Quality control in veterinary blood banks: evaluation of canine platelet concentrates stored for five days.

BACKGROUND: Platelets undergo structural, biochemical and functional alterations when stored, and platelet storage lesions reduce platelet function and half-life after transfusion. The objective of this study was to evaluate stored canine platelet concentrates with platelet aggregation, flow cytometry and biochemistry assays. Twenty-two bags of canine platelet concentrates were obtained by the platelet-rich plasma method and were assessed on days 1, 3 and 5 after collection. Parameters such as platelet counts, residual leukocytes, platelet swirling, glucose, lactate, pH, CD62P expression (platelet activation), JC-1 (mitochondrial function) and annexin V (apoptosis and cell death) were assessed. RESULTS: Over the five days of storage there was a significant decrease in glucose, HCO3, pCO2, ATP, pH, swirling and mitochondrial function, associated with a significant increase in lactate levels and pO2. At the end of storage pH was 5.9 ± 0.6 and lactate levels were 2.8 ± 1.2 mmol/L. Results of the quality parameters evaluated were similar to those reported in human platelets studies. The deleterious effects of storage were more pronounced in bags with higher platelet counts (> 7.49 × 1010/unit), suggesting that canine platelet concentrates should not contain an excessive number of platelets. CONCLUSIONS: Quality parameters of canine platelets under standard storage conditions were similar to those observed in human platelets. Our results have potential to be used for the routine evaluation and quality control in veterinary blood banks.

Comparison of JC-1 and MitoTracker probes for mitochondrial viability assessment in stored canine platelet concentrates: A flow cytometry study.

Mitochondria perform crucial roles in many biochemical processes, and mitochondrial depolarization is an early sign of platelet apoptosis. The mitochondrial membrane potential is usually evaluated through JC-1 probe, but it can also be assessed with MitoTracker probes. Our aim was to evaluate mitochondrial viability in stored canine platelet concentrates (PCs) with the fluorescent probes JC-1 and MitoTracker. Platelets from 22 canine PCs were stained with JC-1 and MitoTracker probes on days 1, 3, and 5 of storage. Data on metabolic parameters were also collected for correlation studies. Results of JC-1 and MitoTracker revealed a decrease in mitochondrial membrane potential in day 5 of storage compared to days 1 and 3, providing evidence of mitochondrial depolarization, a finding that was confirmed by the data on metabolic parameters. MitoTracker probes also added information regarding platelet swelling. In conclusion, MitoTracker probes offered a more complete mitochondrial analysis in the evaluation of stored canine PCs. © 2018 International Society for Advancement of Cytometry.

Effect of fat on serum 25-hydroxyvitamin D levels after a single oral dose of vitamin D in young healthy adults: a double-blind randomized placebo-controlled study.

PURPOSE: This double-blind placebo-controlled trial evaluated serum 25-hydroxyvitamin D [25(OH)D] levels after the oral intake of a single dose of cholecalciferol during one of the three meals, containing different amounts of fat or placebo. METHODS AND RESULTS: Sixty-four healthy medical residents or students of a university hospital in Porto Alegre, latitude 30° S, Brazil, were divided into four groups. Three groups received a single 50,000 IU oral dose of cholecalciferol during a meal containing 0 g (Group 1), 15 g (Group 2) or 30 g (Group 3) of fat, and one group received placebo (Group 4), according to randomization. Serum 25(OH)D, parathyroid hormone, total calcium, albumin, magnesium, and creatinine levels, and urinary calcium, magnesium, and creatinine levels were measured at baseline and after 14 days. Baseline mean serum 25(OH)D levels were low in all groups. Vitamin D given during breakfast increased the mean change of serum 25(OH)D levels, when compared to placebo. Furthermore, the intake of fat with vitamin D increased the mean change of serum 25(OH)D levels. CONCLUSION: A single oral dose of vitamin D given with food increased mean serum 25(OH)D levels, after 2 weeks, and the mean increase was larger, when the meal had at least 15 g of fat. These findings can have important implications to oral vitamin D supplementation.

Blood bank quality control: pH assessment methods in platelet concentrates.

Platelet concentrates undergo progressive changes during storage, such as a decrease in pH. Additionally, pH and lactate production showed the strongest correlation with platelet survival in posttransfusion viability studies. pH measurement is a straightforward method for evaluating the quality control of blood components in blood bank practice. Our aim was to compare three pH assessment methods for canine platelet concentrates. The pH values of the canine platelet concentrates were assessed on the first day of storage using a calibrated pH meter, a portable gas analyzer and pH-indicator strips. The results from the pH meter and portable gas analyzer measurements were similar. The pH indicator strips presented higher average values compared to the other more reliable methods evaluated, which could result in the use of inadequate blood components. In conclusion, it is recommended to implement pH measurements using a pH meter for quality control in veterinary blood banks.

Flow cytometry evaluation of CD14/CD16 monocyte subpopulations in systemic sclerosis patients: a cross sectional controlled study.

BACKGROUND: Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by vasculopathy and fibrosis, which can be subclassified into diffuse cutaneous (dSSc) and limited cutaneous (lSSc) subtypes. Previous studies suggest that an increase in monocytes can be a hallmark of various inflammatory diseases, including SSc. Our aim was to evaluate circulating blood monocyte subpopulations (classical, intermediate and non-classical) of SSc patients and their possible association with disease manifestations. METHODS: Fifty consecutive patients fulfilling the 2013 ACR/EULAR classification criteria for SSc were included in a cross-sectional study. Monocyte subpopulations were identified based on their expression of CD64, CD14 and CD16, evaluated by flow cytometry, and were correlated with the clinical characteristics of the patients; furthermore, the expression of HLA-DR, CD163, CD169 and CD206 in the monocytes was studied. Thirty-eight age- and sex-matched healthy individuals were recruited as a control group. RESULTS: SSc patients had an increased number of circulating peripheral blood monocytes with an activated phenotypic profile compared to healthy subjects. Absolute counts of CD16+ (intermediary and non-classical) monocyte subpopulations were higher in SSc patients. There was no association between monocyte subpopulations and the clinical manifestations evaluated. CONCLUSION: We identified higher counts of all monocyte subpopulations in SSc patients compared to the control group. There was no association between monocyte subpopulations and major fibrotic manifestations. CD169 was shown to be more representative in dSSc, being a promising marker for differentiating disease subtypes.

Dualities of the vitamin D in systemic sclerosis: a systematic literature review.

BACKGROUND: Systemic sclerosis (SSc) is a chronic disease characterized by autoimmunity, vasculopathy, and visceral and cutaneous fibrosis. Vitamin D has several functions in the immunological system, and different studies have suggested a potential role in triggering autoimmune diseases. Patients with SSc may present with low serum levels of vitamin D, but the association between hypovitaminosis D and disease onset or any clinical manifestation is still obscure. Our goal was to verify the causal relationship between hypovitaminosis D and SSc onset or any particular clinical manifestation in the literature. METHODS: A systematic literature review was performed through February 24th, 2021 on Pubmed, Lilacs/BIREME, and Cochrane databases. The eligible studies were read in full text, and, in the absence of exclusion criteria, were included in this review after consensus between two reviewers. RESULTS: Forty articles met the eligibility criteria and the main results of each study are described. In most studies, SSc patients showed a higher prevalence of vitamin D deficiency and insufficiency compared to controls. Additionally, in some reports serum levels of vitamin D were inversely correlated with the severity of SSc. Oral supplementation did not seem to affect serum levels of vitamin D. Four of the included studies were with experimental models. CONCLUSION: In conclusion, vitamin D deficiency seems to have a role in susceptibility to SSc, as well as in the clinical manifestations of the disease.

Polyclonal B-cell lymphocytosis: Report of three cases.

BACKGROUND: Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare benign condition characterized by a polyclonal B-cell lymphocytosis with binucleated lymphocytes. We hereby report three cases of PPBL. METHODS: Flow cytometry immunophenotyping was performed in peripheral blood samples from three patients with clinical suspicion of lymphoproliferative disease. RESULTS: Case 1 was a female middle-aged smoker; Case 2 was an elderly male; and Case 3 was a non-smoker female. Flow cytometry evaluation of all cases revealed an expansion of mature B-cells, with a normal Kappa/Lambda light chain ratio; B-cell lymphocytes of Cases 2 and 3 had CD5 coexpression; Case 3 also had monocytosis. CONCLUSIONS: Diagnose of PPBL is important in order to avoid unnecessary diagnostic procedures and therapy. © 2018 International Clinical Cytometry Society.

Expression of neutrophil surface markers in icteric neonates before and after phototherapy.

BACKGROUND: Jaundice due to indirect hyperbilirubinemia affects more than 60% of neonates and phototherapy is the treatment for severe types. There are no previous studies evaluating the effect of phototherapy on the function of neonates neutrophils. The aim of this study was to assess and compare the function of neutrophils by measuring the expression of neutrophils main surface markers in icteric neonates before and after phototherapy. METHODS: Neonates at a gestational age ≥35 weeks and birth weight ≥2,000 g who met the American Academy of Pediatrics criteria for phototherapy were included. Flow cytometry evaluation of the mean fluorescence intensities of CD10, CD11b, CD11c, CD15, CD16, CD18, CD62L, CD64, and CD66acde was performed before and 24 h after the initiation of phototherapy. RESULTS: Twenty-five neonates at a mean age of 53 h of life were included in the study with a mean bilirubin level of 13.60 ± 2.85 mg/dL. There was no statistical difference in the expression of CD11b, CD15, CD18, CD62L, and CD64 or in the percentage of neutrophils before and after 24 h of phototherapy. There was an increase in the expression of CD10 and CD16 and a decrease in the expression of CD11c and CD66acde after 24 h of phototherapy. CONCLUSIONS: Newborns submitted to phototherapy had an increase in the expression of CD10 and CD16 and a decreased in the expression of CD11c and CD66acde after 24 h of treatment, which may be related to an anti-inflammatory effect of phototherapy. © 2018 International Clinical Cytometry Society.

Expression of Bruton's tyrosine kinase in B-cell neoplasms evaluated by flow cytometry.

Bruton's tyrosine kinase (BTK) is a cytoplasmatic protein that is part of the B-cell antigen receptor signaling pathway. Our aim was to evaluate the expression of BTK in B-cell neoplasms and compare it to normal B-cell lymphocytes. After surface staining with CD19 and CD45, flow cytometry staining for intracellular BTK was performed in leukemic or mature B-cells from bone marrow or peripheral blood samples. No differences in BTK expression were identified between groups, or in comparison to control samples, there was no association between BTK expression and the clinical variables evaluated. BTK expression in B-cell neoplasms was similar to that of normal B-cell lymphocytes.

Quantitative flow cytometric evaluation of CD200, CD123, CD43 and CD52 as a tool for the differential diagnosis of mature B-cell neoplasms.

BACKGROUND: Distinction between mature B-cell neoplasms can be difficult due to overlapping of immunologic features and clinical manifestations. This study investigated whether quantifying mean fluorescence intensity of four monoclonal antibodies in a flow cytometry panel is useful for the differential diagnosis and characterization of these disorders. METHODS: The expressions of CD52, CD200, CD123 and CD43 were analyzed in samples from 124 patients with mature B-cell neoplasms. The quantitative estimation of these antigens was assessed by mean fluorescence intensity. RESULTS: The cases included were 78 chronic lymphocytic leukemias, three atypical chronic lymphocytic leukemias, six marginal zone lymphomas, 11 splenic marginal zone lymphomas, nine lymphoplasmacytic lymphomas, six mantle cell lymphomas, two hairy cell leukemias, two hairy cell leukemias variant, five follicular lymphomas, one Burkitt lymphoma and one diffuse large B-cell lymphoma. The mean fluorescence intensity of CD200 was higher in atypical chronic lymphocytic leukemia, chronic lymphocytic leukemia and hairy cell leukemia cases. CD123 showed higher mean fluorescence intensities in hairy cell leukemia cells. Chronic lymphocytic leukemia, atypical chronic lymphocytic leukemia and mantle cell lymphoma had higher expression of CD43 and all follicular lymphoma cases had very low mean fluorescence intensity values. CD52 expression was consistently positive among all cases. CONCLUSION: Quantitative evaluation of these markers can be a useful additional tool to better identify some types of mature B-cell neoplasms.

Dualities of the vitamin D in systemic sclerosis: a systematic literature review

Abstract Background: Systemic sclerosis (SSc) is a chronic disease characterized by autoimmunity, vasculopathy, and visceral and cutaneous fibrosis. Vitamin D has several functions in the immunological system, and different studies have suggested a potential role in triggering autoimmune diseases. Patients with SSc may present with low serum levels of vitamin D, but the association between hypovitaminosis D and disease onset or any clinical manifestation is still obscure. Our goal was to verify the causal relationship between hypovitaminosis D and SSc onset or any particular clinical manifestation in the literature. Methods: A systematic literature review was performed through February 24th, 2021 on Pubmed, Lilacs/BIREME, and Cochrane databases. The eligible studies were read in full text, and, in the absence of exclusion criteria, were included in this review after consensus between two reviewers. Results: Forty articles met the eligibility criteria and the main results of each study are described. In most studies, SSc patients showed a higher prevalence of vitamin D deficiency and insufficiency compared to controls. Additionally, in some reports serum levels of vitamin D were inversely correlated with the severity of SSc. Oral supplementation did not seem to affect serum levels of vitamin D. Four of the included studies were with experimental models. Conclusion: In conclusion, vitamin D deficiency seems to have a role in susceptibility to SSc, as well as in the clinical manifestations of the disease.

Flow cytometry evaluation of CD14/CD16 monocyte subpopulations in systemic sclerosis patients: a cross sectional controlled study

Abstract Background: Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by vasculopathy and fibrosis, which can be subclassified into diffuse cutaneous (dSSc) and limited cutaneous (lSSc) subtypes. Previous studies suggest that an increase in monocytes can be a hallmark of various inflammatory diseases, including SSc. Our aim was to evaluate circulating blood monocyte subpopulations (classical, intermediate and non-classical) of SSc patients and their possible association with disease manifestations. Methods: Fifty consecutive patients fulfilling the 2013 ACR/EULAR classification criteria for SSc were included in a cross-sectional study. Monocyte subpopulations were identified based on their expression of CD64, CD14 and CD16, evaluated by flow cytometry, and were correlated with the clinical characteristics of the patients; furthermore, the expression of HLA-DR, CD163, CD169 and CD206 in the monocytes was studied. Thirty-eight age- and sex-matched healthy individuals were recruited as a control group. Results: SSc patients had an increased number of circulating peripheral blood monocytes with an activated phenotypic profile compared to healthy subjects. Absolute counts of CD16+ (intermediary and non-classical) monocyte subpopulations were higher in SSc patients. There was no association between monocyte subpopulations and the clinical manifestations evaluated. Conclusion: We identified higher counts of all monocyte subpopulations in SSc patients compared to the control group. There was no association between monocyte subpopulations and major fibrotic manifestations. CD169 was shown to be more representative in dSSc, being a promising marker for differentiating disease subtypes.

Hypovitaminosis D in patients with cystic fibrosis: a cross-section study in South Brazil.

INTRODUCTION: Cystic fibrosis (CF) patients have a susceptibility to vitamin D deficiency because of nutrient malabsorption. OBJECTIVES: To evaluate the prevalence of hypovitaminosis D in CF patients and the factors associated with serum 25-hydroxyvitamin D levels. METHODS: We evaluated the prevalence of vitamin D deficiency defined as 25-hydroxyvitamin D <30 ng/mL, as suggested recently by the Cystic Fibrosis Foundation, and factors associated with its serum levels. Patients with confirmed CF were included. Nutritional status and hospital admissions were evaluated. Serum C-reactive protein, calcium, phosphate, magnesium, albumin, 25-hydroxyvitamin D and parathyroid hormone levels were measured. Lung function was evaluated by spirometry, and clinical and chest radiographic scores were assessed. Statistical significance level was set at P < 0.05. RESULTS: Fifty-nine patients were included. Prevalence of hypovitaminosis D was 61%. Patients with pancreatic insufficiency had a trend to have higher vitamin D levels. Sixteen patients had severe lung disease with percentage of forced expiratory volume in 1 s predicted below 40%. After multivariate analysis, body mass index and hospitalization in the last month remained significantly associated with serum vitamin D levels. CONCLUSIONS: Vitamin D insufficiency is still a problem in CF patients, even in those receiving supplementation.

Quantitative flow cytometric evaluation of CD200, CD123, CD43 and CD52 as a tool for the differential diagnosis of mature B-cell neoplasms

Abstract Background Distinction between mature B-cell neoplasms can be difficult due to overlapping of immunologic features and clinical manifestations. This study investigated whether quantifying mean fluorescence intensity of four monoclonal antibodies in a flow cytometry panel is useful for the differential diagnosis and characterization of these disorders. Methods The expressions of CD52, CD200, CD123 and CD43 were analyzed in samples from 124 patients with mature B-cell neoplasms. The quantitative estimation of these antigens was assessed by mean fluorescence intensity. Results The cases included were 78 chronic lymphocytic leukemias, three atypical chronic lymphocytic leukemias, six marginal zone lymphomas, 11 splenic marginal zone lymphomas, nine lymphoplasmacytic lymphomas, six mantle cell lymphomas, two hairy cell leukemias, two hairy cell leukemias variant, five follicular lymphomas, one Burkitt lymphoma and one diffuse large B-cell lymphoma. The mean fluorescence intensity of CD200 was higher in atypical chronic lymphocytic leukemia, chronic lymphocytic leukemia and hairy cell leukemia cases. CD123 showed higher mean fluorescence intensities in hairy cell leukemia cells. Chronic lymphocytic leukemia, atypical chronic lymphocytic leukemia and mantle cell lymphoma had higher expression of CD43 and all follicular lymphoma cases had very low mean fluorescence intensity values. CD52 expression was consistently positive among all cases. Conclusion Quantitative evaluation of these markers can be a useful additional tool to better identify some types of mature B-cell neoplasms.