A Comparative Study of Erythrocyte Sedimentation Rate using Saline Diluted and Undiluted EDTA with TSC

Erythrocyte sedimentation rate (ESR) is the rate at which RBC sediment in a period of 1 hr. It is a common Haematology test that is a non-specific measure of inflammation and it became a common screening test worldwide for acute phase proteins and chronic diseases. The International Council for Standardization in Haematology (ICSH) recommended the Westergren method as the method of choice for ESR determination. TSC is almost exclusively used as the diluent of choice for setting up ESR, but some contemporary laboratories have resolved to use Normal saline (NS) as the diluent of choice while other set ESR using EDTA anticoagulated Blood (BLD) without any diluent. The objective of this study is to assess the comparison between saline diluted and undiluted EDTA with TSC as an anticoagulant in ESR detection and to find out any gender wise variations by using these anticoagulants. A total of 50 students were participated in this study. From each of the participants 5 ml of BLD was collected and it is then divided into 3 parts. 1.6 ml BLD to 0.4ml 3.8% TSC tube, 1.6 ml EDTA blood to 0.4 ml NS tube. 3rd tube with 2 ml EDTA BLD and set for ESR and obtained result within 1 hour. The Result is the mean +SD value of ESR were 19.48+5.7 mm/hr. in undiluted EDTA, 15.22+4.6 mm/hr. in saline diluted EDTA & 15.36+4.5 mm/hr. in TSC. The mean difference of ESR value between saline diluted EDTA with TSC BLD was 0 and it with undiluted EDTA was 4 mm /hr. The study indicates that there was a significant difference between ESR value with undiluted EDTA and TSC while diluted EDTA and TSC were there is no significant difference. The mean +SD of ESR value using undiluted, diluted EDTA and TSC in males were 16.20+3.3, 11.05+2.8, 11.29+2.6 and while it for females were 21.69+5.2, 17.36+3.8, 17.45+3.8 respectively. In conclusion, TSC is the best diluent to be used in contemporary lab to set ESR as compared to EDTA BLD. But we can use saline diluted EDTA as an alternative to citrate diluted BLD to set ESR and it also showed there is a gender wise variation in ESR using these anticoagulants.

Differential white blood cell counts in rabbits: a comparison of the Advia 2120 and a manual method

We evaluated the performance of the Advia 2120 (Siemens) differential leukocyte count (A-Diff) compared to the manual method (M-Diff) in rabbits. EDTA-anticoagulated blood samples collected for diagnostic purposes were analyzed within 6 h of collection. The M-Diff was performed blindly by 2 observers on blood smears by counting 200 cells. We initially included 117 samples; 25 samples were excluded because of suboptimal gating of leukocytes in the Advia peroxidase cytogram or poor blood smear quality. The correlation between the A-Diff and M-Diff was very high for heterophils (r = 0.924, p < 0.001) and lymphocytes (r = 0.903, p < 0.001), high for basophils (r = 0.823, p < 0.001), moderate for monocytes (r = 0.645, p < 0.001), and low for eosinophils (r = 0.336, p = 0.001). The Passing–Bablok regression analyses revealed a small-to-moderate constant error for lymphocytes and a slight constant error for basophils. Small proportional errors were detected for heterophils, lymphocytes, and eosinophils. The Bland–Altman analyses revealed that the Advia significantly underestimates heterophils and overestimates lymphocytes compared to M-Diff. The biases for the other leukocytes were minimal and likely clinical insignificant; however, our results, particularly for eosinophils, should be interpreted cautiously given the observed low percentages in our samples. Given the observed biases in heterophil and lymphocyte percentages in the Advia 2120 CBC results in rabbits, method-specific reference intervals should be used. The Advia can recognize leporine basophils. Evaluation of blood smears is still recommended to investigate abnormal results and erroneous cytograms reported by the Advia.

Systemic Lupus Erythematosus Progression and role of Genetic Variation in IL-22 and FOXP3 gene in population of Lahore, Pakistan

Systemic Lupus Erythematosus (SLE) is one of autoimmune disorders. It is thought that the deregulation in the inflammatory markers is due to problem in Forkhead box family member (FOXP3) which is involved in tolerance mechanism. One cannot ignore the role of cytokine-mediated signaling pathways like IL-22. This study was done in the Lahore. Pakistan. The main objective of the study was to monitor the patients of SLE. The purpose was to check the alliance of FoxP3 and IL-22 gene polymorphism. Sixty samples (n = 60) were collected from different hospitals of Lahore. DNA was extracted from EDTA anticoagulated blood of SLE patients. After DNA extraction, IL-22 and FoxP3 genes were polymerized through PCR and further sequenced through Sanger Sequencing method. The FOXP3 exon 2 and three SNPs in IL-2 i.e. rs2227491, rs2227485 and rs2227513 which were already identified were confirmed by Chromas 2.6. The mutations were checked with the help of Nucleotide Blast. Our observation showed that there are nine mutations in studied genotyped samples. The frequency of mutation was 27.27%. Allele T in rs2227485 and, allele C in rs2227513 and rs2227491 was identified in the study predominantly. These 9 mutations were found in case of IL-22 gene. No mutation was observed in Exon 2 of FOXP3 gene in SLE patients. It is concluded that that there may be any association between IL-22 gene polymorphism and SLE but FOXP3 gene was not tangled in the progression of SLE in Lahore population.

Investigation of pathological haemorrhage in Maine Coon cats

ObjectiveAfibrinogenaemic haemorrhage was previously reported in a Maine Coon cat. Two littermates subsequently died from surgical non-haemostasis, suggesting a hereditable coagulopathy.MethodsWe prospectively recruited cats which were: a) Maine Coons with pathological haemorrhage (group 1, n=8), b) healthy familial relatives of group 1 (group 2, n=13) and c) healthy Maine Coons unrelated to groups 1 and 2 (group 3, n=12). Coagulation tests: prothrombin time, activated partial thromboplastin time and thrombin clotting time (TCT) were performed on citrated plasma along with quantification of fibrinogen. Routine haematological examination was performed on EDTA-anticoagulated blood collected contemporaneously.ResultsThirty-three blood samples were analysed. Fibrinogen concentrations were significantly reduced in groups 1 (P<0.01) and 2 (P<0.01) compared with group 3. Similarly, TCT was found to be significantly extended in group 1 (P<0.01) and group 2 (P=0.02) with respect to group 3.ConclusionsDysfibrinogenaemia was identified in clinical cases and their healthy relatives, suggesting that this may represent a hereditary condition of Maine Coon cats. Clinicians should be aware of the increased potential for non-haemostasis in this cat breed and consider assessing clotting function before (elective) surgery.

Averrhoa bilimbi Extract as an Alternative Anticoagulant for Manual Complete Blood Count

This study was designed to determine whether Averroha bilimbi extract can be used as an alternative anticoagulant for manual complete blood count (CBC) in the hematology clinical laboratory instead of Ethylenediaminetetraacetic acid (EDTA), the recommended anticoagulant for CBC. Blood from 15 volunteers was extracted and placed in EDTA-anticoagulated tubes and tubes with Averrhoa bilimbi extract. Samples from both tubes were tested for CBC. Using independent t-test the study revealed that there is no difference in the red blood cell (RBC) count, white blood cell (WBC) count, hemoglobin, hematocrit, and a 3-part differential of EDTA anticoagulated blood and blood with Averrhoa bilimbi extract as anticoagulant. The morphology of lymphocytes and monocytes were not affected, however, the granulocytes showed cytoplasmic distortion and vacuolation in the Averrhoa bilimbi extract.

Abstract P139: Stability of DNA Methylation Profiles in Human DNA Samples in Long Term Storage

Introductions: Historic DNA samples represent a potentially highly valuable resource for epigenetic analysis. Reduced representation bisulfite sequencing (RRBS) is a cost-effective, near genome-wide method for quantifying DNA methylation levels at single base resolution. It is unknown whether historic DNA samples stored for a long time (15-20 years) under various conditions could maintain stable methylation profiles as determined by RRBS. Methods: We used 5 groups of DNA samples (n=4 in each group) to compare and evaluate the stability of DNA methylation profiles under standard storage conditions of 4°C since 1996. Group 1 had been extracted from EDTA-anticoagulated blood and stored at 4°C since 1996; Group 2 had been diluted from group 1 to 10ng/μl and stored at -20°C since 2009; Group 3 were extracted from citrate-anticoagulated blood stored at -80°C since 1996; Group 4 were extracted from fresh EDTA-anticoagulated blood; Group 5 were extracted from fresh citrate-anticoagulated blood. The samples in groups 1, 2 and 3 were from the same subjects. All DNA samples were processed for RRBS libraries and then sequenced. Results: The global methylation level of group 1 was lower than group 2 (36.88±4.12% vs 43.66±1.29%, p=0.04), but was not statistically different from group 3 (44.31±1.46%, p=0.07). The methylation level of groups 4 and 5 (40.34±3.73% and 38.64±1.94%) was not significantly different. In addition, group 4 and 5 were not significantly different from group 1. The correlation of methylation levels for all CpG sites between individual samples in group 1 (0.987±0.001) was higher than that in group 2 and group 3 (0.984±0.002, p=0.01, and 0.969±0.015, p=0.01) and not significantly different from group 4 or 5. For CpG sites within CpG islands (CGI), the correlation between individual samples was not significantly different between any of the 5 groups. Moreover, the samples from the same subject show high correlation levels between groups 1, 2, and 3 for all CpG sites (0.987±0.006) and CpG sites within CGI (0.983±0.009). Conclusion: DNA samples stored at 4°C for prolonged periods of time are amenable to RRBS analysis. In addition, there were no significant differences in the DNA methylation profiles between citrate- and EDTA-anticoagulated blood.

The effect of centrifugation speed and time on pre-analytical platelet activation

Background:The results of laboratory analyses are affected by pre-analytical variables, and in particular can platelets be activated by shear handling stress and secrete granular substances. We therefore evaluated the effect of centrifugation speed and time on pre-analytical platelet activation.Methods:Citrate- and EDTA-anticoagulated blood from healthy volunteers were centrifuged at 80–10,000Results:The median percentage of platelets expressing P-selectin in citrate- and EDTA-plasma centrifuged at 2000Conclusions:Proportional to centrifugation speed, platelets in plasma and platelet-rich plasma were activated with centrifugation speed, cell content and composition changed while platelet aggregation was unaltered.