Due to the current pandemic, a global shortage of reagents has drawn interest in developing alternatives to increase the number of coronavirus tests. One such alternative is sample pooling. We compared commercial kits that are used in COVID-19 diagnostics in terms of their sensitivity and feasibility for use in pooling. In this preliminary study, we showed that pooling of up to 80 samples did not affect the efficacy of the kits. Additionally, the RNA-dependent RNA polymerase (RdRp) gene is a more suitable target in pooled samples than the envelope (E) gene. This approach could provide an easy method of screening a large number of samples and help adjust different governmental regulations.

The most frequent mutations in acute myeloid leukemia (AML) – FLT3-ITD and NPM1 – are associated with a specific immunophenotype. We evaluated the levels of surface antigens in an uninvestigated AML patient population according to the combination of FLT3-ITD/NPM1 mutations. Antigen levels were calculated as the geometric mean fluorescence index (MFI) ratio between myeloblasts or monoblasts/monocytes and a negative population for the specific antigen. In myeloblastic populations, FLT3-ITD cases presented CD7^high MFI values (p < .001), while NPM1-MUT cases presented CD33^high (p < .001), and CD34^low (p < .001) MFI values. Within the monoblastic/monocytic populations, CD56^high expression was observed only in the FLT3-WT/NPM1-MUT population (p=.003). The single common antigen expression between myeloblasts and monoblasts/monocytes was CD123^high expression only within the FLT3-ITD/NPM1-MUT subgroup. Our results present a subtle influence of FLT3-ITD/NPM1 mutations upon antigen expression profiles in myeloblasts vs monoblasts/monocytes, and we described a novel correlation between the presence of NPM1 and CD56^high values within bulk leukemic monoblasts/monocytes.

Objectives: To assess the frequency of multiple thrombophilia-associated mutations and polymorphisms in a selected population of high-risk pregnancies. Methods: Thrombophilia screening was performed for 1,500 pregnant women with prior pregnancy complications or thrombotic events. Nine thrombophilia-associated mutations or polymorphisms were screened: factor V Leiden, factor V H1299R, prothrombin G20210A, MTHFR C677T, MTHFR A1298C, factor XIII V34L, PAI-1 4G/5G polymorphisms, EPCR G4600A, EPCR C4678G. Results: Out of the 1,500 patients, 1,291 fulfilled the criteria for data interpretation. All patients had low-risk thrombophilia-associated genetic variants. Only 1.24 % of cases presented high-risk abnormalities (homozygous factor V Leiden/prothrombin G20210A, or both mutations in heterozygous form). Heterozygous factor V Leiden occurred in 10.38 % of cases, while only 5.81 % carried heterozygous prothrombin G20210A mutation. The frequency of prothrombin G20210A mutation was higher (10.37 %) in the subgroup associating factor V Leiden, than in the subgroup lacking it (5.36 %). Low-risk genetic variants occurred with a higher frequency: 23.78 % factor V H1299R, 57.32 % MTHFR C677T, 55.54 % MTHFR A1298C, 44.07 % factor XIII V34L, 73.20 % PAI-1 4G/5G polymorphisms, 69.64 % EPCR G4600A, and 69.63 % EPCR C4678G. Conclusions: All patients had at least one prothrombotic genetic mutation or variant. Our data highlight the need for thrombophilia screening, including low-risk genetic variants, in a high-risk population of pregnant women with a history of pregnancy complications or thrombotic events.

Background: Accurate quantification of BCR::ABL1 transcripts is paramount for the management of chronic myeloid leukemia (CML) and Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL). Despite advancements in real-time reverse transcription PCR (RT-qPCR), the need for more sensitive and precise assays remains. This study evaluated the diagnostic utility of digital reverse transcription PCR (dRT-PCR) for BCR::ABL1 quantification in assessing minimal residual disease (MRD). Material and methods. Twelve patients with CML or Ph+ ALL were included. Peripheral blood or bone marrow samples, collected in PaxGene RNA preservation tubes, were extracted with the automated QIASymphony SP instrument. dRT-PCR conducted on the LOAA analyzer, utilized the Dr. PCR BCR-ABL1 Detection Kit CE_IVD (OPTOLANE Technologies, South Korea). By employing a dual-labeled hydrolysis probe and target-specific primers, this protocol enabled the simultaneous amplification and detection of BCR::ABL1 transcripts e13a2 and e14a2 and ABL1. The resulting copy numbers/reactions were used to calculate the International Scale percentage (%IS) and the molecular reduction (MR) value and analyzed for clinical correlations. Results. Digital RT-PCR demonstrated high precision in quantifying BCR::ABL1 transcripts, the lowest %IS value being 0.02%. A consistent quality RNA sample totalizing 110 ng/reaction was crucial for successful reactions. This assay proved faster and easier than conventional RT-PCR and did not require standards or calibrators. Conclusion. This study supports the feasibility of digital RT-PCR for quantitative analysis. Future objectives for our laboratory include expanding our diagnostic repertoire using this platform, for enhancing patient care.

The BCR::ABL1 translocation and the accompanying Philadelphia chromosome represents the first mutation which defined a disease, chronic myeloid leukemia. It also represents the first druggable target for which a specific compound was developed and accepted in current clinic practice, imatinib. Despite these, there are still areas in which the diagnosis and the best treatment sequence still needs investigation. One such context is the diagnosis and management of BCR::ABL1 positive myeloid neoplasms with ≥20% blasts, more specifically the differentiation between myeloid blast phase chronic myeloid leukemia and BCR::ABL1 positive acute myeloid leukemia. In this paper we present our recent experience with a BCR::ABL1 positive myeloid neoplasms with ≥20% blasts.

Multiple myeloma represents a hematologic malignancy characterized by the abnormal proliferation of plasma cells in the bone marrow. Through this proliferation, the malignant plasma cells disrupt normal blood cell production, subsequently causing the clinical hallmarks of this pathology: bone destruction, anemia, renal impairment and a highly increased susceptibility to infections. Despite the therapeutic options available, multiple myeloma remains an incurable disease (1). The rising use of combination therapies, triplets or quadruplets, by using immunomodulatory drugs, monoclonal antibodies and targeted therapies, elicited improved survival rates and a significant improvement of the quality of life of these patients. However, there is a luring need to precisely quantify the life horizons of these patients, therefore the most recent tool for this estimate has been developed, the R-ISS score. The R-ISS score uses various biomarkers such as serum β2-microglobulin, serum albumin, LDH levels and a more refined tool, the cytogenetic abnormalities list, to predict the outcome of each case. The addition of the precise cytogenetic risk groups boosted the sensitivity of the previous ISS score, but also unveiled the existing discrepancies in accessing modern diagnostic tools across treatment centers (2). Moreover, patient-oriented reports pointed out that from a patient perspective the highest perceived cost had been perceived until the neoplasm diagnostic was established, hence the reluctance for specialized investigations (eg. FISH testing and karyotypes etc.)