A retrospective study on immunohistochemical evaluation of CD34 in the pathogenesis of oral lichen planus.

Context: Oral lichen planus (OLP) is a chronic inflammatory disorder with an autoimmune inflammatory pathogenesis. Although the etiology of OLP is not known, it is considered to be a T-cell-mediated inflammatory disorder. Angiogenesis is the neoformation of anomalous blood vessels in preexisting vascular structures. Chronic inflammatory disease has been related with stimulus of uncharacteristic angiogenesis. Aim: The objective of this study was to analyze and appraise the role of angiogenesis in lichen planus by the expression of CD34 immunohistochemistry. Material and Methods: Group I, the control group, comprised 10 cases. Group II comprised 30 diagnosed cases of OLP. These 40 tissues were gauged for microvessel density (MVD) in four selected areas with high inflammatory infiltrate for the expression of CD34 antibody using immunohistochemistry. Results and Conclusion: Using one-way analysis of variance and Tukey's multiple comparison test we observed a significant difference between the groups (P < 0.0001). Patients with erosive pattern (146.30 ± 16.59) show the highest CD34 MVD than the patients with reticular pattern (104.90 ± 10.61), followed by normal subjects (43.04 ± 8.70). Hence, it can be concluded that angiogenesis is related to the pathogenesis and progression of OLP.

Assessment of dengue virus inactivation in random donor platelets using amotosalen and ultraviolet A illumination.

OBJECTIVES: The study objective was evaluation of amotosalen and ultraviolet A (UVA) illumination-based inactivation of dengue virus (DENV) in blood platelets. MATERIALS AND METHODS: Whole blood was collected from healthy donors and platelet concentrates were prepared at a tertiary care hospital in Gurugram, India. Platelet units collected from five blood group matched individuals were pooled and spiked with DENV. The spiked platelet units were subjected to amotosalen treatment followed by UVA illumination, to evaluate the efficiency of this method for viral inactivation. The treated platelet units were evaluated for the presence of infectious DENV. Amotosalen levels were quantified in the treated samples using high-performance liquid chromatography. RESULTS: The presence of replicating DENV was not observed in spiked platelet units treated with amotosalen and UVA illumination, whereas untreated units contained actively replicating DENV. Amotosalen levels were found to be in the permissible range after photochemical inactivation. CONCLUSIONS: Amotosalen/UVA pathogen inactivation treatment showed efficient inactivation of DENV in platelet components. Therefore, it seems to be a promising method for mitigating the risk of dengue transmission through transfusion of potentially contaminated platelet components in dengue-endemic countries such as India.

Maxillary ameloblastic carcinoma: A diagnostic conundrum.

Ameloblastic carcinoma (AC) is a rare malignant epithelial proliferation that is associated with an ameloblastoma or histologically resembles an ameloblastoma. It is considered to be an aggressive neoplasm that is locally invasive and spread to regional lymph nodes or distant sites. It requires aggressive surgical treatment, and regular follow-up, therefore, differs from ameloblastoma. Sometimes, ameloblastomas exhibit a mild-to-moderate degree of cytological atypia; hence, in such cases, a correlation should be established between the clinical, radiological and histopathological findings, thus detecting the aggressiveness of the tumor. Here, we present the case report of a 52-year-old male patient diagnosed as AC based on histopathological and immunohistochemical findings.

Differential Wnt-ß- catenin pathway activation in HPV positive and negative oral epithelium is transmitted during head and neck tumorigenesis: clinical implications.

The aim of this study is to understand the association of HPV infection and wnt-ß-catenin self-renewal pathway in development of head and neck squamous cell carcinoma (HNSCC). For this reason, the molecular profiles (methylation/deletion/expression) of antagonists (SFRP1/2 and DKK1), agonists (FZD7 and LRP6) and effector protein ß-catenin of the pathway were analyzed in HPV positive/negative oral epithelium at first, followed by its changes during development of the tumor along with correlations with different clinico-pathological parameters. HPV infection alone or in combination with tobacco habit could activate p- ß-catenin expression in basal/parabasal layers of oral epithelium through high expression of FZD7 and significant down regulation of SFRP1/2 through promoter hypermethylation due to over expression of DNMT1 with ubiquitous down regulation of DKK1 and up-regulation of LRP6. This phenomenon has been seen in respective HPV positive and negative HNSCC tumors with additional deletion/microsatellite size alterations in the antagonists. Overall alterations (methylation/deletion) of SFRP1/2, DKK1 gradually increased from Group I (HPV-/Tobacco-) to Group IV(HPV+/Tobacco+) tumors, leading to the worst prognosis of the patients. Thus, the transmission of differentially activated wnt-ß-catenin pathway from HPV positive/negative basal/parabasal layers of oral epithelium to HNSCC tumors determines differences in molecular pathogenesis of the disease.

Clinico-pathological significance of Drp1 dysregulation and its correlation to apoptosis in oral cancer patients.

Dysregulation in mitochondrial dynamics has been associated with several diseases including cancer. Present study assessed the alteration in mitochondrial fission protein (Drp1) in oral epithelial cells collected from clinically confirmed pre-cancer and cancer patients and further correlates it with the cellular apoptosis signaling. Results indicate the ROS accumulation in OSCC patients is accompanied by several changes including increase in mitochondrial mass, expression of mitochondrial fission protein (Drp1) and alteration in apoptotic signaling. The positive co-relation has been observed between the expressions of anti-apoptotic Bcl-2proteinswith mitochondrial fission protein Drp1. Higher mitochondrial fission in oral cancer cells was also correlated with the increased expression of cell cycle marker CyclinD1 indicating highly proliferative stage of oral cancer cells. The clinical correlation can be extended to develop biomarker for diagram and program in oral cancer management.

Characterization of biological variation of peripheral blood immune cytome in an Indian cohort.

Immune parameters show characteristic normal baseline levels and variance in the population. We characterised the degree of inter-individual and within-individual variation over one-year time period in 33 immune cell subsets by flow cytometry in peripheral blood mononuclear cells from 43 healthy young adult volunteers. Our analysis revealed that immune subsets that showed low variability between individuals also showed low short-term fluctuations within-individuals, as well as concordance in siblings. However, when baseline levels and degree of fluctuation were considered together, individuals failed to cluster into discreet groups. Together, the data reveal complex inter-relationships between immune subsets in individuals, and provide insights into the observed heterogeneity between individuals and between multiple immune subsets.

Frequent plateletpheresis donations & its effect on haematological parameters: An observational study.

Background & objectives: The well-being of donors undergoing frequent plateletpheresis has been a matter of concern. The aim of this study was to analyze the effect of frequent plateletpheresis on the haematological parameters (HP) of repeat donors. Methods: The study was conducted during February 2016 to March 2017 on all the repeat plateletpheresis donors undergoing the 2nd plateletpheresis within a month of the first in a tertiary care centre. Donors repeating plateletpheresis 3rd and 4th times were also studied. The values of the HP observed on follow up after plateletpheresis done on three different separators were compared. Results: HPs of the 98 donors were similar at follow up except mean platelet volume (P <0.05). Of the 98 donors, 35 were followed up within a week and 63 were followed up within 8-30 days. No significant alteration was found in the HPs except a significant difference in the variation of platelet counts of the two groups (P=0.025). In 34 donors who presented 3rd time for plateletpheresis (mean gap between 1st and 3rd plateletpheresis=31 days), no significant differences in the HPs were found except the platelet distribution width (P <0.05). Minimal difference in the HP was found in the baseline and the follow up of 3rd plateletpheresis i.e., at 4th plateletpheresis donation. Plateletpheresis through all the three cell separators used had similar effects on the follow up HPs. Interpretation & conclusions: Repeated plateletpheresis can be done without any detrimental effects on the cell counts of the plateletpheresis donors. The three cell separators yielded similar post-donation follow up haematological parameters.

Chemometric analysis of integrated FTIR and Raman spectra obtained by non-invasive exfoliative cytology for the screening of oral cancer.

FTIR spectroscopy and Raman spectroscopy of biological analytes are increasingly explored as screening tools for early detection of cancer. In the present study, an integrated analysis of the FTIR and Raman spectra obtained from exfoliated cells is adopted to improve discrimination of normal, pre-cancerous and cancerous conditions. Multiple spectra were obtained from 13 normal, 13 pre-cancer and 10 cancer patients in both modes. Compared to normal patients, significant differences were observed at 1550, 1580, 1640, 2370, 2330, 2950-3000 and 3650-3750 cm-1 (FTIR) and 520, 640, 785, 827, 850, 935, 1003, 1175, 1311 cm-1 and 1606 cm-1 (Raman) vibrations of the other two. The increase in DNA, protein and lipid content with malignancy was more clearly elucidated by examining both spectra. Principal component analysis (PCA)-linear discriminant analysis (LDA) with 10-fold cross validation of the FTIR and Raman spectral data sets showed efficient discrimination between normal and pathological conditions while overlapping was seen between the two pathologies. The PCA-LDA model of the dual spectra yielded a classification accuracy of 98% in comparison with either FTIR (85%) or Raman (82%) in a spectrum-wise comparison. In the patient-wise approach (mean of all spectra from a patient), the overall classification efficiency was 73%, 80% and 87% for FTIR, Raman and integrated spectral approaches respectively. Moreover, the efficiency of the integrated FTIR-Raman PCA-LDA model as a prediction tool was tested to screen susceptible individuals (11 cigarette smokers) using the dual spectra acquired from these individuals. The study presents proof-of-concept for adopting a large-scale, follow-up trial of the approach for mass screening purposes.

Cell-intrinsic regulation of peripheral memory-phenotype T cell frequencies.

Memory T and B lymphocyte numbers are thought to be regulated by recent and cumulative microbial exposures. We report here that memory-phenotype lymphocyte frequencies in B, CD4 and CD8 T-cells in 3-monthly serial bleeds from healthy young adult humans were relatively stable over a 1-year period, while Plasmablast frequencies were not, suggesting that recent environmental exposures affected steady state levels of recently activated but not of memory lymphocyte subsets. Frequencies of memory B and CD4 T cells were not correlated, suggesting that variation in them was unlikely to be determined by cumulative antigenic exposures. Immunophenotyping of adult siblings showed high concordance in memory, but not of recently activated lymphocyte subsets. To explore the possibility of cell-intrinsic regulation of T cell memory, we screened effector memory-phenotype T cell (TEM) frequencies in common independent inbred mice strains. Using two pairs from these strains that differed predominantly in either CD4 TEM and/or CD8 TEM frequencies, we constructed bi-parental bone marrow chimeras in F1 recipient mice, and found that memory T cell frequencies in recipient mice were determined by donor genotypes. Together, these data suggest cell-autonomous determination of memory T niche size, and suggest mechanisms maintaining immune variability.

Differential transmission of the molecular signature of RBSP3, LIMD1 and CDC25A in basal/ parabasal versus spinous of normal epithelium during head and neck tumorigenesis: A mechanistic study.

Head and neck squamous cell carcinoma (HNSCC) is a global disease and mortality burden, necessitating the elucidation of its molecular progression for effective disease management. The study aims to understand the molecular profile of three candidate cell cycle regulatory genes, RBSP3, LIMD1 and CDC25A in the basal/ parabasal versus spinous layer of normal oral epithelium and during head and neck tumorigenesis. Immunohistochemical expression and promoter methylation was used to determine the molecular signature in normal oral epithelium. The mechanism of alteration transmission of this profile during tumorigenesis was then explored through additional deletion and mutation in HPV/ tobacco etiological groups, followed byclinico-pathological correlation. In basal/parabasal layer, the molecular signature of the genes was low protein expression/ high promoter methylation of RBSP3, high expression/ low methylation of LIMD1 and high expression of CDC25A. Dysplastic epithelium maintained the signature of RBSP3 through high methylation/ additional deletion with loss of the signatures of LIMD1 and CDC25A via deletion/ additional methylation. Similarly, maintenance and / or loss of signature in invasive tumors was by recurrent deletion/ methylation. Thus, differential patterns of alteration of the genes might be pre-requisite for the development of dysplastic and invasive lesions. Etiological factors played a key role in promoting genetic alterations and determining prognosis. Tobacco negative HNSCC patients had significantly lower alterations of LIMD1 and CDC25A, along with better survival among tobacco negative/ HPV positive patients. Our data suggests the necessity for perturbation of normal molecular profile of RBSP3, LIMD1 and CDC25A in conjunction with etiological factors for head and neck tumorigenesis, implying their diagnostic and prognostic significance.

Liaison between heme metabolism and bioenergetics pathways-a multimodal elucidation for early diagnosis of oral cancer.

Metabolic alterations of oral epithelial cells under oxidative stress are important signatures for early diagnosis of oral cancer. Amongst different metabolic alterations, non-invasive photo-diagnostic methods have been extensively used for determining cellular heme metabolism and accumulation of protoporphyrin IX (PpIX) under administration of suitable photosensitizer. In this study, we report these metabolic alterations by direct analysis of oral exfoliated cells obtained from individuals with prolonged smoking habit without the exogenous administration of any photosensitizer. The relative expression level of relevant biomolecules of study groups were compared with clinically diagnosed and histopathologically confirmed leukoplakia (OLPK) and oral squamous cell carcinoma (OSCC) patients. The energy imbalance and variation in 'redox ratio' were examined through spectroscopic studies which showed an increasing trend (p < 0.001) in smokers to OSCC groups in comparison to nonsmoker control. Gene expression of important intermediates of the heme metabolic pathway (viz. 5-aminolevulinate synthase 1 (ALAS1), Ferrochelatase (FECH), hemeoxygenase 1 (HO-1) and ATP binding cassette subfamily G member 2 (ABCG2)) which affect production of PpIX was assessed. Relative mRNA level of ALAS1 and HO1 was upregulated whereas mRNA level of other genes (viz. FECH and ABCG2) were found to be downregulated in smokers as well as in cancer groups. Outcome of different spectroscopic studies on exfoliated cells (viz. fluorescence, atomic absorption and Fourier transform infrared spectroscopy) corroborated with the expression of biomarkers related to cellular endogenous metabolism related to heme cycle. This study indicates significant alterations in endogenous metabolic products, and cellular functional groups in oral epithelial cells among the study groups. Our study reports a strong possibility of diagnosis of early cancer signatures amongst habitual smokers by direct and non-invasive assessment of metabolic status of oral epithelial cells without exogenous administration of photosensitizers. The knowledge accrued from the study may guide clinicians in precise detection of precancer trend in the susceptible population through a noninvasive rapid screening method.

Expression of P53 Protein and Ki-67 Antigen in Oral Leukoplakia with Different Histopathological Grades of Epithelial Dysplasia.

AIMS AND OBJECTIVE: The aim of this study is to investigate the expression of p53 protein and Ki-67 antigen in normal oral mucosa and oral leukoplakia with different grades of dysplasia using immunohistochemistry and to clarify the correlation of the expression of these cell cycle regulatory proteins. MATERIALS AND METHODS: A total of 20 archival tissue blocks obtained from our department which were diagnosed as mild (n = 5), moderate (n = 5), and severe dysplasia (n = 5) with normal mucosa (n = 5) as a control. Positivity of Ki-67 and p53 was analyzed using Statistical Package for the Social Sciences (IBM Corp. Released 2013. IBM SPSS Statistics for Windows, Version 22.0 Armonk, NY:IBM Corp) software. RESULTS: All samples showed positive staining for p53 and Ki-67. Statistically significant difference (P < 0.05) was seen between the frequency of occurrence of p53 and Ki-67 pattern of expression among all the groups. The intensity of staining was mild to intense in basal layer as there was a progression toward the severity of the disease. Almost 81.1% correlation existed between p53 and Ki-67 with high correlation and marked relationship. CONCLUSION: Oral leukoplakia represents the most common oral potentially malignant disorder (OPMD). Molecular biological markers such as p53 and Ki-67 are considered to be of great value in the diagnosis and prognostic evaluation of OPMD. Our results emphasize the potential use of p53 protein and Ki-67 antigen as significant molecular markers for early detection of PMDs and its risk of developing oral squamous cell carcinoma.

Discrepancy in Blood Grouping: Subgroups of B-Challenges and Dilemma.

Though not very frequent, blood group discrepancies are of common occurrence which must be promptly resolved for safe transfusion practices. Blood group discrepancy is defined as the non-corroboration between cell grouping and serum grouping. Variable erythrocyte antigenic expressions (qualitative and/or quantitative difference) owing to allelic heterogeneity, give rise to subgroups. Problems during blood grouping due to weakened expression of antigens are a rare cause for ABO discrepancies. Of note, subgroups of 'B' with decreased expression of the B antigen are of very rare occurrence in the general population. Serologically, the variants of 'B' can be classified into B3, Bx, Bm and Bel. Serological confirmation of such subgroups requires special immuno-haematological procedures. The current study reports 3 cases of subgroups of B (2 cases of Bm and 1 case of Bw) detected at our centre. It is important to detect and resolve discrepant ABO grouping results so as to prevent ABO mis-match transfusions. Such individuals should also be pre-emptively informed of their respective blood donor and transfusion recipient status, as well.

Autologous Blood Transfusion as a Life Saving Measure for a Trauma Patient with Fracture Femur and Drug Induced Hemolytic Anemia: A Case Report.

A positive direct antiglobulin test has been reported in 1:1000 to 1:14,000 blood donors and 1-15 % of hospital patients. Drugs may cause a positive direct antiglobulin test result and/or immune-mediated haemolysis with an incidence of approximately 1 in a 1 million population. Our aim is to highlight the importance of following strict transfusion protocols and management insight in a direct antiglobulin test positive patient showing incompatibility with multiple units possibly due to drug induced immune haemolytic anaemia (DIIHA). We also aim to highlight importance of autologous blood transfusion in an orthopaedic procedure in which homologous transfusion may be needed. We are presenting case of a 36 year old male with alleged h/o of road traffic accident with comminuted intra-articular fracture of the distal femur. He had h/o of ankylosing spondylitis since last 20 years on medication with indomethacin and methotrexate. A review of the literature was performed which showed use of drug methotrexate as an uncommon clinical entity for DIIHA; sporadic reports exist in the medical literature to support this view. The review of the literature in combination with our own data showed methotrexate can be a cause of DIIHA. We therefore advocate proper immunohaematological work up and use of autologous blood for management of at-risk-patients of DIIHA.

Computer-assisted morphometric image analysis of cells of normal oral epithelium and oral squamous cell carcinoma.

CONTEXT: Oral squamous cell carcinoma (OSCC) is a malignant neoplasm of epithelial tissue origin. OSCC is traditionally graded into well, moderate and poorly differentiated. The assessment of cellular and nuclear features is very important in the histological grading of OSCC. AIMS: To establish morphometric study as an important prognostic indicator of OSCC. SETTINGS AND DESIGN: The present study is undertaken for computer-assisted morphometric evaluation of the following parameters: changes in cell perimeter, nuclear perimeter (NP), cell area (CA), nuclear area (NA) and nuclear-cytoplasmic ratio (N:C) in twenty cases of OSCC and ten cases of normal oral mucosa. SUBJECTS AND METHODS: The hematoxylin- and eosin-stained sections were observed under microscope for dysplastic features. For morphometric analysis, images were captured with a 2MP camera attached to a NLCD 307 microscope (Lawrence and Mayo India Pvt., Ltd.,) with a ×40 objective used for morphometric analysis. The images were classified, transferred and stored in the computer for image analysis. STATISTICAL ANALYSIS USED: One-way analysis of variance is used for comparing the parameter for multiple groups followed by Mann-Whitney U-test for pair-wise comparisons. RESULTS: Highly significant difference was seen between cases and controls with respect to CA, NP and NA. Highly significant difference is seen in N:C ratio between the means of cases and control groups. CONCLUSIONS: Techniques of image analysis offer an opportunity to quantify the nuclear and cell changes associated with malignancy and provide an objective basis for grading dysplasia and tumors.

Decreased Spontaneous Baroreflex Sensitivity as an Early Marker for Progression of Haemorrhage.

INTRODUCTION: Blood donation provides an ideal setup for assessment of cardiovascular responses to mild hypovolemia for understanding the underlying mechanisms. AIM: To evaluate cardiovascular responses in time and magnitude by estimating the spontaneous baroreflex sensitivity (BRS) during and after donation of 450 ml of blood. METHODS: Continuous beat-to-beat blood pressure and lead II ECG was recorded before, during and after blood donation in 54 healthy volunteers (age 34.7 ± 5.08 years; weight 77.9 ± 8.20 kg), followed by offline analyses of baroreflex sensitivity. RESULTS: The systolic, diastolic or mean blood pressures did not change during or after the blood donation. Decrease in pulse pressure and increase in heart rate was observed post donation. The spontaneous BRS decreased during [8.68 (6.038-12.69) ms/mmHg] and after blood donation [9.401 (6.396-11.59) ms/mmHg] as compared to the baseline [12.83 (6.884-18.18) ms/mmHg] with a significant decrease in α-HF on spectral analysis. CONCLUSION: Mild blood loss (450 ml) results in non-hypotensive haemorrhage with a decrease in spontaneous BRS before the rise of heart rate during blood donation.

Low-density lipoprotein apheresis in a pediatric patient of familial hypercholesterolemia: Primi experientia from a tertiary care center in North India.

Familial hypercholesterolemia (FH) is an autosomal dominant disorder due to mutation of apolipoprotein-B receptor gene causing severe dyslipidemia. Lifestyle modification and medical treatment attenuate the disease progression, but as these fail to control the blood cholesterol levels, low-density lipoprotein (LDL) apheresis comes forth as a treatment option. To the best of our knowledge, the following is the very first case of pediatric FH being treated by LDL-apheresis to be reported from India. A severely malnourished female child presented with yellowish skin lesions over different parts of the body, viz., bilateral Achilles tendon, both knees, elbows, both pinnae, and outer canthus of both eyes. She had a strong family history of borderline hypercholesterolemia and was diagnosed as a case of FH. She was maintained on diet modification. LDL-apheresis was planned as the cholesterol levels were not controlled with the diet modificationt. However, unavailability of an appropriate kit in India for LDL-apheresis led to the use of the modified PL1 kit meant for therapeutic plasma exchange procedures. We conducted two sessions of LDL-apheresis. After the first session, the LDL-cholesterol (LDL-C) level fell by 75.9% and the total cholesterol fell by 73.5%. A second procedure led to a decline in total cholesterol level by 18.6% and LDL-C by 19.46%. Subsequently, she was advised diet modification and statin therapy with regular follow-up after every 6 months. Thus, the cascade filtration technique is a safe and effective treatment option for removing the undesired lipoproteins.

Adverse Events During Apheresis Procedures: Audit at a Tertiary Hospital.

Apheretic platelets are better quality blood components which reduce donor exposure and chances of TTIs to transfusion recipients when compared to the whole blood derived components. Though safe, these apheretic donations are associated with donor adverse events. We evaluated the incidence of such adverse events associated with the modern apheresis procedures that would provide an insight as well as help formulating preventive steps to avoid frequent occurrences of such events. This prospective audit-based observational study was conducted over 1 year. Donors for plateletpheresis were selected as per the standard operating procedure of the Apheresis Lab. The apheresis procedures were done on the MCS+ (Haemonetics Corp.), Trima Accel (Terumo BCT) and COM.TEC (Fresenius Kabi AG). 1740 apheresis procedures were performed, out of which 1708 were plateletpheresis and 32 therapeutic plasma exchange (TPE) procedures for 7 patients. A total of 102 adverse events were noted; of which, 80 (78.43 %) events were associated with donors, 15 (14.71 %) were owed to equipment related problems and 7 (6.86 %) were technical aberrations. All the events associated with donors were mild. No adverse events were reported with any of the 32 TPEs. Apheresis procedures are associated with adverse events which can be reduced by meticulous donor-vigilance, superior training modules for the technical personnel and continued supervision of experienced transfusion medicine specialists. Continued efforts towards making the donor's experience with apheresis more pleasant give a forward thrust to the noble vision of preparing a voluntary apheresis donor pool in India.