Volume 52, Issue 8

Inherited platelet disorders (IPDs) are the general and common denomination of a broad number of different rare and congenital pathologies affecting platelets. Even if these disorders are characterized by widely heterogeneous clinical presentations, all of them are commonly present as defects in hemostasis. Platelet number and/or function are affected by a wide spectrum of severity. IPDs might be associated with defects in bone marrow megakaryocytopoiesis and, rarely, with somatic defects. Although in the last few years new insights in the genetic bases and pathophysiology of IPDs have greatly improved our knowledge of these disorders, much effort still needs to be made in the field of laboratory diagnosis. This review discusses the laboratory approach for the differential diagnosis of the most common IPDs, suggesting a common multistep flowchart model which starts from the simpler test (platelet count) ending with the more selective and sophisticated analyses.

Reticulated platelets are immature platelets circulating in blood; they reflect the activity of megakaryopoiesis in the bone marrow. Therefore, they can be used as a non-invasive test in patients with thrombocytopenia in various clinical conditions. The preferred method of analysis is by flow cytometry. However, there is an evident lack of analytical standardization, making it difficult to compare results obtained in different laboratories. Currently, two types of hematology analyzers are on the market offering fully automated measurement of reticulated or immature platelets: the high end analyzers manufactured by Sysmex (XE- and XN-series) and Abbott (CELL-DYN Sapphire). Although the methods are essentially different and cannot be used interchangeably, both have been proven to have clinical utility. Reticulated or immature platelet assays are useful for the differential diagnosis of thrombocytopenia and for monitoring bone marrow recovery after chemotherapy or stem cell transplantation. These assays may aid clinicians in platelet transfusion decisions when recovery from thrombocytopenia is imminent. In addition, preliminary findings indicate that there is a rationale for reticulated or immature platelets for risk stratification in acute coronary syndromes and for monitoring the effect of treatment with antiplatelet drugs in patients with coronary artery diseases. The aim of this paper is to present the present technology available for measuring reticulated platelets as well as an overview of the current status of clinical application. This overview also indicates that more research is needed before reticulated or immature platelet assays can be applied in other clinical conditions than thrombocytopenia and after transplantation.

Background: Currently, multiplex ligation-dependent probe amplification (MLPA) is the most commonly used technique for the detection of large genomic rearrangements (LGRs) in the BRCA1/2 genes. However, a very fast assay, the BRCA1/2 multiplex amplicon quantification (MAQ), has been recently developed by Multiplicom. Methods: As no data regarding the application of MAQ method to BRCA1/2 genes are available in literature, here we compared for the first time the performance of the MAQ assay with MLPA by using several positive BRCA1/2 LGRs DNA samples (previously tested by MLPA). Results: MAQ method was able to detect all BRCA1/2 LGRs and no false-positive or -negative results were obtained in independent repetitive experiments. Conclusions: We can affirm that MAQ, as well as MLPA method, results to be valid and reproducible tools for molecular diagnostics and we are confident that this assay can be used for BRCA1/2 mutational screening as a fast and safe alternative to MLPA, particularly in the first line of analysis.

Background: No report on establishing critical values (CVs) lists has described a process for harmonizing different lists in a large academic health center or validation on follow-up after 5 years. Methods: A definition of a critical value was adopted. CVs in use and reporting times for chemistry, hematology and coagulation (CH&C) tests during a 1-week period at one hospital were analyzed prior to the revision and again 5 years later. Results: CVs lists in use by the different campus hospital laboratories were reviewed for compliance with the definition for a critical value. Lists were harmonized with a total of 37 CH&C tests, five of these included adult and either cord blood or neonatal values. Overall, 26 tests were eliminated, 61 individual values were changed and two tests were added. The revised CVs list reduced the number of calls at one primary teaching hospital by 33%. In the next 5-year period, value thresholds changed (n=2) and one value was re-instated (n=1). When retrospectively examined for impact, one value change was considered appropriate. Conclusions: CVs lists were harmonized among campus hospitals. Tests not considered critical were removed and values adjusted for uniformity. Changes in CVs lists should be evaluated for appropriateness. A process is now in place for periodic review and considerations related to CVs lists.

Background: Standardization of parathyroid hormone (PTH) assays is a major issue, especially in hemodialyzed (HD) patients. Two automated third generation PTH assays (Roche Elecsys and DiaSorin Liaison) are now available. These assays are specific for the (1-84) PTH and do not cross-react with the (7-84) fragment, contrary to second generation (intact) assays. We aimed to calibrate the two methods against the WHO International PTH Standard (IS) 95/646 to see if the two assays could provide comparable results in a population of healthy subjects, HD patients and patients suffering from primary hyperparathyroidism (PHP). Methods: We selected 79 healthy subjects and two populations of patients presenting PTH disorders: 56 HD and 27 PHP patients. We reconstituted the IS in a pool of human serum containing undetectable levels of 1-84 PTH and prepared 13 serum standards ranging from 0 to 2000 pg/mL. The standards were run on the two instruments to calibrate the assays on the IS. The different populations were run before and after restandardization. Results: As these kits were differently calibrated, the results obtained after restandarization were significantly different. Restandardization process improved concordance between assays and, taking the analytical variability of the two kits into account, the results could be considered to be similar. Conclusions: Restandardization of automated third generation PTH assays with the WHO 1-84 PTH Standard significantly reduces inter-method variability. Reference ranges and raw values are totally transposable from one method to the other in healthy subjects, but also in diseased patients, e.g., with HD or those suffering from PHP.

Background: The increasing importance of anti-Müllerian hormone (AMH) for the assessment of ovarian reserve requires accurate AMH measurements. There have been conflicting results about the reliability of currently existing manual AMH assays. Methods: Development of a high sensitive, fast and fully automated AMH assay on the Elecsys ® / cobas e electrochemiluminescence immunoassay platform. Results: Elecsys ® AMH is a monoclonal two-site assay used to measure AMH in 50 µL of serum or lithium heparin plasma in about 18 min. Its measuring range is from 0.01 to 23 ng/mL. The assay detects primarily 140 kDa total AMH (proAMH and AMHN,C). Standardization was against the Beckman AMH Gen II. Recovery against the highly cited Immunotech calibration was about 90%. Within-run imprecision coefficient of variation (CV) calculated on 10 serum samples were between 0.5% and 1.8%. Repeatability and intermediate precision calculated on 14 serum samples ranged from 2.6% to 1.7% and 2.9% to 2.1%, respectively. Limit of detection (LoD) [limit of quantitation (LoQ)] was 0.01 ng/mL (0.03 ng/mL). Percent recovery in dilution studies was <10% and after mixing of high and low AMH pools 94% to 103%. Elecsys ® AMH, when compared to revised AMH Gen II (or Ansh Labs ultrasensitive AMH ELISA) using 57 female serum samples yielded a correlation coefficient of 0.98 (0.97) and a slope of 0.81 (0.73). There was no evidence for sample instability or variability. Samples stored at 20–25 °C or 2–8 °C up to 7 days showed no significant storage issues. Freeze-thaw cycles or sample storage at −20 °C and −80 °C for up to 9 months was without any effect on measured AMH. Conclusions: Availability of an automated Elecsys ® AMH assay offers an attractive alternative to the current manual AMH assays.

Background: In the region Limburg (The Netherlands) almost all of the five participating laboratories use a different immunoassay platform to determine thyroid stimulating hormone (TSH) and free thryoxine (FT4). With the frequent transfer of patients within the region, harmonization of test result interpretation is necessary. In this study, we investigated dysthyroxinemia classification between participating laboratories and developed procedures for improvement. Methods: Two ring surveys with an interval of 2 years were performed. Four patient groups (n=100) with different dysthyroxinemia classification were based on biochemical results of the Autodelphia analyzer. Samples were tested in five participating laboratories. In each group the percentage of patients classified with dysthyroxinemia was calculated and differences were analyzed by the Fisher’s exact test. Results: After the first survey, the percentage of patients with hyperthyroxinemia was more than 20% lower in three laboratories compared to the other two. Bhattacharya analysis revealed that the upper reference limit of FT4 was 20%–30% too high in two laboratories. Adjustments of reference ranges appeared to be effective in the second survey. The third laboratory reported significantly lower percentages of patients with hyperthyroxinemia in the second survey. New FT4 reference ranges were determined for this laboratory, resulting in adequate classification of hyperthyroxinemia. Conclusions: This study illustrates the potential of a multicenter evaluation of dysthyroxinemia in a biochemical-defined patient cohort. In particular, classification of hyperthyroxinemia differed between laboratories. Adjustments of reference ranges resulted in better agreement of dysthyroxinemia classification. Even using internal and external quality assurance programs, application of multicenter ring surveys is advised to prevent inadequate reference ranges.

Background: Several studies have revealed a high soluble fms-like tyrosine kinase 1 (sFlt-1) to placental growth factor (PlGF) ratio in preeclamptic women. However, its role in patients with suspected preeclampsia (PE) at triage in the emergency department remains an issue and a controversial unique cutpoint of 85 has been proposed regardless of gestational age. A new cutpoint for sFlt-1/PlGF ratio was investigated to rule out PE at obstetric triage, and to assess its prognostic value for risk of imminent delivery. Methods: Blood samples from 257 pregnant women with suspected PE were obtained at obstetric triage admission. Serum PlGF and sFlt-1 were measured by an electrochemoluminiscence immunoassay (ECLIA) on the immunoanalyzer Cobas e601 (Roche Diagnostics) and the corresponding ratio was calculated. Final outcomes (mainly development of PE) were reviewed and time between clinical presentation and delivery was calculated. Results: The best ratio cutpoint to diagnose PE changed according to gestational age: 23 (92.0% sensitivity, 81.1% specificity) and 45 (83.7% sensitivity, 72.6% specificity) for women <34 and ≥34 weeks’ gestation, respectively. Furthermore, sFlt-1/PlGF ratio inversely correlated with time elapsed between clinical presentation and delivery, and a cutpoint of 178 could predict complications such as imminent delivery or fetal/neonatal death with a sensitivity of 70.6% and a specificity of 97.8%. Conclusions: The new cut-off values for the sFlt-1/PlGF ratio adjusted by the gestational age at clinical presentation can be used to rule out PE at obstetric triage and to predict imminent delivery with better accuracy than the cutpoint currently accepted.

Background: This study aims to evaluate the performance of the soluble Fms-like tyrosine kinase-1/placental growth factor (sFlt-1/PlGF) ratio to predict early-onset, preterm and severe preeclampsia at mid-pregnancy in asymptomatic women. Methods: Based on a prospective cohort of 7929 pregnant women from the Quebec City metropolitan area, a nested case-control study was performed including 111 women who developed preeclampsia and 69 women with gestational hypertension matched with 338 normotensive women. Serum sFlt-1 and PlGF were measured between 20 and 32 weeks of gestation. The performance of the sFlt-1/PlGF ratio, expressed as raw values and multiples of the median (MoM) for the prediction of early-onset, preterm and severe preeclampsia was evaluated. Results: Women who developed preeclampsia had significantly higher MoM sFlt-1/PlGF ratio (p<0.001). In the early-onset preeclampsia group, the median of the MoM distribution was 24.0 and the area under the receiver operating characteristic curve (AUC) was 0.977, giving sensitivities of 77.8% and 88.9% at false-positive rates of 5% and 10%. Positive predictive values (PPV) were 2.5% and 1.5%, respectively. In a subset between 26 and 32 weeks of gestation, at a threshold of 30, the sFlt-1/PlGF ratio yielded 100% specificity and identified, respectively, 85.7% and 65.2% of women who developed early-onset and preterm preeclampsia. Conclusions: The sFlt-1/PlGF ratio has the potential to predict early-onset and preterm preeclampsia at mid-pregnancy in asymptomatic women. However, care must be paid to the prevalence of early-onset preeclampsia in the population since low prevalence reduces PPV and may hamper clinical utility.

Background: Ethyl glucuronide (EtG) measurement in neonatal meconium has emerged as a reliable marker to objectively assess prenatal exposure to maternal ethanol complementary to fatty acid ethyl ester (FAEEs) measurement. The detection of EtG in meconium is currently a lengthy, difficult and expensive process using liquid chromatography tandem mass spectrometry (LC-MS/MS) as the analytical procedure. An enzyme-linked immunosorbent assay (ELISA) for the identification of EtG in meconium was developed, validated and applied to authentic meconium specimens from newborns collected in Europe. Methods: The ELISA procedure was calibrated using 0.45, 0.9, 1.35 and 1.8 nmol/g (100, 200 300 and 400 ng/g) standards. Meconium (0.25 g) was mixed thoroughly, with extraction buffer (pH 7.3; 0.5 mL). The tube was capped, sonicated, centrifuged and the supernatant was decanted. An aliquot of the extract (50 μL) was placed in the well of the microplate followed by enzyme conjugate (150 μL). The plate was incubated for 1 h, washed with deionized water, dried and substrate (200 μL) was added. After 30 min incubation, stop solution was added and the plate was read at 450 nm and 650 nm. Samples were also analyzed for EtG and FAEEs by validated LC-MS/MS assays. Results: Using an EtG cut-off of 0.9 nmol/g for both ELISA screening test and confirmatory LC-MS/MS, immunoassay sensitivity was 100%; specificity 78%; positive-predictive value (PPV) 29% and negative-predictive value (NPV) 100%. Conclusions: The assay is proposed as a preliminary screening test for the meconium of newborns suspected of being born to mothers drinking alcohol during pregnancy.

Background: High acceleration centrifugation conditions are used in laboratory automation systems to reduce the turnaround time (TAT) of clinical chemistry samples, but not of coagulation samples. This often requires separate sample flows. The CLSI guideline and manufacturers recommendations for coagulation assays aim at reducing platelet counts. For measurement of prothrombin time (PT) and activated partial thromboplastin time (APTT) platelet counts (Plt) below 200×10 9 /L are recommended. Other coagulation assays may require even lower platelet counts, e.g., less than 10×10 9 /L. Unifying centrifugation conditions can facilitate the integration of coagulation samples in the overall workflow of a laboratory automation system. Methods: We evaluated centrifugation conditions of coagulation samples by using high acceleration centrifugation conditions (5 min; 3280× g ) in a single and two consecutive runs. Results of coagulation assays [PT, APTT, coagulation factor VIII (F. VIII) and protein S] and platelet counts were compared after the first and second centrifugation. Results: Platelet counts below 200×10 9 /L were obtained in all samples after the first centrifugation and less than 10×10 9 /L was obtained in 73% of the samples after a second centrifugation. Passing-Bablok regression analyses showed an equal performance of PT, APTT and F. VIII after first and second centrifugation whereas protein S measurements require a second centrifugation. Conclusions: Coagulation samples can be integrated into the workflow of a laboratory automation system using high acceleration centrifugation. A single centrifugation was sufficient for PT, APTT and F. VIII whereas two successive centrifugations appear to be sufficient for protein S activity.

Background: Automated analyzers are an important component of modern laboratories. As a representative of the newest generation of coagulation analyzers, the CS-5100 features several technical refinements including a pre-analytical assessment unit as well as multi-wavelength optical detection units. Therefore, the CS-5100 is supposed to rapidly and accurately perform a broad panel of coagulation tests. In the current study, the CS-5100 was evaluated regarding its precision and practicability in a clinical laboratory setting. Methods: The CS-5100 was evaluated regarding its intra- and inter-assay precision using commercially available control samples. Results of patient samples, including hemolytic, icteric and lipemic specimens, measured on the CS-5100 were compared to reference analyzers, which are used in our accredited laboratory. Results: The coefficients of variation, assessed in the intra- and inter-assay precision analyses were below 5% representatively for most parameters. Results, obtained by the CS-5100 showed predominantly a high comparability to used reference analyzers, with correlation coefficients ranging from 0.857 to 0.990. Only minor ranged systemic or proportional differences were found in Passing-Bablok regression between the CS-5100 and reference analyzers regarding most of the tested parameters. Lipemic samples had a tendency to deteriorate correlation coefficients, but an overall effect of the sample’s triglyceride level could be ruled out. In a routine setting, the analyzer reached a sample throughput rate of 160 tests per hour. Conclusions: The CS-5100 is able to rapidly and precisely measure patient samples. No considerable influence on test comparability was found for elevated levels of free hemoglobin, bilirubin or triglycerides.

Background: The detection of faecal occult blood is a fundamental step in making an early diagnosis of colorectal cancer. The aim of the present study was to evaluate the stability of haemoglobin in faeces collected with two sampling devices specific for faecal immunochemical testing (FOB Gold Tube Screen and FOB Gold Tube NG) that contain different preservative buffers (buffer H, BH, and buffer N, BN, respectively). Methods: Fifteen true positive faecal samples were collected with both devices. A pool from each sample was made. Each pool was portioned and stored at +4°C, +21°C and +32°C for 10 days. One aliquot of each pool stored at each of the respective temperatures was tested at five time intervals between sampling and analysis. The same procedure was followed for three synthetic haemoglobin solutions in both buffers. Results: The percentage of cumulative faecal haemoglobin decrease (HbCD%) was evaluated. No significant difference was found between BH and BN in HbCD% at +4°C (p=0.106); at +21°C and +32°C, HbCD% was lower in BH than in BN samples (p=0.002 and p=0.001, respectively) whereas no difference was found between samples stored in BH at +4°C and +21°C. The synthetic haemoglobin degradation percentage was always ≤7.1% for both buffers except for BN at +32°C (about 60%). Conclusions: Synthetic haemoglobin solutions behave differently from the true faecal samples. At +21°C and +32°C BH preserves the haemoglobin better than BN, independent of the haemoglobin concentration. BH, allowing sample stability at both +4°C and +21°C, is more suitable for screening procedures.

Background: Faecal immunochemical tests (FIT) are becoming widely used in colorectal cancer (CRC) screening. Availability of data on faecal haemoglobin concentrations (f-Hb) in three countries prompted an observational study on sex and age and the transferability of data across geography. Methods: Single estimates of f-Hb in large groups were made in Scotland, Taiwan and Italy using quantitative automated immunoturbidimetry on the Eiken OC-Sensor. Distributions were examined for men and women overall and in four different age groups. Results: The distributions of f-Hb were not Gaussian and had kurtosis and positive skewness. The distributions were different in the three countries: f-Hb varies with sex and age in all countries, being higher in men and the elderly, but the degree of variation is inconsistent across countries, f-Hb being higher in Scotland than in Taiwan than in Italy, possibly due to different lifestyles. At any cut-off concentration, more men are declared positive than women and more older people are declared positive than younger individuals. Conclusions: Our analysis supports the view that setting and using a single f-Hb cut-off in any CRC screening programme is far from ideal. We suggest that individualisation is the optimum approach with f-Hb, alone or with other important factors such as sex and age, used to determine important personal issues such as need for colonoscopy, screening interval between tests and risk of future CRC. Whether there is merit in monitoring f-Hb in individuals over time remains an interesting research question for the future.

Background: Colorectal cancer is the second most frequent cause of cancer-related death in the developed world. Recent studies have tried to associate colorectal cancer with the aberrant expression of several microRNAs. The aim of the present study was the development of a highly sensitive quantitative real-time PCR which can be used to evaluate the miR-182 expression levels in colorectal adenocarcinoma and adjacent non-cancerous tissue specimens and associate them with several clinicopathological characteristics, aiming to examine the prognostic potential of miR-182. Methods: Total RNA was isolated from 116 malignant colorectal adenocarcinoma specimens and 60 paired non-cancerous tissues. Then, polyadenylation of 2 μg total RNA by poly(A) polymerase and reverse transcription with suitable oligo-dT-adapter followed. miR-182 levels were quantified by real-time PCR based on SYBR Green chemistry. The results were analyzed by the comparative quantification cycle method and by extensive biostatistical analysis. Results : miR-182 was found to be significantly upregulated in colorectal adenocarcinoma specimens compared to their non-cancerous counterparts (p<0.001). miR-182 expression increases as the histological grade increases (p=0.013). miR-182 overexpression is associated with high depth of tumor invasion, positive regional lymph node status, and advanced TNM stage of patients. Therefore, miR-182 is an unfavorable prognostic marker in colorectal adenocarcinoma, predicting poor overall survival (p=0.007). Most importantly, miR-182 expression retained its unfavorable prognostic significance among patients with well- or moderately differentiated colorectal adenocarcinoma (p=0.006) and among metastasis-free patients (p=0.025). Conclusions: The increased levels of the oncogene-like miR-182 increase the risk for disease progression and predict poor overall survival for colorectal adenocarcinoma patients.

Background: Epigenetic changes are considered to be a frequent event during tumor development. Various methylation changes have been identified and show promise as potential cancer biomarkers. The aim of this study was to investigate promoter methylation of GATA4 and TP53 genes in endometrioid carcinoma of endometrium. Methods: To search for promoter methylation of GATA4 and TP53 genes we used methylation-specific PCR (MSP) to compare the methylation status of 54 patients with endometrioid carcinoma of endometrium and 18 patients with normal endometrial tissue. Results: In our study MSP revealed GATA4 promoter methylation in 44 of 54 in the carcinoma group (81.5%), and in none of the control group. No methylation was observed in TP53 gene. Conclusions: In conclusion, our study showed that there is significantly higher methylation in GATA4 gene in the endometrial cancer group compared with samples of non-neoplastic endometrium. The finding suggests the importance of hypermethylation of this gene in endometrial carcinogenesis and could have implications for future diagnostic and therapeutic strategies for endometrial cancer based on epigenetic changes.

Background: Aberrant DNA methylation is associated with the development of hepatocellular carcinoma (HCC), suggesting that gene methylation could be a potential biomarker for detection of HCC. The aim of this study is to identify potential biomarkers in HCC. Methods: We used the Infinium methylation array and a DNA-pooling strategy to analyze the genome-wide methylation profile in HCC. Quantitative methylation-specific PCR (Q-MSP) was used to validate homeobox A9 ( HOXA9) methylation in 29 normal controls, 100 HCC samples and adjacent non-tumor tissues and in 74 plasma samples, including 40 patients with HCC. Results: Ten genes ( HOXA9, NEUROG 1, TNFRSF10C, IRAK3, GFPT2, ZNF177, DPYSL4, ELOVL4, FSD1 , and CACNA1G ) showed differences in methylation between controls and HCCs. Of these, HOXA9 was significantly hypermethylated in HCCs (76.7%; 23/30) compared with controls (3.4%; 1/29). In addition, combination analysis of two- and three-gene sets for HCC detection showed greater sensitivity (90%–96.7%) and comparable specificity (93.1%–96.6%) to each individual gene (33.3%–76.7% and 55.2%–100.0%). HOXA9 methylation was further validated by Q-MSP in two independent set of clinical samples including 100 HCC and paired non-tumor tissues. Further, HOXA9 methylation could be detected in plasma from HCC patients (n=40) but not in normal plasma (n=34) (p<0.0005). Combined testing (either parameter positive) for α-fetoprotein (AFP, a plasma protein biomarker) and HOXA9 methylation showed greater sensitivity (94.6%) for detection of HCC than AFP alone (75.7%). Conclusions: These data suggest that methylation of HOXA9 could be a helpful biomarker to assist in HCC detection.