Development and Evaluation of a Set of Spike and Receptor Binding Domain-Based Enzyme-Linked Immunosorbent Assays for SARS-CoV-2 Serological Testing.

The implementation and validation of anti-SARS-CoV-2 IgG serological assays are reported in this paper. S1 and RBD proteins were used to coat ELISA plates, and several secondary antibodies served as reporters. The assays were initially validated with 50 RT-PCR positive COVID-19 sera, which showed high IgG titers of mainly IgG1 isotype, followed by IgG3. Low or no IgG2 and IgG4 titers were detected. Then, the RBD/IgG assay was further validated with 887 serum samples from RT-PCR positive COVID-19 individuals collected at different times, including 7, 14, 21, and 40 days after the onset of symptoms. Most of the sera were IgG positive at day 40, with seroconversion happening after 14-21 days. A third party conducted an additional performance test of the RBD/IgG assay with 406 sera, including 149 RT-PCR positive COVID-19 samples, 229 RT-PCR negative COVID-19 individuals, and 28 sera from individuals with other viral infections not related to SARS-CoV-2. The sensitivity of the assay was 99.33%, with a specificity of 97.82%. All the sera collected from individuals with infectious diseases other than COVID-19 were negative. Given the robustness of this RBD/IgG assay, it received approval from the sanitary authority in Mexico (COFEPRIS) for production and commercialization under the name UDISTEST-V2G®.

Poor Survival in COVID-19 Associated with Lymphopenia and Higher Neutrophile-Lymphocyte Ratio.

BACKGROUND: Immune cell counts in blood in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection may be useful prognostic biomarkers of disease severity, mortality, and response to treatment. OBJECTIVES: To analyze sub-populations of lymphocytes at hospital admission in survivors and deceased from severe pneumonia due to coronavirus disease-2019 (COVID-19). METHODS: We conducted a cross-sectional study of healthcare workers confirmed with SARS-CoV-2 in convalescents (control group) and healthy controls (HC) diagnosed with severe COVID-19. Serum samples were taken at hospital admission and after recovery. Serum samples ≥ 25 days after onset of symptoms were analyzed for lymphocyte subpopulations through flow cytometry. Descriptive statistics, Kruskall-Wallis test, receiver operating characteristic curve, calculation of sensitivity, specificity, predictive values, and Kaplan-Meier analysis were performed. RESULTS: We included 337 patients: 120 HC, 127 convalescents, and 90 severe COVID-19 disease patients (50 survivors, 40 deceased). For T cells, total lymphocytes ≥ 800/µL, CD3+ ≥ 400/µL, CD4+ ≥ 180/µL, CD8+ ≥ 150/µL, B cells CD19+ ≥ 80/µL, and NK ≥ 34/µL subsets were associated with survival in severe COVID-19 disease patients. All subtypes of lymphocytes had higher concentrations in survivors than deceased, but similar between HC and convalescents. Leukocytes ≥ 10.150/µL or neutrophils ≥ 10,000/µL were associated with increased mortality. The neutrophil-to-lymphocyte ratio (NLR) ≥ 8.5 increased the probability of death in severe COVID-19 (odds ratio 11.68). CONCLUSIONS: Total lymphocytes; NLR; and levels of CD3+, CD4+, CD8+, and NK cells are useful as biomarkers of survival or mortality in severe COVID-19 disease and commonly reach normal levels in convalescents.

Important decrease in invariant natural killer T, CD4+ regulatory T cells, CD8+ regulatory T cells, gamma-delta T cells, and CD4+ T lymphocytes in HIV-negative patients with hemophilia.

Hereditary hemophilias are X-linked inherited bleeding disorders defined as deficiencies of the coagulation factors VIII or IX. They are characterized by easy to provoke or spontaneous bleeding. HIV infection in hemophilic patients is a risk factor for the reduction of CD4+ T cells. There is no information regarding the cellular immune function in HIV-negative patients with hemophilia. To evaluate the number of lymphocyte subsets in adult patients with hemophilia A or B as compared with healthy donors. 39 Adult hemophilics and 27 healthy donors were included. Lymphocyte subsets [CD4 and CD8 T cells, natural killer cells, natural killer T (NKT) cells, invariant NKT (iNKT) cells, gamma-delta T (γδT) cells, type 1 and 2 dendritic cells, CD14 monocytes, CD4 and CD8 regulatory T cells (Tregs), and B cells], were analyzed by flow cytometry. A significant decrease of CD4+ T lymphocytes, γδT cells, iNKT cells, CD4+ and CD8+ Tregs was observed in patients with hemophilia. Those patients having factor VIII inhibitor had the lowest CD4+ Treg and CD8+ Treg counts. CD14 monocytes were increased, as well as iNKT and type 2 dendritic cells in obese-overweight hemophilics. CD4+ lymphocytes, iNKT, γδT cells, and Tregs (CD4+ and CD8+), are significantly decreased in patients with hemophilia. Depletion of Tregs is more important in patients with factor VIII inhibitor. Physicians caring for hemophilia patients should realize that, even when they are not suffering infections frequently, may have early evidence of cellular immunodeficiency.

Overexpression of CD158 and NKG2A Inhibitory Receptors and Underexpression of NKG2D and NKp46 Activating Receptors on NK Cells in Acute Myeloid Leukemia.

BACKGROUND AND AIMS: Natural killer (NK) cells are innate immune system cells that are actively involved in immune-surveillance of tumor cells. Recognition of tumors by NK cells occurred via natural cytotoxicity receptors and killer cell immunoglobulin-like receptors. Some ligands of the activating receptors seem to be present on malignant cells from patients with acute myeloid leukemia. The aim of the study was to evaluate the expression of activating receptors such as NKG2D, DNAM-1, NKp30, and NKp46, and inhibitory receptors such as NKG2A, CD158b, CD158a, and CD158e1 on NK cells from patients with newly diagnosed acute myeloid leukemia before and after stimulation with IL-2 and IL-12. METHODS: Patients were divided into two groups: group 1 AML M3, and group 2 non-M3 AML. Flow cytometry was performed on whole PBMC to evaluate NK cell receptors. RESULTS: Twenty one AML patients, aged 26-78 years, and 11 matched healthy individuals were studied. NKG2D, and NKp46 expression was decreased in group 1 (p <0.019). Patients in Group 2 showed underexpression of the activating receptors NKp46. Differences after stimulation of NK cells with IL-2 and IL-12 were observed only in Group 2, in which a significant decrease in the expression of NKp46 receptor was found (p <0.0016). Patients in groups 1 and 2 showed overexpression of the inhibitory receptors CD158b (p <0.007) and NKG2A (p <0.01). CONCLUSIONS: NKG2D receptor expression is decreased in patients with AML M3. In addition, patients with all FAB types of AML have overexpression of inhibitory receptors such as CD158b and NKG2A and decreased expression of the activating receptor NKp46.

CD133+CD34+ and CD133+CD38+ blood progenitor cells as predictors of platelet engraftment in patients undergoing autologous peripheral blood stem cell transplantation.

BACKGROUND: Hematopoietic stem cells (HSC) have been characterized by CD34+ expression and an adequate dose of CD34+ cells is associated with a complete engraftment. CD133 is a more specific marker of HSC. MATERIALS AND METHODS: We studied the relationship between graft content of CD34+, CD133+, and CD38+ cells and trilineage engraftment after autologous stem cell transplantation in patients with different hematological disorders. Blood samples were obtained before and after mobilization with recombinant granulocyte-colony stimulating factor (G-CSF, 16 µg/kg), from apheresis collections, and after transplantation. RESULTS: Cell subsets were quantified by flow cytometry, and the dose of each population infused was correlated with success of engraftment. G-CSF induced mobilization of CD133+CD38+ cells (12.6-fold) and CD133+CD34+ cells (14.7-fold). A correlation was observed between the infused dose of CD133+CD34+ and CD133+CD38+ cells and platelet engraftment. CONCLUSION: CD133+CD34+ and CD133+CD38+ cells were mobilized with G-CSF and these cell subsets were correlated with platelet engraftment.

Programa de evaluación de la calidad entre laboratorios. XIV. Un año de evaluación en Citometría Hemática / Evaluation of quality among laboratories XIV. One year evaluation in haematic citrometic

En el primer estudio de la calidad en la sección de Hematología se utilizó sangre humana estabilizada, preparada según la técnica propuesta por la OMS. Sin embargo, la morfología celular no resistió las condiciones del transporte de las muestras. En este artículo se presentan únicamente los resultados de la evaluación de la calidad, en la cuenta diferencial, utilizando extendidos de sangre. En doce ocasiones se proporcionó a cada participante, un frote de sangre periférica, recién extraída de donadores sanos. Los participante tiñeron la laminilla, realizaron una cuenta diferencia, e informaron los resultados a los organizadores. Para evaluación de la calidad, se establece una puntuación del índice de varianza (PIV), utilizando un coeficiente de variación seleccionado (CVS) variable cuando los valores de consenso son menores de 2.5, y un CVS fijo para cifras mayores. Los resultados muestran una clara tendencia hacia la disminución del promedio del PIV, en todos los componentes hamtológicos y en el conjunto siendo mayor para los linfocitos y los segmentados. Esto sugiere que ha habido progreso en la capacidad para identificar y distinguir las células blancas normales. Para los granulocitos el promedio del PIV, de los últimos seis ciclos, es infrerior a 100 puntos. Para los mononucleares aún permanece por arriba de esa cifra. Esto coincide con las experiencias de CAP, que señalan que es común la dificultad para distinguir los linfocitos de los monocitos