Clinico-Haematological Profile Of Chronic Lymphoproliferative Disorders In Patients Presenting With Lymphocytosis.

Objectives: To assess the spectrum and clinico-haematological profile of chronic lymphoproliferative disorders in patients presenting with lymphocytosis. METHODS: The cross-sectional, retrospective study was conducted at the Aga Khan University Hospital, Karachi, and comprised data related to cases of bone marrow aspirate and trephine from January to November 2020. Patients for whom the bone marrow was done for lymphocytosis were studied for the presence of lymphoproliferative disorders, sub-types and patients'characteristics. The diagnosis and classification were based on the World Health Organisation criteria for tumours of haematopoietic and lymphoid tissues. Data was analysed using SPSS 21. RESULTS: Of the 3,334 bone marrow specimenstested, 103(3%) were related to lymphocytosis. Of these, 84(82%) were diagnosed with lymphoproliferative disorders, while diagnosisremained undetermined in 19(18%) cases. Male:female ratio was 3.6:1 and median age was 60 years (range: 21-85 years). Constitutional symptoms were found in 61(73%) patients. Median absolute lymphocyte count was 45x109/L (range: 5.3-480). All 84(100%) patients were classified as B-cell lymphoproliferative disorder. Chronic lymphocytic leukaemia wasthe most common form, 61(73%), and 31(51%) of them presented with advanced stage disease. CONCLUSIONS: A huge majority of patients presenting with lymphocytosis had underlying lymphoproliferative disorders of which B-cell chronic lymphocytic leukaemia was found to be the most common.

Plasmodium in the bone marrow: case series from a hospital in Pakistan, 2007-2015.

BACKGROUND: Malaria is a life-threatening, multisystem disease caused by the plasmodial parasite with a global incidence of approximately 229 million annually. The parasites are known to have unique and crucial interactions with various body tissues during its life cycle, notably the liver, spleen, and recent work has shown the bone marrow to be a reservoir of infection. METHODS: This study is a case series of patients in whom examination of bone marrow revealed malarial parasites. A retrospective record review of 35 parasite-positive bone marrow specimens examined at Aga Khan University Hospital (AKUH), Karachi, Pakistan, over the years 2007 to 2015 was conducted. Bone marrow aspirates were collected as per International Council for Standardization in Haematology (ICSH) guidelines. RESULTS: The median age of patients was 22 years (range 1-75), and 60 % (n = 21) were male. 22 patients had evidence of Plasmodium falciparum, 12 had evidence of Plasmodium vivax and 1 patient had a mixed infection. Gametocytes and trophozoites were the most common stages identified on both peripheral blood and bone marrow examinations. Indications for bone marrow examination included fever of unknown origin and the workup of cytopenias and malignancies. CONCLUSIONS: The incidental finding of Plasmodium in samples of bone marrow suggests the reticuloendothelial system may be regularly harbour these parasites, be the infection acute or chronic in character.

Better get moving on laboratory quality assurance.

PURPOSE: With recent advances in laboratory hematology automation, emphasis is now on quality assurance processes as they are indispensable for generating reliable and accurate test results. It is therefore imperative to acquire efficient measures for recognizing laboratory malfunctions and errors to improve patient safety. The paper aims to discuss these issues. DESIGN/METHODOLOGY/APPROACH: Moving algorithm is a quality control process that monitors analyzer performance from historical records through a continuous process, which does not require additional expenditure, and can serve as an additional support to the laboratory quality control program. FINDINGS: The authors describe an important quality assurance tool, which can be easily applied in any laboratory setting, especially in cost-constrained areas where running commercial controls throughout every shift may not be a feasible option. ORIGINALITY/VALUE: The authors focus on clinical laboratory quality control measures for providing reliable test results. The moving average appears to be a reasonable and applicable choice for vigilantly monitoring each result.

Performance evaluation of a coagulation laboratory using Sigma metrics.

Purpose Two-thirds of medical decisions are based on laboratory test results. Therefore, laboratories should practice strict quality control (QC) measures. Traditional QC processes may not accurately reflect the magnitude of errors in clinical laboratories. Six Sigma is a statistical tool which provides opportunity to assess performance at the highest level of excellence. The purpose of this paper is to evaluate performance of the coagulation laboratory utilizing Sigma metrics as the highest level of quality. Design/methodology/approach Quality indicators of the coagulation laboratory from January 1, 2009, to December 31, 2015, were evaluated. These QIs were categorized into pre-analytical, analytical and post-analytical. Relative frequencies of errors were calculated and converted to Sigma scale to determine the extent of control over each process. The Sigma level of 4 was considered optimal performance. Findings During the study period, a total of 474,655 specimens were received and 890,535 analyses were performed. These include 831,760 (93.4 percent) routine and 58,775 (6.6 percent) special tests. Stat reporting was requested for 166,921 (18.7 percent). Of 7,535,146 total opportunities (sum of the total opportunities for all indicators), a total of 4,005 errors were detected. There were 2,350 (58.7 percent) pre-analytical, 11 (0.3 percent) analytical and 1,644 (41 percent) post-analytical errors. Average Sigma value obtained was 4.8 with 12 (80 percent) indicators achieving a Sigma value of 4. Three (20 percent) low-performance indicators were: unacceptable proficiency testing (3.8), failure to inform critical results (3.6) and delays in stat reporting (3.9). Practical implications This study shows that a small number of errors can decrease Sigma value to below acceptability limits. If clinical laboratories start using Sigma metrics for monitoring their performance, they can identify gaps in their performance more readily and hence can improve their performance and patient safety. Social implications This study provides an opportunity for the laboratorians to choose and set world-class goals while assessing their performance. Originality/value To the best of the authors' knowledge and belief, this study is the first of its kind that has utilized Sigma metrics as a QC tool for monitoring performance of a coagulation laboratory.

Use of ISTH bleeding assessment tool to predict inherited platelet dysfunction in resource constrained settings.

BACKGROUND: The International Society of Thrombosis & Hemostasis (ISTH) bleeding assessment tool (ISTH-BAT) is used to record bleeding symptoms in patients with possible bleeding disorders. AIM: To investigate the utility of the ISTH-BAT in predicting platelet dysfunction in individuals with suspected inherited platelet function disorders. METHOD: Individuals with clinical evidence of bleeding and suspected inherited platelet function disorder and healthy volunteers were included in the study. The ISTH-BAT questionnaire was applied prior to light transmission aggregometry (LTA). RESULTS: A total of 261 participants were included (100 healthy volunteers, and 161 with suspected inherited platelet function disorders). The ISTH-BAT score in participants with suspected inherited platelet function disorders (median 2; interquartile range [IQR] 5-1) was significantly higher than in healthy volunteers (median 0; IQR 2-0). There was also a significant difference between participants with suspected inherited platelet function disorders with a platelet defect detected by LTA (median 4; IQR 8-3) and those with normal platelet function (median 2; IQR 3-1) (p < 0.001). The ISTH-BAT score was associated with a demonstrable platelet defect on platelet function testing (area under the receiver operating characteristic curve = 0.8 [95% confidence interval 0.72-0.87, p = < 0.001] and odds ratio 3.25 [95% confidence interval 2.13-4.37, p = < 0.001]). CONCLUSION: The ISTH-BAT is a useful tool for documenting bleeding symptoms and the score obtained is also predictive of the presence of a platelet defect on LTA in patients with suspected inherited platelet dysfunction.

Clinico-Pathological Profile And Outcomes Of Patients With Polycythaemia Vera, Essential Thrombocythaemia And Idiopathic Myelofibrosis: A Tertiary Care Center Experience From Southern Pakistan.

BACKGROUND: The "Philadelphia Negative Classic Myeloproliferative Neoplasms" include polycythaemia vera (PV), essential thrombocythaemia (ET) and idiopathic myelofibrosis (IMF). These three disorders share several clinical and laboratory features including JAK2 V617F mutation. Our objectives were to determine the clinico-pathological profile and outcomes of Pakistani patients with polycythaemia vera (PV), essential thrombocythaemia (ET) and idiopathic myelofibrosis (IMF) in order to have an insight regarding behaviour of these conditions. METHODS: A retrospective analysis of all the cases of PV, ET and IMF diagnosed at our institute from January 1995 to December 2013 was performed. Age, gender, clinical presentation, laboratory investigations, treatment provided and duration of follow-up were included for analysis. Appropriate statistics were utilized for calculation of data. RESULTS: A total of 58 patients were diagnosed as PV, ET or IMF during the study period. Male to female ratio was 1.1:1. Forty five percent (n=27) patients came to medical attention due to abnormal laboratory results, 3 had cerebrovascular events, 3 had pruritus, and 1 patient each with gangrene and Budd-Chiari syndrome. Haemorrhage was not seen in any patient. Sixty percent (n=35) patients were treated with phlebotomy, hydroxyurea and aspirin alone or in combination. None of the patients transformed to myelofibrosis (MF) or myelodysplasia (MDS) during the mean (±SD) follow-up period of 57.2±50 months. One patient with ET transformed to acute myeloid leukaemia 9 years after the diagnosis. CONCLUSIONS: This study demonstrated a relatively more benign form of PV, ET and IMF with lesser frequency of symptoms, good response to treatment and less likelihood of transformation to MF, MDS or AML.

Digital Morphology: Bridging the Final Gap in Automated Haematology Testing.

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Data mining for prothrombin time and international normalized ratio reference intervals in children.

Reference intervals (RIs) help physicians in differentiating healthy from sick individuals. The prothrombin time (PT) and International normalized ratio (INR) fluctuate in coagulation pathway defects and have interlaboratory variability due to the instrument/reagent used. As direct method is difficult in children, we chose an indirect data mining method for the determining PT/INR RIs. The indirect method overcomes the substantial financial and logistic challenges, and ethical restrictions in children, moreover, allows partitioning in more fine-grained age groups. Prothrombin Time/INR measurements performed in patients aged birth-18 years between January 2013 and December 2020, were retrieved from laboratory management system of the Aga Khan Hospital. Reference intervals were computed using an indirect KOSMIC algorithm. The KOSMIC package function on the assumption that the non-pathologic samples follow a Gaussian distribution (after Box-Cox transformation of the data), following an elaborate statistical process to isolate distribution of physiological samples from mixed dataset. A total of 56,712 and 52,245 values were retrieved for PT and INR respectively. After the exclusion of patients with multiple specimens obtained during the study period, RIs were calculated for 37,356 (PT) and 37,192 (INR) children with stratification into 9 age groups. A comparison of 2.5th and 97.5th percentile results with those of established RIs from SickKids Handbook of Pediatric Thrombosis and Hemostasis demonstrated good agreement in between different age groups. This study supports data mining as an alternate approach for establishing PT/INR RIs, specifically in resource-limited settings. The results obtained are specific to studied population and instrument/reagent used. The study also allows understanding of fluctuations in coagulation pathways with increasing age and hence better clinical decision-making based on PT and INR results.

Establishment of Population Specific Reference Intervals in Healthy Pakistani Adults for 21 Routine and Special Haematology Analytes.

Background: The reference interval (RI) is an interval between two limits derived from distribution of the results obtained from a sample of the reference population. These population based RIs are of paramount significance for the accurate clinical understanding of the patient's health status. Haematological RIs are heavily influenced by a variety of geographical and environmental factors. Therefore, accrediting bodies also mandate that each laboratory should establish its own RIs in its own population. Methods: This cross-sectional study was conducted at the Department of Pathology and Laboratory Medicine, the Aga Khan University Hospital, Pakistan.Twenty-one routine and special quantitative analytes were measured in adults aged 18-60 years who passed the initial health screening questionnaire. All samples were handled strictly following standard operating procedures. Microsoft Excel and EP Evaluator software were used for statistical analysis. Nonparametric CLSI EP28-A3C method was used to establish upper and lower confidence limits at 90% significance. Results: A total of 323 participants passed the questionnaire and were short-listed for blood collection. There were 147 males and 176 females. Reference intervals were established in 297 participants after exclusion of 26 outliers with grossly abnormal test results. Analytes included: 8 red, and 12 white blood cell parameters, platelet count, immature platelet fraction, erythrocyte sedimentation levels, haemoglobin A and A2 levels and glucose-6-phosphatase dehydrogenase levels. Conclusion: Routine and special haematology RIs established in this study reflect significant differences from RIs in Caucasian population. For meaningful interpretation of test results, each haematology laboratory should establish or verify RIs in the population it serves.

Flow Cytometric Analysis of ZAP-70 Protein Expression for B-Cell Chronic Lymphocytic Leukemia Prognostication: Usefulness and Limitations.

OBJECTIVES: The heterogenous clinical course in B-cell chronic lymphocytic leukemia (B-CLL) can be linked to several genetic and phenotypic characteristics of malignant B-cells. Prognostic analysis in B-CLL is routinely carried out to assist patient management; particularly to predict the time to initiate treatment. Increased ZAP-70 expression is a surrogate marker for unmutated immunoglobulin genes and inferior clinical outcomes which can be quantified to predict future outcomes in B-CLL patients. The study determined the ZAP-70 expression pattern using Z-index in Pakistani patients with B-CLL. METHODS: A retrospective analysis of B-CLL cases diagnosed and confirmed on flow cytometry at Aga Khan University Hospital for the last six years which had also undergone ZAP-70 analysis were included. In all these cases, ZAP-70 expression was quantified by measuring mean fluorescence intensities (MFIs) of normal B-cells, T-cells, and CLL-cells (CD19 and CD5 double-positive population). ZAP-70 expression was divided into high, low, and negative categories based on Z-index calculation. Mann-Whitney U test was utilized to determine the significance of ZAP-70 variations in different age groups and genders. P-value <0.05 was considered significant. RESULTS: A total of 120 patients of B-CLL had ZAP-70 analysis during the study period. The median age was 62 with an interquartile range of 35-87 and male to female ratio of 2:1. ZAP-70 expression was high in 18 (15%), low in 52 (43.3%) and negative in 50 (41.7%) cases. No significant difference in ZAP-70 expression with respect to the age or gender of the study population was identified using appropriate statistical calculations. CONCLUSIONS: This study showed only 15% of B-CLL cases showing high ZAP-70 expression, a surrogate biomarker for possible aggressive behavior which may necessitate therapeutic intervention and close surveillance.

Disseminated histoplasmosis in an immuno-competent young male: Role of bone marrow examination in rapid diagnosis.

Fungal infections are usually seen in elderly or immuno-compromised individuals particularly with human immunodeficiency virus infection. In immuno-competent individuals, they seldom present with overt clinical symptoms. In such cases diagnosis is made by combination of tests along with direct microscopic visualization of the organism. We present a case of immuno-competent individual who presented with unexplained fever and found to have Histoplasma capsulatum infection on bone marrow examination.

Distribution of Chromosomal Abnormalities Commonly Observed in Adult Acute Myeloid Leukemia in Pakistan as Predictors of Prognosis

Objectives: The heterogenous response to treatment in acute myeloid leukemia (AML) can be attributed largely to the difference in cytogenetic features identified in between cases. Cytogenetic analysis in acute leukemia is now routinely used to assist patient management, particularly in terms of diagnosis, disease monitoring, prognosis and risk stratification. Knowing about cytogenetic profile at the time of diagnosis is important in order to take critical decisions in management of these patients. The study was conducted to determine the distribution of cytogenetic abnormalities in Pakistani adult patients with AML in order to have insights regarding behavior of the disease. Methods: A retrospective analysis of all the cases of AML (≥15years old) diagnosed at Aga Khan University from January 2011 to December 2016 was performed. Cytogenetic analysis was made for all cases using the trypsin-Giemsa banding technique. Karyotypes were interpreted using the International System for Human Cytogenetic Nomenclature (ISCN) criteria. Results: A total of 321 patients were diagnosed with AML during the study period, of which 288 samples successfully yielded metaphase chromosomes. The male to female ratio was 1.7:1. A normal karyotype was present in 61% (n=176) of the cases whereas, 39% (n=112) had an abnormal karyotype. Of the abnormal cases, t (8;21) (q22;q22) and t (15;17) (q22;q12) were identified in 8.3% and 4.9% cases respectively. Adverse prognostic cytogenetic subgroups including complex karyotype, monosomy 7 and t(6;9)(p23;q34) were identified in 9%, 1% and 0.7% patients respectively. Conclusions: This largest cytogenetic data in adult AML from Pakistan showed comparable prevalence of favorable prognostic karyotype to international data. The prevalence of specific adverse prognostic karyotype was low.

Assessment of WT1 Expression as a Marker of Treatment Outcome in Karyotype Normal Acute Myeloid Leukemia Patients in Pakistan.

Currently, there is an effort to predict relapse by follow-up monitoring of MRD and subsequently to begin the treatment of the patients during their clinical and hematological remission prior to overt hematological relapse. Expression of WT1 in AML is known to be independently associated with significant inferior response to therapy and short survival outcome. Follow-up monitoring of WT1 gene expression during or after therapy would be a valuable predictive marker for early recurrence or relapse of AML disease. This pilot study evaluated newly diagnosed and post-induction or consolidation chemotherapy of AML patients who were registered with the Oncology Clinics of the Aga Khan University Hospital, Karachi. High WT1 burden (> 5000 copies/ml) in 2 patients was indicative of early recurrence of the disease along with shorter disease-free and overall survival. Low WT1 expression (< 200 copies/ml) in 2 patients after induction and consolidation therapy, respectively, was suggestive of better prognosis.

Aleukemic Myeloid Sarcoma of the Breast.

Myeloid sarcoma is a solid collection of leukemic blast cells outside bone marrow. It is seen infrequently in association with overt Acute Myeloid Leukemia (AML), however, it invariably transforms into this aggressive condition. A28-year woman presented with a lump in left breast for last 2 years. Morphological and immunophenotypic results of breast mass were consistent with myeloid sarcoma. Bone marrow aspirate and biopsy were normal, however, patient died after one and a half years of diagnosis. Myeloid sarcoma at any extramedullary site heralds development of overt acute myeloid leukemia and should be appropriately managed at the time of diagnosis.

Frequency of chromosomal abnormalities in Pakistani adults with acute lymphoblastic leukemia.

BACKGROUND: The difference in prognosis of adult and childhood acute lymphoblastic leukemia (ALL) can be attributed largely to variation in cytogenetic abnormalities with age groups. Cytogenetic analysis in acute leukemia is now routinely used to assist patient management, particularly in terms of diagnosis, disease monitoring, prognosis and risk stratification. Knowing about cytogenetic profile at the time of diagnosis is important in order to take critical decisions in management of the patients. AIM AND OBJECTIVES: To determine the frequency of cytogenetic abnormalities in Pakistani adult patients with ALL in order to have insights regarding behavior of the disease. MATERIALS AND METHODS: A retrospective analysis of all the cases of ALL (≥15years old) diagnosed at Aga Khan University from January 2006 to June 2014 was performed. Phenotype (B/T lineage) was confirmed in all cases by flow cytometry. Cytogenetic analysis was made for all cases using the trypsin-Giemsa banding technique. Karyotypes were interpreted using the International System for Human Cytogenetic Nomenclature (ISCN) criteria. RESULTS: A total of 166 patients were diagnosed as ALL during the study period, of which 151 samples successfully yielded metaphase chromosomes. The male to female ratio was 3.4:1. The majority (n=120, 72.3%) had a B-cell phenotype. A normal karyotype was present in 51% (n=77) of the cases whereas 49% (n=74) had an abnormal karyotype. Of the abnormal cases, 10% showed Philadelphia chromosome; t(9;22)(q34;q11.2). Other poor prognostic cytogenetic subgroups were t(4;11)(q21;q23), hypodiploidy (35-45 chromosomes) and complex karyotype. Hyperdiploidy (47-57 chromosomes) occurred in 6.6%; all of whom were younger than 30 years. CONCLUSIONS: This study showed a relatively low prevalence of Philadelphia chromosome in Pakistani adults with ALL with an increase in frequency with age (p=0.003). The cumulative prevalence of Philadelphia- negative poor cytogenetic aberrations in different age groups was not significant (p=0.6).