Samuel Ricardo Comar

Effect of vitamin C, deferoxamine, quercetin and rutin against tert-butyl hydroperoxide oxidative damage in human erythrocytes

Abstract The mature human erythrocyte, when submitted to oxidative stress, can demonstrate depletion of reduced glutathione, oxidation of the hemoglobin molecule and aggregation of complexes of iron close to the membrane. These can produce abnormalities in the erythrocyte membrane and hemolysis. The aim of this work was to study the antioxidative action of vitamin C (vit. C), deferroxamine (DFO) and the flavonoids quercetin and rutin in normal human erythrocytes, submitted to in vitro oxidative stress induced by tert-butylhydroperoxide (tBHP). Venous blood was colleted in citrate–phosphate–dextrose (CPD) solution, as anticoagulant, from healthy adult individuals after informed consent. The erythrocytes were resuspended in PBS to obtain 35% globular volume, and then submitted to the oxidative action of tBHP for up to 30 min, with or without previous incubation for 60 min with vit. C, DFO, quercetin and rutin. Decrease in the GSH concentration, G6-PD and GR activities, and increase in the methemoglobin and Heinz bodies (HB) formation, occurred with the increase in tBHP concentration. tBHP did not effect on the membrane proteins detected by SDS-PAGE. Quercetin, partially prevented the GSH decrease and the formation of HB, but did not prevent MetHb formation from oxidative damage by tBHP. Rutin, after tBHP induction, prevented the GSH decrease and the formation of HB. Vit. C, had no influence on the depletion of GSH, inhibited partially the metHb formation, and it protected GR, but not G6-PD from oxidative damage by tBHP. DFO partially inhibited the metHb formation and GSH decrease, but it did not protect GR and G6-PD from oxidative damage by tBHP. The results obtained suggest that vit. C, DFO and the flavonoids quercetin and rutin contribute to the decrease in the oxidative stress caused by tBHP.

Are the review criteria for automated complete blood counts of the International Society of Laboratory Hematology suitable for all hematology laboratories

Objective to verify whether the review criteria for automated blood counts suggested by the International Consensus Group for Hematology Review of the International Society for Laboratory Hematology are suitable for the Hematology Laboratory of Hospital de Clinicas, Universidade Federal do Paraná. Methods initially, the review criteria of the International Society for Laboratory Hematology were adapted due to limitations in the Institutions electronic hospital records and interfacing systems. The adapted review criteria were tested using 1977 samples. After this first assessment, an additional 180 inpatient samples were analyzed to evaluate the screening criteria of the review criteria in conjunction with positive smear findings established by the institution. The performance of the review criteria was verified by determining false positive, false negative, true positive and true negative rates, sensitivity, specificity, positive predictive value, negative predictive value, microscopic review rate and efficiency. Results initial analysis showed false negatives = 6.73%, false positives = 23.27%, microscopic review rate = 46.03% and efficiency = 70.0%. An evaluation of the screening criteria adapted from the review criteria together with the positive smear findings of the institution showed false negatives = 15.5%, false positives = 10.5%, microscopic review rate = 37.3% and efficiency = 73.8%. In both situations the safety limit (false negative <5%) recommended by the review criteria was exceeded. Conclusions the review criteria adapted from the International Society for Laboratory Hematology are neither suitable nor safe for use in the hematology laboratory of the Hospital de Clinicas. This implies a need to develop and validate institution-specific review criteria in order to decrease false negative results to an acceptable and safe rate for patients.

Performance evaluation of the Sysmex® XE-2100D automated hematology analyzer

Introduction: The Sysmex® XE-2100D is a multiparameter hematology analyzer designed for hematology testing in samples with ethylenediamine tetraacetic acid (EDTA). Objectives: Considering the importance of this hematology analyzer for clinical and laboratory practice, the objective of this study was to evaluate its analytical performance, comparing the obtained results with quality specifications described in literature. Material and method: In the evaluation of analytical performance, according to recommendations of the document H26-A2 of the Clinical and Laboratory Standards Institute (CLSI), intra-run imprecision, inter-run imprecision, linearity, carryover, autosampler evaluation, clinical sensitivity of the atypical lymphocytes flag (n = 400 samples) were included, as well as the comparison between automated and manual leukocyte differential count (n = 400 samples), based on an adaptation of the document H20-A2 of CLSI. Results: Repeatability, reproducibility, linearity and carryover were satisfactory according to the manufacturers specifications. The clinical sensitivity of the atypical lymphocytes flag showed efficiency, sensitivity and specificity of 92.5%, 65.2% and 94.1% respectively. The correlation coefficients between the automated and manual differential counts of neutrophils, lymphocytes, monocytes, eosinophils and basophils were 0.991, 0.99, 0.872, 0.974 and 0.557, respectively. Conclusions: The results were in accordance with quality specifications described in literature, indicating reliability in Sysmex® XE-2100D. This fact ensures certainty to both laboratory professionals and medical staff. We conclude that the Sysmex® XE-2100D showed excellent analytical performance, and is useful to provide reliable hematology data.

Evaluation of RDW-CV, RDW-SD, and MATH-1SD for the detection of erythrocyte anisocytosis observed by optical microscopy

INTRODUCTION AND OBJECTIVE: To evaluate the performance of red cell distribution width reported statistically as coefficient of variation (RDW-CV), standard deviation (RDW-SD), and mathematical deduction of 1 standard deviation (SD) around mean corpuscular volume (MATH-1SD) in identifying anisocytosis in automated blood counts when compared with the manual quantification of erythrocyte anisocytosis in peripheral blood smears. MATERIAL AND METHODS: 806 routine samples obtained from the hematology laboratory of Hospital de Clinicas da Universidade Federal do Parana (HC-UFPR) were analyzed. Performance evaluations were carried out by dividing samples into microcytic, normocytic and macrocytic mean corpuscular volume (MCV). For each MCV range, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and efficiency were calculated. In addition, the Youden index (Y) was obtained and a comparative analysis with receiver operating characteristic (ROC) curves was done to evaluate the performance of RDW-SD, RDW-CV, and MATH-1SD on different MCV ranges. RESULTS AND DISCUSSION: RDW-CV had the best sensitivity (86.8%) and efficiency (86.8%) in detecting anisocytosis in microcytic MCV ranges. RDW-SD and MATH-1SD were more sensitive and efficient in normocytic (82.9% and 83.3%; 92.1% and 92.3%, respectively) and macrocytic (90.2% and 90.2%; 95.1% and 95.1%, respectively) MCV ranges. A ROC curve analysis indicated that RDW-CV was more efficient in detecting anisocytosis in microcytic MCV ranges (p < 0.05 vs. RDW-SD and MATH-1SD). In normocytic and macrocytic MCV ranges, RDW-SD and MATH-1SD showed similar efficiency in detecting anisocytosis (p < 0.05 vs. RDW-CV). CONCLUSION: RDW-SD, RDW-CV, and MATH-1SD deliver different performances in detecting blood smear anisocytosis according to MCV values. They are parameters that complement each other and should be used together to identify erythrocyte size heterogeneity.

Evaluation of criteria of manual blood smear review following automated complete blood counts in a large university hospital

Background There is great interest in reducing the number of automated complete blood counts requiring manual blood smear reviews without sacrificing the quality of patient care. This study was aimed at evaluating and establishing appropriate screening criteria for manual blood smear reviews to improve the performance in a hematology laboratory. Method A total of 1977 consecutive samples from the daily workload were used to evaluate four sets of screening criteria for manual blood smear reviews to identify samples with positive smear findings. Three sets of screening criteria were arbitrarily proposed in this study: Group 1 (narrow ranges), Group 2 (intermediate ranges), and Group 3 (wide limits) and one set (Group 4) was adapted from the International Society for Laboratory Hematology. All samples were run on Sysmex hematology analyzers and were investigated using manual blood smear reviews. Diagnostic accuracy and agreement were performed for each set of screening criteria, including an investigation of microscopic review rate and efficiency. Results The microscopic review rates for Groups 1, 2, 3 and 4 were 73.85%, 54.52%, 46.33% and 46.38%, respectively; the false-negative rates were 0.50%, 1.97%, 2.73% and 3.95%, respectively. The efficiency and negative predictive values of Group 3 were 73.04% and 4.91%, respectively. Conclusions Group 3 had the best relationship between safety (false-negative rate: ≤3%) and efficiency to estimate the limits of automation in performing complete blood counts. This study strengthens the importance of establishing screening criteria for manual blood smear reviews, which account for the different contexts in which hematological determinations are performed. Each laboratory should optimize the screening criteria for manual blood smear reviews in order to maximize their efficiency and safety.

To follow or not to follow the recommendations regarding microscopic analysis of the Clinical and Laboratory Standards Institute H20-A2 to validate the criteria for blood smear review?

e read with great interest the Letter to the Editor by Grotto1 n the need to follow the Clinical and Laboratory Standards nstitute 2007 H20-A22 guidelines during microscopic analyis in reply to the study by Comar et al.3 We appreciate the omments that ensured extensive discussion on this subject. The work of Barnes et al.,4 which represents the core of he criteria for blood smear review (BSR) recommended by he International Society for Laboratory Hematology (ISLH), manates from an international consensus among 20 experts n 2002 during a conference in Indian Wells, CA, USA. In onsensus, Barnes et al.4 proposed a nine-step protocol for alidating the BSR criteria in routine laboratory practices. Step of this protocol is as follows: “Perform a slide review of all amples. Limit the reviews to only one or two senior techologists for consistency. Manual differentials should only be erformed if there is a specific need to do so (e.g., Vote out, bnormal cell-type flags, etc.)” The step-wise protocol by Barnes et al.4 does not mention he NCCLS H20-A5 as a sine qua non condition for the microcopic review of blood smears. In their work, Barnes et al.4 did ot mandate the application of the NCCLS H20-A5 guidelines, s interpreted by Grotto.1 Thus, Barnes et al.4 did not exclude he possibility of one observer counting 100 cells to validate he BSR criteria. We therefore understand that counting perormed by either one or two observers is equally acceptable. The CLSI H20-A22 (formerly NCCLS H20-A)5 is a reference ocument to evaluate hematology analyzers that perform utomated leukocyte differential counts and consider the isual leukocyte differential count as the gold standard. Most tudies that rigorously followed this guideline specifically valuated the automated leukocyte differential count and the uspect flags of the hematology analyzers.6,7 On the other and, studies evaluating sets of criteria for BSR did not necssarily follow the recommendations of the NCCLS H20-A5 or w?

Four cases of Chédiak-Higashi syndrome

Chediak-Higashi Syndrome (CHS) is a rare autosomal recessive disorder, with fewer than 500 cases published over the last 20 years.(1) The clinical features of this syndrome include partial oculocutaneous albinism, photosensitivity, grayish hair and skin,(1,2) severe recurrent bacterial infections, bleeding diathesis and neurological manifestations (central and peripheral neuropathies, sensory loss, muscle weakness, cerebellar ataxia and cognitive impairment).(1,3)

Evaluation of pleural and ascitic fluid analysis on the Sysmex XE-5000 hematology analyzer

Introduction: Body fluid (BF) analysis is critical to the diagnosis and monitoring of several pathological conditions. The limitations of manual cell counts have led to greater interest in the development of automated BF analysis. Objective: To evaluate the analytical performance of the Sysmex XE-5000 hematology analyzer in the analysis of pleural and ascitic fluids in the laboratory routine of a large university hospital. Methods: A total of 56 samples (35 ascitic and 21 pleural fluids) were analyzed by manual optical microscopy (OM) and XE-5000. Analytical performance includes linearity, carryover, functional sensitivity and comparison of patient samples. Results: Performance studies showed linearity up to 25,825 WBC-BF/μl (r = 0.999), WBC-BF showed carryover of 0.18%, and the lower limit of quantitation was set at 22 WBC-BF/μl. Good correlations between the methods were observed just for total cell (TC-BF) and white blood cell (WBC-BF) counts in pleural and ascitic fluids. The high-fluorescence cell count (HF-BF) showed poor correlation but high positive predictive value (PPV) for both fluids (94.74% for pleural and 96.97% for ascitic fluid). Conclusion: XE-5000 provides accurate and precise count for TC-BF, WBC-BF and polymorphonuclear cells (PMN-BF) in pleural and ascitic fluids in medical decision levels, but the morphological differentiation should continue to be held by OM. Histogram and scattergram displayed on XE-5000 must always be analyzed to assess if there is any interference or flag. The HF-BF parameter is a potential tool for screening.

Erratum to “Are the review criteria for automated complete blood counts of the International Society of Laboratory Hematology suitable for all hematology laboratories?” [Rev. Bras. Hematol. Hemoter. 2014;36(3):219–225]

In the article “Are the review criteria for automated complete blood counts of the International Society of Laboratory Hemato-logy suitable for all hematology laboratories?”, published in Rev Bras Hematol Hemoter. 2014;36(3):219–25, please consider thefollowing corrections:Page 221, Table 1, Criteria or rule 12 “>100×10