reliable analytical phase. In addition, the pressure for expert advice on test selection and interpretation of results has increased hand in hand with the ever-increasing complexity of tests and diagnostic fields. Finally, the data on diagnostic errors and inappropriate clinical decisions made due to delay or misinterpretation of laboratory data underscore the current need for greater collaboration at the clinical-laboratory interface.

This document is based on the original recommendation of the Expert Panel on the Theory of Reference Values of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), updated guidelines were recently published under the auspices of the IFCC and the Clinical and Laboratory Standards Institute (CLSI). This document summarizes proposals for recommendations on: (i) The terminology, which is often confusing, noticeably concerning the terms of reference limits and decision limits. (ii) The method for the determination of reference limits according to the original procedure and the conditions, which should be used. (iii) A simple procedure allowing the medical laboratories to fulfill the requirements of the regulation and standards. The updated document proposes to verify that published reference limits are applicable to the laboratory involved. Finally, the strengths and limits of the revised recommendations (especially the selection of the reference population, the maintenance of the analytical quality, the choice of the statistical method used…) will be briefly discussed.

The provision of interpretative advice on laboratory results is a post-analytic activity and an integral part of clinical laboratory services. It is valued by healthcare workers and has the potential to prevent or reduce errors and improve patient outcomes. It is important to ensure that interpretative comments provided by laboratory personnel are of high quality: comments should be patient-focused and answer the implicit or explicit question raised by the requesting clinician. Comment providers need to be adequately trained and qualified and be able to demonstrate their proficiency to provide advice on laboratory reports. External quality assessment (EQA) schemes can play a part in assessing and demonstrating the competence of such laboratory staff and have an important role in their education and continuing professional development. A standard structure is proposed for EQA schemes for interpretative comments in clinical chemistry, which addresses the scope and method of assessment including nomenclature and marking scales. There is a need for evidence that participation in an EQA program for interpretative commenting facilitates improved quality of comments. It is proposed that standardizing goals and methods of assessment as well as nomenclature and marking scales may help accumulate evidence to demonstrate the impact of participation in EQA for interpretative commenting on patient outcome.

Background: The results of laboratory analyses are affected by pre-analytical variables, and in particular can platelets be activated by shear handling stress and secrete granular substances. We therefore evaluated the effect of centrifugation speed and time on pre-analytical platelet activation. Methods: Citrate- and EDTA-anticoagulated blood from healthy volunteers were centrifuged at 80–10,000 g for 5–15 min to prepare plasma and platelet-rich plasma. Pre-analytical platelet activation was assessed by flow cytometric measurement of platelet P-selectin (CD62p) expression. Blood cell counts, mean platelet volume (MPV), immature platelet fraction (IPF), and platelet distribution width (PDW) were measured. Platelet aggregation in platelet-rich plasma induced by arachidonic acid (AA), ADP or thrombin receptor activator peptide-6 (TRAP) was tested by 96-well aggregometry. Results: The median percentage of platelets expressing P-selectin in citrate- and EDTA-plasma centrifuged at 2000 g for 10 min were 43% [interquartile range (IQR), 38%–53%] and 56% (IQR, 31%–78%), respectively (p=0.82). Platelet-rich plasma prepared at 100–250 g for 10 min had significantly lower platelet P-selectin expression (11%–15%), p<0.001. Platelet count in plasma samples decreased with increasing speed but platelets were only completely removed if plasma was re-centrifuged. In platelet-rich plasma, increasing centrifugation speed significantly increased platelet yield but decreased contamination from other blood cells, platelet composition was altered as platelet parameters (MPV, IPF, and PDW) was lowered. Platelet aggregation was not affected by the centrifugation speed platelet-rich plasma was prepared. Conclusions: Proportional to centrifugation speed, platelets in plasma and platelet-rich plasma were activated with centrifugation speed, cell content and composition changed while platelet aggregation was unaltered.

Background: Proficiency testing or external quality assessment schemes (PT/EQASs) are an important method of assessing laboratory performance. As each scheme establishes assigned values and acceptable ranges for the analyte according to its own criteria, monitoring of participant performance varies according to the scheme and can lead to conflicting conclusions. Methods: Standard deviations (SDs) for PT were derived from Thompson’s and biological variation models applied to blood and urine manganese (Mn) robust data from four EQASs from North America and Europe. The fitness for purpose was verified by applying these SDs to individual results. Results: Using Thompson characteristic function the relationship between SD and Mn concentration, expressed in nmol/L was the square root of [19.7 2 +(0.0771 2 ×Mn concentration 2 )] for blood and the square root of [6.77 2 +(0.0985 2 ×Mn concentration 2 )] for urine. While the biological variation model was not suitable for urine, it produced an acceptable range for blood as ±54.4 nmol/L (assigned value ≤320 nmol/L) or 17% (assigned value >320 nmol/L). For blood, individual performance evaluated by the two approaches led to similar conclusions. Conclusions: The biological variation model can be used to propose quality specifications for blood, however it could not be applied to urine. The Thompson characteristic function model could be applied to derive quality specifications for Mn in urine and, to a lesser extent in blood. The more lenient quality specifications for blood highlight the difficulty of determining Mn in this matrix. Further work is needed to harmonize PT, such as using assigned ranges for the specimens.

Background: Method evaluation of new assays for the detection of antiphospholipid antibodies (aPL) such as anti-cardiolipin (aCL) or anti-β 2 -glycoprotein I (aβ2-GPI) is challenging, as no internationally accepted reference material is available yet. Besides a lack of standardization, unacceptable inter-laboratory comparability of established tests is regularly observed. Owing to the absence of a commonly accepted reference standard, the evaluation of two research surface plasmon resonance (SPR) biosensor assays was performed using statistical methods from latent class analysis (LCA). Methods: aCL and aβ2-GPI IgG and IgM were measured in sera from 63 antiphospholipid syndrome patients, fulfilling the Sydney criteria, and in 34 healthy controls with four commercial assays. LCA was performed on the results and sera were assigned to the antibody-positive or antibody-negative group. Sera were subsequently evaluated in the SPR assays for aCL and aβ2-GPI. Optimal cutoffs and diagnostic performances of the research systems were established employing the LCA-derived gold standard. Results: With area under the curve results of 0.96 and 0.89 for the detection of aCL and aβ2-GPI, the research SPR assays discriminated well between antibody-positive and antibody-negative sera. Their sensitivities and specificities were comparable to the investigated commercial immunoassays. Conclusions: SPR assays are a suitable tool for the detection of aCL and aβ2-GPI with diagnostic performances not different from currently available commercial tests. LCA enabled the calculation of sensitivities and specificities for aPL assays in absence of a reference standard.

Background: Iron deficiency represents the most frequent cause of anemia. To diagnose iron deficiency some biochemical tests such as serum ferritin and the transferring saturation percent (TSAT%) are usually used. Recently, some hematological parameters such as mean reticulocyte hemoglobin content (CHr or Ret-He) and percentage of hypochromic RBCs (Hypo% or %Hypo-He) were proposed as alternative to biochemical tests. In this study, the analytic performance and the diagnostic efficiency of these two parameters provided by Sysmex XE5000 analyzer on iron deficiency patients with or without anemia (IDA and ID, respectively) were evaluated. Methods: One hundred and sixty-four healthy adults, 58 with IDA, 21 with iron depleted stores (ID), 23 with β-thalassemia trait, and 24 with non iron deficiency anemia were selected. The gold standard used to define iron deficiency was the coexistence of serum ferritin below 15 μg/L (12 in women) and TSAT <16%. Results: For %Hypo-He, the best cut-off value for both IDA and ID is 0.9% while for Ret-He is 30.6 pg. For both parameters the performance was better to diagnose IDA (AUC, 0.96 and 0.98) than ID (AUC, 0.93 and 0.95). The Ret-He behavior was always slightly better than that of %Hypo-He. Conclusions: The use of these two parameters is useful to detect iron deficiency conditions if the hemoglobin synthesis has already been compromised.

Background: Tumor markers (TM) are a routine test that are not always used well, and can lead to unnecessary additional tests, which are not without risks for the patients. So, to implement appropriate strategies to improve the adequate use of TM and, therefore, improve patient safety, is required to analyze the use of TM, identifying risks and establishing if there are differences in their use as a function of their utility. Methods: The study was a descriptive, longitudinal, retrospective and systematic study in the area covered by the University Hospital of Padua. In the follow-up 2-year study, 23,059 analytical requests of TM, corresponding to 14,728 patients, were analyzed. For the level of statistical significance it applies an approximation of the normal law (Z statistic) and χ 2 -test. Results: Only 9196 requests (39.88%) out of a total of 23,059 on 5080 patients with neoplastic diseases have been classified as adecuate according to current guidelines. The number of requests per patient was variable (1.57±1.35). In patients with neoplastic diseases this increased to 1.80±1.68. The mean of number of TM per request was 2.4±1.73. The analysis showed an association between the number of requests and the type of marker used. Conclusions: The use of TM is variable, mainly of the follow-up markers, when they are used as screening. This inappropriate use, minimizes their utility favoring erroneous interpretations and increases the risk of damage to the patient. So it is essential to implement safe practices in the use of TM.

Background: Infections from microorganisms and parasites have been connected with either increased or decreased cancer risk. The objective of this study was to investigate whether infection by Echinococcus granulosus is associated with cancer risk. Methods: We assembled a pilot retrospective cohort of patients who were diagnosed as being infected by E. granulosus in Cyprus between 1930 and 2011. Age/gender-matched non-infected family members and neighbors were selected as references. Medical history was ascertained from each study subject through in-person interview. Cox proportional hazards regression analysis was performed to assess the association of being infected by E. granulosus with cancer risk. Results: Individuals with prior infection by E. granulosus (n=249) were more likely to have cancer compared to those without infection (n=753), 11.65% vs. 8.37% (p=0.0492). Survival analysis also showed that subjects with prior infection had a higher risk for developing cancer. The hazards ratio (HR) was 1.595, [95% confidence interval (CI) between 1.008 and 2.525]. The risk ratio did not change significantly (HR=1.536; 95% CI: 0.965–2.445) after adjusting for gender, year of birth, smoking status, alcohol consumption, and family history of cancer. Conclusions: Our study suggests that infection by E. granulosus may increase cancer risk. If this observation can be confirmed independently, further investigation of the mechanisms underlying the association is warranted.

Background: The aim of the study was to determine the utility of the dynamic change and serial monitoring of the systemic immune inflammation index (SII), which was based on the numbers of patients’ lymphocytes (L), platelets (P), neutrophils (N) and defined as P*N/L, for predicting prognosis of patients with hepatocellular carcinoma (HCC) after curative resection. Methods: We conducted a prospective study of 163 patients with HCC who underwent curative resection at Zhongshan Hospital from January 2012 to May 2013. SII was calculated using data acquired before and approximately 1 month after surgery. An optimal cutoff value stratified patients into groups with high or low SII. Patients were classified into unfavorable and favorable groups using the dynamic change of the SII. Two groups that were further divided into four categories within the entire cohort and the low-risk subgroups were serially monitored for ≥6 months. Prognostic values of the SII and other factors were determined using the Kaplan-Meier method, the Cox proportional hazards model, and the receiver operating characteristics (ROC) curve. Results: The favorable group was likely to have cirrhosis, and the unfavorable group was likely to have larger tumors and a higher recurrence rate. Multivariate analysis revealed that tumor size and dynamic change of the SII were independent risk factors for early recurrence. Moreover, the predictive value of the SII was retained in α-fetoprotein (AFP)-negative and HBeAg-negative-HBV-DNA <2000 IU/mL subgroups. Further, the serial changes of the SII for recurrence and no recurrence groups were statistically significant. Conclusions: The dynamic change and serial monitoring of the SII represent new indicators for predicting the early recurrence of HCC determining advance optimal therapy in advance.

Background: Aberrant hypermethylation of tumour suppressor genes (TSGs) occurring in hepatocellular carcinoma (HCC) could provide a mean of molecular characterisation of this cancer. The aim of this study was to investigate promoter methylation and gene expression of selected TSGs in HCC to identify candidate genes for further validation as potential biomarkers. Methods: Methylation-specific multiplex ligation-dependent probe amplification method was used to measure the methylation status of 25 TSGs in 49 HCC samples and 36 corresponding non-cancerous liver tissue samples. Relative expression of the differentially methylated genes was assessed at the mRNA level using quantitative PCR. Results: We observed a significantly higher methylation in genes WT1 , PAX5 , PAX6 , PYCARD and GATA5 in HCC compared with control samples. The expression of PAX5 was significantly decreased by methylation; conversely methylation of WT1 was associated with higher mRNA levels. Methylation of GATA5 was significantly associated with overall survival and methylation of WT1 and PAX5 significantly varied between patients with ALBI score 1 vs. 2+3. Moreover, PAX5 was significantly more methylated in patients with tumour grade 2+3 vs. grade 1, and methylation of the PAX5 correlated with the patient’s age at the time of diagnosis. Conclusions: HCC evince aberrant promoter methylation of WT1 , PAX5 , PAX6 , PYCARD and GATA5 genes. Correlation between GATA5 , WT1 and PAX5 methylation and clinical/histological parameters is suggestive of applicability of these markers in non-invasive (epi)genetic testing in HCC.

Background: Previous studies have shown that homocysteine and folate levels in plasma are associated with risk for cardiovascular events and mortality. The aim of this study was to investigate if plasma concentrations of total homocysteine and folate can predict major bleeding, cardiovascular events, and all-cause mortality in patients being treated with warfarin. Methods: In a longitudinal cohort study, 719 patients who were taking warfarin were followed for 3001 treatment years. The following were recorded and classified: major bleeding; cardiovascular events including stroke, arterial emboli, and myocardial infarction (MI); and mortality. Blood samples collected at baseline were analysed for plasma homocysteine and folate levels. Results: After adjustment for age, C-reactive protein, and creatinine, high homocysteine levels were associated with cardiovascular events [hazard ratio (HR) 1.23 per standard deviation (SD); 95% confidence interval (CI): 1.03–1.47], MI (HR 1.38 per SD; 95% CI: 1.03–1.85), and all-cause mortality (HR 1.41 per SD; 95% CI: 1.19–1.68). The highest tertile of folate compared to the lowest tertile was associated with decreased risk for both cardiovascular events (HR 0.64; 95% CI: 0.43–0.91) and MI (HR 0.45; 95% CI: 0.21–0.97). There was no association between major bleeding and homocysteine or folate levels. Conclusions: In patients receiving warfarin treatment, high homocysteine and low folate plasma concentrations are associated with increased risk for cardiovascular events but not major bleeding. For homocysteine levels, there is also an association with all-cause mortality.

Background: Hemolytic samples are one of the most challenging preanalytical issues in laboratory medicine. Even causes leading to hemolytic specimen are various, including phlebotomy practices. Respective educational interventions as well as the reduction of the number of people involved in blood collections are claimed to influence the sample quality for the better. In our hospital 70 junior doctors were in charge of routine phlebotomy until 2012, when this task was shifted to 874 nurses, including a preceding training in phlebotomy and preanalytics. Our aim was to evaluate the impact of this training effect and the increase of people involved on sample quality. Methods: The hemolysis index (HI) of 43,875 samples was measured before (n=21,512) and after (n=22,363) the switch of blood collection responsibilities. Differences in overall hemolysis rates and the amount of plasma samples with a concentration of free hemoglobin (fHb) above 0.5 g/L and 1 g/L were calculated. Results: Overall HI as well as the percentage of samples with an fHb concentration >0.5 g/L decreased after the responsibility for phlebotomy changed. The rate of samples with an fHb concentration >1 g/L remained unchanged. Conclusions: Hemolysis rates were reduced upon passing phlebotomy tasks from untrained physicians on to a trained nursing staff. We therefore conclude that the number of people performing phlebotomy seems to play a minor role, compared to the effect of a standardized training. However, whether a reduction in the number of people involved in blood collection could lead to further improvement of sample quality, remains to be investigated in future studies.