B and T cell responses to the 3rd and 4th dose of the BNT162b2 vaccine in dialysis patients.

Patients on dialysis (PoD) are at high risk of severe morbidity and mortality from COVID-19. Characterizing long-term vaccine immune responses in these patients will help optimize vaccine schedule for PoD. This study aimed to determine whether long-term humoral and B and T cell-responses post 3rd and 4th dose of the BNT162b2 vaccine differed between PoD and controls. Non-infected PoD and controls vaccinated with BNT162b2 were recruited in Ziv Medical Center, Israel, between 2021 and 2022. Specimens were collected 1-2 months pre 3rd dose; 1-3 months post 3rd dose; 4-5 months post 3rd dose and 3-5 months post the 4th dose. Anti-SARS-CoV-2 spike (spike) specific antibodies, spike specific memory B cells, and spike specific CD154+ T cells as well as cytokines producing CD4+/CD8+ T cells were measured using standardized assays and compared between PoD and controls at each time point using Mann Whitney and Fisher's exact tests. We recruited 22 PoD and 20 controls. Antibody levels in PoD were lower compared to controls pre 3rd dose but not post 3rd and 4th doses. Frequencies of spike specific memory B cell populations were similar between PoD and controls overall. Frequencies of spike specific T cells, including those producing IFNγ and TNFα, were not lower in PoD. B and T cell mediated immune response in PoD following a 3rd and a 4th dose of the BNT162b2 vaccine was not inferior to controls up to 5 months post vaccination. Our results suggest that standard BNT162b2 vaccination is suitable for this group.

Comparison of a modified flow cytometry osmotic fragility test with the classical method for the diagnosis of hereditary spherocytosis.

BACKGROUND: Hereditary spherocytosis (HS) is the most common inherited hemolytic anemia. The flow cytometric test using eosin-5'maleimide (EMA) is a well-established diagnostic method. However, in order to improve HS detection, it is recommended that EMA and an osmotic fragility test (OFT) both be performed. OFT is time consuming and labor intensive. We used a flow cytometric (FOFT) adaptation of the classical OFT reported by Yamamoto. We compare the FOFT to the classical OFT including practical data and propose options for simplifying this method. METHODS: Suspected and known HS patients and controls were tested by the following methods: EMA, OFT, and FOFT including some modifications. RESULTS: The FOFT method is robust and correlates to loss of red blood cells. OFT and FOFT gave similar results in healthy controls and four HS patients. Normal range for FOFT in 70 adults is shown and can be used as a reference value. Neonates should have their own normal range defined. Overnight sample incubation at 37°C did not add information to the FOFT results. CONCLUSION: Our modified Yamomoto FOFT can replace the classic OFT as the addition to EMA for the diagnosis of HS. The use of flow cytometry in both these methods requires small sample volume, is reproducible, simpler, and produces results more rapidly.

Flow cytometry aneuploidy and cell cycle indexing as a possible tool for differentiating between CD10+ diffuse large B-cell lymphoma and follicular lymphoma.

BACKGROUND: Differential diagnosis between diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) becomes a challenge when adequate biopsy is unavailable. The present study aimed to investigate the applicability of DNA cell cycle analysis by flow cytometry (FC) for differentiating between CD10+ DLBCL and FL. METHODS: Data were collected from 57 specimens where CD5- /CD10+ /light chain restricted B cells were detected. DNA staining was performed using the Coulter DNA Prep Kit. Cell cycle fractions were evaluated by automatic analysis using the ModFit LT software. RESULTS: Histopathological analysis confirmed the diagnosis of CD10+ FL in 30 specimens (52.6%), CD10+ DLBCL in 24 specimens (42.1%), and CD10+ Burkitt lymphoma in 3 specimens (5.3%). A significantly higher rate of DNA aneuploidy was detected among CD10+ DLBCL than FL specimens (50 vs. 13.3% respectively, p = .003). Likewise, DNA index was significantly higher in CD10+ DLBCL relative to FL (1.26 ± 0.35 vs. 1.04 ± 0.16 respectively, p = .004). Notably, the proportion of cells in the S-phase and proliferative fraction was significantly higher in CD10+ DLBCL than in CD10+ FL (S-phase: 15.97 ± 13.94 vs. 4.43 ± 4.41 mean ± SD, respectively, p < .0001; proliferative fraction: 18.87 ± 15.17 vs. 5.78 ± 7.04 mean ± SD, respectively, p = .0001). Using a receiver operating characteristic analysis, optimal cutoffs for S-phase ≥7% and proliferative fraction ≥9% were determined. These values could be used to differentiate between CD10+ DLBCL and CD10+ FL. CONCLUSION: Including DNA cell cycle analysis in the FC lymphoma assessment panel may be of diagnostic value in differentiating between CD10+ DLBCL and FL when adequate biopsy is unavailable.

Ectopic Expression of Snail and Twist in Ph+ Leukemia Cells Upregulates CD44 Expression and Alters Their Differentiation Potential.

Philadelphia chromosome-positive (Ph+) leukemia is characterized by reciprocal translocation between chromosomes 9 and 22. The resultant BCR/ABL fusion protein displays constitutive tyrosine kinase activity, leading to the induction of aberrant proliferation and neoplastic transformation. The bone marrow (BM) microenvironment is tumor-promoting, and contributes to disease recurrence in Ph+ leukemia. Activity in the BM microenvironment is mediated by several cellular compartments, extracellular matrix, various soluble factors including transforming growth factor beta 1 (TGF-ß1), and the hypoxic conditions in the BM niche. TGF-ß1 is released during bone remodeling and plays a role in maintaining leukemic stem cells, as well as being implicated in the epithelial-mesenchymal transition (EMT) process in most solid tumors. Although EMT is largely implicated in epithelial tumors, recent findings argue for an EMT-like process in leukemia as well. The surface receptor CD44 is involved in cell adhesion, cell migration, and homing of normal and malignant hematopoietic stem cells. Elevation of CD44 expression is considered a marker for a worse prognosis in most hematological malignancies. We explored the functions of Snail and Twist1 in Ph+ leukemia. We showed that ectopic expression of Snail and, to a lesser extent, Twist1, upregulates CD44 expression that is ß-catenin-dependent. Moreover, the presence of Snail or Twist1 partially blocked phorbol 12-myristate 13-acetate-induced megakaryocyte differentiation, while that of Twist significantly altered imatinib-induced erythroid differentiation. Thus EMT modulators affected proliferation, CD44 gene expression and differentiation ability of Ph+ leukemia cells.

The neutrophil/lymphocyte ratio is a better addition to C-reactive protein than CD64 index as a marker for infection in COPD.

BACKGROUND: This study investigated the importance of neutrophil/lymphocyte ratio (NLR) and other new inflammatory markers including CD64 expression in patients with chronic obstructive pulmonary disease (COPD) for identifying the severity of inflammation and recognition of acute exacerbation and infection. METHODS: Seventy-two patients with a diagnosis of COPD exacerbation who were admitted to the Department of Internal Medicine B, 13 with stable COPD, and control group of 15 healthy people were enrolled in the study. Complete blood count (CBC), measurement of C-reactive protein (CRP), mean platelet volume (MPV), red blood cell distribution width (RDW) and CD64 expression were determined within 2 hours of hospital admission. RESULTS: NLR and other inflammatory markers, such as RDW, CRP, and CD64 were found to be significantly elevated in exacerbated COPD compared to stable COPD and control participants. There was a significant correlation of NLR with CRP (r=0.309, P<0.001), For an NLR cutoff of 7.3, sensitivity for detecting exacerbation of COPD was 0.768 and specificity was 1-0.269 (AUC=0.793, P=0.001) RDW was significant as NLR. CD64 is statistically significant (P=0.002) the lack of significance was between acute exacerbation of COPD and stable COPD, but indexes were higher in the group of COPD patients with complications. CONCLUSIONS: Elevated NLR can be used as a marker similar to CRP, and RDW, in the determination of increased inflammation in acutely exacerbated COPD. NLR could be beneficial for the early detection of potential acute exacerbations in patients with COPD who have normal levels of traditional markers; CD64 was higher but did not reach statistical significance. MPV was not helpful.

Differentiating ischemic from non-ischemic chest pain using white blood cell-surface inflammatory and coagulation markers.

Chest pain is one of the most common complaints seen in emergency departments (ED), up to 5-8 % of all ED visits. About 50-60 % of chest pain patients presenting to the ED are hospitalized. Seventy percentage of those patients not discharged from the ED are subsequently shown to not have acute cardiac disease. It has been estimated that emergency physician miss 2-6 % of acute coronary syndrome (ACS) that present to ED. While admitting a non-ACS patient is a financial burden on the medical system, releasing to home an undiagnosed ACS patient has life-threatening consequences. This study used flow cytometry to evaluate a panel of mononuclear cells, neutrophils, cytokines and fibrinolytic activation markers in patients presenting in ED with acute chest pain. The goal was to add diagnostic tools to the differentiation between true ischemic cardiac and non-ischemic chest pain in the process of triage. The study population consisted of 74 consecutive patients presenting with acute chest pain to the emergency department of Ziv Medical Center and were admitted to Intensive Cardiac Care Unit or Internal Wards of our hospital during the period September 2009 to February 2010. ACS has been clearly associated with a decrease in CD89+/CD62L+ population, an increase in percentage of cytotoxic T-cell subset, and an increase in platelet marker. Differences in thrombin receptor surface expression were also noted. The combination of multiple biomarkers may help to enhance diagnostic accuracy.