Comparative analysis of immunohistochemistry and flow cytometry in the diagnosis of acute leukaemia: a single centre study

Background: Morphological evaluation and immunophenotyping are the major diagnostic modalities of acute leukaemia (AL). Although immunohistochemistry (IHC) and flow cytometry (FCM) are necessary for lineage assessment, but in many cases the use of these modalities alone might possess a diagnostic challenge. The study was aimed to analyse the diagnostic utility of IHC and FCM in the diagnosis of AL. Methods: This cross-sectional hospital-based study was done for one year and included 55 cases. Following peripheral blood examination and bone marrow study, IHC and FCM analysis was done using CD34, anti-MPO, CD3 and CD20. Results: There were 74.5% acute myeloid leukaemia (AML) and 25.5% acute lymphoblastic leukaemia (ALL) cases. By IHC, CD34 was positive in 94.5% cases, anti-MPO in 69.1%, CD3 in 3.6% and CD20 in 12.7% cases. But by FCM, CD34 was positive in 96.2% cases, anti-MPO in 61.5%, CD3 in 3.8% and CD20 in 19.3% cases. FCM could not be done for 3 cases as there was dry tap with pancytopenia and lineage assessment was done by IHC. On comparative analysis, CD34 was found to be better expressed by FCM. Anti-MPO and CD20 were better expressed by IHC and CD3 was equally expressed by both. Conclusions: IHC is an easy and cost-effective technique which gives an accurate characterization of the lineage and subtype of AL, especially in cases where use of FCM is limited such as cases with dry tap and pancytopenia and in limited resource centers

Transfusion-associated graft-vs-host disease – A case report

Transfusion-associated graft-vs-host disease (TAGVHD) is a rare complication of blood transfusion. Unlike GVHD associated with hematopoietic stem cell transplantation TA-GVHD involves the patient's bone marrow and leads to bone marrow aplasia. We report a case of TA-GVHD in a 45-year-old post-hysterectomy patient after packed red blood cell transfusion from a sibling donor. The patient had fever, maculopapular rashes all over the body, elevated transaminases, and hyperglycemia after a week of the blood transfusion. Severe pancytopenia and bone marrow aplasia followed and she succumbed to her disease after 3 weeks of onset

Management of B-cell lineage acute lymphoblastic leukemia: expert opinion from an Indian panel via Delphi consensus method

IntroductionCurrently, there are no guidelines for the management of B-cell lineage acute lymphoblastic leukemia (B-ALL) from an Indian perspective. The diagnostic workup, monitoring, and treatment of B-ALL vary among different physicians and institutes.ObjectiveTo develop evidence-based practical consensus recommendations for the management of B-ALL in Indian settings.MethodsModified Delphi consensus methodology was considered to arrive at a consensus. An expert scientific committee of 15 experts from India constituted the panel. Clinically relevant questions belonging to three major domains were drafted for presentation and discussion: (i) diagnosis and risk assignment; (ii) frontline treatment; and (iii) choice of therapy (optimal vs. real-world practice) in relapsed/refractory (R/R) settings. The questionnaire was shared with the panel members through an online survey platform. The level of consensus was categorized into high (≥ 80%), moderate (60%–79%), and no consensus (< 60%). The process involved 2 rounds of discussion and 3 rounds of Delphi survey. The questions that received near or no consensus were discussed during virtual meetings (Delphi rounds 1 and 2). The final draft of the consensus was emailed to the panel for final review.ResultsExperts recommended morphologic assessment of peripheral blood or bone marrow, flow cytometric immunophenotyping, and conventional cytogenetic analysis in the initial diagnostic workup. Berlin–Frankfurt–Münster (BFM)–based protocol is the preferred frontline therapy in pediatric and adolescent and young adult patients with B-ALL. BFM/German Multicenter Study Group for Adult Acute Lymphoblastic Leukemia–based regimen is suggested in adult patients with B-ALL. Immunotherapy (blinatumomab or inotuzumab ozogamicin) followed by allogeneic hematopoietic cell transplantation (allo-HCT) is the optimal choice of therapy that would yield the best outcomes if offered in the first salvage in patients with R/R B-ALL. In patients with financial constraints or prior allo-HCT (real-world practice) at first relapse, standard-intensive chemotherapy followed by allo-HCT may be considered. For subsequent relapses, chimeric antigen receptor T-cell therapy or palliative care was suggested as the optimal choice of therapy.ConclusionThis expert consensus will offer guidance to oncologists/clinicians on the management of B-ALL in Indian settings

Mesenchymal Stem Cells from Human Extra Ocular Muscle Harbor Neuroectodermal Differentiation Potential

<div><p>Mesenchymal stem cells (MSC) have been proposed as suitable candidates for cell therapy for neurological disorderssince they exhibit good neuronal differentiation capacity. However, for better therapeutic outcomes, it is necessary to isolate MSC from a suitable tissue sourcethat posses high neuronal differentiation. In this context, we isolated MSC from extra ocular muscle (EOM) tissue and tested the <i>in vitro</i> neuronal differentiation potential. In the current study, EOM tissue derived MSC were characterized and compared with bone marrow derived MSC. We found that EOM derived MSC proliferated as a monolayer and showed similarities in morphology, growth properties and cell surface marker expression with bone marrow derived MSC and expressed high levels of NES, OCT4, NANOG and SOX2 in its undifferentiated state. They also expressed embryonic cell surface marker SSEA4 and their intracellular mitochondrial distribution pattern was similar to that of multipotent stem cells. Although EOM derived MSC differentiated readily into adipocytes, osteocytes and chondrocytes, they differentiated more efficiently into neuroectodermal cells. The differentiation into neuroectodermal cellswas confirmed by the expression of neuronal markers NGFR and MAP2B. Thus, EOM derived MSC might be good candidates for stem cell based therapies for treating neurodegenerative diseases.</p></div

CFU-F ability and mesodermal differentiation of EOM-MSC.

<p>(A) CFU-F (colony forming unit-fibroblasts) assay was performed to analyse the colony forming ability of EOM-MSC and BM-MSC. Mean±SEM, n = 6–12, *p<0.05. The cells were from passage 3–4. (B-G) EOM-MSC and BM-MSC were differentiated into adipocytes, osteocytes or chondrocytes for 21–28 days. (B, D) Adipogenic differentiation was determined by staining with oil red O, (C, D) osteogenic differentiation by staining with alkaline phosphatase and alizarin red and (D) chondrogenic differentiation was determined by safranin O staining. The number of oil red O (ORO) positive cells per 2000 cells and alkaline phosphatase (ALP) positive cells per 1000 cells were calculated for EOM-MSC and BM-MSC after differentiation into respective lineages. Values are Mean±SEM, n = 4–7. (E-G) Real-time PCR was performed to determine the expression levels of ADIPOQ, OSTEOCALCINor SOX9 in undifferentiated (CONTROL), adipo-differentiated (AD), osteo-differentiated (OS) or chondro-differentiated (CH) EOM-MSC and BM-MSC. Values are Mean±SEM, n = 3. The expression levels were normalized to GAPDH expression levels in the respective samples. EOM-MSC and BM-MSC were from passages 3–5 *p<0.05. ORO-Oil red O, ALP-alkaline phosphatase, AZR-Alizarin red, SFO-Safranin O.</p

Gene expression analysis in EOM-MSC.

<p>(A) Expression levels of transcription factors OCT4, NANOG and SOX2 and neuronal lineage related gene NES in EOM-MSC was analysed by real-time PCR and compared with BM-MSC. mRNA expression levels of (B) apoptosis related genes BAD, cIAP1, cIAP2 and (C) other factors HIF1α, FGF2, IL6, TNFα as well as calcium channel related genes ORAI1, STIM1, TRPC1 were determined in EOM-MSC and BM-MSC by real-time PCR. The expression levels of the genes were normalized to GAPDH expression levels in the respective samples. Values are mean±SD, n = 3–5* p<0.05.</p

Neuronal differentiation of EOM-MSC.

<p>(A) Real-time PCR analysis of neuronal specific genes NES, NEUROD1, PAX6 and TUBB3 in EOM-MSC cultured in growth media and neuronal differentiation media for 14 days. The expression levels of the genes were normalized to GAPDH expression levels in the respective samples. Values are Mean±SEM, n = 5, *p<0.05. Immunoflurescence analysis of neuronal differentiated EOM-MSC showing downregulation of CD146 in cells cultured in (B) neuronal media compared to cells grown in (C) normal growth media. (D) CD90 expression was unaffected in EOM-MSC cultured in neuronal media. Neuronal differentiated cells exhibited upregulation of neuronal lineage markers (E) GFAP, (F) NGFR and (G) MAP2B. CD90, CD146 and GFAP staining were performed 9–10 days after the addition of neuronal induction media whereas NGFR and MAP2B staining were performed after 21 days of neuronal induction. Representative microscopic images are shown.</p

Isolation and expansion of EOM-MSC.

<p>(A) EOM-MSC were isolated from freshly resected EOM tissue from the human eye. The tissue was mechanically dissociated and plated in growth media. Phase-contrast microscopic images of (B) EOM-MSC and (C) BM-MSCshowing spindle shaped adherent cells, passage 2. (D) FESEM analysis of <i>in vitro</i> expanded EOM-MSC (passage 3). (E, e’) CD90 expression in <i>in vitro</i> expanded EOM-MSC determined by immunocytochemical staining. Representative photomicrographs are shown. EOM-MSC were from passage 2–4.</p