Diagnostic accuracy of flow cytometry in detecting malignant epithelial cells in serous effusions.

INTRODUCTION: This study aims to evaluate diagnostic accuracy of flow cytometry (FCM) in detecting malignant epithelial cells in serous effusions. MATERIALS AND METHODS: Flow cytometric assessment of 96 serous fluids (86 ascitic, 10 pleural) was performed by using epithelial cell adhesion molecule (EpCAM) (in all 96 fluids) and MUC-1 (in a subgroup of 40 fluids) as epithelial markers and CD45 and CD14 as leucocyte markers. The percentage of EpCAM positivity and MUC-1 positivity was calculated in the CD14 and CD45 dual negative population by selective gating. The findings were then correlated with the defined gold standard criteria. RESULTS: The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy for EpCAM was found to be 92.06%, 96.96%, 98.31%, 86.48%, and 93.75%, respectively, while that for MUC-1 was 79.16%, 93.75%, 95%, 71.4%, and 85%, respectively. The sensitivity, specificity, PPV, NPV, and diagnostic accuracy for dual positivity for EpCAM and MUC-1 was found to be 83.3%, 100%, 100%, 80%, and 90% respectively. On combining FCM with cytomorphology the sensitivity, specificity, PPV, NPV, and diagnostic accuracy all increased greatly to 95.3%, 100%, 100%, 91.4%, and 96.8%, respectively. CONCLUSIONS: This study highlights the importance of multicolored flow cytometric analysis in detecting epithelial malignancies in effusions specially in cases belonging to the atypia of undetermined significance and suspicious for malignancy categories and in cases with strong clinical suspicion of malignancy with negative fluid cytology. We recommend the combined use of FCM and cytology for this specific subgroup of patients in routine clinical practice for fast and accurate reporting.

Sequential Shifting in T-helper and T-cytotoxic Subset Cell Population in Mild and Severe COVID-19 Patients Infected With Variant B.1.61.

AIM: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) modulates antiviral immunity via T cells, but whether these cells are active or abundant in coronavirus disease 2019 (COVID-19) patients is unknown. The present study aimed to investigate the temporal shifting in the T-cell population and their subsets, T-Helper (Th) cell (CD4) and T-Cytotoxic (Tc) cell (CD8) in COVID-19 patients. METHOD: Thirty confirmed COVID-19 patients (nasal swab reverse transcription-polymerase chain reaction (RT-PCR) confirmed) were enrolled. On the basis of oxygen saturation (SpO2) levels, patients were stratified into two categories: (i) mild (n=11) having fever and SpO2 level >95%, and (ii) severe (n=19) on the ventilator, and in the intensive care unit (ICU) as per the Indian Council of Medical Research (ICMR) guidelines. Thirty age-sex-matched controls without infectious diseases unrelated to COVID-19 were also enrolled in the study. Patients with inflammatory diseases and severe comorbidities that compromise immunity were excluded from the study. Immunophenotyping flow cytometry assay was used to evaluate T-cell viability, Th, and Tc cells population in mild and severe COVID-19 patients on day 1 (at admission) and day 4 (decreasing the infection load) in the second COVID-19 wave (variant: B.1.61).  Categorical variables were expressed as frequency and percentage and p-values were calculated by Chi-square test. All the variables were represented in median and Q1 (25 percentile) and Q3 (75 percentile). The Mann-Whitney test was used to compare the study groups. The Δ mean differences were calculated by using the Paired samples t-test. The statistically significant level was taken as p<0.05. RESULTS: Hemoglobin, total leukocyte count (TLC), lymphocytes, monocytes, and eosinophils were significantly reduced in patients (p<0.05). A significant decrease of CD4 and CD8 cells in severe COVID-19 patients vs. controls (CD4, median 49; CD8, 40.12; p>0.05) was seen. Th-EM (effector memory)-Tim-3 (T-cell immunoglobulin domain and mucin domain 3)+ was significantly higher (p=0.002) however, Tc-EMRA (effector memory cells re-expressing)-Tim-3+, Tc-Naive-Tim-3+, Tc-EM-PD1+ and Tc-CM (central memory)-Tim-3+ significantly reduced (p<0.05) in mild COVID-19 patients than controls. Similarly, in severe COVID-19 patients, Th-EMRA-Tim-3+, Th-Naive-PD1+, Th-EM-PD1+, Th-EM-Tim 3+ and Th-CM-Tim-3+ showed a significant reduction (p<0.05) and Tc-EMRA-Tim-3+, Tc-Naive-Tim-3+, Tc-EM-PD1+, and Tc-CM-Tim-3+ showed similar results. In mild vs. severe group, decreased T-cells (p=0.001), Th-EMRA-Tim-3+ (p=0.024), and Th-Navie-Tim-3+ (p=0.005), and significantly increased (p<0.05) Tc-Naive-Tim3+ (p=0.001), Tc-EM-Tim-3+ (p=0.031), and Tc-CM-Tim-3+ (p=0.08) were observed. Severe COVID-19 patients showed a significant increase in Th-Naive-Tim3+ (day 4-day 1; δ43, p=0.019), Th-EM-Tim3+ (δ 16.24, p=0.033), and Th-CM-Tim3+ (δ 13.57, p=0.041). CONCLUSION: T-cell populations and CD8 subset help to differentiate the mild and severe COVID-19 patients. Monitoring T cells, especially CD8 subset changes, has important implications for diagnosing and treating mild and severe patients being critically ill.

Extraskeletal Ewing sarcoma presenting as an axillary mass with pulmonary metastases.

Extraskeletal Ewing sarcoma (EES) is a tumour of rare variant of the Ewing sarcoma family of tumours. This family of tumours can have different features; however, these tumours are categorised on the basis of genetic translocation, specific molecular and immunohistochemical features. EES is seen commonly affecting young adults with poor prognosis and high mortality rates. It can be detected in various locations making its diagnosis more difficult. It can present with varied imaging features, often non-specific. However, imaging plays a vital role in the primary tumour assessment, local staging, preoperative management and surveillance. Management involves surgery with chemotherapy. Long-term prognosis in cases of metastatic disease is very poor. In literature, only three cases of axillary EES have been reported so far. Here, we report the fourth case of large EES originating in the left axillary region in a woman in her 20s. The patient was given neoadjuvant chemotherapy; however, the size of the tumour increased, which was later surgically treated with complete excision of the tumour. Unfortunately, the tumour metastasised to the lung for which the patient was irradiated. Afterwards, the patient presented to the emergency room with respiratory distress for which she was on ventilator support; sadly, the patient died after 1 week.

Leukoerythroblastosis - An unsusal presentation of COVID 19 infection.

Objectives: Although several peripheral blood abnormalities have been reported in COVID 19,Leukoerythroblastosis is an unusual finding. We report 33 COVID19 cases presenting with leukoerythroblastosis. We intend to describe its incidence in this novel viral infection and correlate it with the clinical outcome. Methods: It is a Prospective study done at a Level 3 COVID 19 hospital of LUCKNOW, INDIA. Hematologic test records of day 1 of admission of COVID 19 cases admitted from 20th August 2020 to 30th September 2020 were reviewed. Peripheral blood smear examination was performed on test results that were flagged for abnormalities. Leukoerythroblastosis was reported when the smears showed presence of granulocyte left shift and nucleated red blood cells. Follow up smears were examined on Day 7. The findings were correlated with the clinical outcome. Results: Out of 274 slides reviewed, 33 (12%) showed a leukoerythroblastic picture on day 1 of admission. Follow up smears on day 7 were available in 76% (25/33) cases. The follow up smears showed improvement in 13 cases, worsening in 9 cases and no changes in 3 cases. There were total 19 (58%) deaths. 12 patients (36%) recovered and 2 patients (6%) were shifted to other hospitals whose further follow up was not available. Conclusions: Leukoerythroblastosis is an unusual presentation of COVID 19. Although rare, this peripheral blood abnormality can provide insight into the underlying pathophysiologic processes. Furthermore, it seems to be an adverse prognostic factor, so examination of follow up smears may help clinicians and intensivists to make prompt management decisions.

The Utility of Ancillary Techniques in the Cyto-Diagnosis of Malignant Scalp Lesions.

BACKGROUND: Fine needle aspiration cytology (FNAC) is rapid, inexpensive, and easy technique to establish the diagnosis of scalp lesions. The use of ancillary techniques such as immunocytochemistry (ICC), immunohistochemistry (IHC), and flow cytometry on aspiration material aids in accurate diagnosis which is additionally beneficial for management and prognosis. AIMS: The objective of this prospective case series was to evaluate the utility of ancillary techniques in the accurate cyto-diagnosis of malignant scalp lesions. MATERIALS AND METHODS: This study was a prospective case series that included 64 cases of scalp lesions in which FNAC had been performed for diagnosis. The lesions were categorized as Non-diagnostic/Inadequate, Inflammatory, Benign and Malignant. In all the cases that were categorized as malignant additional material was collected for ancillary testing that included ICC, cell block preparation followed by IHC and flow cytometry. RESULTS: Non-diagnostic/inadequate aspirates were 17.19% (n = 11/64), 25% (n = 16/64) aspirates were inflammatory, 35.93% (n = 23/64) aspirates were benign and 21.87% (n = 14/64) aspirates were categorized as malignant. With the aid of ancillary techniques, 57.14% malignant scalp aspirates were accurately categorized as epithelial origin. Lesions of bone and soft tissue constituted 28.57% (n = 4/14) of cases and lesions of hematolymphoid origin constituted 14.29% (n = 2/14) of all cases. CONCLUSION: This is a novel study where accurate categorization of malignant scalp tumors has been done with the use of ancillary techniques. This is useful as it may help in defining the tumor type, may aid in patient management. The material obtained can also be triaged for molecular testing.

Evaluation of the relationship between blood cell parameters and vascular calcification in dialysis-dependent end-stage renal disease patients.

Coronary artery calcification is an acceptable tool for cardiovascular risk assessment in end-stage renal disease (ESRD) population. We aimed to identify the association and predictive value of components of blood cell parameters with coronary and thoracic aorta vascular calcification (VC) in ESRD population on dialysis. All ESRD patients receiving hemodialysis or peritoneal dialysis aged between 18 and 60 years were included in the study. Exclusion criteria comprised patients with active infection or inflammatory disease, autoimmune disease, congestive heart failure, angina pectoris and/or documented coronary artery disease, thyroid disease, and hepatic dysfunction. Agatston scoring was used for the evaluation of coronary aorta calcification (CAC) score (CACS) and thoracic aorta calcification (TAC) score (TACS). Compared to participants with no VC, those who had VC were statistically significantly older (P <0.001) and had higher neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) (P = 0.02 and <0.001, respectively). On multivariate logistic regression analysis, increasing age (P = 0.00) and higher PLR (P = 0.04) were associated with an increased likelihood of exhibiting VC (CAC or TAC). There was a positive correlation between CACS and age (rs = 0.495, P = 0.00). A statistically significant positive correlation existed between TACS and age (rs = 0.516, P = 0.00). Similarly, a positive correlation was found between NLR, PLR, and TACS (rs = 0.334, P = 0.001, and rs = 0.438, P = 0.00, respectively). On multivariate linear regression analysis, increased age and red cell distribution width were found to be significant predictors of log(n) TACS. PLR of 135 gave a sensitivity of 80% and a specificity of 50% for predicting VC. Being a cost-effective and easily available investigation, the utilization of the correlation of NLR and PLR with CAC and TAC appears promising, particularly in the age group of 30-60 years.

Mixing-based inhibitor screening in haemophilia A: challenges in interpretation.

: Inhibitor development in haemophilia A patients is a dreaded complication of factor VIII (FVIII) replacement therapy. With increasing use of FVIII replacement therapy, there is an imperative need for cost-effective and standardized screening. To evaluate the efficacy of mixing-based inhibitor screening (MBIS) in the detection of FVIII inhibitors and to assess the best cut-off values for MBIS. Forty inhibitor positive and 40 inhibitor negative haemophilia A patients, diagnosed by standard criteria, with detailed clinical, haematological and on-demand treatment records were included. MBIS was evaluated in all 80 cases and a classical Bethesda assay and Nijmegen modification of Bethesda assay (NBA) were used as gold standards for inhibitor diagnosis. Classical Bethesda assay missed eight cases, most with low titres, which were confirmed by NBA. A systematic analysis of cut-offs for MBIS using a receiver operating characteristic curve fixed the cut-off at more than 5 s. MBIS detected 36 out of 40 inhibitor positive haemophilia A patients with a sensitivity, specificity, PPV and NPV of 90.0, 95, 94.7, 90.5%, respectively, whereas at the conventional cut-off of more than 10 s, MBIS detected only 25 of 40 cases with a low sensitivity of 62.5%. The likelihood ratio of a positive test was 11. The false-negative haemophilia A patients had low titres from 1.6 to 4.2 BU/ml. MBIS at a cut-off of 5 s can be considered as an effective screening test in low-resource situations. In clinical situations and in cases with clinical evidence of inhibitors we recommend that a direct NBA should be done.

Liquid-based cytology: do ancillary techniques enhance detection of epithelial abnormalities?

PURPOSE: Cervical cancer is the fourth most common cancer in women worldwide with very high incidence in India. Liquid-based cytology (LBC) provides the use of ancillary techniques in addition to a good morphology and detection of cytologic abnormalities. The current study was designed to assess the diagnostics of P16INK4a immunoexpression, p16 promoter hypermethylation, human papilloma virus (HPV), and DNA ploidy in LBC samples with cervical precancer and cancer. METHODS: A series of LBC samples categorised by Bethesda system including 22 atypical squamous cells of undetermined significance (ASC-US), 21 low-grade squamous intraepithelial lesion (LSIL), 41 high-grade squamous intraepithelial lesion (HSIL), 54 squamous cell carcinoma (SCC), and 26 controls with normal cytology were included. Ancillary techniques evaluated included P16INK4a immunoexpression, p16 promoter methylation DNA ploidy by flow cytometry, and HPV was detected using PGMY09/PGMY11 primers. RESULTS: The test positivity rate of p16 expression in women with ASC-US, LSIL, HSIL, and SCC was 21.1, 39.0, 67.7, and 85.4%. For the p16 methylation the corresponding test positivity rate was 36.4, 76.2, 92.7, and 92.6%. The test positive rate of HPV in women with ASC-US, LSIL, HSIL, and SCC was 45.5, 76.2, 87.8, and 92.6%. Diploid G1 and diploid S values significantly (p < 0.05 or p < 0.01) discriminate LSIL versus HSIL and LSIL versus. SCC. CONCLUSIONS: P16 gene promoter methylation and HPV seem more sensitive in detection of ASC-US and LSIL cytology with higher specificity. Diploid G1 and diploid S phase study provides progressive change in parameters with progression from LSIL to HSIL and SCC.

Flow Cytometric Analysis of DNA Ploidy in Liquid Based Cytology for Cervical Pre-Cancer and Cancer

Background: DNA ploidy analysis of cervical intraepithelial neoplasia (CIN) and invasive cervical cancer samples by flow cytometry (FCM) has been established as an aid to prognostic assessment. Liquid based cytology (LBC) increases diagnostic specificity by using ancillary techniques that provide information beyond morphology. The present study was undertaken to assess DNA ploidy in LBC samples as an adjunct for early detection of cervical pre-cancer and cancer. Methods: DNA ploidy assessment was performed on LBC samples of 50 cases and 31 controls. Cell pellets were obtained by centrifugation and stained with Telford reagent. At least 20,000 R1 gate (G0-G1) events were acquired on a BD FACSCalibur by using a 575±10 nm filter. Results: Mean diploid G1 values were lowered significantly (p<0.01) while diploid S values were significantly elevated (p<0.01) in both high grade squamous intraepithelial lesions (HSILs) and squamous cell carcinomas (SCCs) as compared to controls. Receiver operating curve (ROC) analysis of the diploid G1 value was found to have significant diagnostic potential (AUC=0.682, Z=2.00, p=0.046) for distinction between control and low grade squamous intraepithelial lesion (LSIL) at a cut off value of ≤91.6 with a sensitivity and specificity of 50.0 and 87.1%, respectively. Conclusions: ROC analysis of diploid G1 and diploid S values allows discrimination between LSIL and HSIL with sensitivities and specificities of 65 and 100% and 70 and100%, respectively, and between LSIL and SCC cases with values of 71.4 and 100% and 64.3 and 100%, respectively.

Chondrosarcoma of chest wall metastasising to the larynx: Case report and review of literature.

Chondrosarcoma is the most common malignant tumor of the chest wall. Most patients present with painful progressive swelling in the anterior chest wall arising from the costochondrosternal junction. CT scan with intravenous contrast is the investigation of choice. Wide excision with adequate margins is the standard treatment for localized disease after image guided biopsy. The role of chemotherapy and radiotherapy is limited. Lung is the most common site for metastasis. Metastasis to the larynx from chondrosarcoma has not been reported in the literature though primary chondrosarcoma can occur in the larynx. We hereby report a case of laryngeal metastasis from chondrosarcoma of the chest wall as a part of disease failure.

Cystic microfilarial infestation of the liver: a rare presentation.

Cystic liver lesions in an adult may occur for a variety of reasons, most of which are benign in nature. Infiltrating benign lesions in the liver parenchyma may pose a clinical challenge in diagnosis and management. In the case presented herein, a cystic lesion adjacent to the gall bladder and involving the liver parenchyma had to be differentiated from gall bladder carcinoma, which is quite common in India. Parasitic infestation of the liver is an extremely rare presentation and may pose a significant challenge in its diagnosis and management. This case highlights an infrequent presentation and the challenges in the clinical approach and subsequent management.

Sézary syndrome presenting with 'leonine facies'.

A 71-year-old man presented with erythroderma and multiple nodular skin lesions over the face, scalp, upper limbs and trunk. The facial skin was thickened, producing the rare 'leonine facies' appearance. Investigations revealed the presence of atypical lymphoid cells in the peripheral blood, bone marrow and skin. The atypical lymphoid cells in the peripheral blood and bone marrow were positive for helper T-cell antigens (CD4, CD2, CD5 and CD7) on immunophenotyping by flow cytometry. The histopathology of skin showed dermal infiltration by atypical small lymphocytes with epidermotropism. These cells were positive for helper T-lymphocyte antigens on immunohistochemistry. A diagnosis of Sézary syndrome was made based on clinical, peripheral blood and immunophenotypical findings.