Tathagata Chatterjee

Minimal residual disease detection using flow cytometry: Applications in acute leukemia

Minimal residual disease (MRD) describes disease that can be diagnosed by methodologies other than conventional morphology, and includes molecular methods (like polymerase chain reaction (PCR)) or flow cytometry (FCM). Detection and monitoring of MRD is becoming the standard of care, considering its importance in predicting the treatment outcome. MRD aids in identifying high-risk patients and hence therapy can be intensified in them while deintensification of therapy can prevent long-term sequelae of chemotherapy in low-risk category. FCM is considered as a less labor-intensive and faster MRD technique as compared to PCR although it has its own share of disadvantages. Current immune-based methodologies for detection of MRD depend on establishing leukemia-associated aberrant immunophenotype (LAIP), at diagnosis or relapse and use this information at specified time points for detection of MRD, or apply a standardized panel of antibody combinations for all MRD cases, in a different-from-normal approach. This review highlights MRD detection by FCM and its application in acute leukemia.

Optimizing cord blood collections: Assessing the role of maternal and neonatal factors

Background: As processing and cryopreservation of cord blood is time consuming and costly, it is essential to select units with optimal CD34+ cells, total nucleated cell (TNC) number and colony forming units (CFUs). These are the most important factors affecting outcome of UCB transplantation and are influenced by various maternal and neonatal factors. Aim and objectives: To determine the maternal and neonatal factors affecting TNC and CD34+ cell counts in cord blood so as to aid in proper selection of cord blood units for cryopreservation. Materials and Methods: A total of 100 UCB units were collected from normal vaginal deliveries, processed and assessed for volume, TNC, CD34+ cell count and CFU-GM. These parameters were then analyzed to find out whether they correlated with maternal and neonatal characteristics such as mother′s age, parity, gestational age, baby′s birth weight, and sex. Results: The volume of CB collected significantly correlated with the TNC, CD34+ cell, and CFU-GM yields (P < 0.02). A heavier placenta (P < 0.05), and a heavier baby (P < 0.002) were associated with a significantly greater volume of CB whereas the age, parity of mother and the sex of the baby had no significant effect. Conclusion: The only factors found to affect the TNC and CD34+ cell counts significantly were weight of the baby and placenta and the volume of cord blood collected. Since these factors are of prognostic significance, their analysis will aid in deciding which UCB unit should be processed and cryopreserved for UCB banking and subsequent transplantation.

A Rare Case of a Mixed Phenotypic Acute Leukemia (Mpal) - Report From a Tertiary Care Hospital

The term mixed phenotype acute leukemia (MPAL) applies to both acute bilineal leukemia, that is, leukemias containing a separate population of blasts of more than one lineage and biphenotypic leukemia containing a single population of blasts co expressing antigens of more than one lineage. Here we report a rare case of MPAL –NOS T/Megakaryocytic type in a 4 year old female child which has not been reported so far. She presented with fever, generalized weakness and right calf pain. Her peripheral blood picture revealed a leuco-erythroblastic blood picture with two population of blasts .Bone marrow biopsy revealed scattered lymphoblast amidst clusters of megakaryoblast. Immunohistochemical examination was done which revealed one population to be positive for CD3 and other population to be CD61 positive. Patient was diagnosed as a case of MPAL-NOS T/megakaryocytic type and was subsequently given AML chemotherapy. Patient achieved complete morphological remission and thereafter received an allogeneic bone marrow transplant. Patient is presently doing well after eighteen months of bone marrow transplant and has had no episode of relapse. This case report highlights the shortcomings of the WHO 2008 requirements for assigning more than one lineage to a leukemia as there is no inclusion criteria for megakaryocytic lineage which is myeloperoxidase negative.

Comparative Analysis of Various Aspects of Plateletpheresis on the Fenwal Amicus and Fresenius COM.TEC Cell Separator Instruments

OBJECTIVE To compare the Fenwal Amicus and the Fresenius COM.TEC apheresis instruments regarding donor peripheral blood parameters, operational variables of the instruments, and quality control parameters of the product obtained. METHODS We performed 100 platelet collections from 100 voluntary donors using the 2 studied devices. We measured platelet count using an automated analyzer and analyzed the activation statuses using a flow cytometer. RESULTS The median time needed to perform the procedures was significantly longer with the COM.TEC. However, the product we obtained using the Amicus instrument showed higher degrees of platelet-activation. All products we obtained with both instruments had white blood cell counts of less than 5 × 10(6) per bag. We observed no statistical difference regarding collection efficiency and collection rates between the devices. CONCLUSION Both instruments collected platelets efficiently, with minimal donor discomfort. Compared with the COM.TEC instrument, the Amicus reached the platelet target yield more quickly; however, it displayed an increase in platelet activation.

Summary and Review of the Abstracts on Philadelphia-Negative Myeloproliferative Neoplasms Presented at Haematocon 2017

There are lot of grey zones in Philadelphia negative chronic myeloproliferative neoplasms (CMPNs) and that’s the reason they are in hit list of researchers. Having a spectrum of disorders their diagnosis is very important and especially to differentiate from each other since they overlap with each other in many ways. Diagnosis doesn’t start from lab but with clinical phenotype. Clinical phenotype not only able to provide us the diagnosis but also helps in management of the disease per se. When diagnosis comes, the old timer but an evergreen morphology plays an important role which along with the newer generation tool “molecular” helps in differentiating these disorders. Lot of studies have already come up from the world. Indian data has also started coming up. When we say about the Indian data nothing holds more important role than Indian Society of Haematology and Blood Transfusion, ISHBT. This small review will cover all papers with BCR-ABL negative CMPNs which were presented at the annual national conference of the ISHBT (Haematocon 2017) which was conducted at Guwahati. These abstract papers from various reputed institutes and centres will provide a short academic journey towards ongoing research activities at these places and will able to guide us regarding Philadelphia negative CMPNs and also stimulate our brain for some left or conflicted areas.

Exteriorisation of the Ommaya Reservoir Secondary to Wound Dehiscence in a Case of ALL

A 41-year-old male a case of acute lymphoblastic leukaemia with C3 disease (CNS involvement) was being managed with modified BFM-90 protocol. For his CNS positive disease, he was planned for intensive triple intrathecal therapy. For the ease of administration of intrathecal and to ensure adequate distribution of chemotherapeutic agents, he was subjected to Ommaya reservoir placement. There were no immediate postoperative complications. The patient received 04 intrathecal therapies through the reservoir as inpatient and was subsequently discharged after CSF was negative and managed as an outpatient. He presented to OPD on D40 of Ommaya insertion with complaints of wetness of the scalp near the site of insertion. Physical examination revealed exteriorisation of Ommaya with CSF leak. CSF examination revealed no evidence of meningitis. Patient hasn’t received any radiation to lead to wound dehiscence. An Ommaya reservoir is a synthetic dome that is surgically placed beneath the scalp and attached to a catheter directed into a ventricle. Complications associated with Ommaya reservoir include technical complications such as misplacement of the catheter, intraventricular haemorrhage, malfunctioning reservoirs, and bacterial meningitis [1]. Other complications included wound dehiscence, ventricular catheter associated cerebral oedema and leakage of cerebrospinal fluid. With wound dehiscence, a superficial wound infection can easily track to the CSF and intracranial cavity resulting in serious intracranial complications. Countersinking is a good technique to prevent wound dehiscence, device extrusion and in irradiated patients with very thin skin, it also enables tension-free closure of the wound [2]. Our patient had wound dehiscence and CSF leak with our associated meningitis in the absence of radiation to scalp at D40 which is an uncommon complication. It is thus mandatory to do a regular local physical examination of the scalp in a patient with Ommaya reservoir (Figs. 1, 2).

Postmortem diagnosis of fulminant infectious mononucleosis

Fulminant infectious mononucleosis (FIM) is a rare but life-threatening complication of Epstein-Barr virus infection that usually affects immunodeficient individuals. It is a hyperinflammatory syndrome caused due to release of massive amount of various cytokines, leading to hemophagocytic lymphohistiocytosis on histopathology, and a plethora of clinical manifestations due to simultaneous involvement of multiple organs of the body. The case of a young male who presented with fever and dyspnea and died within 12 h of presentation to the hospital was taken for autopsy. The postmortem evidence of hemophagocytic lymphohistiocytosis in the spleen, bone marrow, lymph nodes, liver, and lungs and the presence of large, basophilic intranuclear inclusions that stained latent membrane protein-1 positive on immunohistochemistry led to the diagnosis of FIM.

Antiphospholipid syndrome: A diagnostic challenge

The antiphospholipid syndrome (APS) is an acquired autoimmune thrombophilic disorder that is characterized by thrombosis (venous, arterial and microvascular) and obstetric morbidity due to a diverse family of antibodies against phospholipid-binding proteins present in plasma. The term antiphospholipid antibody is actually a misnomer as the antibodies are not against the phospholipid per se, but target the plasma protein co-factors, which bind to anionic PLs. The exact etiology has not been elucidated and is multifactorial. The initial guidelines for the diagnosis of APS were laid down in Sapporo, 1999, which were subsequently revised as the Sydney Consensus Conference criteria in 2006. Major changes were the inclusion of β2GPI as independent laboratory criteria, addition of ischemic stroke and transient cerebral ischemia as established clinical criteria and the requirement of repeating the test after 12 weeks. The laboratory tests recommended are coagulation assays, which study the effect of lupus anticoagulant on the clotting time and immunological assays, mostly ELISAs to detect IgG and IgM antibodies against cardiolipin and/or β2 glycoprotein I. For the diagnosis of APS, at least one clinical criterion and one laboratory criterion should be present. Limitations pertaining to the standardization, reproducibility and robustness of the currently recommended diagnostic tests still remain. Despite elaborate guidelines and syndrome defining criteria, the diagnosis of APS still remains a challenge. A greater interaction between the clinicians and the laboratory professionals is necessary for arriving at the correct diagnosis as a misdiagnosis of APS can have grave consequences.

Flowcytometric comparative analysis in acute leukemias between Indian and proposed minimal screening panel

BACKGROUND Acute myeloid leukemia and acute lymphoid leukemia differ substantially in response to therapy and course, and accurate differentiation of the two is fundamental to therapeutic decisions. Immunophenotyping is used for this purpose, and various guidelines have been proposed regarding a minimal screening antibody panel. Most of them have been found inefficient. METHODS Eighty-two cases of consecutive acute leukemias reporting to this hospital over a period of two years were included in the study. Peripheral blood smear, bone marrow aspirate, and bone marrow biopsy were studied using morphology, cytochemical stains, and relevant immunohistochemical stains on selected biopsy specimens. Flowcytometry analysis was carried out using Indian consensus screening panel and our proposed minimal screening panel (PMSP) for comparison. RESULT Immunophenotyping using PMSP resulted in 95.12% accurate diagnosis versus Indian consensus minimal screening panel (ICMSP) with an accuracy of 92.68%. This result was statistically significant as per Chi Square tests. CONCLUSION PMSP can be used as a substitute for ICMSP, since it includes lineage-specific cytoplasmic antibodies, as well as lesser number of monoclonal antibodies, and enables us to diagnose mixed lineage leukemia. Fewer markers can be linked to a lower cost as well, which is relevant in a developing economy.

Comparing flow cytometry immunophenotypic and immunohistochemical analyses in diagnosis and prognosis of chronic lymphoproliferative disorders: Experience from a Tertiary Care Center

Background: The latest World Health Organization classification incorporates extensive description of immunophenotype of the neoplastic cells while describing chronic lymphoproliferative disorders (CLPDs). The present study was undertaken with an aim to identify and compare the roles of flow cytometry (FCM) and immunohistochemistry (IHC) as modalities of immunophenotyping in the diagnosis of CLPDs. Materials and Methods: Thirty untreated cases of CLPDs were enrolled in the study. Twenty eight cases of B-CLPD were divided into two groups - chronic lymphocytic leukemia (CLL) (21 patients) and non-CLL (7 patients). Peripheral blood/bone marrow aspirate samples were analysed by FCM using various panels of monoclonal antibodies. Immunohistochemical analysis of bone marrow biopsies obtained from these patients was also performed. Results: Panel A of monoclonal antibodies comprising CD5, CD23, CD22, surface membrane immunoglobulin (SmIg), FMC7 and Panel B comprising CD5, CD23, CD22, SmIg, FMC7, CD79b were useful (P < 0.01 and <0.001 respectively) while Panel C comprising CD5, CD23, SmIg, FMC7 and CD79b was not found to be useful in distinguishing CLL from non-CLL (P > 0.05) The concordance rate between FCM and IHC ranged from 80% to 100% for all comparable immunological markers. In all cases of CLPDs, we propose a screening panel comprising 9 markers including CD19, CD5, CD23, FMC7, CD10, CD20, CD3, kappa and lambda, which are important for specifying the lineage (B or T), to differentiate CLL from non-CLL group and for deciding the secondary panel. Conclusion: Scoring system using CD5, CD23, CD22, FMC7, CD79b, and SmIg is useful in differentiating CLL from non-CLL cases. Concordance rate of FCM and IHC in CLPDs is 93.3%. Using a panel comprising CD19, CD5, CD23, FMC7, CD10, CD20, CD3, kappa and lambda, a diagnosis of CLL, mantle cell, and follicular lymphoma, the three most common CLPDs can be made. Secondary panels for diagnosis of hairy cell leukemia and T-cell CLPD should be utilized.