Routine Peripheral Blood Research Articles

Multi-drug algorithm to accurately predict best first-line treatments in newly-diagnosed acute myeloid leukemia (AML).

6525 Background: AML is a heterogeneous hematological malignancy with poor prognosis. Several treatments are approved for AML, but clinical trials have shown that current stratification approaches to determine patients’ eligibility produce false positives (treated patients that fail to respond) and negatives (patients not treated but could have responded). Venetoclax + azacitidine (VA) treatment is currently reserved for unfit patients, with younger patients stratified based on their FLT3status, and treated with either intensive chemotherapy (IC), or IC plus midostaurin (MIC). Here, we used phosphoproteomics to build a signature and algorithm that accurately predict which of these approved therapies may be more efficacious for a given patient. Methods: Routine bone marrow and peripheral blood diagnosis samples (s, n=182) were collected across the UK, Austria, Canada and the USA from 138 patients (p) subsequently treated with MIC (n=44/64 p/s), VA (n=40/48 p/s) or IC (n=54/70 p/s). Patients were grouped into Good Responders (GR) and Poor Responders (PR) based on treatment response. For VA, patients that achieved complete remission (CR) were considered GR, while refractory patients were considered PR. For MIC and IC, we considered patients that achieved CR without relapse within 6 months as GR, and those refractory or relapsed within 6 months as PR. Samples were processed for mass spectrometry-based phosphoproteomics. Phosphopeptide abundance data, generated with in-house PiQuant software, was used to identify phosphopeptides that distinguish GR and PR groups in each cohort. Statistical models based on these features were assessed via cross-validation. Results: We compared phosphoproteomes of 182 diagnosis samples from 138 AML patients, from three treatment cohorts (treated with IC, MIC or VA), with each cohort stratified by patients’ response to their respective treatment. Enrichment analysis in each cohort identified several phosphopeptides specific to one of the responder groups, mapping both to proteins with known roles in AML biology (e.g. DNMT3A or RUNX1) and proteins not yet implicated. Next, using machine learning, we identified phosphopeptides that could distinguish between PR and GR, and trained drug response prediction models based on the abundance of these phosphopeptides. In cross-validation, each model stratified patients with log rank p<0.001, HR<0.1 and more than 90% accuracy, greatly outperforming all currently-used stratification methods for first-line AML therapies. Conclusions: We built a suite of predictive models that accurately predict patient response to first-line AML treatment using phosphoproteomic data from routine diagnosis samples. Following validation in independent patient cohorts, this tool will be developed into a single test that predicts treatment response for AML patients, thus addressing an unmet clinical need in this disease.

Acute paroxysmal cold hemoglobinuria upon dengue fever: A case report

Rationale: Dengue fever is capable of inciting the formation of transient polyclonal antibodies directed at red blood cell antigens, resulting in complement-mediated hemolysis, leading to intravascular hemolysis and hemoglobinuria. Patient’s concern: A 12-year-old male patient who recovered from dengue fever a week ago had red blood cell agglutination, spherocytes, and engulfment of red blood cells (erythrophagocytosis) by monocytes and neutrophils on routine hematological peripheral blood smear. The unexpected blood smear results prompted the lab physicians to investigate autoimmune hemolytic anemia, which revealed a monospecific positive direct antiglobulin test for complement (C3d, C3b) and the presence of Donath-Landsteiner antibody. Diagnosis: Paroxysmal cold hemoglobinuria (PCH), secondary to dengue fever. Interventions: Oxygen supplements, antibiotics, intravenous immunoglobulins, steroid therapy, and packed cell transfusions were administered. Outcomes: The patient’s condition was improved following the therapy. Lessons: Post-dengue PCH is a rare complication that requires a thorough peripheral smear examination for erythrophagocytosis, as advanced hematology analyzers fail to detect such findings.

Characteristic changes in blood routine and peripheral blood lymphocyte subpopulations in recipients of different types of rejection.

Rejection remains the most important factor limiting the survival of transplanted kidneys. Although a pathological biopsy of the transplanted kidney is the gold standard for diagnosing rejection, its limitations prevent it from being used as a routine monitoring method. Recently, peripheral blood lymphocyte subpopulation testing has become an important means of assessing the body's immune system, however, its application value and strategy in the field of kidney transplantation need further exploration. Additionally, the development and utilization of routine test parameters are also important methods for exploring diagnostic strategies and predictive models for kidney transplant diseases. This study aims to explore the correlation between peripheral blood lymphocyte subpopulations and T cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR), as well as their diagnostic value, in conjunction with routine blood tests. A total of 154 kidney transplant recipients, who met the inclusion and exclusion criteria and were treated at the Second Xiangya Hospital of Central South University from January to December, 2021, were selected as the study subjects. They were assigned into a stable group, a TCMR group, and an ABMR group, based on the occurrence and type of rejection. The basic and clinical data of these recipients were retrospectively analyzed and compared among the 3 groups. The transplant kidney function, routine blood tests, and peripheral blood lymphocyte subpopulation data of the TCMR group and the ABMR group before rejection treatment were compared with those of the stable group. The stable, TCMR group, and ABMR group showed no statistically significant differences in immunosuppressive maintenance regimens or sources of transplanted kidneys (all P>0.05). However, the post-transplant duration was significantly longer in the ABMR group compared with the stable group (P<0.001) and the TCMR group (P<0.05). Regarding kidney function, serum creatinine levels in the ABMR group were higher than in the stable group and the TCMR group (both P<0.01), with the TCMR group also showing higher levels than the stable group (P<0.01). Both TCMR and ABMR groups had significantly higher blood urea nitrogen levels than the stable group (P<0.01), with no statistically significant difference between TCMR and ABMR groups (P>0.05). The estimated glomerular filtration rate (eGFR) was lower in both TCMR and ABMR groups compared with the stable group (both P<0.01). In routine blood tests, the ABMR group had lower hemoglobin, red blood cell count, and platelet count than the stable group (all P<0.05). The TCMR group had higher neutrophil percentage (P<0.05) and count (P<0.05) than the stable group, and the ABMR group had a higher neutrophil percentage than the stable group (P<0.05). The eosinophil percentage and count in the TCMR group were lower than in the stable and ABMR groups (all P<0.05). Both TCMR and ABMR groups had lower basophil percentage and count, as well as lower lymphocyte percentage and count, compared with the stable group (all P<0.05). There were no significant differences in monocyte percentage and count among the 3 groups (all P>0.05). In lymphocyte subpopulations, the TCMR and ABMR groups had lower counts of CD45+ cells and T cells compared with the stable group (all P<0.05). The TCMR group also had lower counts of CD4+ T cells, NK cells, and B cells than the stable group (all P<0.05). There were no significant differences in the T cell percentage, CD4+ T cell percentage, CD8+ T cell percentage and their counts, CD4+/CD8+ T cell ratio, NK cell percentage, and B cell percentage among the stable, TCMR, and ABMR groups (all P>0.05). The occurrence of rejection leads to impaired transplant kidney function, accompanied by characteristic changes in some parameters of routine blood tests and peripheral blood lymphocyte subpopulations in kidney transplant recipients. The different characteristics of changes in some parameters of routine blood tests and peripheral blood lymphocyte subpopulations during TCMR and ABMR may help predict and diagnose rejection and differentiate between TCMR and ABMR.

Deep learning-assisted smartphone-based quantitative microscopy for label-free peripheral blood smear analysis.

Hematologists evaluate alterations in blood cell enumeration and morphology to confirm peripheral blood smear findings through manual microscopic examination. However, routine peripheral blood smear analysis is both time-consuming and labor-intensive. Here, we propose using smartphone-based autofluorescence microscopy (Smart-AM) for imaging label-free blood smears at subcellular resolution with automatic hematological analysis. Smart-AM enables rapid and label-free visualization of morphological features of normal and abnormal blood cells (including leukocytes, erythrocytes, and thrombocytes). Moreover, assisted with deep-learning algorithms, this technique can automatically detect and classify different leukocytes with high accuracy, and transform the autofluorescence images into virtual Giemsa-stained images which show clear cellular features. The proposed technique is portable, cost-effective, and user-friendly, making it significant for broad point-of-care applications.

A novel and apparent de novo ALAS2 missense variant associated with congenital sideroblastic anemia.

Congenital sideroblastic anemia (CSA) constitutes a group of inherited erythropoietic disorders. Some affect mainly or exclusively erythroid cells; other syndromic forms occur within multisystem disorders with extensive nonhematopoietic manifestations. In this study, we have performed clinical and molecular investigations on a 10-year-old boy suspected of having CSA. Routine blood examination, peripheral blood and bone marrow smears, and serum iron tests were performed. Gene mutation analysis was conducted using whole-exome sequencing (WES) and the results were confirmed using Sanger sequencing. Furthermore, the functional impact of the identified variant was assessed/predicted with bioinformatics methods. The patient presented with severe microcytic anemia (hemoglobin, 50 g/L), iron overload and ring sideroblasts in the bone marrow. Moreover, WES revealed the presence of a hemizygous missense variant in ALAS2 (c.1102C > T), changing an encoded arginine to tryptophan (p. Arg368Trp). This variant was verified via Sanger sequencing, and neither of the parents carried this variant, which was suspected to be a de novo variant. Using in silico analysis with four different software programs, the variant was predicted to be harmful. PyMol and LigPlot software showed that the p. Arg368Trp variant may result in changes in hydrogen bonds. The patient was treated with vitamin B6 combined with deferasirox. After 6 months, the hemoglobin increased to 99 g/L and the serum ferritin decreased significantly. We report a novel pathogenic variant in the ALAS2 gene (c.1102C > T:p. Arg368Trp), which caused CSA in a 10-year-old boy. Mutational analysis is important in patients with CSA when family history data are unavailable. Anemia due to the ALAS2 Arg368Trp variant responds to pyridoxine supplements.

Umbilical Cord Blood-Derived Cells Can Reconstruct Hematopoiesis in an Aplastic Anemia Animal Model.

To explore the efficacy and the mechanism of the umbilical cord-derived cells combined with cyclosporine A (CsA) in treating aplastic anemia (AA) in mice. Immune-mediated AA model mice were treated with CsA + UC mesenchymal stem cells (UC-MSC), CsA + umbilical cord blood regulatory T cells (UCB-Treg), UC-MSC, UCB-Treg, CsA alone, or blank control, respectively (n = 9 mice/group). CsA and the cell infusion was administered on d0. Routine peripheral blood testing was performed once weekly; bone marrow colony culture, bone marrow cell flow cytometry, peripheral blood T cell subsets, and serum inflammatory cytokines tests were performed on d14. Transcriptome sequencing was performed for cells from CsA + UC-MSC, CsA + UCB-Treg, and CsA groups to detect the possible related genes. Gene function cluster and signal pathway enrichment analysis were also performed. Blank control mice died due to pancytopenia within 21 days, whereas mice in other groups survived for >28 days. On d14, the CsA + UC-MSC and CsA + UCB-Treg groups had higher white blood cell (WBC) counts than the other groups (p < 0.05), along with higher burst-forming unit (BFU) and colony-forming unit-granulocyte, macrophage (CFU-GM) counts (p < 0.01). The CsA + UC-MSC group had the highest BFU count (p < 0.01). The CsA + UC-MSC and CsA + UCB-Treg groups exhibited the highest bone marrow CD34+ cell proportion (9.68% ± 1.35% and 8.17% ± 0.53%, respectively; p < 0.01). Tumor necrosis factor (TNF)-α and interleukin (IL)-2 levels in the CsA + UC-MSC group (p < 0.05) and TNF-α, interleukin-2, and interferon (INF)-γ levels in the CsA + UC-Treg group (p < 0.01) were lower than those in the CsA group. Compared with CsA treatment, CsA + UC-MSC significantly downregulated the histone methylation pathway (p < 0.05), whereas CsA + UCB-Treg significantly upregulated energy metabolism processes (p < 0.05). Treatment with CsA + UC-MSC upregulated superoxide dismutase activity compared with CsA + UCB-Treg treatment. Adding UC-MSC or UCB-Treg to CsA markedly enhanced the reconstruction of hematopoiesis in AA mice, with UC-MSC eliciting greater efficiency than UCB-Treg. Accordingly, the addition of these cells could further improve immune abnormalities.

Analysis of lymphocyte subsets in patients with sepsis and its impact on prognosis

To explore the characteristics of changes in peripheral blood lymphocyte subsets in patients with sepsis in intensive care unit (ICU) and analyze their predictive value for prognosis. The clinical data of sepsis patients admitted to the surgical intensive care unit (SICU) of the First Affiliated Hospital of Zhengzhou University from January 2020 to December 2021 were analyzed retrospectively. The patients met the diagnostic criteria of Sepsis-3 and were ≥ 18 years old. Peripheral venous blood samples were collected from all patients on the next morning after admission to SICU for routine blood test and peripheral blood lymphocyte subsets. According to the 28-day survival, the patients were divided into two groups, and the differences in immune indexes between the two groups were compared. Logistic regression analysis was used to analyze the risk factors of immune indexes that affect prognosis. (1) A total of 279 patients with sepsis were enrolled in the experiment, of which 198 patients survived at 28 days (28-day survival rate 71.0%), and 81 patients died (28-day mortality 29.0%). There were no significant differences in age (years old: 57.81±1.71 vs. 54.99±1.05) and gender (male: 60.5% vs. 63.6%) between the death group and the survival group (both P > 0.05), and the baseline data was comparable.(2) Acute physiology and chronic health evalution II (APACHE II: 22.06±0.08 vs. 14.08±0.52, P < 0.001), neutrophil percentage [NEU%: (88.90±1.09)% vs. (84.12±0.77)%, P = 0.001], procalcitonin [PCT (μg/L): 11.97±2.73 vs. 5.76±1.08, P = 0.011], platelet distribution width (fL: 16.81±0.10 vs. 16.57±0.06, P = 0.029) were higher than those in the survival group, while lymphocyte percentage [LYM%: (6.98±0.78)% vs. (10.59±0.86)%, P = 0.012], lymphocyte count [LYM (×109/L): 0.70±0.06 vs. 0.98±0.49, P = 0.002], and platelet count [PLT (×109/L): 151.38±13.96 vs. 205.80±9.38, P = 0.002], and thrombocytocrit [(0.15±0.01)% vs. (0.19±0.07)%, P = 0.012] were lower than those in the survival group. (3) There was no statistically significant difference in the percentage of lymphocyte subsets between the death group and the survival group, but the absolute value of LYM (pieces/μL: 650.24±84.67 vs. 876.64±38.02, P = 0.005), CD3+ absolute value (pieces/μL: 445.30±57.33 vs. 606.84±29.25, P = 0.006), CD3+CD4+ absolute value (pieces/μL: 239.97±26.96 vs. 353.49±18.59, P = 0.001), CD19+ absolute value (pieces/μL: 111.10±18.66 vs. 150.30±10.15, P = 0.049) in the death group was lower than those in the survival group. Other lymphocyte subsets in the death group, such as CD3+CD8+ absolute value (pieces/μL: 172.40±24.34 vs. 211.22±11.95, P = 0.112), absolute value of natural killer cell [NK (pieces/μL): 101.26±18.15 vs. 114.72±7.64, P = 0.420], absolute value of natural killer T cell [NKT (pieces/μL): 33.22±5.13 vs. 39.43±2.85, P = 0.262], CD4-CD8- absolute value (pieces/μL: 41.07±11.07 vs. 48.84±3.31, P = 0.510), CD4+CD8+ absolute value (pieces/μL: 3.39±1.45 vs. 3.47±0.36, P = 0.943) were not significantly different from those in the survival group. (4)Logistic regression analysis showed that lymphocyte subsets were not selected as immune markers with statistical significance for the prognosis of sepsis. The changes of immune indexes in sepsis patients are closely related to their prognosis. Early monitoring of the above indexes can accurately evaluate the condition and prognosis of sepsis patients.

Comparison of Clinical Characteristics and Peripheral Blood Tests of COVID-19 and Influenza B Patients.

This study aimed to investigate the differences in clinical features and routine peripheral blood testing between coronavirus disease 2019 (COVID-19) infection and influenza B infection. Patients with COVID-19 and influenza B admitted to our fever clinic from January 1, 2022 to June 30, 2022 were recruited. A total of 607 patients were included (301 with COVID-19 infection and 306 with influenza B infection). The results of a statistical analysis showed that 1) patients with COVID-19 infection were older and had a lower temperature and shorter duration from fever onset to clinic visit than patients with influenza B infection; 2) apart from fever, viral infection symptoms appeared to be more common in patients with influenza B infection than in patients with COVID-19 infection, including sore throat, cough, muscle aches, weeping, headache, fatigue, and diarrhea (P < 0.001); and 3) compared with patients with influenza B infection, patients with COVID-19 infection had higher numbers of white blood cells and neutrophils but lower numbers of red blood cells and lymphocytes (P < 0.001). In summary, several important differences were identified between COVID-19 and influenza B, which may help to guide clinicians in their initial diagnosis of these two respiratory viral infections.

Abstract CT101: Phase I study of the T-cell receptor-like antibody Hu8F4 in patients with advanced hematologic malignancies

Abstract Background: Despite recent advances in the treatment of AML, most approaches are rarely curative and most patients (pts) succumb to relapsed disease. The effectiveness of stem cell transplant and associated graft vs. leukemia effect implies an important role for immune-based therapy in producing long lasting remissions. Traditional approaches using immunotherapy have failed to establish a suitable surface target or treatment paradigm that is effective in myeloid malignancies. Hu8F4 is a humanized T-cell receptor-like monoclonal antibody that binds to the conformational epitope of PR1 bound to HLA-A2, which is highly, differentially expressed on the surface of AML compared to normal progenitors. Methods: We conducted a first in human, phase I dose escalation trial of Hu8F4 in pts with myeloid malignances. Pts with R/R AML, MDS, CMML, and myeloid blast phase of CML with adequate organ function and PS ≤ 2 were eligible. Pts were treated on 7 escalating dose levels, ranging from 0.01 mg/kg to 10 mg/kg IV on D1 &amp; 15. Initial dose levels required 1 pt per dose (0.01, 0.03, 0.1, 0.3, 1), followed by 3 pts per dose (3, 10). Results: 10 pts with R/R AML have been enrolled, with a median age of 65 years (range, 23-77), including 6 females (60%). Pts had received a median of 4 (1-4) prior therapies; 5 pts (50%) had a PS of 2. At enrollment, the median WBC was 1.9 (0.1 - 18.4), median BM blasts were 32% (8 - 76); 9 (90%) pts had complex karyotype and 3 (30%) had a TP53 mutation. All pts had &amp;gt; 98% surface expression of PR1 on the myeloid blasts. Hu8F4 Cmax ranged up to 160,000 ng/mL with t1/2 of 48 hours and clearance of 2.61 hr*ng/mL at the highest dose. Weak anti-drug antibodies were observed after week 4 in 2 of 3 pts treated at 3 mg/kg. With a median follow up of 3.5 months (1.1 - 9.9), pts have received a median of 1 (1-4) cycle of therapy. Two pts had decline in BM blasts and 4 had stable disease. Routine peripheral blood testing revealed sharp decline in peripheral blasts immediately after infusion of Hu8F4 on D1 and 15 with associated elevation in serum LDH in some pts and a rise in normal granulocytes, consistent with on-tumor effects. The pharmacokinetic parameters and transient blast reduction indicated a possible sink effect mediated by high levels of circulating blasts. SAEs documented on study were mostly disease-related and included infections, cytopenias, hemoptysis, pneumonia, and GI bleeding. Treatment related AEs were temporally related to the infusion included hypotension (Grade 2: N=2), rigors (Grade 2: N=2; Grade 1: N=1). All infusion reactions were observed at dose levels of 3 and 10 mg/kg, but were transient, and managed with steroids and antihistamines. All pts proceeded with their next dose without further issues. No cytokine release syndrome or neurologic toxicity was observed. Correlative studies support antibody dependent cellular cytotoxicity and phagocytosis as important mechanisms of anit-AML activity. Conclusion: Hu8F4 was well tolerated with no dose-limiting toxicities observed at the maximum planned dose. On-target peripheral blast reduction temporally related to infusion suggests biological activity. Real-time pharmacokinetic data on study indicate a possible sink effect that may be overcome by a more frequent dosing strategy. Citation Format: Tapan M. Kadia, Hagop Kantarjian, Gheath Alatrash, Anna Sergeeva, Hong He, Lisa St. John, Priya Koppikar, Celine Kerros, Abhishek Maiti, Courtney Dinardo, Elias Jabbour, Serge Vesrstovsek, Naveen Pemmaraju, Nitin Jain, Ghayas Issa, Guillermo Montalban-Bravo, Aditi Shastri, Daniel Couriel, Rhona Pinsoy, Sapna Parshottam, Richard Champlin, Jorge Cortes, Jeffrey Molldrem. Phase I study of the T-cell receptor-like antibody Hu8F4 in patients with advanced hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT101.

Association of peripheral blood neutrophil-lymphocyte ratio (NLR) and red cell distribution width (RDW) with immune checkpoint inhibitors (ICIs) for metastatic urothelial carcinoma (mUC).

540 Background: Determining a benefit from ICIs radiographically may take months and may be confounded by pseudoprogression. The NLR &amp; RDW are readily accessible inflammatory markers from routine peripheral blood analysis and have been associated with outcomes in malignancies. We evaluated the association of early changes in NLR and RDW within 4 weeks after initiating ICI with any regression of tumor (ART) and overall survival (OS) in mUC. Methods: This study included de-identified data from Dana-Farber Cancer Institute in pts with mUC who were treated with ICIs from 2015 to 2020. Age, gender, setting (untreated vs. post-platinum), sites of metastasis (mets), performance status (PS), platelet count, RDW, and NLR at baseline and 3-4 weeks after initiating the ICI were collected. The primary objective was to assess the association of early increase in NLR by ≥1.0 from baseline or RDW at 3-4 weeks (as a continuous variable) post-ICI therapy with ART and OS. ART was assessed via subsequent imaging at any time point while on ICI. A multivariable logistic regression model and Cox proportional-hazards model were employed to identify the association of NLR changes with ART and OS, respectively, using backward selection. Results: A total of 330 pts were evaluable. The median age was 70 years, 72.1% (n = 298) were male and 69.4% (n= 227) received post-platinum ICI (remaining were platinum naïve). 46.5% (n = 146) experienced ART and the median OS was 13 months (11.14-16.14). An increase in NLR ≥ 1 in 3-4 weeks was associated with a lower rate of ART (OR: 0.41, 95% CI 0.24-0.70; p: 0.001) and worse OS (hazard ratio [HR]: 1.84, 95% CI 1.37-2.46; p &lt; 0.0001) on multivariable analysis (Table). Higher RDW values at 3-4 weeks were associated with worse OS (HR: 1.08, 95% CI 1.03-1.14; p: 0.0004) but were not associated with ART. Conclusions: In pts with mUC initiating ICI, an early increase in NLR ≥ 1 and higher RDW in 3-4 weeks were associated with poor outcomes. Early changes in these readily available variables may enable early modification or intensification of therapy to improve outcomes. External validation of these findings is warranted. [Table: see text]

Five Years’ Experience with Gene Panel Sequencing in Hereditary Hemolytic Anemia Screened by Routine Peripheral Blood Smear Examination

Hereditary hemolytic anemia (HHA) is defined as a group of heterogeneous and rare diseases caused by defects of red blood cell (RBC) metabolism and RBC membrane, which leads to lysis or premature clearance. The aim of this study was to investigate individuals with HHA for potential disease-causing variants in 33 genes reported to be associated with HHA. A total of 14 independent individuals or families diagnosed with suspected HHA, and in particular, RBC membranopathy, RBC enzymopathy, and hemoglobinopathy, were collected after routine peripheral blood smear testing. A custom designed panel, including the 33 genes, was performed using gene panel sequencing on the Ion Torrent PGM™ Dx System. The best candidate disease-causing variants were confirmed by Sanger sequencing. Several variants of the HHA-associated genes were detected in 10 out of 14 suspected HHA individuals. After excluding those variants predicted to be benign, 10 pathogenic variants and 1 variant of uncertain significance (VUS) were confirmed in 10 individuals with suspected HHA. Of these variants, the p.Trp704Ter nonsense variant of EPB41 and missense p.Gly151Asp variant of SPTA1 were identified in two out of four hereditary elliptocytoses. The frameshift p.Leu884GlyfsTer27 variant of ANK1, nonsense p.Trp652Ter variant of the SPTB, and missense p.Arg490Trp variant of PKLR were detected in all four hereditary spherocytosis cases. Missense p.Glu27Lys, nonsense p.Lys18Ter variants, and splicing errors such as c.92 + 1G > T and c.315 + 1G > A within HBB were identified in four beta thalassemia cases. This study provides a snapshot of the genetic alterations in a cohort of Korean HHA individuals and demonstrates the clinical utility of using gene panels in HHA. Genetic results can provide precise clinical diagnosis and guidance regarding medical treatment and management for some individuals.

Determination of Platelet Count Estimation Factor on Peripheral Blood Smear Confirmation Using Field Number 22 Microscope

The automatic platelet count sometimes requires confirmation on the peripheral blood smear. Platelet count estimation can also be used for reporting platelet count if an automatic cell counter is not available, with an estimation factor according to the Field Number (FN) of the microscope used. This study aimed to determine the platelet count estimation factor based on peripheral blood smear confirmation using an FN 22 microscope. An observational cross-sectional study was carried out in patients who had routine hematological and peripheral blood smear examinations during September 2021 by determination of platelet count using the automatic cell counter method and an average number of platelet counts per field of view with 100x objective magnification. The estimation factor is the total ratio divided by sample size. The total ratio of 254 samples was 4.086. The platelet count estimation factor was 16, indicating that 1 platelet per field of view was equivalent to 16x103/µL. There was a very strong significant correlation between mean platelet count per field of view and platelet count using the automatic cell counter (p&lt;0.001, R&gt;0.750). The field number is the image diameter of the microscope eyepiece. The latest generations of microscope use FN 20 or more, which provides a wider field of view, enabling the observation of more platelets. Factor estimation was used to determine the estimated platelet count on a peripheral blood smear. A big difference between automatic cell counter and peripheral blood smear might indicate pre-analytic, analytic, and post-analytic errors. The platelet count estimation factor based on peripheral blood smear confirmation using the FN 22 microscope was 16. Each laboratory needs to determine the estimation factor according to the FN microscope used.

Abrocitinib Attenuates Microglia-Mediated Neuroinflammation after Traumatic Brain Injury via Inhibiting the JAK1/STAT1/NF-κB Pathway.

Neuroinflammation has been shown to play a critical role in secondary craniocerebral injury, leading to poor outcomes for TBI patients. Abrocitinib, a Janus kinase1 (JAK1) selective inhibitor approved to treat atopic dermatitis (AD) by the Food and Drug Administration (FDA), possesses a novel anti-inflammatory effect. In this study, we investigated whether abrocitinib could ameliorate neuroinflammation and exert a neuroprotective effect in traumatic brain injury (TBI) models. First, next-generation sequencing (NGS) was used to select genes closely related to neuroinflammation after TBI. Then, magnetic resonance imaging (MRI) was used to dynamically observe the changes in traumatic focus on the 1st, 3rd, and 7th days after the induction of fluid percussion injury (FPI). Moreover, abrocitinib's effects on neurobehaviors were evaluated. A routine peripheral blood test was carried out and Evans blue dye extravasation, cerebral cortical blood flow, the levels of inflammatory cytokines, and changes in the numbers of inflammatory cells were evaluated to investigate the function of abrocitinib on the 1st day post-injury. Furthermore, the JAK1/signal transducer and activator of transcription1 (STAT1)/nuclear factor kappa (NF-κB) pathway was assessed. In vivo, abrocitinib treatment was found to shrink the trauma lesions. Compared to the TBI group, the abrocitinib treatment group showed better neurological function, less blood-brain barrier (BBB) leakage, improved intracranial blood flow, relieved inflammatory cell infiltration, and reduced levels of inflammatory cytokines. In vitro, abrocitinib treatment was shown to reduce the pro-inflammatory M1 microglia phenotype and shift microglial polarization toward the anti-inflammatory M2 phenotype. The WB and IHC results showed that abrocitinib played a neuroprotective role by restraining JAK1/STAT1/NF-κB levels after TBI. Collectively, abrocitinib treatment after TBI is accompanied by improvements in neurological function consistent with radiological, histopathological, and biochemical changes. Therefore, abrocitinib can indeed reduce excessive neuroinflammation by restraining the JAK1/STAT1/NF-κB pathway.

Elevated high-sensitivity C-reactive protein and the risk for cardiovascular events in chronic cardiac disease

Abstract Background High sensitivity C-reactive protein (hs-CRP) is a biomarker used for risk prediction for cardiovascular disease by assessing low concentration of inflammation. This study aims to assess the event-free time for the composite outcome between patients of different hs-CRP risk groups and the possible predictive value of hs-CRP for event occurrence in patients with chronic cardiac disease. Methods Data from 607 consecutive patients referred for cardiovascular risk assessment with hs-CRP from November 2017 to October 2018 were reviewed retrospectively. Routine peripheral venous blood samples were taken on the day of study inclusion and sent to the local laboratory, where laboratory parameters were analyzed and processed in accordance with local laboratory standards. 570 patients who had hs-CRP measurement by immunoturbidimetric assay were included in the analysis and classified into three (low-, medium- and high-risk) groups (hs-CRP cut-off: &amp;lt;1, 1–3, &amp;gt;3 mg/L). Association between hs-CRP and occurrence of the composite outcome (acute myocardial infarction, stroke, coronary intervention (percutaneous coronary intervention or bypass surgery) or death) was determined with Cox regression analysis and visualized with Kaplan Meier curves. Results In total, 570 patients from our cardiology outpatient clinic were included in this study. Cohorts were formed according to hs-CRP risk groups, 209 (36.7%), 226 (39.6%) and 135 (23.7%) patients were classified as low-, medium- and high-risk, respectively. The composite endpoint occurred in 93 (19.1%) of the 486 patients with available follow-up. Events occurred in 29 (16.3%), 30 (15.7%), 34 (29.1%) patients of the low-, medium- and high-risk group, respectively (p=0.016). There was a significant difference in the event-free survival time patients of the low- and medium-risk groups compared with patients in the high-risk group (p=0.015). The difference between groups is shown by the Kaplan-Meier plot (log rank test, p=0.01) (Figure 1). Univariate Cox proportional-hazard analysis identified age, hs-CRP risk group, hypertension, diabetes, hyperlipidemia, coronary artery disease, peripheral artery disease, cerebrovascular disease log(NT-proBNP) and creatinine as significant predictors for the primary study outcome. In multivariable analysis coronary artery disease and age were found to be highly significant predictors for the occurrence of an composite event during follow-up, while patients categorized in the low- and medium-risk groups appeared to predict a lower likelihood for events (Table 1). Conclusions Cardiovascular events were more likely to occur in patients who were older, with hs-CRP &amp;gt;3 mg/L and a history of coronary artery disease. However, assessment of inflammation markers alone may play a secondary role compared to other established cardiovascular risk factors, elevated CRP appears helpful to detect higher risk and in prediction of further cardiovascular events and mortality. Funding Acknowledgement Type of funding sources: None.

Evaluation of lymphocyte count, T-cell subsets and neutrophil-to-lymphocyte ratio as early predictors for severity and outcome of COVID-19 disease–a report from a highly complex hospital in Brazil

IntroductionLymphopenia is a laboratory marker of poor prognosis and severity of disease in the SARS-CoV-2 infection. This study aims to describe the immune profile of a Brazilian population. MethodsA total of 121 consecutive patients with severe acute respiratory syndrome (SARS) were analyzed between April and June 2020. Routine peripheral blood counts and multiparametric flow cytometry were performed on admission to assess lymphocytes and subsets (CD3, CD4, CD8). Demographic, clinical and laboratory data were collected from hospital sources. ResultsThe total of 116 patients included 63 (54.3%) males; 76 (62.8%) COVID-19 patients were divided, based on clinical characteristics and mechanical ventilation (MV) use, into moderate (n = 41; no MV) and severe (n = 35; MV) groups. The control group (n = 40) was comprised of patients with SARS of different etiologies. All patients had lymphopenia, with overall lymphocyte counts and their subsets considerably lower in severe patients, when compared to the moderate and controls. Patients with a high neutrophil-to-lymphocyte ratio (> 15.2) and T-cell lymphopenia (CD3 < 593 cells/µL, CD4 < 326 cells/µL, CD8 < 121 cells/µL) had a higher risk of being intubated and progressing to death. A total of 39 patients (95.1%) in the moderate group and 54.3% (n = 19) in the severe group were discharged; 28 patients died. ConclusionLaboratory assessment of the neutrophil/lymphocyte ratio and T-cell subsets may be predictive of mortality and may be useful for stratifying COVID-19 patients.

Performance evaluation and validation of automated digital image analysis in peripheral blood cells morphology examination

Objective: To verify and evaluate the performance of automated digital image(DIA) for peripheral blood cell morphology examination. Methods: Three hundred and seventy-nine routine peripheral blood smears and 18 plasmodium positive peripheral blood smears were collected. Blood smears were made and stained by Wright -Giemsa method.White blood cell (WBC) differentiation of blood smears were pre-classified by DIA (DIA direct classification), re-classified (manually reviewed after DIA classification), and artificially classified under microscope. the inter-assay and intra-assay coefficients of variation (CV) of DIA were respectively calculated for repeatability verification. Taking the artificial microscopy as the gold standard, the sensitivity、specificity and accuracy of DIA were calculated. The DIA ability of peripheral blood blast cell morphological count, platelet (PLT) morphological count and morphological examination of plasmodium were also verified. Results: Except for eosinophils and basophils, the inter-assay and intra-assay CV of WBC classification by DIA in normal samples were < 10%. The CV of WBC classification in abnormal samples increased with the decrease of cell percentage. The sensitivity, specificity and accuracy of DIA pre-classification were 90.5%, 99.2%, 98.2%. Through pre-classification and re-classification by DIA,the results of the blood smears which triggered blast cell alarm had a good correlation with manual classification(r=0.812, 0.983, both P<0.01). The PLT morphological count by DIA had high correlation with hematology analyzer (r=0.946, P<0.01). The deviation absolute value of two methods of PLT count was < 15%, while in PLT aggregation or giant thrombocytosis samples,the deviation absolute value of PLT count by two methods was > 15%. After image acquisition by DIA, 17 plasmodium trophozoites were detected in 18 plasmodium-positive peripheral blood smears, and the images were clear. Conclusions: The DIA system has good repeatability, high sensitivity, specificity and accuracy in peripheral blood WBC classification. Its pre-classification and re-classification results have high correlation with the manual classification results.

Neutrophil Lymphocyte Ratio, Monocyte Lymphocyte Ratio, Platelet Lymphocyte Ratio in Covid-19 Patients.

Covid-19 is a pandemic viral infection with high pathogenicity and contagiousness. Our aim is to evaluate the preliminary hematological findings analyzed during admission in order to determine the diagnostic value of hematological parameters in Covid-19 patients and to reveal their relationship with the severity of the disease. Our study includes a total of 169 patients, whose diagnosis was confirmed and 93 of whom were treated in the ward, 76 of whom were treated in the Intensive Care Unit (ICU), and 67 control patients. Neutrophil-to-lymphocyte ratio (NLR), monocyte-lymphocyte (MLR) ratio, platelet-lymphocyte ratio (PLR), mean platelet volume/platelet count ratio (MPV/PLT) data on admission were analyzed retrospectively and compared. ICU patients had significantly higher values of NLR, MLR, PLR, and MPV/PLT (p < 0.001 for each) but had lower values of lymphocyte count and hemoglobin (p < 0.001 for each) compared to that of ward patients. According to the results of ROC analysis, the diagnostic values of NLR, MLR, PLR, and MPV/PLT parameters were statistically significant (p < 0.05). According to the results of our study, abnormal routine peripheral blood examination results were detected in Covid-19 patients. NLR, MLR, and PLR can be considered as independent, reliable biomarkers for assessing disease severity, hospitalization, and clinical classification in Covid-19. Therefore, it was concluded that fast, cost-effective, easily accessible admission hemogram parameters are reasonably important to predict the prognosis of Covid-19 patients.

Study on plasma Golgi protein 73 and related models in the diagnosis of nonalcoholic fatty liver disease

Objective: To investigate the relationship between plasma Golgi protein 73 (GP73) levels and the occurrence and development of non-alcoholic fatty liver disease (NAFLD), and to establish a diagnostic model based on this combination with lipid metabolism indicators to clarify its diagnostic efficacy and clinical application value for NAFLD. Methods: 225 cases with NAFLD [diagnosed by ultrasound, transient elastography (FibroScan502) and liver biopsy (some patients)] and 108 healthy controls were selected from the Department of Hepatology and Physical Examination Center of Integrated Traditional Chinese and Western Medicine, The Third Hospital of Hebei Medical University. Clinical data, routine peripheral blood and serum biochemical test results were collected. The plasma GP73 level was detected by enzyme-linked immunosorbent assay. SPSS 21.0 statistical software was used for statistical analysis. Binary logistic regression model was used to calculate the NAFLD diagnostic model. Receiver operating characteristic curve was used to evaluate the NAFLD constructed model diagnostic efficacy. Results: NAFLD incidence was significantly reduced in younger age group, mostly in young and middle-aged male. However, the NAFLD incidence was increased with increasing age in female. The analysis of age ratio composition showed that the average age for NAFLD onset was 20 ~ 50 years old, and the incidence rate was as high as 47% in among 30 ~ 39 years old, but the incidence rate was significantly decreased in over 60 years old (4.00%). GP73 was an independent risk factor for the occurrence and development of NAFLD. The diagnostic models of GBT, GB and GT were established by GP73 (G) combined with body mass index (BMI, B) and serum triglyceride (TG, T), and the results showed that the areas under the curves of GBT, GB and GT models were 0.969, 0.937 and 0.909, respectively. The sensitivity and the specificity were 84.90%, 77.80% and 84.00%, and 95.40%, 95.40% and 82.40%, respectively, P < 0.05. The GBT model had efficacy of best diagnostic performance. Conclusion: NAFLD is more common in young and middle-aged male, but with advanced age, the incidence of female patients gradually increases. Plasma GP73 levels are related to the occurrence and development of NAFLD. The GBT model can be used as a new model for non-invasive diagnosis and one of the indicators for clinical evaluation of diagnostic efficacy of NAFLD.

Jaundice in a Preterm Infant Twelve Hours After Birth.

Jaundice in a Preterm Infant Twelve Hours After Birth.

Utilisation of Deep Learning-Enabled Image Analysis for Detection and Enumeration of Plasmodial Forms in Red Cells

Abstract Introduction/Objective Objective: This work investigates utilisation of deep learning enabled peripheral blood smear image analysis for automated detection and enumeration of red cell parasites. Methods/Case Report Methods: Peripheral blood smear red cell images from 30 individuals identified as positive and/or negative for plasmodilum falciparum forms were used. Blood submitted to hematology laboratory for complete blood count was evaluated on Sysmex XN 3100 analyzer with routine peripheral blood slides performed on instrument associated SP50 stainer. Images of red cells obtained with Xfinity DX40 microscope mounted camera were subjected to classification using deep learning software (Cognex, ViDi Suite 4.1). The training of the classification tool was performed on 200 peripheral blood smear images divided in two dataset classes: normal/negative and abnormal/positive for plasmodia, 50% of training images represented positive data set. Performance of the developed model was tested on 300 images including 66% positive for plasmodia obtained from 20 patients. Enumeration of parasitic forms was performed for each case. Model performance was compared to expert hematopathology reviewer which was used as gold standard. Results (if a Case Study enter NA) Results: Overall, Cognex ViDi Suite 4.1 demonstrates the effectiveness in discriminating between images positive and negative for red cell plasmodial forms as well as enables parasite quantification. Following performance specifications were determined for parasite detection: sensitivity (0.969230769), specificity (0.99383217). High correlation coefficients (0.9961) between automatically detected parasites and ground truth, on both image level and patient level, demonstrate the practicality of our method. Conclusion Deep learning enabled image analysis of peripheral blood smears is a promising alternative to manual identification and enumeration of red cell plasmodial forms with performance comparable to expert hematopathology reviewer.