Algorithm of differential diagnosis of anemia involving laboratory medicine specialists to advance diagnostic excellence.

OBJECTIVES: Anemia is a severe global public health issue. Testing practices for anemia suggest overuse of screening laboratory tests and misinterpretation of studies even in "easy-to-diagnose" underlying causes, leading to late diagnoses and missed treatment opportunities. We aimed to develop a complete and efficient algorithm for clinical pathologists and laboratory medicine physicians for the differential diagnosis of anemia. METHODS: Comprehensive literature search encompassing original articles, studies, reviews, gold standard books, and other evidence. RESULTS: We created a complex algorithm, primarily for clinical pathology/laboratory use, that explores all major and several rare causes of anemia in an efficient and evidence-based manner. The algorithm includes gold-standard diagnostic laboratory tests available in most clinical laboratories and indices that can be easily calculated to provide an evidence-based differential diagnosis of anemia. CONCLUSIONS: The diagnostic strategy combines previously available diagnostic tests and protocols in an efficient order. Clinical pathologists following the algorithm can independently provide valuable diagnostic support for healthcare providers. Clinical pathologists providing complete differential diagnostic services with the proposed algorithm may create an opportunity for an advanced diagnostic service that supports diagnostic excellence and helps patients receive a timely diagnosis and early treatment opportunities.

Monoclonal whole IgG impairs both fibrin and thrombin formation: hemostasis and surface plasmon resonance studies.

OBJECTIVES: Monoclonal gammopathies frequently associate with hemostatic alterations. Thrombotic events occur with high incidence particularly upon treatment, while in rarer cases hemorrhagic diathesis can be observed. The pathology of these tendencies could be caused by thrombocytopenia or hyperviscosity burden of circulating monoclonal antibodies. Studies also suggest interference of monoclonal antibodies with primary hemostasis. We isolated monoclonal whole IgG paraproteins from two myeloma patients to observe their effect on thrombin formation and fibrin polymerization. METHODS: Monoclonal whole IgG was prepared from sera of two newly diagnosed untreated multiple myeloma patients and control normal plasma samples. Fibrin formation was measured using thrombin time and dilute prothrombin time tests and thrombin formation was detected with a fluorimetric thrombin generation assay. In addition, molecular interactions were investigated by surface plasmon resonance (SPR). RESULTS: Thrombin time was prolonged upon addition of monoclonal IgG even at 30 g/L by 12 %, increasing up to 36 % at 60 g/L concentration. Dilute prothrombin time was prolonged by 20 % even at 30 g/L. Thrombin generation assay indicated an impairment in thrombin formation at the presence of monoclonal IgG compared to polyclonal at equivalent concentration. By an SPR assay we determined that both clonality IgG preparations interacted with fibrinogen, however interaction with human thrombin was only detected with monoclonal immunoglobulins (KD=1.03 × 10-7 M). CONCLUSIONS: Here we provide evidence that isolated monoclonal whole IgG from myeloma patients can impair both fibrin and thrombin formation and we demonstrate by SPR assay that it interacts with components of the final phase of the coagulation system.

Elevated level of serum human epididymis protein 4 (HE4) predicts disease severity and mortality in COVID-19 pneumonia.

BACKGROUND: We retrospectively analyzed serum level of human epididymis protein 4 (HE4) as a pulmonary inflammatory biomarker in patients with COVID-19 pneumonia in association with disease severity and outcome. METHODS: Ninety-nine (40 critically ill, 40 severe and 19 mild) COVID-19 patients and as controls 25 age- and sex-matched non-COVID-19 bacterial sepsis subjects were included. Serum HE4 was measured by an immunoassay (Architect® i1000SR, Abbott) in the baseline samples of all study participants obtained at intensive care unit (ICU) admission or during outpatient clinic visit and follow-up sera were available in case of 30 COVID-19 subjects with life-threating conditions. Associations were studied between serum HE4, routinely available laboratory parameters, clinical characteristics, and disease progression. RESULTS: Baseline HE4 level was significantly higher (P < 0.0001) in critically ill (524.7 [300.1-1153.0] pmol/L) than severe COVID-19 subjects (157.4 [85.2-336.9] pmol/L) and in mild SARS-CoV-2 infection (46.7 [39.1-57.2] pmol/L). Similarly increased HE4 concentrations were found in bacterial sepsis (1118.0 [418.3-1953.0] pmol/L, P = 0.056) compared to critically ill COVID-19 individuals. Serum HE4 levels significantly correlated with age, SOFA-score, inflammation-dependent biomarkers, and the degree of lung manifestation evaluated by chest CT examination in ICU COVID-19 individuals. Based on ROC-AUC curve analysis, baseline HE4 independently indicated the severity of COVID-19 with an AUC value of 0.816 (95% CI [0.723-0.908]; P < 0.0001), while binary logistic regression test found HE4 as an independent prognostic parameter for death (OR: 10.618 [2.331-48.354]; P = 0.002). Furthermore, COVID-19 non-survivors showed much higher baseline HE4 levels without a substantial change under treatment vs. survivors (P < 0.0001). Finally, pre-treatment HE4 level of ≥ 331.7 pmol/L effectively predicted a larger risk for mortality (Log-Rank P < 0.0001) due to severe COVID-19 pneumonia. CONCLUSION: Elevated serum HE4 level at ICU admission highly correlates with COVID-19 severity and predicts disease outcome.

Laboratory Approaches to Test the Function of Antiphospholipid Antibodies.

Antiphospholipid syndrome (APS) is a systemic autoimmune disorder caused by the presence of aPLs (antiphospholipid antibodies, i.e., anti-ß2-glycoprotein I and anti-cardiolipin). Everyday practice in terms of laboratory diagnostics of APS includes determination of aPLs and well-known functional assays assessing for lupus anticoagulant (LA), in turn using various tests. According to recent guidelines, the recommended method for LA identification or exclusion is based on the Russell Viper Venom test and a sensitive activated partial thromboplastin time assay. Despite the fact that LA can be quantified in laboratory practice in this way, LA is still used as a binary parameter that is just one of the risk factors of thrombosis in APS. As of today, there are no other functional assays to routinely assess the risk of thrombosis in APS. It is well-known that APS patients display a wide range of clinical outcomes although they may express very similar laboratory findings. One way to solve this dilemma, could be if antibodies could be further delineated using more advanced functional tests. Therefore, we review the diagnostic approaches to test the function of aPLs. We further discuss how thrombin generation assays, and rotational thromboelastometry tests can be influenced by LA, and how experimental methods, such as flow cytometric platelet activation, surface plasmon resonance, or nano differential scanning fluorimetry can bring us closer to the puzzling interaction of aPLs with platelets as well as with their soluble protein ligand. These novel approaches may eventually enable better characterization of aPL, and also provide a better linkage to APS pathophysiology.

The Proteasome Inhibitor Bortezomib Induces Apoptosis and Activation in Gel-Filtered Human Platelets.

Bortezomib (BTZ) has demonstrated its efficacy in several hematological disorders and has been associated with thrombocytopenia. There is controversy about the effect of BTZ on human platelets, so we set out to determine its effect on various types of platelet samples. Human platelets were investigated in platelet-rich plasma (PRP) and as gel-filtered platelets (GFPs). Mitochondrial inner membrane potential depolarization and phosphatidylserine (PS) and P-selectin expression levels were studied by flow cytometry, while thrombin generation was measured by a fluorescent method. In PRP, BTZ caused negligible PS expression after 60 min of treatment. However, in GFPs, PS expression was dose- and time-dependently increased in the BTZ-treated groups, as was P-selectin. The percentage of depolarized cells was also higher after BTZ pretreatment at both time points. Peak thrombin and velocity index increased significantly even with the lowest BTZ concentration (p = 0.0019; p = 0.0032) whereas time to peak and start tail parameters decreased (p = 0.0007; p = 0.0034). The difference between PRP and GFP results can be attributed to the presence of plasma proteins in PRP, as the PS-stimulating effect of BTZ could be attenuated by supplementing GFPs with purified human albumin. Overall, BTZ induces a procoagulant platelet phenotype in an experimental setting devoid of plasma proteins.

Preterm Intraventricular Hemorrhage-Induced Inflammatory Response in Human Choroid Plexus Epithelial Cells.

Following an intraventricular hemorrhage (IVH), red blood cell lysis and hemoglobin (Hb) oxidation with the release of heme can cause sterile neuroinflammation. In this study, we measured Hb derivates and cellular adhesion molecules ICAM-1 and VCAM-1 with cell-free miRNAs in cerebrospinal fluid (CSF) samples obtained from Grade-III and Grade-IV preterm IVH infants (IVH-III and IVH-IV, respectively) at multiple time points between days 0-60 after the onset of IVH. Furthermore, human choroid plexus epithelial cells (HCPEpiCs) were incubated with IVH and non-IVH CSF (10 v/v %) for 24 h in vitro to investigate the IVH-induced inflammatory response that was investigated via: (i) HMOX1, IL8, VCAM1, and ICAM1 mRNAs as well as miR-155, miR-223, and miR-181b levels by RT-qPCR; (ii) nuclear translocation of the NF-κB p65 subunit by fluorescence microscopy; and (iii) reactive oxygen species (ROS) measurement. We found a time-dependent alteration of heme, IL-8, and adhesion molecules which revealed a prolonged elevation in IVH-IV vs. IVH-III with higher miR-155 and miR-181b expression at days 41-60. Exposure of HCPEpiCs to IVH CSF samples induced HMOX1, IL8, and ICAM1 mRNA levels along with increased ROS production via the NF-κB pathway activation but without cell death, as confirmed by the cell viability assay. Additionally, the enhanced intracellular miR-155 level was accompanied by lower miR-223 and miR-181b expression in HCPEpiCs after CSF treatment. Overall, choroid plexus epithelial cells exhibit an abnormal cell phenotype after interaction with pro-inflammatory CSF of IVH origin which may contribute to the development of later clinical complications in preterm IVH.

Elevated Pro-Inflammatory Cell-Free MicroRNA Levels in Cerebrospinal Fluid of Premature Infants after Intraventricular Hemorrhage.

Intraventricular hemorrhage (IVH) represents a high risk of neonatal mortality and later neurodevelopmental impairment in prematurity. IVH is accompanied with inflammation, hemolysis, and extracellular hemoglobin (Hb) oxidation. However, microRNA (miRNA) expression in cerebrospinal fluid (CSF) of preterm infants with IVH has been unknown. Therefore, in the present study, candidate pro-inflammatory cell-free miRNAs were analyzed in CSF samples from 47 preterm infants with grade III or IV IVH vs. clinical controls (n = 14). miRNAs were quantified by RT-qPCR, normalized to "spike-in" cel-miR-39. Oxidized Hb and total heme levels were determined by spectrophotometry as well as IL-8, VCAM-1, ICAM-1, and E-selectin concentrations by ELISA. To reveal the origin of the investigated miRNAs, controlled hemolysis experiments were performed in vitro; in addition, human choroid plexus epithelial cell (HCPEpiC) cultures were treated with metHb, ferrylHb, heme, or TNF-α to replicate IVH-triggered cellular conditions. Levels of miR-223, miR-155, miR-181b, and miR-126 as well as Hb metabolites along with IL-8 were elevated in CSF after the onset of IVH vs. controls. Significant correlations were observed among the miRNAs, oxidized Hb forms, and the soluble adhesion molecules. During the post-IVH follow-up, attenuated expression of miRNAs and protein biomarkers in CSF was observed upon elimination of Hb metabolites. These miRNAs remained unaffected by a series of artificially induced hemolysis, which excluded red blood cells as their origin, while stimulation of HCPEpiCs with oxidized Hb fractions and heme resulted in increased extracellular miRNA levels in the cell culture supernatant. Overall, the hemorrhage-induced CSF miRNAs reflected inflammatory conditions as potential biomarkers in preterm IVH.

Reduced miR-26b Expression in Megakaryocytes and Platelets Contributes to Elevated Level of Platelet Activation Status in Sepsis.

In sepsis, platelets may become activated via toll-like receptors (TLRs), causing microvascular thrombosis. Megakaryocytes (MKs) also express these receptors; thus, severe infection may modulate thrombopoiesis. To explore the relevance of altered miRNAs in platelet activation upon sepsis, we first investigated sepsis-induced miRNA expression in platelets of septic patients. The effect of abnormal Dicer level on miRNA expression was also evaluated. miRNAs were profiled in septic vs. normal platelets using TaqMan Open Array. We validated platelet miR-26b with its target SELP (P-selectin) mRNA levels and correlated them with clinical outcomes. The impact of sepsis on MK transcriptome was analyzed in MEG-01 cells after lipopolysaccharide (LPS) treatment by RNA-seq. Sepsis-reduced miR-26b was further studied using Dicer1 siRNA and calpain inhibition in MEG-01 cells. Out of 390 platelet miRNAs detected, there were 121 significantly decreased, and 61 upregulated in sepsis vs. controls. Septic platelets showed attenuated miR-26b, which were associated with disease severity and mortality. SELP mRNA level was elevated in sepsis, especially in platelets with increased mean platelet volume, causing higher P-selectin expression. Downregulation of Dicer1 generated lower miR-26b with higher SELP mRNA, while calpeptin restored miR-26b in MEG-01 cells. In conclusion, decreased miR-26b in MKs and platelets contributes to an increased level of platelet activation status in sepsis.

The Fungal Iron Chelator Desferricoprogen Inhibits Atherosclerotic Plaque Formation.

Hemoglobin, heme and iron are implicated in the progression of atherosclerosis. Therefore, we investigated whether the hydrophobic fungal iron chelator siderophore, desferricoprogen (DFC) inhibits atherosclerosis. DFC reduced atherosclerotic plaque formation in ApoE-/- mice on an atherogenic diet. It lowered the plasma level of oxidized LDL (oxLDL) and inhibited lipid peroxidation in aortic roots. The elevated collagen/elastin content and enhanced expression of adhesion molecule VCAM-1 were decreased. DFC diminished oxidation of Low-density Lipoprotein (LDL) and plaque lipids catalyzed by heme or hemoglobin. Formation of foam cells, uptake of oxLDL by macrophages, upregulation of CD36 and increased expression of TNF-α were reduced by DFC in macrophages. TNF-triggered endothelial cell activation (vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecules (ICAMs), E-selectin) and increased adhesion of monocytes to endothelium were attenuated. The increased endothelial permeability and intracellular gap formation provoked by TNF-α was also prevented by DFC. DFC acted as a cytoprotectant in endothelial cells and macrophages challenged with a lethal dose of oxLDL and lowered the expression of stress-responsive heme oxygenase-1 as sublethal dose was employed. Saturation of desferrisiderophore with iron led to the loss of the beneficial effects. We demonstrated that DFC accumulated within the atheromas of the aorta in ApoE-/- mice. DFC represents a novel therapeutic approach to control the progression of atherosclerosis.

A novel flow cytometric method for enhancing acute promyelocytic leukemia screening by multidimensional dot-plots.

Acute promyelocytic leukemia (APL) is generally characterized by t(15;17)(q24;q21). In some cases, the classic translocation cannot be identified by conventional methods, since the PML-RARA fusion protein results from complex, variant, or cryptic translocation. The diagnostic algorithm of APL starts with screening methods, such as flow cytometry (FC), followed by fluorescence in situ hybridization or polymerase chain reaction to confirm the diagnosis. Our aim was to develop a novel protocol for analyzing APL samples based on multidimensional dot-plots that can provide comprehensive information about several markers at the same time. The protocol included four optimized multidimensional dot-plots, which were tested by retrospective reanalysis of FC results in APL (n = 8) and non-APL (n = 12) acute myeloid leukemia (AML) cases. After predicting the potential position of hypergranular- and microgranular-type aberrant promyelocytes, the percentages of blast populations were examined within the gates in all AML cases. The percentage of blasts in each predefined gate was well above the cut-off value (95%) in APL cases in all tubes. In non-APL AML cases, the percentage of blasts in the same gates never reached the cut-off value in all investigated tubes, and even when it did in a single tube, the pattern was markedly different from that observed in APL cases. In conclusion, multidimensional dot-plots can be used for screening APL even in cryptic APL cases, although reproducibility across several laboratories would require standardization of antibodies and fluorochromes. This easy-to-use and quick method can support the diagnosis of APL and the prompt initiation of the appropriate treatment.

Dasatinib inhibits coated-platelet generation in patients with chronic myeloid leukemia.

Since the introduction of tyrosine kinase inhibitors, the overall survival of patients with chronic myeloid leukemia has markedly improved. However long term use of these drugs results in various adverse events. Treatment with second generation dasatinib is often complicated by hemorrhagic events. Previous lumi-aggregometry studies have shown impaired platelet function in patients on dasatinib therapy. Dual agonist activated platelets (coated-platelets) are also sensitive indicators of platelet function. We hypothesized that dual activation with convulxin and thrombin of platelets in a flow cytometric assay could be a more sensitive method for detecting platelet dysfunction as compared to single agonist studies used in lumi-aggregometer. Platelets of healthy volunteers incubated with dasatinib as well as platelets from patients on dasatinib therapy were investigated. Low therapeutic plasma level dasatinib concentrations at which a considerable reduction in coated-platelet generation was observed in vitro, did not cause detectable change in platelet aggregation response. Coated-platelet assay and lumi-aggregometry were also investigated at 0, 1 and 4 hours after drug administration in dasatinib treated CML patients. Significant decrease was observed at 1 hour in maximal aggregation by collagen. Although the aggregation curves became normalized by 4 hours, coated-platelet generation was still inhibited in dasatinib treated patients. Nilotinib, another second generation tyrosine kinase inhibitor, had no effect on aggregation and on coated-platelet formation neither in vitro nor in ex vivo samples. At therapeutic plasma levels coated-platelet assay is more sensitive than lumi-aggregometry studies for the demonstration of the inhibitory effect of dasatinib on platelet function.

Dasatinib Inhibits Procoagulant and Clot Retracting Activities of Human Platelets.

Tyrosine kinase inhibitors (TKI) such as the BCR-ABL inhibitor dasatinib and nilotinib are highly effective therapies for chronic myeloid leukemia (CML). However, several lines of evidence suggest that dasatinib can induce bleeding which may be due to impaired collagen-induced platelet adhesion, aggregation, and secretion. Sarcoma family kinases (SFK) play central role in the GPVI-induced signaling pathway. We aimed to investigate whether and how dasatinib can modulate SFK-mediated platelet procoagulant activity in a purified system and in dasatinib/nilotinib treated CML patients. In platelet rich plasmas of healthy volunteers, dasatinib dose-dependently reduced convulxin-induced phosphatidylserine exposure and attenuated thrombin formation. Similarly to these changes, integrin activation and clot retraction were also significantly inhibited by 100 nM dasatinib. Platelets isolated from dasatinib treated patients showed a significantly lower phosphatidylserine expression upon convulxin activation compared to premedication levels. In these samples, thrombin generation was significantly slower, and the quantity of formed thrombin was less compared to the trough sample. Western blot analyses showed decreased phosphorylation levels of the C-terminal tail and the activation loop of SFKs upon dasatinib administration. Taken together, these results suggest that dasatinib inhibits the formation of procoagulant platelets via the GPVI receptor by inhibiting phosphorylation of SFKs.

Cellular Factor XIII, a Transglutaminase in Human Corneal Keratocytes.

Cellular factor XIII (cFXIII, FXIII-A2), a transglutaminase, has been demonstrated in a few cell types. Its main function is to cross-link proteins by isopeptide bonds. Here, we investigated the presence of cFXIII in cells of human cornea. Tissue sections of the cornea were immunostained for FXIII-A in combination with staining for CD34 antigen or isopeptide cross-links. Isolated corneal keratocytes were also evaluated by immunofluorescent microscopy and flow cytometry. FXIII-A in the corneal stroma was quantified by Western blotting. FXIII-A mRNA was detected by RT-qPCR. The cornea of FXIII-A-deficient patients was evaluated by cornea topography. FXIII-A was detected in 68 ± 13% of CD34+ keratocytes. Their distribution in the corneal stroma was unequal; they were most abundant in the subepithelial tertile. cFXIII was of cytoplasmic localization. In the stroma, 3.64 ng cFXIII/mg protein was measured. The synthesis of cFXIII by keratocytes was confirmed by RT-qPCR. Isopeptide cross-links were detected above, but not within the corneal stroma. Slight abnormality of the cornea was detected in six out of nine FXIII-A-deficient patients. The presence of cFXIII in human keratocytes was established for the first time. cFXIII might be involved in maintaining the stability of the cornea and in the corneal wound healing process.

Analytical parameters and validation of homopolymer detection in a pyrosequencing-based next generation sequencing system.

BACKGROUND: Current technologies in next-generation sequencing are offering high throughput reads at low costs, but still suffer from various sequencing errors. Although pyro- and ion semiconductor sequencing both have the advantage of delivering long and high quality reads, problems might occur when sequencing homopolymer-containing regions, since the repeating identical bases are going to incorporate during the same synthesis cycle, which leads to uncertainty in base calling. The aim of this study was to evaluate the analytical performance of a pyrosequencing-based next-generation sequencing system in detecting homopolymer sequences using homopolymer-preintegrated plasmid constructs and human DNA samples originating from patients with cystic fibrosis. RESULTS: In the plasmid system average correct genotyping was 95.8% in 4-mers, 87.4% in 5-mers and 72.1% in 6-mers. Despite the experienced low genotyping accuracy in 5- and 6-mers, it was possible to generate amplicons with more than a 90% adequate detection rate in every homopolymer tract. When homopolymers in the CFTR gene were sequenced average accuracy was 89.3%, but varied in a wide range (52.2 - 99.1%). In all but one case, an optimal amplicon-sequencing primer combination could be identified. In that single case (7A tract in exon 14 (c.2046_2052)), none of the tested primer sets produced the required analytical performance. CONCLUSIONS: Our results show that pyrosequencing is the most reliable in case of 4-mers and as homopolymer length gradually increases, accuracy deteriorates. With careful primer selection, the NGS system was able to correctly genotype all but one of the homopolymers in the CFTR gene. In conclusion, we configured a plasmid test system that can be used to assess genotyping accuracy of NGS devices and developed an accurate NGS assay for the molecular diagnosis of CF using self-designed primers for amplification and sequencing.

Optimized angiotensin-converting enzyme activity assay for the accurate diagnosis of sarcoidosis.

BACKGROUND: Serum angiotensin-converting enzyme (ACE) activity determination can aid the early diagnosis of sarcoidosis. We aimed to optimize a fluorescent kinetic assay for ACE activity by screening the confounding effects of endogenous ACE inhibitors and interfering factors. Genotype-dependent and genotype-independent reference values of ACE activity were established, and their diagnostic accuracies were validated in a clinical study. METHODS: Internally quenched fluorescent substrate, Abz-FRK(Dnp)P-OH was used for ACE-activity measurements. A total of 201 healthy individuals and 59 presumably sarcoidotic patients were enrolled into this study. ACE activity and insertion/deletion (I/D) genotype of the ACE gene were determined. RESULTS: Here we report that serum samples should be diluted at least 35-fold to eliminate the endogenous inhibitor effect of albumin. No significant interferences were detected: up to a triglyceride concentration of 16 mM, a hemoglobin concentration of 0.71 g/L and a bilirubin concentration of 150 µM. Genotype-dependent reference intervals were considered as 3.76-11.25 U/L, 5.22-11.59 U/L, 7.19-14.84 U/L for II, ID and DD genotypes, respectively. I/D genotype-independent reference interval was established as 4.85-13.79 U/L. An ACE activity value was considered positive for sarcoidosis when it exceeded the upper limit of the reference interval. The optimized assay with genotype-dependent reference ranges resulted in 42.5% sensitivity, 100% specificity, 100% positive predictive value and 32.4% negative predictive value in the clinical study, whereas the genotype-independent reference range proved to have inferior diagnostic efficiency. CONCLUSIONS: An optimized fluorescent kinetic assay of serum ACE activity combined with ACE I/D genotype determination is an alternative to invasive biopsy for confirming the diagnosis of sarcoidosis in a significant percentage of patients.

Comprehensive genetic testing in children with a clinical diagnosis of ARPKD identifies phenocopies.

BACKGROUND: Autosomal recessive polycystic kidney disease (ARPKD) is genetically one of the least heterogeneous ciliopathies, resulting primarily from mutations of PKHD1. Nevertheless, 13-20% of patients diagnosed with ARPKD are found not to carry PKHD1 mutations by sequencing. Here, we assess whether PKHD1 copy number variations or second locus mutations explain these cases. METHODS: Thirty-six unrelated patients with the clinical diagnosis of ARPKD were screened for PKHD1 point mutations and copy number variations. Patients without biallelic mutations were re-evaluated and screened for second locus mutations targeted by the phenotype, followed, if negative, by clinical exome sequencing. RESULTS: Twenty-eight patients (78%) carried PKHD1 point mutations, three of whom on only one allele. Two of the three patients harbored in trans either a duplication of exons 33-35 or a large deletion involving exons 1-55. All eight patients without PKHD1 mutations (22%) harbored mutations in other genes (PKD1 (n = 2), HNF1B (n = 3), NPHP1, TMEM67, PKD1/TSC2). Perinatal respiratory failure, a kidney length > +4SD and early-onset hypertension increase the likelihood of PKHD1-associated ARPKD. A patient compound heterozygous for a second and a last exon truncating PKHD1 mutation (p.Gly4013Alafs*25) presented with a moderate phenotype, indicating that fibrocystin is partially functional in the absence of its C-terminal 62 amino acids. CONCLUSIONS: We found all ARPKD cases without PKHD1 point mutations to be phenocopies, and none to be explained by biallelic PKHD1 copy number variations. Screening for copy number variations is recommended in patients with a heterozygous point mutation.

[Alteration in the expression of platelet microRNAs in diseases with abnormal platelet activation]. / Vérlemezke-mikro-RNS-ek expressziójának változása thrombocytaaktivációval járó betegségekben.

MicroRNAs (miRNA) are short, non-coding RNAs consisting of 18-25 nucleotides that regulate posttranscriptionally the gene expression involved in the regulation of physiological processes of the cells. Their key role is to modulate the translation of target mRNAs via binding to complementary sequences within the 3' UTRs of mRNAs resulting in altered protein synthesis or even the degradation of mRNAs. miRNAs are carried not only by cells with nucleus, but also in platelets, red blood cells, and they are present in the circulation, in urine and in other body fluids as well. The fact about functional miRNAs in platelets without nucleus having a half-life of 8-12 days was questioned for a long time, thus it was also obscure whether platelets are able to produce proteins de novo when being exposed to different challenges. In the last few years, several publications have described the expression and function of certain platelet mRNAs with their regulatory miRNAs in terms of regulation of cell activation, especially in diseases in which platelet activation status is elevated, such as in type 2 diabetes mellitus or in sepsis. Apart from their pathophysiological role, miRNAs may be applied as potential new biomarkers in the investigation or differential diagnosis of these clinical conditions. This review article sought to summarize the recent findings about platelet miRNAs focusing on their altered expression in diabetes and sepsis. Orv Hetil. 2018; 159(47): 1962-1970.

[Laboratory diagnostics of lung neuroendocrine tumors]. / Neuroendokrin tüdõtumorok laboratóriumi kivizsgálása.

Perhaps innovations in protein and peptide analysis utilizing immunochemistry methodology have been the most demarcating in the field of routine laboratory diagnostics in the past few decades. Presently, the state of art immunochemistry (e.g., immunofluorimetry, electrochemiluminescence) and separation techniques (e.g., high pressure liquid chromatography) facilitate achievement of detection limits well below the nmol/L range. These techniques have allowed reproducible and high throughput analysis with a short turnaround time of tumor markers, among others, in the routine diagnostic laboratory setting. Tumor marker determination is an integral part of the diagnostic work-up of tumors, and is indispensable in therapeutic monitoring and clinical decision-making. This article presents the utility of tumor markers in the diagnostics of lung neuroendocrine tumors (NET). Furthermore, since the majority of the tests used in lung NET diagnostics are infrequently requested, quite a few practical issues are also discussed. The generally applicable rule pertaining to tumor markers is also valid for NET diagnostics, i.e., improved sensitivity and specificity is widely achieved with a combination of tumor markers, as such, promoting diagnosis, efficient therapeutic monitoring and defining the prognosis.

Laboratory characterization of leukemic cell procoagulants.

BACKGROUND: In acute myeloid leukemias, there is an increased chance to develop thrombotic disorders. We hypothesized that in addition to leukemic promyelocytes, monocytic leukemia cells may also have a higher procoagulant activity. METHODS: Fibrin formation was assessed by a one-stage clotting assay using a magnetic coagulometer. The thrombin generation test (TGT) of magnetically isolated normal human monocytes, intact leukemic cells and their isolated microparticles was performed by a fluorimetric assay. Phosphatidylserine (PS) expression of leukemic cells and microparticle number determinations were carried out by flow cytometry. RESULTS: All cell lines displayed a significant procoagulant potential compared to isolated normal human monocytes. In the TGT test, the mean of lagtime and the time to peak parameters were significantly shorter in leukemic cells (3.9-4.7 and 9.9-10.3 min) compared to monocytes (14.9 and 26.5 min). The mean of peak thrombin in various monocytic leukemia cell lines was 112.1-132.9 nM vs. 75.1 nM in monocytes; however, no significant difference was observed in the ETP parameter. Factor VII-deficient plasma abolished all procoagulant activity, whereas factor XII-deficient plasma did not affect the speed of fibrin formation and thrombin generation but modulated the amount of thrombin. Factor XI-deficient plasma affected the time to peak values in one leukemic cell line and also attenuated peak thrombin. Leukemia cell-derived microparticles from all three cell lines exerted a procoagulant effect by significantly shortening the lagtime in TGT; there was a nonsignificant difference in case of ETP parameter. CONCLUSIONS: All investigated monocytic leukemia cell lines exhibited significant thrombin generation. This phenomenon was achieved by the procoagulants on the surface of leukemic cells as well as by their microparticles.

[Laboratory aspects of novel oral anticoagulant treatment]. / Az új orális antikoagulánsokkal történo kezelés laboratóriumi vonatkozásai.

The introduction of novel oral anticoagulants (NOAC) have long been expected drugs and they quickly became used widespread as their clinical effectiveness was as good as, or even better than the previously used only oral anticoagulant drug, the coumarins. Thus, the direct thrombin inhibitor dabigatran and the activated factor X inhibitors (rivaroxaban, apixaban, edoxaban) have become the part of daily therapeutic practice. Their permeation was facilitated by the guideline which suggested that no laboratory monitoring was required during NOAC treatment and this was very convenient for both patients and doctors. The clinical experience obtained in the past years, however have proved that the 'one size fits all' view is oversimplified and there are numerous situations when the determination NOAC levels is unavoidable or highly recommended. This review discusses the laboratory aspects of NOAC treatment, primarily summarizing their effect on the screening tests and special assays of hemostasis and we also describe the correct methods to determine their plasma concentrations. Orv Hetil. 2017; 158(49): 1930-1945.