Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: The immunomodulatory drugs (IMiDs) have made a profound difference in the treatment of multiple myeloma (MM). Lenalidomide (LND) is chiral compound with two enantiomers and one enantiomer may have different pharmacologic action than the other. For a given chiral drug, until proven otherwise, it is right to think of the two enantiomers as two different drugs with unique properties. Aims: This study aimed to explore efficacy of lenalidomide (R) - (+) and (S) - (-) enantiomers in human multiple myeloma cell line. Methods: H929 myeloma cell line model was designed to understand the potential differences in chirality of the R and S form of LND enantiomers and racemate in multiple myeloma cell line. We report the first biological evaluation of lenalidomide in racemic and in both (R)- and (S)-enantiomerically pure forms against (in vitro) H929 cells of multiple myeloma (MM) using an annexin V assay. Results: It was observed that at 48 hours S-LND treated cells demonstrated significant reduction in the metabolic activity at 10 and 20 µg/mL and profound morphological changes. Cell cycle analysis showed cells in S and G2/M phase were decreased suggesting that all of the drugs can arrest cells at G1 cell cycle phase. We demonstrate (S) - (-) enantiomers of lenalidomide inhibited the growth of H929 MM cells without any in-vivo activation. Summary/Conclusion: The study concludes that (S) - (-) enantiomers of lenalidomide noted more potent agent which could have both the anti-cancerous and immunomodulatory activity.Keywords: Imids, Cell line, Multiple myeloma
Background: FLT3-ITD and NPM1 mutations are considered to be the major determinants of the patient response to therapy and outcome. The primary aim of this study was to establish the correlation between these molecular mutations and the clinico-hematologic parameters as well as the prognostic outcome of the Indian acute myeloid leukemia (AML) patients. Materials and Methods: This prospective study involved newly diagnosed nonpromyelocytic AML patients who had undergone complete diagnostic workup, including immunophenotyping, conventional cytogenetics and molecular analysis for NPM1 and FLT3-ITD mutation by reverse transcriptase polymerase chain reaction at presentation. Results: Overall, the prevalence of NPM1 and FLT3-ITD mutations was found to be 14.4% and 10.8%, respectively. Among patients with normal karyotype, leukocytosis was significantly associated with NPM1+ group than the NPM1− group (P = 0.0019) and more severe degree of anemia was observed in the FLT3-ITD+ patients than the other groups (P = 0.025). No significant correlation was found in terms of age at presentation (P = 0.56), sex ratio (P = 0.467), median platelet count (P = 0.27), and blast percentage between NPM1+ and FLT3-ITD+ groups. Complete remission (CR) rates were better in the NPM1+/FLT3-ITD− group than the other three groups. Unlike most other studies, improved CR rates as well as disease-free survival were observed in the NPM−/FLT3-ITD− group than the FLT3-ITD+ groups although not reaching statistically significant levels. Conclusion: Some differences in the clinical behavior of the Indian AML patients in comparison to that of the West in the presence of NPM1 and FLT3-ITD suggests that comprehensive studies are required to confirm the definitive role of these mutations among AML patients, especially with normal karyotype.
Aim: We investigated our previous finding of increased retention of poly(lactic-co-glycolic) acid nanoparticles (PLGA-NPs) with metabolic inhibitors (MI) and studied the effect of some small molecule inhibitors on PLGA-NP assimilation. Materials & methods: Intracellular PLGA-NP colocalization in the presence of MI was investigated by confocal microscopy. Intracellular retention of PLGA-NPs by some small molecules was estimated by fluorescence microscopy and flow cytometry after Pulse/Chase experiments. Results: MI caused PLGA-NP colocalization in intracellular membranous structures, mainly endosomes and lysosomes. Some small molecule inhibitors demonstrated increased intracellular PLGA-NP accumulation. Conclusion: This study elucidates the movement of PLGA-NP in cells and suggests that clinically used small molecules can reduce their extrusion by enhancing their stay within intracellular vesicles, with possible clinically beneficial consequences.
Aims and Objectives: Total quality management is an essential component of laboratory to maintain quality and includes pre-analytical, analytical and post-analytical phases. Pre-analytical phase starts from ordering investigation by the clinician till it is analysed in the laboratory. It is essential that a pathologist is aware of the confounding factors that may lead to pre-analytical errors with the knowledge of reducing them to improve the quality of the lab. The present study was conducted to evaluate the pre-analytical errors in the haematology laboratory and steps taken to minimise it. Materials and Methods: The study was conducted in the Haematology Laboratory of Pathology Department, All India Institute of Medical Sciences, Rishikesh, over a period of 4 years and included all the blood samples analysed in the lab. The pre-analytical variables including requisition form, patient preparation, labelling, sample collection, quality and transportation were analysed for the errors along with the corrective actions taken to minimise them. Results: A total of 25,15,583 investigations were received in the haematology laboratory over a period of 4 years. The total errors observed in the haematology lab in pre-analytical phase were 13,031 comprising 0.51% of total haematological investigations during the study period. The most common error observed was due to clotted sample (40.3%), followed by haemolysed samples (15%) and incomplete or wrongly filled requisition forms (10%). Conclusion: Pre-analytical phase forms an integral part of total quality management system and its analysis is essential to minimise the errors in haematology lab. The quality of blood sample with completely filled requisition forms and prompt transportation of sample to the lab are essential components to avoid pre-analytical errors. In addition, continuous training and acknowledgement of non-conformance with root cause analysis is the basic key for reducing errors and improving quality of haematology lab.
Somatic mutations in Calreticulin (CALR) have been recently discovered in JAK2/MPL unmutated patients with primary myelofibrosis (PMF) or essential thrombocythemia.Clinical and hematologic features were obtained for 80 patients with PMF. JAK2V617F mutation was analyzed by DNA tetra-primer amplification refractory mutation system (ARMS-PCR). CALR and MPL mutations were identified by bi-directional Sanger sequencing.CALR mutations were detected in 11.2% (9/80) of all PMF patients and 25.7% (9/35) of all JAK2V617F and MPL unmutated patients all of which were Type I mutation or deletions. A novel CALR mutation pattern (c.1241_1288del) was identified in one (1/9) patient. No case of Type II mutations or scattered point mutations was found in any of these patients. Uni-variate analysis at presentation showed that CALR mutations were significantly associated with younger age (P = 0.003) and larger spleen size (P = 0.001). No significant correlation was found between CALR mutation and clinico-hematologic characteristics or international prognostic scoring system (IPSS) scoring of the PMF patients.CALR mutations have a distinct molecular profile in Indian patients, different from that of other studies worldwide. Larger prospective studies need to be designed to establish the impact of paucity of Type II mutations in contributing to disease phenotype and prognostic outcome of patients.
The process of programmed cell death or apoptosis has, in the decade preceding the publication of this 2005 book, been shown to be centrally involved in the pathogenesis of the significant majority of human illnesses and injury states. The cellular attrition observed in most degenerative conditions is apoptotic in nature; conversely a failure of apoptosis has been proposed to underlie many forms of cancer. The central role of apoptosis in human disease clearly brings with it clinical promise; for example, the strong possibility exists that attenuation of apoptotic death will significantly modulate the severity of degenerative disorders. Similarly, conditions, such as cancer, autoimmune disease, psoriasis and endometriosis, in which aberrant cellular proliferation is observed, may benefit from enhanced rates of apoptosis. This book surveys the underlying molecular mechanisms of apoptosis, investigates its role in degenerative and other diseases, and evaluates potential therapies that will permit appropriate activation or inhibition of apoptosis in disease and injury states.
Abstract Study question Does immune response to COVID-19 vaccination affect the clinical outcome in fully vaccinated infertile women undergoing IVF/ICSI cycles? Summary answer COVID-19 IgG antibodies are present in follicular fluid post vaccination and higher immune response increases duration of gonadotrophins required and negatively impacts the IVF outcome. What is known already Recent studies assessed the influence of COVID-19 infection and mRNA COVID-19 vaccine on the stimulation cycle characteristics and embryological variables of patients undergoing IVF cycle and found no effect on the IVF outcome in their immediate IVF cycle after recovery, except for a decreased number of top quality embryos. One study reported infection or mRNA vaccine results in rapid formation of anti-COVID IgG which can be detected in follicular fluid. This immune response did not lead to any significant negative effect on ovarian follicular function. There is a possibility that COVID-19 infection might affect numerous fertility-linked proteins. Study design, size, duration Prospective observational study, conducted at Division of Reproductive Medicine of tertiary care institute. After taking informed consent, 32 patients who satisfy the inclusion and exclusion criteria with history of receiving two doses of Covishield or Covaxin vaccine with at-least 2 weeks from last dose, were recruited for IVF/ICSI cycles from December 2021 to January 2022, for assessing COVID-19 IgG antibodies in their follicular fluid. Participants/materials, setting, methods Women of 21-40 years with normal ovarian reserve and normal uterine cavity were included, those with history of COVID infection were excluded. All patients underwent GnRH antagonist protocol. Follicular fluid was collected at time of oocyte retrieval. After collecting oocytes, 400 microlitre of follicular fluid was stored at -80 and later thawed and analysed for SARS-CoV-2 IgG antibodies (ADVIA Centaur COV2G assay, Germany) which are expressed in index value and reported as reactive (≥1 index). Main results and the role of chance Out of 32, 21 (65.6%) of the participants had received COVISHIELD (V1)and 11 (34.3%) received COVAXIN (V2). The mean gap between vaccine and the IVF cycle was 84.94 ± 52.65 days. The mean COVID IgG antibody titres (Index) were significantly higher in V1, 28.77±33.50 (0.34 -100), than V2 2.28±3.74(0.05-13.23), p<0.001. Patients with higher antibody titres, required longer duration of ovarian stimulation, rho=0.42, p = 0.017. Patients with higher COVID IgG antibodies were negatively correlated with clinical pregnancy rate (20.9 0± 29.68 vs 4.60 ± 6.28, p = 0.153). The time gap from the last dose of vaccine to IVF cycle had moderate negative correlation with percentage of grade-I embryos out of the total embryos fertilised (%), rho= -0.33, p = 0.068. Furthermore, higher gonadotropins doses were required in patients with high antibody titres, rho=0.25, p = 0.160, and amongst V1 vs V2, total dose of gonadotropins required was 3802.38±742.92 vs 3422.73±564.52, respectively, p=0.115. COVID IgG antibody titres had weak negative correlation with number of grade-I embryos, rho= -0.16, p = 0.396. The time gap from the last dose of vaccine to IVF cycle had a weak negative correlation with number of grade-I embryos, rho=-0.28, p = 0.124. Limitations, reasons for caution The main limitation of this study is small sample size. However, the study is currently ongoing, and these are the interim results of the same. As prospective studies with larger sample size would be required to assess the effect of different COVID-19 vaccines in different populations on the IVF outcomes. Wider implications of the findings The present study confirms the presence of COVID IgG antibodies in follicular fluid in vaccinated women, and proves that COVISHIELD vaccinated patients had higher antibody titres. Higher antibody titres require longer duration of stimulation and result in poorer outcomes so a longer interval from vaccine to IVF should be recommended. Trial registration number NA
A retrospective analysis of eleven pregnancies complicated by isolated fetal congenital complete heart block (CCHB) in anti-SSA/Ro antibody positive women was carried out at a tertiary hospital in India to study the perinatal outcome. The mean gestational age at the time of detection of fetal CCHB was 24.5 ± 3.1weeks. Six mothers were asymptomatic; two had Sjögren's syndrome and three had systemic lupus erythematosus. Oral dexamethasone was given to all the patients after the diagnosis was made. There was one case of intrauterine death. Seven (63.6%) neonates needed a permanent pacemaker. There was no significant difference in the perinatal outcome in asymptomatic women with fetal CCHB and in women with connective tissue disorder and fetal CCHB. To conclude, fetal CCHB is associated with high morbidity but the presence of underlying connective disorder in the mother does not worsen the prognosis of the affected neonate.
Flowcytometry has an essential role in the diagnosis and classification of acute leukemias. However, there exists a great degree of inter-laboratory variability on issues like panel selection, antibody combinations, gating strategies, fluorochromes, and clonal selection.The primary aim of this study was to derive a minimal panel of antibodies and evaluate its diagnostic usefulness in acute leukemias by flowcytometry by using the detailed immune-phenotype of different lineage-specific or non-specific markers.This prospective observational study involved 400 newly diagnosed cases of acute leukemias. Bone marrow aspirate samples were subjected to morphological evaluation, cytogenetics and flow cytometric immunophenotyping.A minimal panel of eight antibodies comprising of CD45/CD34/CD19/MPO/cytoCD3/CD64/CD117/CD79a was derived by applying different permutations and combinations with a diagnostic yield of 97.5%. The minimal panel was further validated by testing in an independent cohort of patients with similar demographic characteristics, where it showed a high diagnostic yield of 98% in comparison with the screening panels proposed by other recently published studies.It may be concluded that the diagnostic performance of the eight antibody panel is better than most other panels used across the different laboratories in terms of yield, number of antibodies used and the scientific approach used to derive and validate the results and so henceforth may be applied in any setting with limited resources for better diagnostic accuracy.
Myomectomies are increasingly being performed by a minimal access route. However, laparoscopic myomectomy remains a technical challenge, especially when the fibroid burden is high because of either a very large myoma or the presence of multiple myomas. These technically difficult cases increase the operating time which in turn increases blood loss, suboptimal uterine reconstruction, and length of stay in hospitals. Laparoscopic-assisted myomectomy addresses all of these difficulties, making myomectomy a quick and feasible method for large and multiple fibroids. Since this surgery involves morcellation by knife, large myomas can be removed safely and swiftly. Uterine reconstruction is carried out in multiple layers with the aid of open surgical instruments. Suturing is simple and easy. Length of hospital stay and overall recovery are similar to those of total laparoscopic myomectomy. One can avoid open myomectomy even in patients with large and multiple fibroids. This chapter describes the stepwise technique and the outcome of 50 cases that underwent this procedure at our unit at Apollo Hospitals in Hyderabad, India.
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