Testing for Factor V Leiden (FVL) and Prothrombin G20210A Genetic Variants.

Laboratory testing for Factor V Leiden and Prothrombin G20210A genetic variants permits defining the increased relative risk for venous thromboembolism in selected patients. Laboratory DNA testing for these variants may be undertaken by a variety of methods, including fluorescence-based quantitative real-time PCR (qPCR). This is a rapid, simple, robust, and reliable method to identify genotypes of interest. This chapter describes the method that employs PCR amplification of the patient DNA region of interest and genotyping by allele-specific discrimination technology on a quantitative real-time PCR (qPCR) instrument.

Is It Possible to Predict Clonal Thrombocytosis in Triple-Negative Patients with Isolated Thrombocytosis Based Only on Clinical or Blood Findings?

JAK2, MPL, and CALR mutations define clonal thrombocytosis in about 90% of patients with sustained isolated thrombocytosis. In the remainder of patients (triple-negative patients) diagnosing clonal thrombocytosis is especially difficult due to the different underlying conditions and possible inconclusive bone marrow biopsy results. The ability to predict patients with sustained isolated thrombocytosis with a potential clonal origin has a prognostic value and warrants further examination. The aim of our study was to define a non-invasive clinical or blood parameter that could help predict clonal thrombocytosis in triple-negative patients. We studied 237 JAK2 V617-negative patients who were diagnosed with isolated thrombocytosis and referred to the haematology service. Sixteen routine clinical and blood parameters were included in the logistic regression model which was used to predict the type of thrombocytosis (reactive/clonal). Platelet count and lactate dehydrogenase (LDH) were the only statistically significant predictors of clonal thrombocytosis. The platelet count threshold for the most accurate prediction of clonal or reactive thrombocytosis was 449 × 109/L. Other tested clinical and blood parameters were not statistically significant predictors of clonal thrombocytosis. The level of LDH was significantly higher in CALR-positive patients compared to CALR-negative patients. We did not identify any new clinical or blood parameters that could distinguish clonal from reactive thrombocytosis. When diagnosing clonal thrombocytosis triple-negative patients are most likely to be misdiagnosed. Treatment in patients with suspected triple negative clonal thrombocytosis should not be delayed if cardiovascular risk factors or pregnancy coexist, even in the absence of firm diagnostic criteria. In those cases the approach "better treat more than less" should be followed.

Identification of Variants Associated With Rare Hematological Disorder Erythrocytosis Using Targeted Next-Generation Sequencing Analysis.

An erythrocytosis is present when the red blood cell mass is increased, demonstrated as elevated hemoglobin and hematocrit in the laboratory evaluation. Congenital predispositions for erythrocytosis are rare, with germline variants in several genes involved in oxygen sensing (VHL, EGLN1, and EPAS1), signaling for hematopoietic cell maturation (EPOR and EPO), and oxygen transfer (HBB, HBA1, HBA2, and BPGM) that were already associated with the eight congenital types (ECYT1-8). Screening for variants in known congenital erythrocytosis genes with classical sequencing approach gives a correct diagnosis for only up to one-third of the patients. The genetic background of erythrocytosis is more heterogeneous, and additional genes involved in erythropoiesis and iron metabolism could have a putative effect on the development of erythrocytosis. This study aimed to detect variants in patients with yet unexplained erythrocytosis using the next-generation sequencing (NGS) approach, targeting genes associated with erythrocytosis and increased iron uptake and implementing the diagnostics of congenital erythrocytosis in Slovenia. Selected 25 patients with high hemoglobin, high hematocrit, and no acquired causes were screened for variants in the 39 candidate genes. We identified one pathogenic variant in EPAS1 gene and three novel variants with yet unknown significance in genes EPAS1, JAK2, and SH2B3. Interestingly, a high proportion of patients were heterozygous carriers for two variants in HFE gene, otherwise pathogenic for the condition of iron overload. The association between the HFE variants and the development of erythrocytosis is not clearly understood. With a targeted NGS approach, we determined an actual genetic cause for the erythrocytosis in one patient and contributed to better management of the disease for the patient and his family. The effect of variants of unknown significance on the enhanced production of red blood cells needs to be further explored with functional analysis. This study is of great significance for the improvement of diagnosis of Slovenian patients with unexplained erythrocytosis and future research on the etiology of this rare hematological disorder.

The Contemporary Approach to CALR-Positive Myeloproliferative Neoplasms.

CALR mutations are a revolutionary discovery and represent an important hallmark of myeloproliferative neoplasms (MPN), especially essential thrombocythemia and primary myelofibrosis. To date, several CALR mutations were identified, with only frameshift mutations linked to the diseased phenotype. It is of diagnostic and prognostic importance to properly define the type of CALR mutation and subclassify it according to its structural similarities to the classical mutations, a 52-bp deletion (type 1 mutation) and a 5-bp insertion (type 2 mutation), using a statistical approximation algorithm (AGADIR). Today, the knowledge on the pathogenesis of CALR-positive MPN is expanding and several cellular mechanisms have been recognized that finally cause a clonal hematopoietic expansion. In this review, we discuss the current basis of the cellular effects of CALR mutants and the understanding of its implementation in the current diagnostic laboratorial and medical practice. Different methods of CALR detection are explained and a diagnostic algorithm is shown that aids in the approach to CALR-positive MPN. Finally, contemporary methods joining artificial intelligence in accordance with molecular-genetic biomarkers in the approach to MPN are presented.

Genetic analysis of 39 erythrocytosis and hereditary hemochromatosis-associated genes in the Slovenian family with idiopathic erythrocytosis.

BACKGROUND: Erythrocytosis is a condition with an excessive number of erythrocytes, accompanied by an elevated haemoglobin and/or haematocrit value. Congenital erythrocytosis has a diverse genetic background with several genes involved in erythropoiesis. In clinical practice, nine genes are usually examined, but in approximately 70% of patients, no causative mutation can be identified. In this study, we screened 39 genes, aiming to identify potential disease-driving variants in the family with erythrocytosis of unknown cause. PATIENTS AND METHODS: Two affected family members with elevated haemoglobin and/or haematocrit and negative for acquired causes and one healthy relative from the same family were selected for molecular-genetic analysis of 24 erythrocytosis and 15 hereditary haemochromatosis-associated genes with targeted NGS. The identified variants were further analysed for pathogenicity using various bioinformatic tools and review of the literature. RESULTS: Of the 12 identified variants, two heterozygous variants, the missense variant c.471G>C (NM_022051.2) (p.(Gln157His)) in the EGLN1 gene and the intron variant c.2572-13A>G (NM_004972.3) in the JAK2 gene, were classified as low-frequency variants in European population. None of the two variants were present in a healthy family member. Variant c.2572-13A>G has potential impact on splicing by one prediction tool. CONCLUSION: For the first time, we included 39 genes in the erythrocytosis clinical panel and identified two potential disease-driving variants in the Slovene family studied. Based on the reported functional in vitro studies combined with our bioinformatics analysis, we suggest further functional analysis of variant in the JAK2 gene and evaluation of a cumulative effect of both variants.

Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis.

High resolution melting analysis (HRM) is a powerful method for genotyping and genetic variation scanning. Most HRM applications depend on saturating DNA dyes that detect sequence differences, and heteroduplexes that change the shape of the melting curve. Excellent instrument resolution and special data analysis software are needed to identify the small melting curve differences that identify a variant or genotype. Different types of genetic variants with diverse frequencies can be observed in the gene specific for patients with a specific disease, especially cancer and in the CALR gene in patients with Philadelphia chromosome-negative myeloproliferative neoplasms. Single nucleotide changes, insertions and/or deletions (indels) in the gene of interest can be detected by the HRM analysis. The identification of different types of genetic variants is mostly based on the controls used in the qPCR HRM assay. However, as the product length increases, the difference between wild-type and heterozygote curves becomes smaller, and the type of genetic variant is more difficult to determine. Therefore, where indels are the prevalent genetic variant expected in the gene of interest, an additional method such as agarose gel electrophoresis can be used for the clarification of the HRM result. In some instances, an inconclusive result must be re-checked/re-diagnosed by standard Sanger sequencing. In this retrospective study, we applied the method to JAK2 V617F-negative patients with MPN.

Improved survival of AML patients by addition of cladribine to standard induction chemotherapy.

One hundred and eight consecutive acute myeloid leukemia (AML) patients aged 60 or less treated with two different induction regimens were retrospectively analyzed. Induction regimen for the first 50 consecutive patients was DA3+7, and the following 58 patients received cladribine 5 mg/m2 on days 1 through 5 in addition to DA3+7 (DAC). There were no significant differences in the median age and the proportion of patients with unfavorable characteristics between the two groups. Remission after induction chemotherapy was achieved in 30/50 (60%) patients in DA3+7 and in 46/58 (79%) in DAC group (p = 0.028). The median survival in the DA3+7 group was 18 months, while in the DAC group it was not reached (p = 0.034). We confirmed results from other research groups by demonstrating improved remission induction rate and overall survival of AML patients aged 60 years or less treated with DAC induction compared with standard DA3+7 induction chemotherapy.

CALR mutations in a cohort of JAK2 V617F negative patients with suspected myeloproliferative neoplasms.

Suspicion of myeloproliferative neoplasms (MPNs) and especially essential thrombocythemia (ET) in primary care is often based solely on blood counts, with patients referred to a haematologist without a thorough evaluation. We retrospectively assessed the role of calreticulin gene (CALR) mutations in the diagnosis of MPN in this population. We studied CALR mutations in 524 JAK2 V617F-negative patients with suspected MPN. Uncommon CALR mutations were confirmed by Sanger sequencing and searched for in the COSMIC or HGMD database. Mutations were defined as frameshift or non-frameshift mutations. CALR mutations were detected in 23 patients (23/524 = 4.4%). Four mutations detected in our study were newly identified mutations. Non-frameshift mutations were detected in two patients. Most patients (380/524 = 72.5%) were diagnosed with secondary conditions leading to blood count abnormalities such as iron deficiency, inflammatory and infectious diseases, malignancy and hyposplenism. Nine patients (9/23 = 39%) were retrospectively diagnosed with ET based on CALR mutation confirmation. Two patients with non-frameshift CALR mutations were diagnosed with reactive thrombocytosis and MPN unclassifiable, respectively. Our study showed that CALR mutations are important, non-invasive diagnostic indicators of ET and can aid in its diagnosis. Moreover, the type of CALR mutation must be accurately defined, as non-frameshift mutations may not be associated with ET. Finally, CALR mutation detection should be reserved for patients with high suspicion of clonal haematological disease.

Concurrent acquired inhibitors to factor VIII and IX, a laboratory artifact: a case report.

Acquired inhibitors to coagulation factors other than factor VIII are extremely rare. We describe a case of a 59-year-old woman with abnormal bleeding, diagnosed with concurrent inhibitor antibodies to factor VIII and IX by Bethesda testing. We demonstrate that anti-FVIII antibodies of a very high titre are capable of disturbing the aPTT-based Bethesda assay, resulting in falsely-positive antibodies to factor IX. The case also illustrates the usefulness of the immunological assay (ELISA) in complementing the inhibitor diagnosis.

Monitoring of imatinib targeted delivery in human leukocytes.

The success of imatinib therapy in chronic myeloid leukemia is highly influenced by its active transport into target cells. However, the methodology for analytical evaluation of intracellular drug concentration is rare and usually reliant upon the use of radioactively labeled drugs. More specifically, there is no published method available in the literature for the determination of imatinib concentration in granulocytes. To gain further insight into the intracellular drug uptake a very reliable two-stage sample concentration procedure was devised and coupled with a sensitive ultra-high performance liquid chromatography tandem mass spectrometry. The reliability of this sample preparation and sensitivity of the analysis was confirmed by a successful validation of all necessary method parameters to an impressive lower limit of quantification of 0.5 ng imatinib per 10(6) cells still at the signal to noise ratio of 670. The usefulness of the method is further improved with only 6 mL of blood being necessary for patient analysis. The method has been applied to blood samples of 13 CML patients treated with imatinib and all the measured intracellular drug concentrations were within the validated range. These and further measurements will enable the research of factors which may, besides blood plasma concentration, influence the individual's response to imatinib therapy. Furthermore, individualisation of dosing based on the directly measured targeted drug delivery could be possible.

Simultaneous measurement of imatinib, nilotinib and dasatinib in dried blood spot by ultra high performance liquid chromatography tandem mass spectrometry.

Imatinib, dasatinib and nilotinib are three tyrosine kinase inhibitors currently used to treat Bcr-Abl1 positive chronic myelogenous leukaemia (CML). However, achieving maximum benefit with these drugs may require optimal dosing and adherence to therapy. In those cases, therapeutic drug monitoring (TDM) can be a useful tool in managing patients with CML. The paper presents simple and high throughput method for simultaneous determination of all three TKIs in dried blood spot (DBS) samples from CML patients. DBS samples were prepared by applying 10 µL of spiked whole blood onto an Agilent DBS cards. Whole blood spot was punched out of the card, transferred to a well in a 96-well Captiva ND Lipids filter plate. After the addition of isotopically labelled internal standard, the drug was extracted with 0.1% formic acid in methanol. The collected extract (1 µL) was injected onto a Phenomenex Kinetex 50 mm × 2.1 mm C18 column and eluted with acetonitrile gradient into a triple quadrupole ESI-MS/MS Agilent 6460 operated in positive mode. The total run time was only 2.6 min. The method was validated in terms of linearity, selectivity, specificity, accuracy, precision, absolute and relative matrix effect and stability. The effect of haematocrit (Hct) on the accurate concentration determination was also examined. The method was linear in the range of 50-5000 µg/L for imatinib and nilotinib and in the range of 2.5-250 µg/L for dasatinib, with correlation coefficient values higher than 0.997. Lower limits of quantification (LLOQ) were 50 µg/L for imatinib and nilotinib and 2.5 µg/L for dasatinib. The method proved to be accurate (% bias < 13.2) and precise (CV < 10.3%) on intra- as well as on inter-day basis. Sample matrix (% ME=94.5-106.7) and different Hct values had no significant effect on the accuracy of measured concentrations. Samples proved to be stable whilst stored on DBS cards at room temperature or in the refrigerator; however, at 40 °C the stability of dasatinib was compromised. The method presented was successfully applied to clinical samples.

Factor V Leiden and FII 20210 testing in thromboembolic disorders.

Factor V Leiden and prothrombin (F2) c.20210G>A mutation detection are very important in order to define the increased relative risk for venous thromboembolism in selected patients. Use of DNA-based methods to detect both mutations has become widely available in clinical diagnostic laboratories, including fluorescence-based quantitative real-time PCR (qPCR). The latter is a rapid, simple, robust and reliable method to identify genotypes of interest. There are several chemistries used for qPCR; this article describes their principles and applicability for Factor V Leiden and prothrombin (F2) c.20210G>A mutation detection.

Glutamate dehydrogenase activity in lymphocytes of B-cell chronic lymphocytic leukaemia patients.

OBJECTIVES: To investigate the pattern of glutamate dehydrogenase (GLDH) activity, GLUD1 and GLUD2 expressions in peripheral blood mononuclear cells (PBMC) of untreated B-chronic lymphocytic leukemia (B-CLL) in healthy individuals (HI) and patients with infectious mononucleosis (IM). DESIGN AND METHODS: GLDH activity was determined in a supernatant obtained from pelleted PBMC. GLUD1 and GLUD2 mRNA expression was determined using a quantitative real-time polymerase chain reaction. CD19(+) B cells from PBMC were purified by using positive selection. RESULTS: The highest GLDH activity was found in PBMC of the B-CLL group followed by the HI group and IM group. The PBMC GLDH activity was higher in 60% of the B-CLL patients according to the established reference interval for our HI (2.17-5.70 microkat/g protein). The greater GLDH activity was also found in the CD19(+) cell preparation of the B-CLL patients (two of the three) but not in HI (n=3). The median value of GLUD1 expression was highest in the IM group (n=11), followed by the HI (n=14) and B-CLL groups (n=59) (median 4.69/3.78, P<0.005 and 4.69/2.91, P<0.0005, respectively). GLUD2 expression was not significantly different between groups. CONCLUSIONS: The increased GLDH activity is specific for the PBMC of B-CLL patients. The GLUD1 but not the GLUD2 gene expression pattern is different between the PBMC of IM and B-CLL patients.