Impaired Akt phosphorylation in B-cells of patients with common variable immunodeficiency.

Common variable immunodeficiency (CVID) is a heterogeneous group of primary immunodeficiency characterized by recurrent infections. We evaluated whether defective PI3K/Akt/FoxO pathway could influence B-cell fate. Determination of B-cell subsets in CVD patients and healthy donors (HDs) were performed using flow cytometry. We evaluated mRNA and protein expression of PI3K, Akt and FoxO using real-time PCR and flow cytometry, respectively. Moreover, phosphorylated Akt (pAkt) expression in B-cells has been measured by flowcytometry. We identified a significant reduction in the percentage of marginal zone like B-cells, memory B-cells (total, switched and unswitched) and plasmablasts in patients, as these decreased B-cell subsets had a significant negative correlation with increased apoptosis in patients. Surprisingly, we identified decreased pAkt expression in B-cells of patients than HDs. We described for the first time impaired pAkt expression in B-cells of CVID patients that had a significant correlation with antibody response to the vaccine and worse clinical complications.

In vitro assessment of platelet lesions during 5-day storage in Iranian Blood Transfusion Organization (IBTO) centers.

BACKGROUND AND OBJECTIVES: Platelet concentrates (PC) are used in thrombocytopenia and inherited or acquired platelet dysfunction disorders. Thus, retaining the platelets quality and function during storage will lead to desirable outcomes in treatment of such patients.  METHODS:  In this study, we evaluated 40 PC bags, prepared by PRP method in IBTO centers. We applied an array of assays, on first, third and fifth days of storage for PC quality control, including swirling, cell counting, bacterial contamination, measurement of CD62P, pH, and platelet aggregation test, to evaluate platelet lesion during storage.  RESULTS:  All units were negative for bacterial contamination. Swirling was positive for all units on various days; platelet count was in the acceptable range. Measurement of CD62P on fifth day was not significantly higher than third or first day (P > 0.15) (P > 0.05). pH on fifth day was significantly lower than first day (P < 0.01) (P < 0.05). Platelet aggregation with arachidonic acid and ristocetin showed significant decrease on fifth day compared to third day (P < 0.01) (P < 0.05). CONCLUSIONS: CD62P associated with other platelet function tests can be used as an activation marker in evaluation of PC functions during storage.

The caspase-3 inhibitor (peptide Z-DEVD-FMK) affects the survival and function of platelets in platelet concentrate during storage.

BACKGROUND: Although apoptosis occurs in nucleated cells, studies show that this event also occurs in some anucleated cells such as platelets. During storage of platelets, the viability of platelets decreased, storage lesions were observed, and cells underwent apoptosis. We investigated the effects of caspase-3 inhibitor on the survival and function of platelets after different periods of storage. METHODS: Platelet concentrates were obtained from the Iranian Blood Transfusion Organization in plastic blood bags. Caspase-3 inhibitor (Z-DEVD-FMK) was added to the bags. These bags along with control bags to which no inhibitor was added were stored in a shaking incubator at 22℃ for 7 days. The effects of Z-DEVD-FMK on the functionality of platelets were analyzed by assessing their ability to bind to von Willebrand factor (vWF) and to aggregate in the presence of arachidonic acid and ristocetin. Cell survival was surveyed by MTT assay. RESULTS: At day 4 of storage, ristocetin-induced platelet aggregation was significantly higher in the inhibitor-treated (test) than in control samples; the difference was not significant at day 7. There was no significant difference in arachidonic acid-induced platelet aggregation between test and control samples. However, at day 7 of storage, the binding of platelets to vWF was significantly higher in test than in control samples. The MTT assay revealed significantly higher viability in test than in control samples at both days of study. CONCLUSION: Treatment of platelets with caspase-3 inhibitor could increase their functionality and survival.

The Expression and Prognostic Impact of CD95 Death Receptor and CD20, CD34 and CD44 Differentiation Markers in Pediatric Acute Lymphoblastic Leukemia.

OBJECTIVE: This study investigated the expression and prognostic significance of the CD95 death receptor and CD20, a B cell-lineage associated marker, along with CD34 and CD44 non-lineage associated molecules in Iranian children with acute lymphoblastic leukemia (ALL). METHODS: We performed immunophenotyping for expressions of the molecules in blood samples from children diagnosed with ALL by using a panel of monoclonal antibodies for flow cytometry analysis. The expression of markers was evaluated in relation to clinical and paraclinical features as well as response to treatment in the patients. Findings : CD95 showed a higher expression in T-ALL compared to B-ALL (P<0.001). Analysis of the clinical and laboratory findings at diagnosis in the group of B-ALL patients revealed an association between CD95 expression with lower white blood cell (WBC) numbers and bone marrow blasts (P<0.05). We detected a positive correlation between the expressions of CD95 and CD44 (r=0.445, P<0.01) in B-ALL patients. There was an association between CD20 expression and several poor prognostic factors that included increased extramedullary involvement (EMI) and decreased platelet numbers (P<0.008). The mean expression of CD34 in B-ALL was higher than T-ALL (P=0.004). At follow-up, complete remission duration (CRD) and survival duration did not significantly differ between patients who were positive or negative for each marker. CONCLUSION: Association of the studied molecules with several prognostic factors implies the significance of CD95 molecule as favorable and CD20 as unfavorable prognostic markers for childhood ALL.

CD44 and CD27 expression pattern in B cell precursor acute lymphoblastic leukemia and its clinical significance.

The expression of CD44 and CD27 molecules correlates with the differentiation stage of B cell precursors. The present study was designed to investigate the prognostic relevance of CD44 and CD27 molecules in patients with B cell acute lymphoblastic leukemia (ALL). CD27 and CD44 expression was determined in 58 patients by flow cytometry and their relation to established prognostic factors and response to therapy was investigated. Four patterns of expression were found; CD27 single positive (SP) in 20.7 % of patients, CD44SP in 25.8 %, CD27CD44 double positive (DP) in 20.7 %, and CD27CD44 double negative (DN) in 32.8 %. CD27 expression and the CD27SP pattern correlated directly with TEL/AML1 genotype (P = 0.012). Conversely, CD44 expression and the CD44SP pattern correlated inversely with this genotype (P = 0.016). Patients with the DP pattern had a lower WBC count (P = 0.03), lower percentage of blasts in their bone marrow (P = 0.028), and higher platelet count, whereas CD44SP patients had a higher WBC count and higher percentage of bone marrow blasts. Moreover, a negative association between DN pattern and complete remission (CR) rate was detected (P = 0.03). Mean CD27 expression was significantly higher in low-risk group and in patients who achieved CR (P = 0.001), and in those with a higher platelet number (P = 0.046) and less extramedullary involvement (P = 0.008). Although survival and CR duration were longer in patients with DP pattern and shorter in those with DN pattern, the result did not reach statistical significance. The expression of CD27 together with CD44 showed a relationship with several established risk factors as well as response to therapy, indicating the biological significance of these molecules in ALL.

Fine-needle aspiration cytology and flow cytometric immunophenotyping in diagnosis and classification of non-Hodgkin lymphoma in comparison to histopathology.

This prospective study aimed to compare the value of fine needle aspiration (FNA) cytology (FNAC) and flow cytometric immunophenotyping (FCI) with histopatopathology (HP) in the diagnosis and classification of non-Hodgkin lymphoma (NHL). Twenty-nine excised lymph nodes suspected of NHL were evaluated using FNAC, FCI, and HP. Specimens were divided into two equal parts; one for HP and the other for FNAC and FCI. Results were compared in terms of diagnosis (malignant, benign or reactive, and metastatic) and NHL class. With combined FNAC/FCI, 11 (37.9%) cases were diagnosed as NHL, 11 cases (37.9%) as reactive lymph node, six cases (20.6%) as Hodgkin's lymphoma, and one case (3.4%) as metastasis. HP revealed nine cases (31%) of NHL, five cases (17.2%) of reactive lymph nodes and all the diagnosed metastatic and Hodgkin's lymphoma. Considering histology as a gold standard method in diagnosis, the sensitivity, specificity, PPV and NPV of FNAC/FCI in differentiate malignant and benign lesion were 73.9%, 83.3%, 94.4%, and 45.5%, respectively and in differentiate NHL from others were 75%, 93.8%, 90%, and 83.3%, respectively. Cytology and HP in addition to FCI and HP are significantly different from determination of NHL lesions point of view (P = 0.001 and P < 0.0001, respectively). However, FCI can be considered as an adjunctive method for Cytology especially because Cytology is not competent enough to differentiate between benign lesions and Lymphoma. Additionally, FCI is shown to be an accurate method in classifying NHL.

HLA Antigens Shed from the Surface of Synthetic or Naturally Occurred Platelet-Derived Microparticles During Storage of Platelet Concentrate.

The demand for standard platelet concentrates (PCs) has continued to increase in the recent years. Infusible platelet membranes (IPM) prepared from new or outdated human platelets have been developed as an alternative to standard PCs, with the additional advantage of long shelf life and increased viral safety. Reduction of HLA antigens on the IPM has been assigned as one of the probable advantages of this product. In re-examining this issue, we studied the existence of HLA class I on the surface of IPM microparticles. In comparison we also surveyed HLA expression on the surface of the naturally occurred platelet-derived microparticles (nPMPs) during 7 days storage. Intended for producing IPM, PCs obtained from Iranian blood transfusion organization were lysed; virally inactivated with wet heat in the presence of a heat stabilizer and then sonicated. IPMs were separated using centrifugation and liquid-stored in 4°C. The expression of HLA class I antigens was surveyed using flow cytometry technique. HLA molecules were present on the microparticles. Shedding of HLA antigens was demonstrated from the surface of the both liquid-stored IPM and nPMPs during storage. Storage of IPM in 4°C was accompanied with significant reduction of HLA molecules. It seemed that achievement of HLA-free IPM could be impossible unless chloroquine treated platelets were used to prepare these microvesicles.