Simple and rapid flow cytometry assay for the detection of malignant epithelial cells in body fluids.

INTRODUCTION: Morphology is routinely used for detecting malignant cells in body fluids, but it has limitations. Recently, flow cytometry (FCM) is used as an effective tool for studying non-haematological malignancies. The main objective of this study is to standardize a simple and rapid FCM test for the detection of malignant epithelial cells in body fluids. MATERIALS AND METHODS: Body fluids that had been processed for cytology/cytology and FCM were enrolled in this prospective study. We developed a fluorescent-labelled, monoclonal antibody panel composed of cell surface markers for this FCM assay. We compared the results of cytology/cell block and FCM. RESULTS: A total of 121 fluid samples were studied. Comparing the diagnostic performance of cytology/cell block and FCM, 52 (43%) cases were positive and 60 (49.5%) cases were negative for carcinoma cells by both techniques. Nine cases showed discordant results between the two techniques. Six cases were cytology+ but FCM- and three cases were FCM+ cytology-. Clustered Epithelial Cell Adhesion Molecule (EpCAM)-positive events with high scatter properties were definitive for positive diagnosis by FCM. We studied PD-L1 expression in 13 cases by FCM. Six cases were reported as false negative by this FCM assay due to hypocellularity and lack of EpCAM expression in malignant cells. CONCLUSIONS: This FCM assay is simple, easier and cost-effective yielding sensitive results with no inter-observer variability. FCM would become a valuable tool to complement routine diagnostic cytology and reduces misdiagnosis.

Deciphering stage 0 hematogones by flow cytometry in follow-up bone marrow samples of pediatric B-Acute lymphoblastic leukemia cases: A potential mimicker of residual disease after anti CD19 therapy.

CD19 is frequently targeted for immunotherapy in B cell malignancies, which may result in loss of CD19 expression in leukemic cells as an escape mechanism. Stage 0 hematogones (Hgs) are normal CD19-negative very early B cell precursors that can be potentially mistaken for CD19 negative residual leukemic cells by flow cytometry (FCM) in B cell acute lymphoblastic leukemia (BCP-ALL) cases treated with anti CD19 therapy. Our main objective was to characterize and study the incidence of stage 0 hematogones in follow-up bone marrow samples of pediatric BCP-ALL cases. We analyzed the flow cytometry standard files of 61 pediatric BCP-ALL cases treated with conventional chemotherapy and targeted anti-CD19 therapy, for identifying the residual disease and normal B cell precursors including stage 0 Hgs. A non-CD19 alternate gating strategy was used to isolate the B cells for detecting the residual disease and stage 0 Hgs. The stage 0 Hgs were seen in 95% of marrow samples containing CD19+ Hgs. When compared with controls and posttransplant marrow samples, the fraction of stage 0 Hgs was higher in patients receiving anti CD19 therapy (p = 0.0048), but it was not significant when compared with patients receiving chemotherapy (p = 0.1788). Isolated stage 0 Hgs are found in samples treated with anti-CD19 therapy simulating CD19 negative residual illness. Our findings aid in understanding the stage 0 Hgs and its association with CD19+ Hgs in anti CD19 therapy and conventional chemotherapy. This is crucial as it can be potentially mistaken for residual disease in patients treated with anti CD19 therapy.

Biclonal Low-Grade B Cell Lymphoma as Detected by Multiparametric Flow Cytometry in Blood and Marrow.

Biclonal B cell lymphomas (BCL) of two different low-grade components are rare. Multiparametric flow cytometry (MFC) is an integral tool in lymphoma diagnosis and very efficient in picking up BCL cases, especially when the associated second component is small. We studied the incidence of BCL in the routine blood and marrow samples sent for immunophenotyping by MFC. A total of 376 cases were analyzed retrospectively. We studied their clinical, immunophenotypic, molecular, and cytogenetic findings with literature review. We found five cases of BCL with two different clonal low-grade B cell components at an incidence of 1.3%. All cases were males with a median age of 68 years. The chronic lymphocytic leukemia (CLL) clone was present in all 5 cases. The other associated component in cases 1 to 5 were lymphoplasmacytic lymphoma, follicular lymphoma, splenic marginal zone lymphoma, hairy cell leukemia, and monoclonal B cell population of non-CLL type respectively. Diagnosing BCL may be challenging and may also go undiagnosed when the second component is petite and overshadowed by the more significant component. It may be critical if the small, unnoticed lymphoma component is of a more aggressive type. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-022-01625-y.

Role of high acquisition flow cytometry in the detection of marrow involvement in patients with extramedullary B cell non-Hodgkins lymphoma: a comparison with marrow aspirate cytology, trephine biopsy, and PET.

Detection of marrow infiltration is crucial in extramedullary B cell non-Hodgkin lymphoma (B-NHL). We studied the efficiency of high acquisition flow cytometry (FCM) in detecting marrow involvement in B-NHL patients and compared its performance with marrow aspiration (BMA) cytology, marrow biopsy (BMB), and positron emission tomography (PET). No case with marrow infiltration was found to be BMB positive and FCM negative. BMA cytology showed poor sensitivity and specificity. Only 50% of FCM positive cases showed evidence of marrow involvement by PET. Neither the nature of lymphoma nor the burden of the marrow disease showed a correlation with PET positivity. Four cases that were positive only by PET had findings in areas other than the iliac region. We found more cogent and convincing results with high event acquisition in FCM. This study shows that a combination of PET and FCM with high event acquisition is the best way for assessing marrow involvement in B-NHL cases.

Independent diagnostic utility of CD20, CD200, CD43 and CD45 in chronic lymphocytic leukaemia.

Immunophenotyping plays a major role and is essential for establishing the diagnosis of chronic lymphocytic leukemia (CLL). Though CLL has a characteristic phenotype, diagnosis may be challenging due to immunophenotypic overlap with other B cell non-Hodgkin's lymphomas (B-NHL). Markers like CD200, CD43, CD20 and CD45 were found valuable in CLL and we investigated their diagnostic efficiency and accuracy in 174 patients with leukemic B-NHL. On the integration of four markers by a scoring system, 96% (49/51) of CLL cases showed a score of 3 or 4 and 90% (36/40) of non-CLL cases had a score of 0 or 1. This scoring system for CLL diagnosis showed a sensitivity of 98.2% and 96% in the analytical cohort and validation cohort respectively, which was significantly higher than the classical Matutes score. Hence we strongly suggest considering the expression of CD200, CD20, CD43 and CD45 in the diagnosis of B-NHL cases.

Matched Family versus Alternative Donor Hematopoietic Stem Cell Transplantation for Patients with Thalassemia Major: Experience from a Tertiary Referral Center in South India.

Hematopoietic stem cell transplantation (HSCT) is the only curative option available for patients with thalassemia major in India with increasing access to alternate donor transplantation for patients with no matched family donor. We aimed to analyze the impact of family and alternate donor HSCT on morbidity and mortality post-HSCT. We conducted a retrospective study in the department between July 2007 and December 2018 where all children who underwent HSCT for thalassemia major were included. A total of 264 children were included with a median age of 6 years (male/female, 1.4:1). The graft source was matched related donor (MRD) (76%; parent 15%, sibling 85%) and matched unrelated donor (MUD) (22%). All children received a myeloablative conditioning regimen with treosulfan/thiotepa/fludarabine in 93% and busulfan/cyclophosphamide in 7%. The source of stem cells was peripheral blood in 61%, bone marrow in 38%, and umbilical cord blood in 3%. The incidence of bacteremia was 14% versus 25% in MRD versus MUD groups. There was a higher incidence of posterior reversible encephalopathy syndrome (PRES) in the MUD group (10% versus 3%). Engraftment occurred in 97% with a higher trend toward mixed chimerism in the MRD group (12% versus 2%). When indicated, whole-blood donor lymphocyte infusion was used to ensure complete chimerism in children in the MRD group. A statistically significant difference was found in the incidence of graft versus host disease (GVHD), both acute and chronic between the MUD versus MRD groups, 60% versus 20% and 41% versus 17%, respectively (P = .001). Similarly, immune cytopenia and cytomegalovirus reactivation were also significantly higher in the MUD group, 27% versus 1.4% and 25% versus 2%, respectively (P = .001). Thalassemia-free survival in our cohort was 96%, 94%, and 84% with a median follow-up of 65 months in the matched sibling donor, matched family donor, and MUD groups, respectively. Overall survival of 95% and 90% with a median follow-up of 65 months was noted in those who underwent transplantation less than and greater than 7 years of age, respectively. MUD transplantation for patients with thalassemia major involves specific challenges such as PRES and unusual manifestations of GVHD such as immune cytopenia. Early interventions to optimize supportive care and measures to reduce GVHD are required to ensure survival rates of over 90%.

Treosulfan-based reduced toxicity hematopoietic stem cell transplantation in X-linked agammaglobulinemia: A cost-effective alternative to long-term immunoglobulin replacement in developing countries.

X-linked agammaglobulinemia (XLA) is a primary antibody disorder due to a mutation in the Bruton tyrosine kinase gene that requires lifelong immunoglobulin replacement resulting in a significant economic burden and treatment abandonment. Hematopoietic stem cell transplantation (HSCT) offers an alternative option for complete cure. In our series, two children with XLA underwent successful HSCT using a myeloablative conditioning with thiotepa, treosulfan, and fludarabine from a matched sibling donor. The second child had rejected his first graft following a busulfan-based regimen with resultant autologous reconstitution. At 6 months post-HSCT, serum IgG were normal, off IVIG, and had no infections. Both children after a median follow-up of 20 months have 100% chimerism. Treosulfan-based reduced toxicity myeloablative HSCT has encouraging results with a positive impact on the socioeconomics in developing countries.

Haploidentical Stem Cell Transplantation in Children with Benign Disorders: Improved Survival and Cost-Effective Care Over 15 Years from a Single Center in India.

We present our experience in haploidentical stem cell transplantation (haplo SCT) in children with benign disorders. We performed a retrospective study where children aged up to 18 years diagnosed to have benign disorders and underwent haplo SCT from 2002 to September 2017 were included. Of the 54 children, the most common indications were Fanconi anaemia 12 (22%), severe aplastic anaemia 8 (14%) and primary immune deficiency disorders (PID) 25 (46%). Post-transplant cyclophosphamide (PTCy) was used in 41 (75.9%) and ex vivo T depletion in 13 (24.1%). Engraftment rates were 70% with acute graft versus host disease in 36% and cytomegalovirus reactivation in 55% children. There was a statistically significant difference found between survival with siblings as donors as compared to parents (p value 0.018). Overall survival was 60% which is the 1-year survival, with 68% survival among those with PIDs. Cytokine release syndrome was noted in 12/41 (29%) of children who received T replete graft and PTCy. In children over 6 months of age, PTCy at a cost of INR 1200 provides cost effective T cell depletion comparable with TCR α/ß depletion priced at INR 1200,000. Haplo SCT is feasible option for cure in children with benign disorder.

Safety and efficacy of fresh whole blood donor lymphocyte infusion in children.

Donor lymphocyte infusion (DLI) is a form of cellular immunotherapy which is known to be effective in preventing relapse in leukemia by inducing graft versus leukemia (GVL) effect. In hematopoietic stem cell transplantation (HSCT) for benign hematological conditions including primary immune deficiency, mixed chimerism is seen with the use of reduced intensity conditioning. DLI can help prevent graft rejection by boosting the existing graft in these situations. There is scant data on the use of DLI in children who have undergone HSCT for benign hematological disorders. We present our case series with early withdrawal of immunosuppression and DLI as a means to mitigate relapse of leukemia and prevent graft rejection in mixed chimerism in children transplanted for benign hematological disorders. Donor lymphocyte infusion was given in a graded regimen with the cell dose of 1 × 105 CD3 cells/kg (1 × 104/kg in haploidentical transplant), 5 × 105 CD3 cells/kg, 1 × 106 CD3 cells/kg depending on the graft kinetics and the clinical status of the children. A total of fifty eight children including those with haploidentical donors underwent DLI with an overall survival of 81.1%. The use of fresh whole blood in very small aliquots from the donor has made this technique cost effective and an attractive form of immunotherapy.

Haploidentical Stem Cell Transplantation with Post-Transplant Cyclophosphamide for Primary Immune Deficiency Disorders in Children: Challenges and Outcome from a Tertiary Care Center in South India.

Haploidentical stem cell transplantation (haplo SCT) has emerged as an acceptable alternative to matched family donor transplantation for children diagnosed to have primary immune deficiency disorders (PIDs). We present data over 4 years on the challenges and efficacy of unmanipulated T cell replete haplo SCTs with post-transplant cyclophosphamide (PTCy) in children diagnosed to have PIDs. We performed a retrospective study in the pediatric blood and marrow transplantation unit where all children less than 18 years of age diagnosed to have PIDs and who underwent haplo SCT with PTCy from January 2014 to February 2018 were included in the study. Of the 16 transplants included in the study, 5 children were diagnosed to have Wiskott-Aldrich syndrome, 3 with congenital hemophagocytic lymphohistiocytosis, 2 each with Griscelli syndrome and Mendelian susceptibility to mycobacterial diseases, and one each with Chediak-Higashi syndrome, ORAI 1 mutation immune deficiency, severe combined immune deficiency, and Hyper IgM syndrome. The source of stem cells was PBSC in 62.5% and bone marrow in 32.5%. Engraftment by day 16-21 post hematopoietic stem cell transplantation was achieved in 75% transplants with 91% of these remaining in sustained complete chimerism. Acute skin and gut graft versus host disease of grade 2-3 were noted in 50% transplants and cytomegalovirus (CMV) reactivation in 43.7% transplants. One child with congenital HLH succumbed to refractory CMV, adenovirus, and BK virus infection. Cytokine release syndrome (CRS) was noted in 75% transplants with 2 children succumbing to the illness. Tocilizumab was successfully used early in one child. Overall mortality was found to be 37.5% with overall survival of 62.5% with a median follow-up of 23.3 months. In resource limited settings, PTCy has the potential to provide a cost-effective advantage in terms of accessibility of this curative procedure among children with PIDs.

Hematopoietic Stem Cell Transplantation for Primary Immunodeficiency Disorders: Experience from a Referral Center in India.

OBJECTIVE: To share experience of over 15 years in hematopoietic stem cell transplantation in children with primary immunodeficiency disorders. DESIGN: Medical record review. SETTING: A referral center for pediatric hemato-oncological disorders. PARTICIPANTS: Children (<18 y) diagnosed to have primary immune deficiencies who underwent hematopoietic stem cell transplantation between 2002 and August 2017. MAIN OUTCOME MEASURES: Disease-free survival, morbidity and mortality. RESULTS: 85 primary immunodeficiency disorder transplants were performed with engraftment noted in 80 (94%) transplants and an overall survival of 67%. The conditioning regimen was individualized based on the underlying immune defect. Mixed chimerism was noted in 20% children with 56% (9/16) remaining disease-free. Graft versus host disease was noted in 33 (39.2%) children with most seen in children with chronic granulomatous disease. Severe combined immune deficiency transplants were mainly complicated by infections. Immune cytopenias complicated Wiskott Aldrich syndrome and Hemophagocytic lymphohistiocytosis transplants. 29.4% (25/85) children underwent haploidentical transplant in our cohort with a survival of 70% in this group. Infectious complications were the most common cause of death. CONCLUSIONS: Primary immunodeficiency disorders are curable in India when transplanted in centers with experienced and trained pediatric transplant physicians and intensivists.

Changing Trends in the Use of Granulocyte Transfusions in Neutropenic Children with Sepsis in India.

We present our data on granulocyte transfusions in children undergoing treatment for cancer and HSCT at our centre and their changing indications. In this retrospective observational analysis of children who received granulocytes from 2007 to 2015, we divided children receiving granulocytes into two groups-the first from January 2007 till December 2013 and the second from January 2014 till December 2015. This division is based on the change in our policy to use granulocytes within 48 h of septicemia as the incidence of drug resistant bacterial strains had increased at our centre. Data on 72 children with febrile neutropenia treated with 230 granulocyte infusions was analyzed. From 2007 to 2013 (n = 48/72), 27/48 (56 %) had culture proven sepsis of which 14 (51 %) were carbapenem resistant gram negative bacilli. 11 of the 27 children survived the crisis (41 %). We then changed our policy to transfuse granulocytes early during sepsis. From 2014 to 2015 (n = 24/72) 22 patients had culture proven sepsis (91 %) of which 20 had carbapenem resistant gram negative bacilli. 12/22 (54 %) with culture proven sepsis survived the episode. The survival rate improved from 41 % in first group to 54 % after early intervention with granulocytes (P value 0.0347). Despite the increased incidence of resistant bacteria during the period of 2014-2015, early use of granulocytes improved survival rate from 41 to 54 %. This intervention cannot be taken in isolation and needs to be offered early in parallel with appropriate antibiotics.

Cytomegalovirus reactivation posthematopoietic stem cell transplantation (HSCT) and type of graft: A step toward rationalizing CMV testing and positively impacting the economics of HSCT in developing countries.

We aimed to determine a correlation between cytomegalovirus reactivation post hematopoeitic stem cell transplantation (post-HSCT) with the type of graft source, defining children at risk. We analyzed data on children less than 18 years of age undergoing HSCT from 2002 to May 2016 (n = 464). Correlation between reactivation and graft source was analyzed statistically. Reactivation occurred in 3% of children with matched-related donor (MRD) transplants, 33.3% with unrelated peripheral blood stem cells, 17.4% with unrelated cords, and 36.5% (15/41) with mismatched or haploidentical grafts (P = <0.0001). MRD does not warrant weekly PCR, unlike unrelated or haploidentical donors, thus defining protocols for developing countries with limited resources.