Hematological Profile in COVID-19 Infection Among Patients in a Tertiary Care Hospital in Tamil Nadu, South India.

Introduction A wide range of hematological abnormalities has been observed in SARS-CoV-2 infection which is directly related to the disease progression, clinical severity, and mortality among affected individuals. The objective of this study was to evaluate the abnormalities in hematological parameters among severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected patients in a tertiary care hospital in south India. Methods This was a cross-sectional study carried out in the pathology department of Karpaga Vinayaga Institute of Medical Sciences and Research Centre, Chengalpattu, Tamil Nadu, India from 1st May 2021 to 30th June 2021. The hematological reports including complete blood count (CBC), neutrophil-lymphocyte ratio (NLR), serum ferritin, serum C-reactive protein (CRP), serum lactate dehydrogenase (LDH), and D-dimer levels of all the blood samples from COVID-19 positive patients were retrieved from the laboratory records. The Leishman-stained peripheral smear findings were also tabulated and analyzed. Results Out of 65 patients, 38 (58.5 %) were males and 27 (41.5%) were females with a majority (78.4%) of them being more than 40 years of age. The salient hematological abnormalities were leukopenia (21.5%), elevated NLR (43%), and thrombocytopenia (6.2%). Peripheral smear showed schistocytes (15.4%), neutrophils with ring nuclei (84.6%), and toxic granules (81.5%). A statistically significant association between elevated NLR and serum CRP was seen among male patients. The association between the presence of schistocytes with serum LDH and D-dimer levels was statistically insignificant. Conclusions The significant hematological abnormalities in patients with COVID-19 infection were elevated NLR, lymphopenia, thrombocytopenia, and elevated D-dimer levels. Careful evaluation of the hematological parameters will help in categorizing the high-risk cases and thereby initiating early intervention and appropriate intensive care management. This will bring down the morbidity and mortality among COVID-19 patients.

Combining imaging flow cytometry and machine learning for high-throughput schistocyte quantification: A SVM classifier development and external validation cohort.

BACKGROUND: Schistocyte counts are a cornerstone of the diagnosis of thrombotic microangiopathy syndrome (TMA). Their manual quantification is complex and alternative automated methods suffer from pitfalls that limit their use. We report a method combining imaging flow cytometry (IFC) and artificial intelligence for the direct label-free and operator-independent quantification of schistocytes in whole blood. METHODS: We used 135,045 IFC images from blood acquisition among 14 patients to extract 188 features with IDEAS® software and 128 features from a convolutional neural network (CNN) with Keras framework in order to train a support vector machine (SVM) blood elements' classifier used for schistocytes quantification. FINDING: Keras features showed better accuracy (94.03%, CI: 93.75-94.31%) than ideas features (91.54%, CI: 91.21-91.87%) in recognising whole-blood elements, and together they showed the best accuracy (95.64%, CI: 95.39-95.88%). We obtained an excellent correlation (0.93, CI: 0.90-0.96) between three haematologists and our method on a cohort of 102 patient samples. All patients with schistocytosis (>1% schistocytes) were detected with excellent specificity (91.3%, CI: 82.0-96.7%) and sensitivity (100%, CI: 89.4-100.0%). We confirmed these results with a similar specificity (91.1%, CI: 78.8-97.5%) and sensitivity (100%, CI: 88.1-100.0%) on a validation cohort (n=74) analysed in an independent healthcare centre. Simultaneous analysis of 16 samples in both study centres showed a very good correlation between the 2 imaging flow cytometers (Y=1.001x). INTERPRETATION: We demonstrate that IFC can represent a reliable tool for operator-independent schistocyte quantification with no pre-analytical processing which is of most importance in emergency situations such as TMA. FUNDING: None.

[Evaluation of Sysmex XN-3000 for Detection of Schistocyte].

OBJECTIVE: Compared with the method of optical microscopy, to evaluate the accuracy of fragmented red cells(FRC) detection by Sysmex XN-3000. METHODS: A total of 111 samples were collected from patients diagnosed as thrombotic thrombocytopenic purpura, autoimmune disease, hematological disease, malignant tumor and health examination in our hospital from June 2019 to February 2021, including 74 cases in the case group and 37 cases in the healthy control group. All samples were detected by optical microscope and Sysmex XN-3000, respectively. ROC was used to evaluate the detection ability of Sysmex XN-3000 for schistocyte. Bland-Altman method was used to evaluate the consistency of the results of the two methods for detection of schistocyte, and Pearson correlation analysis was conducted for the difference of the results. RESULTS: The area under the ROC curve was 0.890(95% CI: 0.828-0.952, P<0.01). Sysmex XN-3000 count did not quantitatively agree with schistocyte counts by microscopy in the case group(mean of difference:-1.53, 95% limits of agreement: -8.78~5.72). There was a weak positive correlation between platelet count and the difference of analyzer and microscopic results (r=0.32,P<0.05). CONCLUSION: Sysmex XN-3000 can be used as a reference for qualitative determination of schistocyte. However, the sensitivity of Sysmex XN-3000 should be improved. It is still necessary to combine with manual microscopy. The quantitative results are not reliable now and cannot be used as a reference for monitoring the results of schistocyte in clinical patients after treatment.

Evaluation of Sysmex XN-3000 for Detection of Schistocyte / 中国实验血液学杂志

OBJECTIVE@#Compared with the method of optical microscopy, to evaluate the accuracy of fragmented red cells(FRC) detection by Sysmex XN-3000.@*METHODS@#A total of 111 samples were collected from patients diagnosed as thrombotic thrombocytopenic purpura, autoimmune disease, hematological disease, malignant tumor and health examination in our hospital from June 2019 to February 2021, including 74 cases in the case group and 37 cases in the healthy control group. All samples were detected by optical microscope and Sysmex XN-3000, respectively. ROC was used to evaluate the detection ability of Sysmex XN-3000 for schistocyte. Bland-Altman method was used to evaluate the consistency of the results of the two methods for detection of schistocyte, and Pearson correlation analysis was conducted for the difference of the results.@*RESULTS@#The area under the ROC curve was 0.890(95% CI: 0.828-0.952, P<0.01). Sysmex XN-3000 count did not quantitatively agree with schistocyte counts by microscopy in the case group(mean of difference:-1.53, 95% limits of agreement: -8.78~5.72). There was a weak positive correlation between platelet count and the difference of analyzer and microscopic results (r=0.32,P<0.05).@*CONCLUSION@#Sysmex XN-3000 can be used as a reference for qualitative determination of schistocyte. However, the sensitivity of Sysmex XN-3000 should be improved. It is still necessary to combine with manual microscopy. The quantitative results are not reliable now and cannot be used as a reference for monitoring the results of schistocyte in clinical patients after treatment.

Plasmapheresis in Sepsis-induced Thrombotic Microangiopathy: A Case Series.

INTRODUCTION: Cytokines and granulocyte elastase produced in sepsis cleave a disintegrin and metalloprotease with thrombospondin type I motif 13 (ADAMTS13) and deplete its levels. By this mechanism, sepsis results in microangiopathic hemolytic anemia (MAHA) with thrombocytopenia. Hence, the hypothesis is that plasmapheresis may help in sepsis-induced thrombotic microangiopathy (sTMA), by removing the factors responsible for low levels of ADAMTS13. In tropical countries like India, the contribution of sepsis to intensive care unit (ICU) mortality is high; and hence, it is essential to look out for newer modalities of sepsis treatment. There is abundant literature on the use of plasmapheresis in sepsis but data on its use in sTMA are limited, thus necessitating further research in this field. CASE DESCRIPTION: This case series studies the outcomes of five patients admitted with sTMA in the ICU and attempts to evaluate the effectiveness of plasmapheresis in improving their outcomes. All patients diagnosed with sTMA and treated with plasmapheresis, between January 2016 and August 2018 at our tertiary care center, were selected for the study. The diagnosis of sepsis was based on sepsis-3 definition. RESULTS: Four different gram-negative organisms were found to have caused MAHA, with the commonest source being either urinary tract infection (UTI) or lower respiratory tract infection. Three of five patients required hemodialysis and two had disseminated intravascular coagulation (DIC). All five had good outcome and recovered well from the acute episode post plasmapheresis. DISCUSSION: In two of five patients, the initial smear was negative and hence the need for repeated examination of the peripheral blood smear should be kept in mind in cases of sTMAs. The median of the number of plasmapheresis sessions required in sTMA is six, which is lesser than that required for primary thrombotic thrombocytopenic purpura (TTP). Hence, the duration of central line placement and the risk of catheter-related complications are low. Based on the observations made in this case study, further exploratory studies are required to evaluate the efficacy of plasmapheresis in sTMA secondary to tropical infections. HOW TO CITE THIS ARTICLE: Upadhya SRS, Mahabala C, Kamat JG, Jeganathan J, Kumar S, Prabhu MV. Plasmapheresis in Sepsis-induced Thrombotic Microangiopathy: A Case Series. Indian J Crit Care Med 2020;24(3):195-199.

A Fatal Case of Thrombotic Microangiopathy Without Schistocytosis and Absent Biochemical Markers of Hemolysis.

Thrombotic microangiopathy (TMA) is characterized by microangiopathic hemolytic anemia with thrombocytopenia. In addition to the primary TMA syndromes, microangiopathic hemolytic anemia with thrombocytopenia can be seen in many systemic diseases. Transplant associated TMA (TA-TMA) affects patients following stem cell or solid organ transplant. A 48-year-old male who underwent autologous stem cell transplant for nonsecretory multiple myeloma was admitted to our hospital with worsening anemia, thrombocytopenia, renal dysfunction and hepatosplenomegaly. Initial blood work revealed rare schistocytes and normal lactate dehydrogenase and haptoglobin levels. He underwent an extensive workup looking for an infectious, inflammatory or malignant etiology but a definitive diagnosis could not be reached. Over his prolonged stay at the hospital, he suffered from multiorgan failure and eventually passed away. An autopsy revealed TMA involving all clinically affected organ systems and was deemed to be the cause of his demise. The absence of typical blood work suggestive of hemolysis does not rule out a diagnosis of TA-TMA. Knowledge of this rare disease entity will help physicians identify and treat this life-threatening condition early and effectively.

Gastrointestinal infection-related disseminated intravascular coagulation mimicking Shiga toxin-mediated hemolytic uremic syndrome - implications of classical clinical indexes in making the diagnosis: A case report and literature review.

BACKGROUND: Thrombocytopenia associated with acute kidney injury is a challenging disorder. Thrombotic microangiopathy (TMA) is a potentially life- or organ-threatening syndrome that can be induced by several disorders or medical interventions. There is overlap between the clinical presentation and pathophysiology of thrombotic thrombocytopenia purpura and hemolytic uremic syndrome (HUS), and to a lesser extent, disseminated intravascular coagulation (DIC). We describe a case to illustrate the potential diagnostic difficulty, especially at initial presentation. CASE SUMMARY: We reported a case of a 44-year-old woman that presented with diarrhea, thrombocytopenia, schistocytes, elevated serum lactate dehydrogenase (LDH) level and acute kidney injury. While the clinical presentation resembled that of Shiga toxin-induced HUS, the disease course was more consistent with gastrointestinal infection-related DIC. To aid in the accurate diagnosis of TMA and other associated disorders, we have undertaken a review and provided a clear interpretation of some typical biomarkers including schistocytes, LDH and platelet count, coagulation profile and more specific indexes of ADAMTS13, complement profile, and the isolation of Shiga toxin-producing Escherichia coli (commonly referred to as STEC). CONCLUSION: The use and correct interpretation of classical indexes of schistocyte, LDH, and platelet count is vital in diagnosing TMA and associated disorders. Understanding the characteristics of these biomarkers in the context of thrombocytopenia purpura, HUS and DIC will facilitate the accurate diagnosis and early initiation of appropriate treatment.

Erythrocytes morphology in pregnancy.

Morphologic anomalies of the red blood cells (RBCs) during pregnancy are poorly known. Peripheral blood films from 69 healthy pregnant women were investigated for shape, color and content anomalies of the RBCs. A range of minor alterations was observed, without clinical significance. Only a slight increase of polychromatophilic RBCs was regularly observed. However, we would like to stress that spherocytes or schistocytes can occasionally be found, even in the absence of hemolysis.

Automated Quantification of Fragmented Red Blood Cells: Neonatal Reference Intervals and Clinical Disorders of Neonatal Intensive Care Unit Patients with High Values.

BACKGROUND: Schistocytes are circulating erythrocyte fragments. They can be identified microscopically from a blood smear; but automated systems evaluate more cells and avoid inconsistencies in microscopy. Studies using adult subjects indicate that automated quantification of schistocytes can be clinically useful. However, reference intervals for automated schistocyte counts of neonates have not been published, and the relevance of a high automated schistocyte count from neonates has not been reported. OBJECTIVES: Using retrospective automated neonatal complete blood count (CBC) data, we created reference intervals for fragmented red cells (FRCs) and sought to discover the clinical conditions of neonates with high FRCs (above the upper reference interval). RESULTS: We created reference intervals based on 39,949 CBCs from 15,655 neonates 0-90 days old. The lower reference interval was 0 FRC/µL and the upper interval was 100,000/µL. The highest FRCs (96 CBCs from 44 neonates) were > 250,000/µL. These neonates clustered into the following groups: 37% had sepsis, 29% had disseminated intravascular coagulation (DIC), 17% had a genetic syndrome, 14% necrotizing enterocolitis (NEC), and 7% had iron deficiency (some had more than one diagnosis). Based on the reference intervals, we divided the 39,949 FRC values into 3 groups: (1) < 100,000/µL ("normal"), (2) 100,000-200,000/µL ("moderately elevated"), and (3) > 200,000/µL ("extremely elevated"). The odds that a microangiopathic condition (DIC, sepsis, NEC) or a microcytic disorder (iron deficiency) were present were significantly higher in the moderately elevated, and more so in the extremely elevated group. CONCLUSIONS: Our study suggests that a high FRC could prompt investigation into, or inform follow-up of, a neonatal microangiopathic or extremely microcytic disorder.

Fragmented Red Cell as a Possible Favorable Prognostic Marker of Hematopoietic Stem Cell Transplantation Associated Thrombotic Microangiopathy.

BACKGROUND: Fragmented red cell (FRC) by automated hematologic analyzer is known to detect schistocyte. In this study, it is noted that FRC might be a favorable prognostic marker of hematopoietic stem cell transplantation associated thrombotic microangiopathy (TA-TMA). METHODS: The peripheral blood samples and clinical data of 89 patients were collected. The diagnosis of TA-TMA was defined by the Blood and Marrow Transplant Clinical Trials Network's criteria and schistocyte or both schistocyte- and FRC-positive cases and other parameters fulfilled are regarded as TA-TMA. RESULTS: Schistocyte and FRC displayed a correlation coefficient of 0.461 (P < 0.001) by Spearman's method. The diagnostic concordance of TA-TMA using schistocyte and FRC was 92.1% with kappa index of 0.531 (P < 0.001). The number of diagnosed patients and mean survival month were as follows: TA-TMA by schistocyte, 8 (8.9%), 13.5 month; TA-TMA by schistocyte and FRC, 7 (7.8%), 40.4 month; No TMA, 74 (83.1%), 38.3 month, respectively. Kaplan-Meier survival analysis by log-rank method of the patient with TA-TMA by schistocyte and rest of the group showed statistical significance (P < 0.01). CONCLUSION: As evidenced by the data, FRC might be a favorable prognostic marker for TA-TMA, but additional studies with larger patients groups are required for validation of clinical applications.

Schistocytes in disseminated intravascular coagulation.

INTRODUCTION: The presence of schistocytes on the peripheral blood film during disseminated intravascular coagulation (DIC) remains controversial. METHODS: We examined schistocytes count on blood films from 35 DIC patients and checked morphological anomalies of all RBCs. RESULTS: Thirty of 35 patients presented with schistocytes and 22 with acanthocytes, which was the commonest shape anomaly. Mean percentage ± standard deviation was 0.33 ± 0.38%, median value was 0.1%, and range was 0-1.4%. The patients with schistocytes ≥ 1% had circumstances frequently associated with increased schistocytes count (promyelocytic leukaemia, pregnancy, severe infection). DISCUSSION: Schistocytes were thus frequently observed in DIC patients, usually with low percentage, within or close to the reference range (<0.5%). Schistocytes measurement is not a clue test for the initial diagnosis of DIC, but might be of clinical value to suggest an associated or underlying thrombotic microangiopathy if ≥ 1%.

Evaluation of ICSH schistocyte measurement guidelines in France.

INTRODUCTION: The schistocytes are fragmented red blood cells mainly observed in the setting of hemolytic anemias where they remain an important criterion for the diagnosis. As the identification of these cells is still problematic, the International Council for Standardization in Hematology (ICSH) set up a consensus report in November, 2011. The French Group of Cellular Hematology (GFHC) aimed to collect the opinion of French biologists directly confronted to schistocytes measurements, about these guidelines. METHODS: Among the 578 professionals, 169 (29%) answered to the 10 questions dealing with the identification and measurements of schistocytes as proposed by the ICSH. RESULTS: A consensus was reached for the urgent need of such guidelines documents, especially in the current background of the European accreditation EN ISO 15189 rules. A traduction in native (French) language was warmly wished in order to facilitate the diffusion of the information. The pathologic threshold for the diagnosis of thrombotic microangiopathic anemia (TMA) (>1%) remained questionable. For half of the biologists, the new fragmented red blood cell (FRC) parameter recently provided by two manufacturers of automated blood cell counters was still doubtful for routine use. CONCLUSION: This survey assessed the impact of international 'guidelines' on the French biological community. The will to implement validated recommendations was strong, reflecting the awareness of the biologists to standardize the laboratory investigations.

Microscopic schistocyte determination according to International Council for Standardization in Hematology recommendations in various diseases.

INTRODUCTION: Recently, a consensus report for microscopic schistocyte determination was prepared by International Council for Standardization in Hematology (ICSH). ICSH focused on diagnosis of thrombocytopenic purpura (TTP)/hemolytic uremic syndrome (HUS). We aimed to reanalyze schistocytes according to ICSH recommendations, to study diseases other than TTP/HUS related to the schistocytes, and to compare the percentage of schistocytes among the various diseases. METHODS: We retrieved all reported cases of peripheral blood (PB) smear in a single institution during 6 years. Schistocytes on 282 PB smears showing previous peripheral schistocytes and hemoglobin ≤10 g/dL were recounted according to ICSH recommendations. RESULTS: The schistocytes were frequently observed in patients with microangiopathic hemolytic anemia (MAHA), metastatic carcinoma, sepsis, chronic renal failure, preterm infant, and infection. Only two among 34 patients categorized as MAHA were diagnosed as TTP/HUS. Schistocytes were observed with other morphological changes in 169 of 170 cases with schistocyte ≤1% and in 102 of 112 with schistocyte >1%. The median schistocyte percentages of patients with hematologic malignancy, megaloblastic anemia, acute renal failure, and preterm infant were 1.20%, 1.30%, 1.35%, and 1.70%, respectively. CONCLUSION: Schistocytes were observed above 1% in many diseases other than TTP /HUS. Therefore, it is important to understand that schistocytes could be seen in various diseases, and in these cases, schistocytes were usually detected together with other red blood cell morphologic changes. These data support ICSH recommendation that a schistocyte count should be considered clinically meaningful if schistocytes represent the main morphological abnormality in the PB smear.

Fragmented red blood cells automated measurement is a useful parameter to exclude schistocytes on the blood film.

INTRODUCTION:   The diagnosis of thrombotic microangiopathies (TMA) or disorders that may mimic their features remains difficult. Mechanical hemolytic anemia with the detection of shistocytes on the blood smear is a cornerstone finding to assess the diagnosis, but microscopic evaluation of shistocytes is still problematic with wide interobserver variations. Some of the latest generation automated blood cell counters (ABCC) propose an original quantitative approach of fragmented red cells (FRC), aiming to be equivalent to the microscopic count. This parameter has been poorly evaluated. METHODS:   To assess the predictive value (PV) of this test, we conducted studies comparing automated and microscopic counts of FRC/schistocytes, based on the analysis of thousands samples in four university hospitals and using the 2 ABCC currently available (Siemens ADVIA series, Sysmex XE-2100). RESULTS: Reference range for FRC was <0.3% for the ADVIA and <0.5% for the XE-2100. The presence of FRC below a threshold determined at 1% (ADVIA and XE-2100) had a negative PV close to 100% to exclude the presence of schistocyte on the blood smear, but in relationship with a poor PV value. CONCLUSIONS: Our study validated the utility of the immediately available FRC parameter on ABCC to exclude schistocytes and the diagnosis of TMA.