An Overview of Complete Blood Count Sample Rejection Rates in a Clinical Hematology Laboratory Due to Various Preanalytical Errors.

Introduction The Chughtai Laboratory collects blood samples for complete blood counts from various hospitals, emergency departments, ICUs, and through home sampling services all across the country. The preanalytical phase is an integral component of laboratory medicine. A laboratory report has a key role in patient treatment and the clinician's decision in the management of the disease. Preanalytical errors are most frequently caused by the absence of a sample and/or inappropriate understanding of a test request, mislabeling, contamination from the sampling site, hemolyzed, clotted, insufficient samples, storage issues, and inappropriate blood to anticoagulant proportion or inappropriate choice of anticoagulant. Objective To identify the cause of rejection rates of the complete blood count samples and reduce the rejection rates by improving the accuracy of the results and lowering pre-analytical errors. Methods This cross-sectional study was done in the Hematology Department of Chughtai Laboratory's head office in Lahore between 19-06-2021 and 19-10-2021. Simple random sampling was applied to collect the data. About 3 ml of each blood sample was received in an ethylenediaminetetraacetic acid (EDTA) vial, inspected visually, run on Sysmex XN-9000 (Sysmex Corporation, Kobe, Hyogo, Japan), and was reviewed on peripheral smears. Results Out of 231,008 blood samples, 11,897 (5.15%) samples were rejected. The most common pre-analytical mistake was storage issues due to transportation delay (19.45%) followed by wrong medical records (19.16%), diluted samples (16.35%), incorrect tubes (16.01%), hemolyzed samples (15.13%), unlabeled samples (10.01%), and clotted sample (3.88%). Conclusion In the hematology department, the total rejection rate observed during the study period was 5.15%. Recognition of preanalytical errors and avoiding them will help us lower the sample rejection rate and raise the overall quality of laboratory management.

Frequency of Nucleated Red Blood Cells in the Peripheral Blood of ICU-Admitted Patients.

Background Nucleated red blood cells (NRBCs) are not normally found in the peripheral blood of normal healthy individuals. The presence of NRBCs on an adult peripheral blood smear indicates that there is an extremely high demand for the bone marrow to manufacture RBCs and that immature red blood cells are being released into the bloodstream. Anemia, myelofibrosis, thalassemia, miliary tuberculosis, malignancies of the bone marrow (myelomas, leukemias, lymphomas), and prolonged hypoxemia are a few possible pathogenic reasons. Critically ill patients who have NRBCs have a high mortality rate and a worse prognosis. OBJECTIVE: To evaluate the clinical significance of NRBCs in the peripheral blood of critically ill patients admitted to the ICU to find a cut-off to predict mortality. MATERIALS AND METHODS: A cross-sectional study was carried out over a period of six months September 1, 2020, to March 31, 2021, in Lahore, Pakistan. A total of 800 critically ill patients of both sexes in the age group of 18-70 years were included. Patients younger than 18 years and patients who underwent surgery were excluded. A quantity of 3 ml of whole blood sample in an ethylenediamine tetraacetic acid (EDTA) vial from each patient was run on SYSMEX XN-9000 (Sysmex Corporation, Kobe, Hyogo, Japan) and the results were reviewed on peripheral smears. RESULTS: The incidence of NRBCs in ICU-admitted patients was 62.5% (500/800). The total number of NRBC-positive patients recovering after the treatment was 364 (72.8%). The overall mortality of NRBC-positive patients was 30% (150/500). It was significantly higher (p<0.001) than that of NRBC-negative patients (14%; 44/300). During treatment, the highest mortality rate was seen in patients due to malignancy (100%), followed by sepsis (58.8%). It was observed that the disease pattern and number of NRBCs were significantly different (p<0.001) among all disease groups. However, there was no statistically significant difference in NRBCs on the basis of gender (p >0.05). In our study, a cutoff of NRBCs of 2.50 showed a high risk of mortality with a sensitivity of 91%. CONCLUSION: The presence of NRBCs may predict mortality in critically ill ICU-admitted patients. Their presence in the blood may be regarded as a marker of severity suggesting a high risk of ICU death.

Glanzmann Thrombasthenia in Pakistani Patients: Identification of 7 Novel Pathogenic Variants in the Fibrinogen Receptor αIIbß3.

Glanzmann thrombasthenia (GT) is a rare autosomal recessive inherited platelet disorder occurring frequently in populations with high incidence of consanguineous marriages. GT is characterized by quantitative and/or qualitative defect of the platelet αIIbß3 (GPIIb/IIIa) receptor caused by pathogenic variants of the encoding genes: ITGA2B and ITGB3. Patients present with a moderate to severe bleeding tendency with normal platelet count. Platelets show reduced/absent aggregation for all agonists except ristocetin in light transmission aggregometry and reduced/absent αIIbß3 expression in flow cytometry (FC). In this study, we investigated a cohort of 20 Pakistani patients and 2 families collected from the National Institute of Blood Disease, Karachi and Chughtai's Lab, Lahore. Platelet aggregation studies, FC (platelet CD41, CD61, CD42a, CD42b) and direct sequencing of the candidate genes were performed. All patients showed altered platelet aggregation, but normal agglutination after stimulation with ristocetin. Absent/reduced αIIbß3 receptor expression was present in the platelets of 16 patients, in 4 patients expression was borderline/normal. Candidate gene sequencing identified pathogenic/likely pathogenic variants in 15 patients. Seven variants are novel. One patient with absent receptor expression remained without genetic finding. 13 (86.7%) of 15 patients stated consanguinity reflected by homozygosity finding in 14 (93.3%) patients.

Relationship Between High-Density Lipoprotein-Cholesterol and Red Cell Distribution Width in Patients With Coronary Artery Disease.

Background A high red cell distribution width (RDW), which indicates ongoing inflammation, and low levels of high-density lipoprotein-cholesterol (HDL-C) are associated with increased mortality and morbidity in patients with coronary artery disease (CAD). Recent studies have suggested that HDL-C possesses anti-inflammatory and antioxidant effects, which may explain its anti-atherogenic properties. This study aims to determine the relationship between HDL-C levels and RDW in patients with CAD. Materials and methods This cross-sectional study was performed on 120 patients with CAD from July 2020 to June 2021 in the Hematology Department of Chughtai Lab Lahore. Patients were graded according to the degree of coronary artery stenosis as follows: Grade 1,30%-50%; Grade 2, 51%-70%; and Grade 3,>70%. The HDL-C level was measured from venous blood samples by a fully automated Abbot Alinity analyzer. The RDW was measured by Sysmex XN-5000. The sample size was calculated using the Select Statistics calculator. The mean RDW and HDL-C of the patients were calculated, and correlation analyses were performed using the Pearson correlation coefficient. Results The HDL-C level was inversely related to the RDW. Of the 120 patients, 38, 44, and 38 had Grade 1, Grade 2, and Grade 3 stenosis, respectively. The mean HDL-C level and RDW were 30.58 ±3.77 mg/dL and 16.04% ±1.66%, respectively. The value of r was -0.8622 (strongly negative). Data were stratified based on the degree of stenosis. The values of r in Grades 1, 2, and 3 were -0.43 (moderately negative), -0.604 (moderately negative), and -0.27 (weakly negative), respectively. Conclusion The RDW can be used as an additional marker to determine the disease status in CAD patients.

The Frequency of Aberrant CD7 Antigen Expression in Acute Myeloid Leukaemia Patients.

Background Aberrant phenotype expression in acute myeloid leukemia (AML) may be due to genetic defects and is associated with a poor prognosis. CD7 is the first T-cell-associated antigen to be expressed during T-lymphocyte maturation. Aberrant expression of CD7 in AML influences clinical response, remission rate, and overall survival in these patients. Objective To determine the frequency of aberrant CD7 expression in patients with AML. Materials and methods This cross-sectional study was performed over a period of 12 months from July 2020 to June 2021 in the Hematology Department, Chughtai Lab, Lahore. This study included 120 patients who were newly diagnosed with AML. The following tests were performed for included patients: complete blood count (CBC), peripheral blood smear analysis, and flow cytometric analysis using a blood sample or bone marrow aspirate. Blast cells were analyzed for aberrant CD7 expression. Calculation of the sample size was performed by using the Select Statistics calculator. All statistical analyses were performed using SPSS ver. 23 software (IBM Corp., Armonk, NY). Data were expressed as frequencies, means ± standard deviation (SD), and percentages. Results Of 120 patients newly diagnosed with AML, the CD7 antigen was aberrantly expressed in 36 cases (30%). Of these patients, the AML2 subtype was the most common type of AML with aberrant CD7 expression, followed by AML M4, AML M1, M3, AML M5, and AML M0, respectively. Conclusion In our study, aberrant CD7 expression occurred at a high frequency in acute myeloid leukemia. Thus, this marker should be added to the current flow cytometry panels.

Mutation Spectrum and Genotype-Phenotype Analyses in a Pakistani Cohort With Hemophilia B.

This study aimed to (1) identify F9 genetic alterations in patients with hemophilia B (HB) of Pakistani origin and (2) determine the genotype-phenotype relationships in these patients. Diagnosed cases of HB were identified through registries at designated tertiary health-care centers across the country. Consenting patients were enrolled into the study. The factor IX (FIX) coagulation activity (FIX:C) and key clinical features were recorded. Direct sequencing of F9 was carried out in all patients. All the variants identified were analyzed for functional consequences employing in silico analysis tools. Accession numbers from National Center of Biotechnology Information ClinVar database were retrieved for the novel variants. Genotype-FIX:C relationships were determined followed by FIX:C clinical phenotype assessment. A total of 52 patients with HB from 36 unrelated families were identified, which mainly comprised patients with moderate HB (n = 35; 67.3%). Among these, 35 patients from 22 unrelated families could be contacted and enrolled into the study. Missense variants were the most frequent (58.8%), followed by nonsense variants (17.6%). A missense, a short insertion, and a nonsense novel variants in exon 2, 6, and 7, respectively, were also identified. The disease manifested FIX:C heterogeneity in relation to the corresponding mutation in a significant number of cases. Clinical phenotype heterogeneity was also observed in relation to FIX:C-based severity assessment. We concluded that the registered FIX-deficient population of Pakistan mainly comprises moderate HB. F9 mutation spectrum in Pakistani patients with HB is heterogeneous. The HB population of Pakistan manifests a significant amount of genotype-FIX:C and FIX:C-clinical phenotype heterogeneities.

Autosomal recessive inherited bleeding disorders in Pakistan: a cross-sectional study from selected regions.

BACKGROUND: Autosomal recessive bleeding disorders (ARBDs) include deficiencies of clotting factors I, II, V, VII, X, XI, XIII, vitamin K dependent clotting factors, combined factor V & VIII, Von Willebrand Disease (vWD) type 3, Glanzmann's thrombasthenia (GT) and Bernard-Soulier syndrome. Patients with primary bleeding disorders from all the major provincial capitals of Pakistan were screened for ARBDs. Prothrombin (PT), activated partial thromboplastin time (APTT), bleeding time (BT) and fibrinogen levels were measured. Cases with isolated prolonged APTT were tested for factors VIII and IX using factor assays This was followed by FXI:C level assessment in cases with normal FVIII and FIX levels. vWD was screened in patients with low FVIII levels. Factors II, V and X were tested in patients with simultaneous prolongation of PT and APTT. Peripheral blood film examination and platelet aggregation studies were performed to assess platelet disorders. Urea clot solubility testing was done to detect Factor XIII levels where platelet function tests were normal. Descriptive analysis was done using SPSS version 16. RESULTS: Of the 429 suspected bleeding disorder patients, 148 (35%) were diagnosed with hemophilia A and 211 (49.1%) patients had ARBDs. 70 patients (16.3%) remained undiagnosed. Out of 211 patients with ARBD; 95 (33.8%) had vWD type 3. Fibrinogen deficiency was found in 34 patients (12%), GT in 27 (9.6%), factor XIII deficiency in 13 (4.6%), factor VII deficiency in 12 (4.3%), factor V deficiency in 9 (3.2%). Eight patients (2.8%) had vitamin K-dependent clotting factor deficiency, Bernard-Soulier syndrome was diagnosed in seven patients (2.5%), factor X deficiency in 2 (0.7%), factor II deficiency in 2 (0.7%), factor XI deficiency and combined factor V and VIII deficiency in 1 (0.4%) patient each. CONCLUSION: vWD type 3 was the most common ARBD found in our sample of patients in Pakistan, followed by fibrinogen deficiency and GT in respective order.