Impact of Staining Methods and Human Factors on Accuracy of Manual Reticulocyte Enumeration.

Although peripheral blood reticulocyte enumeration reflects bone marrow functional integrity, which is important for differential diagnosis of hematological diseases, the factors affecting its accuracy have not been adequately addressed. Using 100 consecutive venous blood samples being processed with four supravital staining techniques [i.e., brilliant cresyl blue (BCB), new methylene blue (NMB), and BCB/NMB with Liu's stain] for reticulocyte enumeration, two technologists (senior vs. junior) conducted microscopic counting. The results were compared with those obtained with an automated system (Sysmex XE-5000) that served as the standard. The aims of this study were to identify (1) the technique that gave the most reliable outcome, and (2) possible human factors (i.e., seniority, repeated counting) that may affect the counting results. Analysis showed least bias (i.e., deviation from automated counting) associated with BCB staining, followed by NMB. In addition, the senior observer exhibited a higher bias in counting compared with their junior counterpart. Repeated counting also correlated with a higher rate of bias. Nevertheless, inter-observer consistency was high (intraclass correlation coefficient >0.95) and inter-/intra-observer variations were non-significant (both p > 0.05). Our results supported the use of BCB stain for reticulocyte enumeration and the reliability of manual counting despite the involvement of human factors, which had negligible impacts on the final outcomes.

Differential impacts of hemolysis on coagulation parameters of blood samples: A STROBE-compliant article.

ABSTRACT: This study aimed at investigating the impact of hemolysis on different coagulation parameters.A total of 216 venous blood samples without visible hemolysis were collected from adult patients at a tertiary referral center over six months. The plasma obtained was quantified for six coagulation parameters including prothrombin time, activated partial thromboplastin time, fibrinogen, D-dimer, antithrombin III, and protein C. The rest of the plasma from each blood sample was aliquoted into three tubes, each containing 1 mL of plasma with three different volumes of cell-free hemoglobin (i.e., 2, 4, 8 µL) from lysed RBCs to create simulated hemolyzed blood samples with hemoglobin concentration of approximately 0.1, 0.2, and 0.4 g/dL to mimic mild (1+), moderate (2+), and severe (3+) hemolysis, respectively, before repeating the coagulation tests to determine possible correlation between the simulated degree of hemolysis and the changes in test results of the coagulation parameters.Spearman correlation analysis showed significant decreases in the values of activated partial thromboplastin time, fibrinogen, D-dimer, and protein C values with an increasing degree of simulated hemolysis (all P < .01). Comparison of the percentage bias of biological variance showed significant positive associations of cell-free hemoglobin concentrations with the percentage bias of D-dimer and protein C. However, only the former was still within the range of biological variance under condition of simulated hemolysis. Besides, the presence of cell-free hemoglobin regardless of concentration had a notable impact on the percentage bias of activated partial thromboplastin time, whereas the influence was non-significant for prothrombin time, fibrinogen, and antithrombin III.The results showed different impacts of simulated hemolysis on six coagulation parameters, highlighting the dependence of clinical reliability on the coagulation parameter to be investigated in hemolytic blood samples.

Urinary Metabolomics Study of the Intervention Effect of Hypoglycemic Decoction on Type 2 Diabetes Mellitus Rats Model.

The hypoglycemic decoction (HD) is a traditional Chinese medicine (TCM) preparation for the treatment of diabetes mellitus (DM), with a remarkable therapeutic effect. However, its mechanism of action is still unclear at the metabolic level. In this study, the biochemical markers from type 2 DM (T2DM) rats, induced by a high-sugar and high-fat diet combined with streptozotocin (STZ), were detected. The metabolomics-based analysis using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) was conducted to evaluate urine samples from control, model, metformin, and HD groups. After oral administration of HD for 28 days, the general state, weight, fasting blood glucose (FBG), blood lipid level, oral glucose tolerance test (OGTT), fasting insulin (FINS), insulin sensitivity index (ISI), and homeostasis model assessment of insulin resistance (HOMA-IR) were significantly improved (P < 0.01). The western blotting showed that HD can enhance the protein expression of glucose transporter 4 (GLUT4) and adenosine monophosphate-activated protein kinase (AMPK). The metabolomics results revealed that after treatment with HD, the levels of L-carnitine, 1-methyladenosine, 1-methylhistamine, and 3-indoleacrylic acid were upregulated and the levels of riboflavin, phenylalanine, atrolactic acid, 2-oxoglutarate, citrate, isocitrate, cortisol, and glucose were downregulated. The main mechanism may be closely related to the regulation of the tricarboxylic acid (TCA) cycle, phenylalanine metabolism, glyoxylate metabolism, and dicarboxylate metabolism. Additionally, it was also found that HD can regulate the protein expression of GLUT4 and AMPK to interfere with TCA cycle and carbohydrate metabolism to treat T2DM.

Genetic disposition and modifiable factors independently associated with anemia in patients with type 2 diabetes mellitus.

AIMS: Anemia is prevalent but under-recognized in patients with diabetes mellitus (DM). Genetic variants in angiotensin-converting enzyme (ACE), tumor necrosis factor-alpha (TNF-α) and erythropoietin (EPO) have been associated with diabetic nephropathy. In the present study, we investigated the associations between anemia and polymorphisms in EPO promoter (rs1617640), TNF-α G-308A and ACE Insertion/Deletion in Chinese patients with type 2 diabetes. METHODS: Polymorphisms in ACE, TNF-α and EPO were genotyped in 1142 patients. Anemia was defined as hemoglobin (Hb) levels below 12 g/dL for women and 13 g/dL for men. RESULTS: 286 (25%) patients had anemia. Patients with anemia were older, had longer duration of diabetes, worse renal function and more albuminuria. ACE Insertion/Deletion and TNF-a G-308A were not associated with anemia. The frequencies of EPO polymorphism (rs1617640) were significantly different between anemic and nonanemic patients. Patients with TT genotype had higher prevalence of anemia than those with TG and GG. Regression analysis identified EPO SNP, duration of DM, serum albumin, albuminuria and renal function independently associated with anemia. After adjusting for multiple variables, TT and TG genotypes were associated with 3-5-fold increased risk for anemia compared to GG. CONCLUSIONS: The EPO genotype in Chinese patients with type 2 diabetes is associated with anemia and may help to identify those at risk. Further evaluation of its effect on clinical outcomes in prospective studies may be useful to predict the outcomes of erythropoiesis stimulating therapy, and to individualize anemia management.

Enhanced accuracy of optical platelet counts in microcytic anemia.

OBJECTIVE: To determine whether microcytic erythrocytes influence the accuracy of automated platelet (PLT) counting. METHODS: We divided a total of 206 K2 ethylenediaminetetraacetic acid (EDTA)-anticoagulated blood samples into 4 groups, as follows: In group 1 (control group), normal mean corpuscular volume (MCV > 80 fL) and PLT count equal or greater than 140,000/microL (n = 45); group 2, normal MCV, reduced PLT count (< 140 x 10(3)/microL, n = 41); group 3, microcytic samples with normal PLT count (n = 68); and group 4, microcytic samples with reduced PLT count (n = 49). We also compared the platelet counting using electroimpedance (PLT-EI), platelet count using fluorescent optical (PLT-FO), and platelet-count manual (PLT-M) methods, using the Sysmex XE 2100 automatic analyzer. RESULTS: Despite highly significant overall correlations between PLT-EI and PLT-FO, PLT-EI and PLT-M, and PLT-FO and PLT-M (r = 0.95 [all P < .001]), use of the PLT-EI method resulted in widely overestimated PLT counts in microcytic samples (MCV < 80 fL), compared with use of PLT-FO and PLT-M. Our results identify an MCV of 70 fL as the critical threshold below which PLT-EI became unreliable. CONCLUSION: The PLT-EI mode overestimated PLT counts compared with PLT-FO and PLT-M modes in microcytic blood. Therefore, PLT-FO is the preferred method for PLT counting in patients with microcytic anemia when using an automated analyzer.

A large-scale gene discovery for the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae).

The red palm weevil (RPW; Rhynchophorus ferrugineus) is a devastating pest of palms, prevalent in the Middle East as well as many other regions of the world. Here, we report a large-scale de novo complementary DNA (cDNA) sequencing effort that acquired ∼5 million reads and assembled them into 26 765 contigs from 12 libraries made from samples of different RPW developmental stages based on the Roche/454 GS FLX platform. We annotated these contigs based on the publically available known insect genes and the Tribolium castaneum genome assembly. We find that over 80% of coding sequences (CDS) from the RPW contigs have high-identity homologs to known proteins with complete CDS. Gene expression analysis shows that the pupa and larval stages have the highest and lowest expression levels, respectively. In addition, we also identified more than 60 000 single nucleotide polymorphisms and 1 200 simple sequence repeat markers. This study provides the first large-scale cDNA dataset for RPW, a much-needed resource for future molecular studies.

Clinical interpretation of reticulocyte hemoglobin content, RET-Y, in chronic hemodialysis patients.

BACKGROUND: Iron deficiency is the most common factor associated with erythropoietin (EPO) hyporesponsiveness. Current iron indices are inadequate to demonstrate the status or utility of iron in erythropoiesis. The aims of this study are to investigate the value of the reticulocyte hemoglobin content, RET-Y, in hemodialysis (HD) patients and compare the levels with conventional iron indices. METHODS: HD patients (n = 289) were divided into 4 groups according to serum ferritin (cutoff value 100 ng/ml) and transferrin saturation (TSAT, cutoff value 20%). The RET-Y value, hemogram and biochemical data were determined and compared between groups. Factors associated with RET-Y were examined. RESULTS: The mean RET-Y value was 1,716 +/- 125 AU. Patients with absolute iron deficiency had lower RET-Y levels and mean corpuscular volume (MCV). Patients with functional iron deficiency had a lower reticulocyte production index and serum albumin levels. MCV, mean corpuscular hemoglobin concentration (MCHC) and albumin were independently correlated with the RET-Y level (all p < 0.001). EPO-independent patients had low iron indices and low RET-Y levels, but a higher reticulocyte production index and albumin levels were noted. CONCLUSION: RET-Y levels in HD patients were close to that of the normal population. Low RET-Y levels were observed in patients with absolute iron deficiency and also in EPO-independent patients with low ferritin and low TSAT. There was a strong association between the serum albumin and RET-Y levels in chronic HD patients.

Usefulness of brilliant cresyl blue staining as an auxiliary method of screening for alpha-thalassemia.

Hemoglobin (Hb) electrophoresis is widely used in thalassemia screening. Most Hb variants express a specific abnormal band on the cellulose acetate membrane. The technique is useful in the diagnosis of the type of thalassemia but is not sensitive enough to detect alpha-thalassemia minor because the quantity of the HbH is too small to be expressed on the supporting medium. We used simple staining of blood smears rather than the sophisticated molecular method to detect HbH inclusions. To evaluate the effectiveness of this method, we used brilliant cresyl blue (BCB) staining of red blood cells in 509 patients with microcytosis and erythrocytosis caused by various conditions. The results indicate that BCB staining is useful in the identification of subjects who possess alpha-thalassemia traits. Coexisting conditions such as beta-thalassemia and iron-deficiency anemia did not affect the detection of the HbH inclusions with the use of BCB staining. We conclude that BCB staining is helpful and reliable as an auxiliary method of detecting HbH inclusions in the diagnosis of alpha-thalassemia traits, especially in places where medical resources are limited.

Estrogen receptor beta is not increasingly expressed in leiomyoma nodules which show no progressive enlargement in premenopausal women.

BACKGROUND AND PURPOSE: Studies on the expression of estrogen receptors (ERs) in uterine leiomyomata have yielded conflicting results. A controlled study focusing on leiomyomal nodules with same clinical presentations and hormonal milieu was conducted to investigate the role of ER beta (ERbeta) on leiomyomal growth. METHODS: Thirty pairs of leiomyomal and myometrial tissues were included in the study. The specimens were obtained from 30 premenopausal women (40-45 years old) with parity 2, and were sampled during the proliferative menstrual phase. All of the leiomyomal nodules were unifocal, medium sized (4-5 cm), intramurally located, and without progressive enlargement for 6 months. The expressions of ERbeta mRNA and protein in leiomyomata and their adjacent myometria were analyzed by reverse transcriptase-polymerase chain reaction and immunohistochemistry using ERbeta-specific antibody, respectively. ER alpha (ERalpha) was simultaneously studied using similar methods. The results were digitally quantified and compared with each other. RESULTS: All paired tissues (30/30, 100%) showed a higher ERalpha expression level (paired t test, p < 0.05). In most leiomyomal nodules (27/30, 90%), both ERbeta and ERalpha exhibited no differences in mRNA levels compared to their adjacent myometria (p > 0.05), but the ratio of ERbeta/ERalpha (expression index) was lower (p < 0.05) in leiomyomal tissue. Although the immunoreactivity scores for ERbeta were similar in paired tissues, more ERbeta-positive cells were detected in myometrial specimens than in their leiomyoma counterparts (mean of 62% vs 56%). Furthermore, the distribution pattern of ERbeta in leiomyoma (nuclear and cytoplasmic) was different from that in the corresponding myometrium (nuclear). The lower ERbeta/ERalpha expression index in stationary leiomyoma suggests that the relative abundance of ERbeta and ERalpha, rather than their individual amounts, determines the existence and development of leiomyoma. CONCLUSIONS: This study demonstrated that in premenopausal women, ERbeta is not increasingly expressed in the leiomyomal nodules which show no progressive enlargement. The distinctive ERbeta-to-ERalpha ratio rather than their individual amounts, is associated with the stationary status of the leiomyomal nodule.