Volume 53, Issue 8

There are a growing number of studies documenting that, similarly to patients with solid cancers, also patients with hematological malignancies (i.e., acute leukemia, lymphoproliferative and myeloproliferative neoplasms and plasma cell disorders) are at increased risk of thrombosis. The pathogenesis of the hypercoagulable state associated with hematological cancers is often multifactorial. Contributor factors include tumor cell-derived procoagulants, antineoplastic therapies, central venous catheters, concomitant infections and advanced age. In this narrative review, the epidemiology, pathogenesis and management of thrombosis in patients with hematological malignancies are reviewed.

Vitamin B 12 deficit is one of the most common vitamin deficiencies. However, there is no consensus on the cut-off points for vitamin B 12 and its co-markers, such as folate, holotranscobalamin, methylmalonic acid and homocysteine. In order to establish the state of the art about cut-off points used to determine vitamin B 12 deficiency in the last decades, the database MEDLINE was used for searching studies published in adults between December 1992 and May 2014 (69 articles), using search terms like ‘vitamin B 12 ’, ‘cobalamin’, ‘cut-off’, ‘deficiency’ alone or in combinations. Broad ranges of cut-off points for vitamin B 12 and its biomarkers were identified: vitamin B 12 ranged between 100 pmol/L and 350 pmol/L, holotranscobalamin 20–50 pmol/L, methylmalonic acid 0.210–0.470 μmol/L, homocysteine 10–21.6 μmol/L, serum folate 3.7–15.9 nmol/L and red blood cell 124–397 nmol/L. For the majority of studies, the potential influence of age, analytical methods, gender and fortified food consumption was not taken in account when choosing cut-off values. This could explain the discrepancies between studies on vitamin B 12 and folate deficiency prevalences. We conclude that there is inconsistency in the literature regarding vitamin B 12 cut-offs. It would be necessary to establish different reference cut-offs according to age, considering the analytical methods used.

The international standard ISO 15189 requires that medical laboratories estimate the uncertainty of their quantitative test results obtained from patients’ specimens. The standard does not provide details how and within which limits the measurement uncertainty should be determined. The most common concept for establishing permissible uncertainty limits is to relate them on biological variation defining the rate of false positive results or to base the limits on the state-of-the-art. The state-of-the-art is usually derived from data provided by a group of selected medical laboratories. The approach on biological variation should be preferred because of its transparency and scientific base. Hitherto, all recommendations were based on a linear relationship between biological and analytical variation leading to limits which are sometimes too stringent or too permissive for routine testing in laboratory medicine. In contrast, the present proposal is based on a non-linear relationship between biological and analytical variation leading to more realistic limits. The proposed algorithms can be applied to all measurands and consider any quantity to be assured. The suggested approach tries to provide the above mentioned details and is a compromise between the biological variation concept, the GUM uncertainty model and the technical state-of-the-art.

The recent revision of ISO15189 has further strengthened its position as the standard for accreditation for medical laboratories. Both for laboratories and their customers it is important that the scope of such accreditation is clear. Therefore the European co-operation for accreditation (EA) demands that the national bodies responsible for accreditation describe the scope of every laboratory accreditation in a way that leaves no room for doubt about the range of competence of the particular laboratories. According to EA recommendations scopes may be fixed, mentioning every single test that is part of the accreditation, or flexible, mentioning all combinations of medical field, examination type and materials for which the laboratory is competent. Up to now national accreditation bodies perpetuate use of fixed scopes, partly by inertia, partly out of fear that a too flexible scope may lead to over-valuation of the competence of laboratories, most countries only use fixed scopes. The EA however promotes use of flexible scopes, since this allows for more readily innovation, which contributes to quality in laboratory medicine. In this position paper, the Working Group Accreditation and ISO/CEN Standards belonging to the Quality and Regulation Committee of the EFLM recommends using an approach that has led to successful introduction of the flexible scope for ISO15189 accreditation as intended in EA-4/17 in The Netherlands. The approach is risk-based, discipline and competence-based, and focuses on defining a uniform terminology transferable across the borders of scientific disciplines, laboratories and countries.

Background: In large clinical trials, where RNA cannot be extracted immediately after sampling, preserving RNA in whole blood is a crucial initial step in obtaining robust qPCR data. The current golden standard for RNA preservation is costly and designed for time-consuming column-based RNA-extraction. We investigated the use of lysis buffer for long-term storage of blood samples for qPCR analysis. Methods: Blood was collected from 13 healthy adults and diluted in MagMAX lysis/binding solution or PAXgene Blood RNA tubes and stored at –20 °C for 0, 1, or 4 months before RNA extraction by the matching method. RNA integrity, yield and purity were evaluated and the methods were compared by subsequent analyses of the gene expression levels of 18S , ACTB , IL1B , IL1RN , IL1R2 , and PGK1 using qPCR. Results: The MagMAX system extracted 2.3–2.8 times more RNA per mL blood, with better performance in terms of purity, and with comparable levels of integrity relative to the PAXgene system. Gene expression analysis using qPCR of 18S , ACTB , IL1B , IL1RN , IL1R2 , and the promising blood-specific reference gene, PGK1 , revealed negligible differences (<1-fold) between the samples stored in MagMAX lysis/binding solution over time and between samples stored and extracted by the two systems. Conclusions : The MagMAX system can be used for storage of human blood for up to 4 months and is equivalent to the PAXgene system for RNA extraction. It furthermore, provides a means for significant cost reduction in large clinical trials.

Background: Prenatal diagnosis of sex-linked disorders requires invasive procedures, carrying a risk of miscarriage of up to 1%. Cell-free fetal DNA (cffDNA) present in cell-free DNA (cfDNA) from maternal plasma offers a non-invasive source of fetal genetic material for analysis. Detection of Y-chromosome sequences in cfDNA indicates presence of a male fetus; in the absence of a Y-chromosome signal a female fetus is inferred. We aimed to validate the clinical utility of insertion-deletion polymorphisms (INDELs) to confirm presence of a female fetus using cffDNA. Methods: Quantitative real-time PCR (qPCR) for the Y-chromosome-specific sequence, SRY , was performed on cfDNA from 82 samples at 6–39 gestational weeks. In samples without detectable SRY , qPCRs for eight INDELs were performed on maternal genomic DNA and cfDNA. Detection of paternally inherited fetal alleles in cfDNA negative for SRY confirmed a female fetus. Results: Fetal sex was correctly determined in 77/82 (93.9%) cfDNA samples. SRY was detected in all 39 samples from male-bearing pregnancies, and none of the 43 female-bearing pregnancies (sensitivity and specificity of SRY qPCR is therefore 100%; 95% CI 91%–100%). Paternally inherited fetal alleles were detected in 38/43 samples with no SRY signal, confirming the presence of a female fetus (INDEL assay sensitivity is therefore 88.4%; 95% CI 74.1%–95.6%). Since paternally inherited fetal INDELs were not used in women bearing male fetuses, the specificity of INDELs cannot be calculated. Five cfDNA samples were negative for both SRY and INDELS. Conclusions: We have validated a non-invasive prenatal test to confirm fetal sex as early as 6 gestational weeks using cffDNA from maternal plasma.

Background: Manufacturers and laboratories might benefit from using a modern integrated tool for quality management/assurance. The tool should not be confounded by commutability issues and focus on the intrinsic analytical quality and comparability of assays as performed in routine laboratories. In addition, it should enable monitoring of long-term stability of performance, with the possibility to quasi “real-time” remedial action. Therefore, we developed the “Empower” project. Methods: The project comprises four pillars: (i) master comparisons with panels of frozen single-donation samples, (ii) monitoring of patient percentiles and (iii) internal quality control data, and (iv) conceptual and statistical education about analytical quality. In the pillars described here (i and ii), state-of-the-art as well as biologically derived specifications are used. Results: In the 2014 master comparisons survey, 125 laboratories forming 8 peer groups participated. It showed not only good intrinsic analytical quality of assays but also assay biases/non-comparability. Although laboratory performance was mostly satisfactory, sometimes huge between-laboratory differences were observed. In patient percentile monitoring, currently, 100 laboratories participate with 182 devices. Particularly, laboratories with a high daily throughput and low patient population variation show a stable moving median in time with good between-instrument concordance. Shifts/drifts due to lot changes are sometimes revealed. There is evidence that outpatient medians mirror the calibration set-points shown in the master comparisons. Conclusions: The Empower project gives manufacturers and laboratories a realistic view on assay quality/comparability as well as stability of performance and/or the reasons for increased variation. Therefore, it is a modern tool for quality management/assurance toward improved patient care.

Background: Diagnosis of vitamin B12 deficiency is generally based on the measurement of serum vitamin B12 levels. However, in selected cases functional indices of vitamin B12, such as methylmalonic acid (MMA) and homocysteine (HCY), are needed. Here we compare the performance of four automated total vitamin B12 assays and also investigate how these assays relate to functional indices of vitamin B12 status. Methods: Total vitamin B12, MMA and HCY were measured in 69 serum samples from routine vitamin B12 assay requests. Serum vitamin B12 analysis was performed using four different immunoassay autoanalyzers: DxI 800 Unicel (Beckman Coulter, USA), ADVIA Centaur XP (Siemens Diagnostics, Tarrytown, NY, USA), Roche Cobas E601 (Roche Diagnostics, Germany), Architect i2000sr (Abbott Laboratories, Abbott Park, IL, USA). Serum MMA levels were determined by liquid chromatography-mass spectrometry (LC-MS) and serum homocysteine levels were determined by high pressure liquid chromatography (HPLC) methods. Results: Four immunoassay methods were comparable and correlated with each other. Correlation coefficients (r) ranged from 0.898 to 0.987, p<0.001. Highest correlation was observed between Roche Cobas – Architect i2000sr and poorest correlation was observed between DxI 800 Unicel – ADVIA Centaur comparison. DxI 800 Unicel assay demonstrated high mean bias [–122 pg/mL (–616–125 pg/mL)] and a concordance correlation coefficient (CCC) of 0.9161, lower than the others. MMA and HCY were correlated with the vitamin B12 results. The correlation coefficients with their 95% CI indicated that there was no statistically significant difference between the four methods according to their relationship with MMA and HCY. Conclusions: Total B12 assays correlate very well with each other. However, results of DxI 800 Unicel were lower compared to the other three autoanalyzers. All total vitamin B12 methods show similar relationships with HCY and MMA. Standardization of serum vitamin B12 assays is still not completed and further standardization studies are needed. Laboratory professionals and clinicians should be aware of this disagreement between assay methods and they should use these tests as ancillary tests.

Background: A novel approach to determine vitamin B 12 status is to combine four blood markers: total B 12 (B 12 ), holotranscobalamin (holoTC), methylmalonic acid (MMA) and total homocysteine (tHcy). This combined indicator of B 12 status is expressed as cB 12 =log 10 [(holoTC·B 12 )/(MMA·Hcy)]–(age factor). Here we calculate cB 12 in datasets with missing biomarkers, examine the influence of folate status, and revise diagnostic cut-points. Methods: We used a database with all four markers (n=5211) plus folate measurements (n=972). A biomarker Z (assumed missing) was plotted versus X (a combination of other markers) and Y (age). Each chart was approximated by a function Z theor , which predicted the potentially absent value(s). Statistical distributions of cB 12 were aligned with physiological indicators of deficiency and used to determine cut-offs. Results: The predictive functions Z theor allowed assessment of the “incomplete” indicators, 3cB 12 (three markers known) and 2cB 12 (two markers known). Predictions contained a systematic deviation associated with dispersion along two axes Z and X (and unaccounted by the least squares fit). Increase in tHcy at low serum folate was corrected (cB 12 +Δ folate ) based on the function of Δ folate =log 10 (Hcy real /Hcy theor ) versus folate. Statistical distributions of cB 12 revealed the boundaries of groups with B 12 deficiency, i.e., cB 12 <–0.5. Conclusions: We provide equations that combine two, three or four biomarkers into one diagnostic indicator, thereby rescaling unmatched data into the same coordinate system. Adjustment of this indicator is required if serum folate is <10 nmol/L and tHcy is measured. Revised cut-points and guidelines for using this approach are provided.

Background: Prothrombin complex concentrate (PCC) is used to reverse vitamin K antagonist (VKA)-induced anticoagulation. Prothrombin time-derived international normalized ratio (INR) measurements are widely used in determining the required PCC dose, but this approach requires reappraisal. The aim of the present study was to determine the added value of the thrombin generation assay (TGA) compared with the INR in guidance of VKA reversal by PCC. Methods: In an open, observational study, INR and TGA measurements were carried out on plasma samples from phenprocoumon-treated patients receiving VKA reversal. Following both analytical methods, PCC dosing correlates were calculated and compared retrospectively. Alternatively, in vitro PCC spiking experiments were performed. Results: As expected, an exponential relationship between PCC dose and INR was found. For the TGA parameters peak thrombin and endogenous thrombin potential (ETP), however, this relationship was found to be linear throughout the full therapeutic range. Additional computational analysis showed a positive correlation (r 2 =0.7) between the initial INR and PCC dose required for a target INR of 2.1, which was completely lost at a lower target INR. In contrast, a positive correlation (r 2 =0.8) between initial ETP as well as peak height and PCC dose required to obtain parameter normalization was found. These correlates appeared useful for calculating PCC dose. Conclusions: Our results support the current debate questioning the rationale for the use of the INR in the management of anticoagulation by VKA. Compared with INR, TGA-based calculations may enable a more accurate PCC dosing regimen for patients requiring VKA reversal.

Background: Assessing the anticoagulant effect of dabigatran may be useful in certain clinical settings. When plasma sampling is not available, serum or urine samples may provide another option for dabigatran determinations. Methods: Dabigatran was assessed in patients on treatment under real-life conditions in plasma samples by four clotting time-based assays and in plasma, serum, and urine samples by two chromogenic substrate methods. Results: The concentrations of dabigatran in patients’ plasma samples were not different for the Hemoclot test (106.8±89.4 ng/mL) and the ecarin clotting time (ECT, 109.5±74.5 ng/mL, p=0.58). Activated partial thromboplastin time and prothrombinase-induced clotting time showed low correlations with the other assays. Chromogenic assays measured similar concentrations as Hemoclot and ECT. For both chromogenic assays, the concentrations of dabigatran were about 70% lower in serum than in plasma samples (p<0.0001). The intra-class coefficient (ICC, Bland-Altman analysis) was strong comparing ECT, Hemoclot thrombin inhibitor (HTI) assay, and the two chromogenic assays (r=0.889–0.737). The ICC was low for comparisons of the chromogenic assays of serum vs. plasma values (ICC, 0.15 and 0.66). The ICC for the determination of dabigatran in urine samples by the two chromogenic assays (5641.6±4319.7 and 4730.0±3770.2 ng/mL) was 0.737. Conclusions: ECT, HTI, and chromogenic assays can be used to determine dabigatran in plasma samples from patients under real-life conditions. Chromogenic assays require further improvement to reliably measure dabigatran in serum samples. Dabigatran concentrations in urine samples can also be determined quantitatively.

Background: Since the urinary concentration of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a reliable biomarker of exposure to tobacco smoke, we developed a relatively simple high-throughput chromatographic method to quantify total urinary NNAL concentrations in the general population. Methods: The high-throughput analytical method was developed using ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) to identify and quantify total urinary NNAL concentrations in 10 non-smokers and 15 otherwise healthy smokers. Results: Loss of nitric oxide at m/z 30 was found to be the predominant mass transitioned, and therefore was used as the SIM transition to quantify both NNAL and NNAL-methyl- d 3 in urine. The analytical method did not require sample derivatization. Standard curves for total NNAL concentrations were linear between 20 and 1500 pg/mL, with coefficients of determination >0.95. Precision and accuracy ranged from 2.2% to 8.6% (CV) and from –5.6% to 10.9% (percent error), respectively. The lowest limit of quantification was 6.7 pg/mL, and 2.0 pg/mL the lowest limit of detection (LLOD). Total urinary NNAL concentrations in non-smoker subjects were <LLOD, whereas in smokers varied between <LLOD to 112.1 pg/mL. Conclusions: An UPLC-MS/MS analytical method to quantify total urinary NNAL concentrations in smokers that does not require sample derivatization is presented herein. The method could be useful in clarifying the toxicities associated with human exposure to cigarette smoking. However, quantification might be adversely affected by co-eluting interfering compounds or selective ion suppression or enhancement as a result of having only one ion transition to monitor NNAL and NNAL-methyl- d 3 in urine.

Background: Physostigmine, commonly used as an antidote in anticholinergic poisoning, is reported to have additional pharmacological effects, such as activation of the cholinergic anti-inflammatory pathway in sepsis models. Due to the narrow therapeutic range of physostigmine and its metabolite eseroline, however, the plasma concentrations of these substances need to be determined so as to understand their effect and ensure safety in the treatment of septic patients. Methods: To determine physostigmine and its metabolite eseroline, a rapid and sensitive high performance liquid chromatography (HPLC) method has been developed and validated. Spiked plasma samples were cleaned up and concentrated using a simple liquid-liquid extraction (LLE) procedure with N-methylphysostigmine as internal standard. Separation was achieved using reversed-phase HPLC on a Kinetex C18 column with gradient elution and fluorescence detection (254 nm excitation/355 nm emission). Results: LLE produced clean extracts and a mean recovery of 80.3% for eseroline and 84.9% for physostigmine. The HPLC assay revealed a limit of detection (LOD) of 0.025 ng/mL and a lower limit of quantification (LLOQ) of 0.05 ng/mL for both analytes. Linearity was observed at 0.05–10.0 ng/mL (r 2 >0.999). Intra- and inter-day precision ranged from 0.7% to 6.6%, and intra- and inter-day accuracy 97.5%–110.0%. Conclusions: The presented method is useful for human drug level monitoring of physostigmine and eseroline in accordance with current guidelines. Remarkably low plasma concentrations can be quantified after LLE with gradient elution and fluorescence detection, making this a suitable method for pharmacokinetic studies in a clinical setting.

Background: Antiphosphatidylserine/prothrombin complex (aPS/PT) antibodies are emerging as an important marker for antiphospholipid syndrome (APS). We aimed to compare their performance with that of conventional antiphospholipid antibodies (aPL) such as lupus anticoagulant (LA), anticardiolipin (aCL), and anti-β2-glycoprotein I (anti-β2GPI) in APS and to assess their frequency in APS-negative (APS-ne) patients. Methods: We considered 160 APS patients and 128 APS-ne patients with clinical criteria for APS but tested negative for conventional aPL. Immunoglobulin (Ig)G/IgM aPS/PT, IgG/IgM aCL, and IgG/IgM anti-β2GPI were detected using ELISA assay and LA with a series of coagulation tests. Results: IgG aPS/PT were significantly associated with IgG aCL, IgG anti-β2GPI, and LA (p<0.0001 for all). IgM aPS/PT were significantly associated only with LA (p<0.0001) instead. There was a significant correlation between IgG aPS/PT and both IgG aCL and IgG anti-β2GPI levels (ρ=0.42 and ρ=0.40, respectively). Both IgG aPS/PT and IgM aPS/PT positivity significantly correlated with LA (ρ=0.44 and ρ=0.5, respectively). IgG and IgM aPS/PT were significantly more frequent in triple than in double and in single positivity (p<0.0001). According to multivariate analysis, IgG and/or IgM aPS/PT were independent risk factors for LA. APS/PT antibodies were found in 9.4% of the APS-ne patients vs. 2% of healthy control (p=0.043); those antibodies were significantly more frequent in the thrombosis with respect to the pregnancy morbidity subset (p=0.01). Conclusions: Our data attribute a clinical relevance to both IgG and IgM aPS/PT antibodies. In particular, the significant prevalence of aPS/PT in APS-ne patients suggests including them as additional laboratory criterion for APS.

Background: The order of draw is regarded as a preanalytical issue to prevent carryover of additives during blood collection. Our objective was to prove the theory of ethylenediaminetetraacetic acid (EDTA) carryover for a closed vacuum system and the influence of EDTA on concentrations of selected biomarkers. Methods: To test the carryover of EDTA, a blood collection with tripotassium EDTA (K 3 EDTA) and subsequent non-additive tubes was simulated using distilled water as substitute for blood. EDTA concentrations were measured by tandem mass spectrometry. Then we added increasing concentrations of EDTA to heparinized blood and measured routine biomarkers, thereby simulating a carryover of EDTA whole blood and pure EDTA, respectively. Additionally, we tested for EDTA contamination and biomarker alteration in samples collected from 10 healthy volunteers by a syringe with subsequent transfer into sample tubes. Results: No EDTA contamination was detected in samples collected subsequent to a K 3 EDTA tube when adhering to guidelines of blood sampling. Magnesium, calcium, and potassium levels were altered by artificial K 3 EDTA whole-blood contamination as well as when adding 1 μL pure K 3 EDTA. Iron values were altered at EDTA concentrations of 4.4 mmol/L. All other parameters remained unaffected. A slight EDTA carryover was observed in syringe collection and subsequent transfer into EDTA and heparin tubes, however, without any biomarker alteration. Conclusions: An EDTA carryover during blood collection using a closed vacuum system is highly unlikely. Even if carryover of EDTA whole blood occurs, an absolute volume larger than 10 μL would be necessary to alter test results. However, contamination of samples with preloaded pure K 3 EDTA solution by severe neglect of current recommendations in blood collection may significantly alter testing results.

Background: Reliable reference intervals for two novel urinary biomarkers of renal injury, neutrophil gelatinase-associated lipocalin (uNGAL) and kidney injury molecule-1 (uKIM-1) are lacking for infants. Therefore, the aim of our study was to establish reference intervals for urinary NGAL and KIM-1 absolute concentrations as well as normalized to urinary creatinine in young infants categorized in small age intervals. Methods: From June 2010 to March 2014, serum and urine samples of 106 basically healthy infants (born between 37 and 42 weeks of gestation) aged 1 day to 1 year were collected. Blood samples were assayed for serum creatinine levels to confirm a healthy renal status. Urine samples were assayed for creatinine, uNGAL (ng/mL) and uKIM-1 (ng/mL). Results: Two thirds of the study cohort were boys. uNGAL concentrations declined with increasing age (likelihood ratio test, p=0.001). Also, uNGAL concentrations were higher in girls (50th centile uNGAL was 27.1 ng/mL) than boys (50th centile uNGAL was 14.3 ng/mL) (two tailed Wald test, p<0.001) NGAL concentrations were not related to ethnicity. uKIM-1 concentrations were extremely low in almost all 106 subjects [median uKIM-1 was 0.08 (IQR 0.08–0.08) ng/mL] and not related with age, gender or ethnicity (all p>0.05). Conclusions: Our data uniquely provide uNGAL and uKIM-1 reference intervals for the first year of life. Notably, only uNGAL levels decreased with increasing age and were higher in girls. These reference intervals enable future studies to evaluate the performance of both biomarkers in detecting early kidney tubular injury, particularly in the setting of critical care.

Background: The aim of the study was to investigate serum NT-proBNP levels in adult patients with transposition of the great arteries (d-TGA) corrected by atrial switch procedures (Mustard or Senning) operation and to assess the relationship with ventricular impairment and NYHA class. Methods: Serum NT-proBNP levels were measured in a group of 81 consecutive adult patients (59 males, mean age 27 years and 22 females, mean age 28 years) with transposition of the great arteries (TGA) after surgical correction in childhood, and in a control group of 25 healthy individuals (16 males, mean age 32 years, and 9 females, mean age 29 years). Age-matched correlation of NT-proBNP concentrations in TGA patients after Mustard or Senning correction was performed, but this correlation was considered not significant (p=0.08). Results: Concentrations of NT-proBNP in patients with TGA were significantly elevated compared to the control group of healthy individuals (203 ng/L vs. 41 ng/L, p<0.0001). Patients after the Mustard repair had significantly higher NT-proBNP values than patients after the Senning operation (234 ng/L vs. 148 ng/L, p=0.0023). NT-proBNP correlated negatively with the systemic right ventricular ejection fraction with the greatest significance in patients after Mustard correction (r=–0.32, p<0.0001). The concentration of NT-proBNP was also associated with NYHA functional class (p=0.0035) with the greatest significance in patients with Mustard correction (p=0.028). Conclusions: Elevated levels of NT-proBNP appear to be a useful tool in assessing heart failure in patients with transposition of the great arteries after atrial switch correction.