Volume 54, Issue 9

Myelodisplastic syndromes (MDS) are heterogeneous myeloid disorders characterized by peripheral cytopenias and increased risk of transformation into acute myelogenous leukemia (AML). MDS are generally suspected in the presence of cytopenia on routine analysis and the evaluation of bone marrow cells morphology and cellularity leads to correct diagnosis of MDS. The incidence of MDS is approximately five cases per 100,000 people per year in the general population, but it increases up to 50 cases per 100,000 people per year after 60 years of age. Typically MDS affect the elderly, with a median age at diagnosis of 65–70 years. Here the current therapeutic approaches for MDS are evaluated by searching the PubMed database. Establishing the prognosis in MDS patients is a key element of therapy. In fact an accurate estimate of prognosis drives decisions about the choice and timing of the therapeutic options. Therapy is selected based on prognostic risk assessment, cytogenetic pattern, transfusion needs and biological characteristics of the disease, comorbidities and clinical condition of the patients. In lower-risk patients the goals of therapy are different from those in higher-risk patients. In lower-risk patients, the aim of therapy is to reduce transfusion needs and transformation to higher risk disease or AML, improving the quality of life and survival. In higher-risk patients, the main goal of therapy is to prolong survival and to reduce the risk of AML transformation. Current therapies include growth factor support, lenalidomide, immunomodulatory and hypomethylating agents, intensive chemotherapy, and allogenic stem cell transplantation. The challenge when dealing with MDS patients is to select the optimal treatment by balancing efficacy and toxicity.

In the past few years, interest in the development of digital PCR (dPCR) as a direct nucleic acid amplification technique for clinical viral diagnostics has grown. The main advantages of dPCR over qPCR include: quantification of nucleic acid concentrations without a calibration curve, comparable sensitivity, superior quantitative precision, greater resistance to perturbations by inhibitors, and increased robustness to the variability of the target sequence. In this review, we address the application of dPCR to viral nucleic acid quantification in clinical applications and for nucleic acid quantification standardization. Further development is required to overcome the current limitations of dPCR in order to realize its widespread use for viral load measurements in clinical diagnostic applications.

Background: Duchenne muscular dystrophy (DMD) is typically caused by disrupting the reading frame of the dystrophin gene: approximately 70%–80% of mutational events are represented by deletions or duplications of one or more exons in the dystrophin gene, and the remaining cases by subtle mutations, including point mutations, small indels, small inversions, and complex small rearrangements. The dystrophin gene is the largest known gene with one of the highest known rates of new mutations. Methods: Deletions and duplications were detected in the DMD gene of the proband by using multiple ligation-dependent probe amplification (MLPA). Targeted next-generation sequencing (NGS) was used in the subtle mutation detection, followed by Sanger sequencing confirmation. The effect of the mutation on the splicing of the DMD gene was assessed by bioinformatics prediction and hybrid minigene splicing assay (HMSA). Results: Neither duplication nor deletion was found in the DMD gene of the proband. While a novel splice site mutation c.6762+1G>C was identified in the proband by NGS and Sanger sequencing, and his mother was heterozygous at the same site. Bioinformatics predicted that the 5′ donor splice site of intron 46 disappeared because of the mutation, which would lead to aberrant splicing and introduce premature stop codon. The HMSA results were in agreement with the prediction. Conclusions: The novel splice site mutation caused DMD in the proband by aberrant splicing. We suggested that combined applications of MLPA, NGS, HMSA and bioinformatics are comprehensive and effective methods for diagnosis and aberrant splicing study of DMD.

Background: The availability of simple and accurate assays of plasma active renin (DRC) and aldosterone concentration (PAC) can improve the detection of secondary forms of arterial hypertension. Thus, we investigated the performance of an automated chemiluminescent assay for DRC and PAC in referred hypertensive patients. Methods: We prospectively recruited 260 consecutive hypertensive patients referred to an ESH Center for Hypertension. After exclusion of six protocol violations, 254 patients were analyzed: 67.3% had primary hypertension, 17.3% an aldosterone producing adenoma (APA), 11.4% idiopathic hyperaldosteronism (IHA), 2.4% renovascular hypertension (RVH), 0.8% familial hyperaldosteronism type 1 (FH-1), 0.4% apparent mineralocorticoid excess (AME), 0.4% a renin-producing tumor, and 3.9% were adrenalectomized APA patients. Bland-Altman plots and Deming regression were used to analyze results. The diagnostic accuracy (area under the curve, AUC of the ROC) of the DRC-based aldosterone-renin ratio (ARR CL ) was compared with that of the PRA-based ARR (ARR RIA ) using as reference the conclusive diagnosis of APA. Results: At Bland-Altman plot, the DRC and PAC assay showed no bias as compared to the PRA and PAC assay. A tight relation was found between the DRC and the PRA values (concordance correlation coefficient=0.92, p<0.0001) and the PAC values measured with radioimmunoassay and chemiluminescence (concordance correlation coefficient=0.93, p<0.001). For APA identification the AUC of the ARR CL was higher than that of the ARR RIA [0.974 (95% CI 0.940–0.991) vs. 0.894 (95% CI 0.841–0.933), p=0.02]. Conclusions: This rapid automated chemiluminescent DRC/PAC assay performed better than validated PRA/PAC radioimmunoassays for the identification of APA in referred hypertensive patients.

Background: A pilot study showing a decrease in androstenedione concentration in serum collected into gel-containing serum tubes (STs) triggered an investigation of the effect of serum collection tube on steroid hormone stability. Methods: In the main study, two tube types were examined: BD Vacutainer ® SST™II Advance and BD Vacutainer ® Serum Tube. Forty-seven serum samples from apparently healthy volunteers were collected and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for testosterone, androstenedione, 17-hydroxyprogesterone (17-OHP) (n=20); and oestradiol (n=27). Primary specimens were centrifuged once, maintained at room temperature and extracted within 2 h for day zero (d0) results. To assess stability following refrigeration (2–8 °C), aliquots were taken from the primary tube on day one (d1) and day five (d5) and analysed immediately. Differences in measurand concentration between tubes at d0 and following storage (d1 and d5) were evaluated for statistical significance. Results: There was a progressive and statistically significant decrease in androstenedione concentration from d0 to d5 (p<0.001) in the SST™II tubes. In addition, there was a statistically significant reduction in testosterone, 17-OHP and oestradiol concentrations at d5 (p<0.01). Interestingly, oestradiol and testosterone concentrations increased with time in plain STs (p<0.01). The only change likely to have a clinical impact was that of androstenedione in serum gel tubes. Conclusions: To optimise conditions and to reduce pre-analytical error we recommend the use of plain serum collection tubes for androstenedione and rapid separation of serum from cells when oestradiol and testosterone are requested.

Background: Low cholesterol is typically observed in the plasma of patients with cystic fibrosis (CF) contrasting with the subcellular accumulation of cholesterol demonstrated in CF cells and in mice models. However, the homeostasis of cholesterol has not been well investigated in patients with CF. Methods: We studied the plasma of 26 patients with CF and 33 unaffected controls campesterol and β-sitosterol as markers of intestinal absorption and lathosterol as a marker of de novo cholesterol biosynthesis by gas chromatography (GC-FID and GC-MS). Results: Plasma campesterol and β-sitosterol results were significantly (p=0.01) lower while plasma lathosterol was significantly higher (p=0.001) in patients with CF as compared to control subjects. Plasma cholesterol results were significantly lower (p=0.01) in CF patients. Conclusions: Our data suggest that the impaired intestinal absorption of exogenous sterols in patients with CF stimulates the endogenous synthesis of cholesterol, but the levels of total cholesterol in plasma remain lower. This may be due to the CFTR dysfunction that reduces cholesterol blood excretion causing the accumulation of cholesterol in liver cells and in other tissues contributing to trigger CF chronic inflammation.

Background: Investigation of possible B12 and folate deficiencies requires measurement of these vitamins in serum. There is evidence that holotranscobalamin (holoTC), the active portion of B12 available to cells, is a more specific marker of early B12 deficiency than total B12. The availability of immunoassays for holoTC prompted an international collaborative study to assign a holoTC value to the World Health Organization (WHO) 1st International Standard (IS) for vitamin B12 and serum folate, 03/178. Methods: The IS, 03/178, and three serum samples with different holoTC levels were assayed by 12 laboratories in eight countries using manual and automated immunoassays for holoTC; one laboratory additionally performed an in-house assay. Fourteen sets of data were analysed. Results: Overall, the IS, 03/178, and the three serum samples demonstrated assay linearity and parallelism. An overall geometric mean (GM) holoTC value of 106.8 pmol/L was obtained for 03/178, with an inter-laboratory geometric coefficient of variation (GCV) of 10.5%. There was a reduction in inter-laboratory variability when the holoTC levels in the serum samples were determined relative to the IS with an assigned holoTC value rather than to the assays’ calibration. Accelerated degradation studies showed that 03/178 was sufficiently stable to serve as an IS for holoTC. Conclusions: The WHO Expert Committee on Biological Standardization endorsed the proposal to assign a holoTC value of 107 pmol/L to 03/178, corresponding to 0.107 pmol per ampoule, for use as the 1st IS for vitamin B12, serum folate, and holoTC.

Background: The objective of the study was to compare a new AutoDELFIA ® Inhibin A kit (B064-102) with the Access Inhibin A kit (A36097) using clinical specimens and to evaluate the AutoDELFIA ® Inhibin A assay performance in screening for Down syndrome in the second trimester of pregnancy. Methods: Using clinical samples, we performed a method comparison between new and existing inhibin A kits and assessed AutoDELFIA ® Inhibin A kit precision performance. Normal median values for the second trimester of pregnancy were also determined. Finally, we evaluated the screening performance of the AutoDELFIA ® Inhibin A kit together with other second trimester biomarkers for the detection of Down syndrome. Results: The two methods showed a high degree of correlation ( r =0.99, Pearson and Spearman correlation), and the average relative level difference between the methods at a concentration range of 41.7–1925 pg/mL was 19.6% [95% confidence interval (CI) from 17.6% to 21.5%]. The acceptable precision of the AutoDELFIA ® Inhibin A kit was demonstrated: the within-lot CV% varied from 1.9% to 3.9%. The screening performance results show that AutoDELFIA ® Inhibin A when added to a combination of other second trimester serum markers [human alpha foetoprotein (hAFP), free beta subunit of human chorionic gonadotropin (free hCGβ) and unconjugated estriol (uE3) or hAFP and free hCGβ] improves the detection rate of screening in both combinations. Conclusions: The performance of the AutoDELFIA ® Inhibin A assay is highly acceptable for routine laboratory use for screening Down syndrome in the second trimester of pregnancy.

Background: The aim was to investigate whether first trimester glycodelin and angiopoietin-2 can predict small-for-gestational age (SGA) at delivery, individually or in combination. Methods: In this case-control study we measured glycodelin and angiopoietin-2 on serum from 170 singleton pregnant women delivering SGA neonates and 985 singleton pregnant women delivering normal-weighted neonates. All values were converted to multiples of the medians (MoM). Results: Pregnant women delivering SGA neonates had lower first trimester glycodelin and angiopoietin-2 MoM values [median (interquartile range)] compared with pregnant women delivering normal-weighted neonates for glycodelin: 0.86 (0.58–1.24) vs. 1.03 (0.74–1.45), p<0.001, and for angiopoietin-2: 0.89 (0.69–1.19) vs. 1.01 (0.78–1.31), p<0.001. The prediction performances of the biomarkers showed that the areas under the curve (AUC) were 0.59 (glycodelin), 0.58 (angiopoietin-2), and 0.60 (glycodelin and angiopoietin-2). Conclusions: We demonstrated that first trimester glycodelin and angiopoietin-2 were associated with SGA, but they were, individually and in combination, poor predictors of SGA at delivery. The AUCs were low which indicate low detection rates and high false positive rates.

Background: Total C-terminal agrin fragment (tCAF) is a new biomarker that was previously correlated with kidney function. This article studies the validity of tCAF as a biomarker for kidney function in chronic kidney disease (CKD). Methods: Plasma tCAF, serum creatinine (Cr), cystatin C (CyC), blood urea-nitrogen (BUN) concentrations and estimated glomerular filtration rate (eGFR CKD-EPI crea-cystatin ) were assessed in 426 individuals [71 without CKD (CKD 0°) and 355 CKD patients]. In addition to descriptive statistics, univariate correlation between tCAF and biomarkers/eGFR was calculated; multiple linear regression modeling was applied between logarithmic (log) tCAF and log eGFR and adjusted for demographic data. The same methods were used to analyze the association of demographic factors and the different biomarkers adjusted for eGFR. Results: Mean tCAF levels were 1012.2±789.9 pM. tCAF correlated with all biomarkers/eGFR in univariate analysis (eGFR: r=–0.77, Cr: r=0.74, BUN: r=0.66, CyC: r=0.75). Linear regression modeling revealed an excellent coefficient estimate between log tCAF and log eGFR (CKD-EPI crea-cystatin ) (–0.91, p<0.001). tCAF was the parameter least associated with demographic parameters in both univariate and multivariate regression modeling (only with age, coefficient estimate r=–0.159, p=0.001 in multivariate regression). Conclusions: In conclusion, tCAF is a promising biomarker for the assessment of kidney function in CKD patients showing an excellent correlation with eGFR and being less influenced by demographic parameters compared to conventional biomarkers. These preliminary results encourage further evaluation of tCAF in larger CKD cohorts and other clinical settings such as acute renal failure.

Background: Despite accumulating evidence about the negative health effects of exposure to electromagnetic fields emitted by mobile phones, no information is available on the potential impact of radiofrequency (RF) waves on polymorphonuclear leukocytes biology. Methods: Two sequential whole blood tubes were collected from 16 ostensibly healthy volunteers. After placing the former tube of each subject in a plastic rack, 1 cm from a commercial smartphone (carrier frequency, 900 MHz), a call was placed on the smartphone and a communication lasting 30 min was manually activated. The latter blood tube of each volunteer was placed in another plastic rack, for an identical period of time, avoiding close contact with sources of RF waves. A complete blood count was then assessed in all whole blood samples, using Advia 2120. Results: The 30-min exposure of blood to RF waves did not induce significant variations of total and differential leukocyte counts. A significant decrease was however observed for many neutrophils parameters, with median percentage variation of −3.9% for the lobularity index (LI), −29.8% for the myeloperoxidase index (MPXI), −0.6% for the neutrophil cluster mean x (NEUTx) and −0.7% for the neutrophil cluster mean y (NEUTy), respectively. The percentage of blood samples with reduced values after exposure to RF waves was 81% for LI, 88% for NEUTx and 100% for both MPXI and NEUTy. Conclusions: The results of this study show that exposure to smartphone RF waves triggers activation of neutrophils in vitro, as mirrored by the significant variations observed in many activation parameters in Advia 2120.

Background: The distinction between reactive and neoplastic leukocytes, especially atypical lymphocytes suspected to be reactive or neoplastic, is a particular challenge in automated hematological cell differentiation. The aim of the study was to evaluate the performance of the XN analyzer supplemented with the WPC channel for differentiating between reactive and neoplastic leukocytosis. Methods: Blood samples of 253 patients with viral infections, lymphoma or leukemia were analyzed by the Sysmex XN-2000 analyzer equipped with the WPC channel. The results were compared to routine leukocyte differentiation using the routine Sysmex XE-2100 analyzer and automated digital microscopy (DM96). The combined information from standard morphology, immune phenotyping and clinical diagnosis served as a reference. Results: The XN WPC channel demonstrated an excellent performance for differentiating neoplastic (AUC=0.933) and reactive leukocytosis (AUC=0.900) as compared to morphological smear examination (AUC=0.949 and AUC=0.968, respectively) or to the differentiation results of our routine hematology analyzer (AUC=0.630 and AUC=0.635, respectively). Conclusions: Our data show that the combined WDF/WPC of the Sysmex XN-Series analyzer is advantageous in the automated differentiation of neoplastic and reactive leukocytosis, thus supporting the correct diagnostic decision in the daily laboratory routine.

Background: Lupus anticoagulant (LAC) testing includes a screening, mixing and confirmation step. Although recently published guidelines on LAC testing are a useful step towards standardization, a lack of consensus remains whether to express mixing tests in clotting time (CT) or index of circulating anticoagulant (ICA). The influence of anticoagulant therapy, e.g. vitamin K antagonists (VKA) or direct oral anticoagulants (DOAC) on both methods of interpretation remains to be investigated. The objective of this study was to contribute to a simplification and standardization of the LAC three-step interpretation on the level of the mixing test. Methods: Samples from 148 consecutive patients with LAC request and prolonged screening step, and 77 samples from patients non-suspicious for LAC treated with VKA (n=37) or DOAC (n=30) were retrospectively evaluated. An activated partial thromboplastin time (aPTT) and dilute Russell’s viper venom time (dRVVT) were used for routine LAC testing. The supplemental anticoagulant samples were tested with dRVVT only. We focused on the interpretation differences for mixing tests expressed as CT or ICA and compared the final LAC conclusion within each distinct group of concordant and discordant mixing test results. Results: Mixing test interpretation by CT resulted in 10 (dRVVT) and 16 (aPTT) more LAC positive patients compared to interpretation with ICA. Isolated prolonged dRVVT screen mix ICA results were exclusively observed in samples from VKA-treated patients without suspicion for LAC. Conclusions: We recommend using CT in respect to the 99th percentile cut-off for interpretation of mixing steps in order to reach the highest sensitivity and specificity in LAC detection.

Background: Reference change values (RCV) are used for judging the significance of changes between any two measurements. Based on the within-individual CV ( CV I ), RCV is conventionally computed as the 95% confidence limit (CL) of the changes: RCV=1.96×2CVI.$RCV = 1.96 \times \sqrt 2 C{V_I}.$ However, the appropriateness of assuming a constant CV I and using the 95% CL for RCV remains controversial. Methods: The level-specific CV I and RCV were estimated for 20 screening tests using a database composed of results from 13,545 health-screening attendees over a 17-year period, after preliminary exclusion of individuals taking medications or having unusual changes in body mass index (BMI). A rational CL for RCV was explored in reference to a clinical score for the metabolic syndrome, sMS, which was derived based on a logistic regression model consisting of tests related to metabolic syndrome. The effect of adjusting CL for the RCV on diagnostic efficacies of detecting between-year change in sMS was evaluated. Results: Test level dependency of CV I was apparent for some screening tests which have distributions with prominent skewing. The use of level-specific RCV was thus essential for them. The sensitivity for detecting a critical change in sMS based on the RCV set at 95%CL was extremely low in the majority of tests. However, by lowering CL stepwise from 95 to 75%, the sensitivity improved greatly without much change in specificity and positive predictive value. Loss-and-gain analysis showed that CL for RCV set around 80% gave the lowest loss, assuming a policy of reducing false negative judgment. Conclusions: Level specific CV I and RCV were necessary in tests with skewed distributions. RCV using 80%–90% CL is suitable in health screening for diseases that require early intervention for changes.

Background: Risk stratification in patients with papillary thyroid carcinoma (PTC) currently relies on postoperative parameters. Testing for BRAF mutations preoperatively may serve as a novel tool for identifying PTC patients at risk of persistence/recurrence after surgery. Methods: The study involved 185 consecutive patients with a histological diagnosis of PTC and BRAF analysis performed on thyroid fine-needle aspiration biopsy (FNAB). We assessed BRAF status in FNAB specimens obtained before thyroidectomy for PTC, and examined its association with the clinicopathological characteristics identified postoperatively, and with outcome after a mean 55±15 months of follow-up. Results: One hundred and fifteen of 185 (62%) PTCs carried a BRAF mutation. Univariate analysis showed that BRAF status correlated with the histological variant of PTC, cancer size, and stage at diagnosis, but not with gender, age, multifocality, or lymph node involvement. BRAF -mutated cases had a higher prevalence of persistent/recurrent disease by the end of the follow-up (11% vs. 8%), but this difference was not statistically significant. The Kaplan-Meier curve shows that among the patients with persistent/recurrent disease, BRAF -mutated patients needed a second treatment earlier than patients with BRAF wild-type, although the difference did not completely reach the statistical significance. Conclusions: Our study confirmed that preoperatively-identified BRAF mutation are associated with certain pathological features of PTC that correlate with prognosis. We speculate that it has a role in identifying PTCs that would generally be considered low-risk but that may reveal an aggressive behavior during their follow-up.