Effect size and inferential statistical techniques coupled with machine learning for assessing the association between prolactin concentration and metabolic homeostasis.

BACKGROUND AND OBJECTIVE: Recent guidelines classify low prolactin levels as low as <7 ng/mL and high levels as >25 ng/mL, while the "Homeostatically Functionally Increased Transient Prolactinemia" (HomeoFIT-PRL) range (25-100 ng/mL) suggests that a temporary increase in prolactin could be metabolically beneficial if no related health issues are present. The aim of this study was to investigate the association between mean prolactin concentrations and disturbances in glycidic and lipidic metabolism and to identify the gray zone associated with prolactin inflection points that correlate with these metabolic changes. METHODS: This cross-sectional study involved 65,795 adults who underwent HOMA-IR, glucose, insulin, total cholesterol, HDL-c, LDL-c, and triglyceride tests. Data was categorized into 106 partitions based on prolactin results. Employing an approach referred to in this study as "Hierarchical Multicriteria Analysis of Differences Between Groups - Statistical and Effect Size Approach" (HiMADiG-SESA) comparing the mean concentrations of metabolic tests across prolactin ranges. A machine learning model was utilized to determine inflection points and their corresponding confidence intervals (CIs). These CIs helped establish gray zones in mean prolactin results related to metabolic changes. RESULTS: Statistically and clinically, metabolic test means differed for prolactin <7 ng/mL, except insulin. In the HomeoFIT-PRL range, means were lower except for HDL-c. The gray zones of the mean prolactin results associated with changes in glycidic and lipidic metabolism were 9.58-12.87 ng/mL and 13.81-18.73 ng/mL, respectively. CONCLUSION: A strong correlation was identified between mean prolactin concentrations and the results of metabolism tests below the gray zones associated with inflection points, indicating the potential role of prolactin in the appearance of metabolic disorders. Mean prolactin results can provide deeper insight into metabolic balance.

Overcoming Supply Shortage for SARS-CoV-2 Detection by RT-qPCR.

In February 2020, our laboratory started to offer a RT-qPCR assay for the qualitative detection of severe acute respiratory syndrome coronavirus 2. A few months after the assay was released to our patients, some materials, reagents, and equipment became in short supply. Alternative protocols were necessary in order to avoid stopping testing to the population. However, the suitability of these alternatives needs to be validated before their use. Here, we investigated if saliva is a reliable alternative specimen to nasopharyngeal swabs; if 0.45% saline is a reliable alternative to guanidine hydrochloride as a collection viral transport media; the stability of SARS-COV-2 in guanidine hydrochloride and in 0.45% saline for 10 and 50 days at room temperature; and if the primers/probe concentration and thermocycling times could be reduced so as to overcome the short supply of these reagents and equipment, without a significant loss of the assay performance. We found that saliva is not an appropriated specimen for our method-nasopharyngeal swabs perform better. Saline (0.45%) and guanidine hydrochloride have a similar SARS-CoV-2 diagnostic capability as tube additives. Reliable SARS-CoV-2 RNA detection can be performed after sample storage for 10 days at room temperature (18-23 °C) in both 0.45% saline and guanidine hydrochloride. Using synthetic RNA, and decreasing the concentration of primers by five-fold and probes by 2.5-fold, changed the assay limit of detection (LOD) from 7.2 copies/reaction to 23.7 copies/reaction and the subsequent reducing of thermocycling times changed the assay LOD from 23.7 copies/reaction to 44.2 copies/reaction. However, using real clinical samples with Cq values ranging from ~12.15 to ~36.46, the results of the three tested conditions were almost identical. These alterations will not affect the vast majority of diagnostics and increase the daily testing capability in 30% and increase primers and probe stocks in 500% and 250%, respectively. Taken together, the alternative protocols described here overcome the short supply of tubes, reagents and equipment during the SARS-CoV-2 pandemic, avoiding the collapse of test offering for the population: 105,757 samples were processed, and 25,156 SARS-CoV-2 diagnostics were performed from 9 May 2020 to 30 June 2020.

Analytical Sensitivity and Specificity of Two RT-qPCR Protocols for SARS-CoV-2 Detection Performed in an Automated Workflow.

WHO declared the novel coronavirus (COVID-19) outbreak a global pandemic on 11 March 2020. The establishment of standardized RT-qPCR protocols for respiratory secretions testing, as well as sharing of specimens, data, and information became critical. Here, we investigate the analytical performance of two interim RT-qPCR protocols (Charité and Centers for Disease Control (CDC)) for the qualitative detection of SARS-CoV-2 executed in a fully automated platform. Analytical specificity, PCR amplification efficiency, analytical sensitivity (limit of detection), and cross-reactivity were evaluated using contrived samples. The on-going accuracy was evaluated by retrospective analysis of our test results database (real clinical samples). N1, E, and a modified version of RdRP assays presented adequate analytical specificity, amplification efficiency, and analytical sensitivity using contrived samples. The three assays were applied to all individuals who requested the SARS-CoV-2 molecular test assay in our laboratory and it was observed that N1 gave more positive results than E, and E gave more positive results than RdRP (modified). The RdRP and E were removed from the test and its final version, based on N1 assay only, was applied to 30,699 Brazilian individuals (from 19 February 2020 to 8 May 2020). The aggregated test results available in the database were also presented.

Serum Has Higher Proportion of Janus Kinase 2 V617F Mutation Compared to Paired EDTA-Whole Blood Sample: A Model for Somatic Mutation Quantification Using qPCR and the 2-∆∆Cq Method.

Detection of the Janus Kinase-2 (JAK2) V617F mutation is a diagnostic criterion for myeloproliferative neoplasms, and high levels of mutant alleles are associated with worse outcomes. This mutation is usually tested on blood DNA by allele-specific qPCR (AS-qPCR) and measured using absolute quantification. However, some automated DNA extractions co-extracts of PCR inhibitors from blood and qPCR absolute quantification need increased efforts in order to maintain standard curves. JAK2 V617F can also be detected in serum using droplet digital PCR (ddPCR), a specimen with less inhibitors and favorable to automated extractions, but ddPCR instruments are not wide available as qPCR thermocyclers. Here, we evaluate whether JAK2 V617F could be accurately quantified by AS-qPCR using the 2-∆∆Cq method on blood DNA and validate the assay using gold-standard molecular diagnostic protocols. Next, we apply the validated method to assess if the mutation could be reliably detected/quantified in serum. JAK2 V617F could be quantified by AS-qPCR using the 2-∆∆Cq method-the assay was highly accurate (bias of 1.91%) compared to a commercial kit, highly precise (total CV% of 0.40%, 1.92%, 11.12% for samples with 93%, 54%, and 2.5% of mutant allele), highly sensitive (limit of detection of 0.15%), and demonstrated a linear detection response from 1.1% to 99.9%. Serum presented a higher mutant allele burden compared to the paired whole blood (mean of 4%), which allows for an increased JAK2 mutant detection rate and favors increased JAK2 V617F high-throughput analysis.

Genotyping of Single Nucleotide Polymorphisms Using Allele-Specific qPCR Producing Amplicons of Small Sizes Directly from Crude Serum Isolated from Capillary Blood by a Hand-Powered Paper Centrifuge.

The cell-free genomic DNA (gDNA) concentration in serum ranges from 1500 to 7500 copies/mL within 2 h after phlebotomy (6⁻24 times the concentration observed in plasma). Here, we aimed to evaluate the gDNA size distribution in serum with time after coagulation and to test if crude serum can be directly used as a source of gDNA for qPCR. Next, we investigated if single nucleotide polymorphisms (SNPs) could be genotyped directly from the crude serum isolated from capillary blood using a hand-powered paper centrifuge. All tested PCR targets (65, 100, 202 and 688 base pairs) could be successfully amplified from DNA extracted from serum, irrespective of their amplicon size. The observed qPCR quantitation cycles suggested that the genomic DNA yield increased in serum with incubation at room temperature. Additionally, only 65 and 101 base pair qPCR targets could be amplified from crude serum soon after the coagulation. Incubation for 4 days at room temperature was necessary for the amplification of PCR targets of 202 base pairs. The 688 base pair qPCR target could not be amplified from serum directly. Lastly, serum was successfully separated from capillary blood using the proposed paper centrifuge and the genotypes were assigned by testing the crude serum using allele-specific qPCR, producing small amplicon sizes in complete agreement with the genotypes assigned by testing the DNA extracted from whole blood. The serum can be used directly as the template in qPCR for SNP genotyping, especially if small amplicon sizes are applied. This shortcut in the SNP genotyping process could further molecular point-of-care diagnostics due to elimination of the DNA extraction step.

[From the molecular model to the impact on prognosis: an overview on acute promyelocytic leukemia]. / Do paradigma molecular ao impacto no prognóstico: uma visão da leucemia promielocítica aguda.

Acute promyelocytic leukemia (APL) is a model of clinical applicability of the knowledge of molecular physiopathology. It is characterized by recurrent genetic involvement of the retinoic acid alpha receptor. The consequence is a protein with low sensibility to its ligand and a myeloid maturation arrest. However, higher doses of all-trans-retinoic acid (ATRA) are able to supersede this deficiency and this is the mainstay of APL treatment leading to over 80% disease free survival, when adequately treated. Epidemiologically, it differs from other acute myeloid leukemia due to a higher incidence in young adults and in countries of "Latin" colonization. Differing from excellent results observed in developed countries, APL mortality in Brazil is still high, despite the wide availability of drugs.

Flow Cytometric Analysis of Erythrocytes Osmotic Fragility in Hereditary Spherocytosis: A Case-Controlled Study Evaluating the Best Anticoagulant, Sample Pre-Treatment and NaCl Concentration for Reliable Screening of this Red Blood Cell Membrane Disorder.

BACKGROUND: The cytometric flow osmotic fragility test (FC-OFT) was recently introduced. However, the test is still under development and some variables have not yet been fully tested. METHODS: The osmotic fragility of hereditary spherocytosis (HS) cases and healthy controls were evaluated by FC-OFT using a series of tubes containing decreasing concentrations of NaCl. The analyses were executed in fresh and incubated (37°C for 24 h) blood samples anticoagulated with EDTA and heparin. The percentages of residual red blood cells were used to plot the osmotic fragility curves. The OF curves of each tested condition were compared using the median corpuscular fragility (MCF). ROC curve analyses identified the most accurate NaCl concentrations for differentiation between HS cases and healthy controls. RESULTS: FC-OFT curves assumed a sigmoidal dose-response shape and the MCF of cases and controls were different in all instances. MCF comparisons revealed that incubation and anticoagulant have major and minor effects on the FC-OFT, respectively. One hundred percent of sensitivity and specificity was obtained from 5.5 to 6.0 g/L of NaCl in EDTA-treated fresh blood, from 6.0 to 8.0 g/L of NaCl in EDTA-treated incubated blood, and in none of the tested NaCl concentration in heparinized blood. CONCLUSIONS: EDTA is the anticoagulant of choice for the assay. Incubation at 37°C for 24 h increased its diagnostic capability. The most reliable NaCl concentration for the discrimination of HS case from controls was 6.0 g/L of NaCL in fresh EDTA-treated blood, and was 7.5 g/L of NaCl in incubated EDTA-treated blood. © 2018 International Clinical Cytometry Society.

Clinical features and outcomes of 134 Brazilians with acute promyelocytic leukemia who received ATRA and anthracyclines.

We report an increased incidence of high relapse risk features in 157 APL Brazilian patients. Out of 134 patients treated with ATRA and anthracyclines, only 91 (67.9%) achieved remission because 43 (32%) died during induction. The death rate during consolidation was 10.5%. Bleeding complications were the most frequent cause of failure (21.6%).

Acute myeloid leukemia (AML-M2) with t(5;11)(q35;q13) and normal expression of cyclin D1.

We report a case of acute myeloid leukemia (AML) subtype M2, with t(5;11)(q35;q13), in a 30-year-old man. Conventional cytogenetic, spectral karyotyping, and fluorescence in situ hybridization (FISH) studies on bone marrow sample obtained at diagnosis revealed an abnormal karyotype in all cells examined. FISH analysis demonstrated absence of translocations in the region of the cyclin D1 gene and real-time quantitative reverse transcriptase-polymerase chain reaction revealed normal expression of this gene. Similar to the 11q23 region, 11q13 changes can be found in both myeloid and lymphoid neoplasias with different chromosomes serving as donors in translocations.

Do paradigma molecular ao impacto no prognóstico: uma visão da leucemia promielocítica aguda: [revisão] / From the molecular model to the impact on prognosis: an overview on acute promyelocytic leukemia: [review]

A leucemia promielocítica aguda (LPA) é um modelo da aplicabilidade clínica dos conhecimentos moleculares fisiopatológicos. Caracteriza-se por alterações genéticas recorrentes que envolvem o gene do receptor alfa do ácido retinóico. A conseqüência é uma proteína com sensibilidade reduzida ao ligante, com bloqueio da diferenciação mielóide. Entretanto, doses suprafisiológicas do ácido all-trans-retinóico (ATRA) são capazes de suplantar esta deficiência, e este é o princípio fundamental do tratamento da LPA, permitindo uma sobrevida livre de doença acima de 80 por cento quando adequadamente tratada. Epidemiologicamente, difere dos demais subtipos de leucemia mielóide aguda por apresentar incidência predominante em adultos jovens e, aparentemente, maior incidência em países de colonização "latina". Contrastando com os excelentes resultados observados em países desenvolvidos, a mortalidade por LPA no Brasil ainda é alta, apesar da ampla disponibilidade das medicações no país.

Acute promyelocytic leukemia (APL) is a model of clinical applicability of the knowledge of molecular physiopathology. It is characterized by recurrent genetic involvement of the retinoic acid alpha receptor. The consequence is a protein with low sensibility to its ligand and a myeloid maturation arrest. However, higher doses of all-trans-retinoic acid (ATRA) are able to supersede this deficiency and this is the mainstay of APL treatment leading to over 80 percent disease free survival, when adequately treated. Epidemiologically, it differs from other acute myeloid leukemia due to a higher incidence in young adults and in countries of "Latin" colonization. Differing from excellent results observed in developed countries, APL mortality in Brazil is still high, despite the wide availability of drugs.