Immunophenotypic Characterization and Ploidy Analysis of Neoplastic Plasma Cells by Multiparametric Flow Cytometry.

Flow cytometric (FCM) immunophenotyping is an important tool for generating diagnostic and prognostic information in plasma cell dyscrasias. This study aimed to evaluate the immunophenotype and ploidy status of plasma cells (PCs) in patients of myeloma and its correlation with other laboratory parameters. Bone marrow of 70 newly diagnosed cases of myeloma were subjected to FCM using a panel of antibodies; CD138, CD38, CD19, CD45, CD28, CD81, CD56, CD200, and CD229. FxCycle Violet (FCV) dye was used for the ploidy analysis of clonal PCs. Median age was 60 years with M:F ratio of 3.2:1. A positive correlation was noted between the morphological and FCM-based PC enumeration (r = 0.4, p = 0.001). Aberrant expression of CD56, CD200, CD28, CD117, CD81 and CD19 and was observed in 88.5%, 77%, 29%, 37%, 23% and 17% cases respectively. Two aberrant antigens were noted in all cases. CD81 + cases had a relatively higher quantity of monoclonal-protein (> 1 g/dl, p < 0.05) and renal insufficiency (Cr > 2 mg/dl, p < 0.05) as compared to the CD81- cases. CD229 was expressed in all the cases, with a median MFI in PCs significantly higher than other hematopoietic elements. Hyperdiploid PCs (median DI-1.59, range, 1.16-2.6) were noted in 80% cases (n = 48), diploid/ near-hyperdiploid PCs in 8% (n = 5) cases and hypodiploidy in 3% (n = 1) cases. Bright CD56/CD200 and CD45- can identify abnormal PC in the majority of the cases. CD81 appears to correlate with disease burden and might be useful as a prognostic marker. CD229 is a reliable gating marker for plasma cells. Ploidy analysis may be incorporated in routine workup to guide in the identification of patients with poor prognosis. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-021-01477-y.

Utility of a Single-Tube, Six-Color Flow Cytometry Panel for the Diagnosis of Myelodysplastic Syndrome: Experience of a Tertiary Care Centre in India.

Background: Diagnosis of myelodysplastic syndromes (MDS) is challenging in the presence of morphological mimickers. Flow cytometric immunophenotyping (FCI) has been added to the diagnostic armamentarium, but its use in clinical practice is variable. Materials and Methods: Bone marrow aspirate samples from 54 patients with a clinical and/or morphological suspicion of MDS were subjected to FCI using a single-tube, 6-colour panel comprising of monoclonal antibodies against CD13, CD11b, CD16, CD34, CD45 and CD56. Analysis was centered on the abnormal maturation pattern of granulocytes, blast percentage (≥3%) and ratio of side scatter peak channel value (SSC-PCV) of granulocytes and lymphocytes. Each of these parameters was assigned a score of 1. Overall sensitivity and specificity of this panel was ascertained to differentiate cytopenia/s of MDS from non-MDS cases. Results: Forty MDS and 14 non-MDS cases were diagnosed based on morphology and cytogenetic results. Twenty control samples were also processed simultaneously for FCI to assign the cutoff for various flow cytometric parameters. A score ≥2 was defined as positive for MDS. Hypogranularity was present in 62.5% cases of MDS. The median SSC-PCV of granulocytes and lymphocytes was 6.16 in the MDS group, 7.9 in the non-MDS group and 8.90 in the control group (p <0.05). This cut-off value of 6.16 had a specificity of 92.5% based on the ROC curve analysis. Abnormal granulocyte maturation patterns for CD13/16, CD13/11b and CD11b/16 dot plots were observed in 95.3, 69.8 and 74.4% cases, respectively. The overall sensitivity and specificity of the panel was found to be 87.5% and 64.2%, respectively. Conclusion: FCI is now an important tool for diagnostic workup in patients presenting with persistent cytopenia with or without morphological evidence of dyspoiesis. Inclusion of objective parameters like SSC-PCV would also reduce inter-lab variability in MDS diagnosis.

Clinicopathological profile of paroxysmal nocturnal haemoglobinuria clone-positive aplastic anaemia paediatric patients-A single centre study from North India.

INTRODUCTION: There is a paucity of literature related to the prevalence of Paroxysmal Nocturnal haemoglobinuria (PNH) clones in paediatric aplastic anaemia (AA) patients. METHODS: We performed a retrospective analysis over a period of 42 months to study the prevalence of PNH clones in paediatric (age less than 18 years) AA cases, using Fluorescein-labelled proaerolysin-based flow cytometric screening and analysed their clinico-pathological features. RESULTS: PNH clone was identified in 100 (33.2%) of the 301 patients screened. These were comprised of 51 cases of non-severe AA, 33 cases of severe AA and 16 cases of very severe AA. The median age was 13 years with an M:F ratio of 2.5:1. The median clone size (taken as the proportion of PNH-positive neutrophils) was 2.15% (range: 0.05%-93.1%). Although a majority of patients (n = 77) had a clone size of less than 10%, a significant proportion (n = 23) did harbour a clone size of more than 10%. Evidence of haemolysis was observed in 3 patients, all of them having a clone size of more than 10%. Interestingly, 1 patient with dural sinus thrombosis harboured a clone size of 1.25% only. Chromosomal breakage analysis was performed in 61 patients, none of which was positive. Complete and partial response to immunosuppressive therapy was found in 55.1% patients (16/29). CONCLUSION: There is a high prevalence of PNH clones in paediatric AA patients, which in a majority of cases are of small clone sizes. The use of immunosuppressive therapy does not show a better outcome as compared to PNH-negative cases.

The triple-negative (CD34-/HLA-DR-/CD11b-) profile rapidly and specifically identifies an acute promyelocytic leukemia.

INTRODUCTION: The genetic testing to confirm or rule out an acute promyelocytic leukemia (APL) typically takes a minimum of 24-72 hours. Flow cytometric immunophenotyping (FCI) on the other hand provides rapid and objective information to differentiate APL from non-APL. METHODS: FCI features, with single-tube 8-color combination using CD45, CD34, HAL-DR, CD11b, CD13, CD33, and CD117 and CD64, were compared for the 30 consecutive APL and 30 non-APL acute myeloid leukemia (AML) cases which morphologically mimicked an APL. The diagnosis was confirmed by cytogenetic or molecular genetic testing in the form of t (15:17) (q22; q21)/variant translocations or PML-RARA fusion transcript analysis. RESULTS: The APL cells lacked CD34, HLA-DR, and CD11b in 90%, 90%, and 93.3% cases, respectively. Myeloid antigens such as CD33, CD13, CD117, and CD64 were expressed in 96.7%, 96.7%, 76.7%, and 70% cases, respectively. The dual negative profiles, CD34-/HLA-DR- or HLA-DR-/CD11b-, were noted in 90% and 93.3% cases. The triple-negative (CD34-/HLA-DR-/CD11b-) profile was noted in 90% of the cases. The sensitivity, specificity, and positive predictive value (PPV) of CD34-/HLA-DR- and HLA-DR-/CD11b- profiles for the diagnosis of APL were found to be 90%, 80% & 81.1% and 93.3%, 86.7%& 87.5%, respectively. Combining the above two profiles resulted in a triple-negative profile (CD34-, HLA-DR- and CD11b-), which had a better specificity (93.3%) and positive predictive value (93.1%), with similar sensitivity. CONCLUSION: FCI is a rapid and reliable modality for the diagnosis of an APL. The triple-negative profile (CD34-/HLA-DR-/CD11b-) rapidly and specifically identifies an APL case.

Fluorescent Aerolysin (FLAER)-based paroxysmal nocturnal hemoglobinuria (PNH) screening: a single center experience from India.

BACKGROUND: Fluorescent aerolysin (FLAER) has been recommended as an important part of antibody panel used for flow cytometric detection of paroxysmal nocturnal hemoglobinuria (PNH) clone. This study was aimed to observe the frequency of PNH-positive clones and their sizes in patients screened for various indications. METHOD: A retrospective analysis of 624 patients screened over a period of 30 months. Frequency and size of clone sizes noted, and laboratory parameters were compared among different groups of patient being screened. RESULTS: There were 445 adults and 179 pediatric patients. Indications for screening included AA (n = 433), myelodysplastic syndrome (MDS) (n = 34), hemolytic anemia (n = 84), and thrombophilia workup group (n = 63). PNH clones were found in 39.03%, 5.88%, 26.19%, and 1.59% cases, respectively. No significant difference among adult or pediatric population was noted. The bone marrow failure (BMF) group [AA and MDS] with PNH clone had a significantly lower clone size (Median- 2.7%) as compared to classic PNH group (Median-77.2%). Most of the classic PNH cases (78.26%) and a small proportion of AA (9.9%) showed a large clone size (>50%). In spite of having large clone size, there was a significant difference between the median LDH values of these two groups (2511.5 vs 593 U/L). CONCLUSION: FLAER-based screening detects the presence of PNH clone in a high proportion of AA patients and some MDS patients. These patients usually have a small clone size. Even if they have a large clone, it does not get translated into a high LDH or severe clinical symptoms.

Role of CD200 in differential diagnosis of mature B-cell neoplasm.

INTRODUCTION: CD 200 is a type I immunoglobulin super family membrane glycoprotein, which is expressed in various mature B-cell neoplasm (MBN). This study aimed at analyzing the expression pattern of CD200 by flow cytometry immunophenotyping (FCI) and to evaluate its utility in narrowing down the differential diagnosis of MBN, particularly in low-grade lymphomas. METHODS: A total of 160 samples were evaluated by FCI over a period of 2 years (July 2014-June 2016), by a panel of antibodies including CD200. The mean fluorescence intensity (MFI) of CD200 in the neoplastic population was noted and compared among several groups of MBN. RESULTS: All the 98 cases of chronic lymphocytic leukemia (CLL), five being CD23-negative, expressed CD200 with moderate-to-bright intensity (median MFI: 1174). None of the 24 mantle cell lymphoma (MCL) cases, two being CD23-positive, expressed CD200 (median MFI: 10). All six hairy cell leukemia (HCL) cases expressed CD200. CD200 expression in HCL was brightest among all the MBNs, with a median MFI of 5050. Other MBN (n = 32) expressed CD200 in a proportion of cases and with variable intensity, usually dimmer than CLL. CONCLUSION: CD200 has a valid role in differentiating CLL from MCL, especially in the cases with immunophenotypic overlap. HCL has a consistent and brightest expression of CD200. It would be prudent to include CD200 in the primary panel of antibodies for MBN analysis.

Design and development of controlled porosity osmotic tablet of diltiazem hydrochloride.

The present work aims towards the design and development of extended release formulation of freely water-soluble drug diltiazem hydrochloride (DLTZ) based on osmotic technology by using controlled porosity approach. DLTZ is an ideal candidate for a zero-order drug delivery system because it is freely water-soluble and has a short half-life (2-3 h). Sodium chloride (Osmogen) was added to the core tablet to alter the solubility of DLTZ in an aqueous medium. Cellulose acetate (CA) and sorbitol were used as semipermeable membrane and pore former, respectively. The effect of different formulation variables namely concentration of osmogen in the core tablet, % pore former, % weight gain, pH of the dissolution medium and agitation intensity on the in vitro release was studied. DLTZ release was directly proportional to % pore former and inversely proportional to % weight gain. The optimized formulation (F8) delivered DLTZ independent of pH and agitation intensity for 12 h at the upper level concentration of % pore former (25% w/w) and middle level concentration of % weight gain (6% w/w). The comparative study of elementary osmotic pump (EOP) and controlled porosity osmotic pump revealed that it superior than conventional EOP and also easier and cost effective to formulate.

Establishment of a rat model of myocardial infarction with a high survival rate: A suitable model for evaluation of efficacy of stem cell therapy.

The most common rat model of myocardial infarction (MI) is by ligation of left anterior descending (LAD) coronary artery but it is associated with high mortality and large variations in the infarct size. We evolved certain innovations/modifications in the existing technique including immobilization of the heart without exteriorization, identification of the LAD by pressing it proximal to the site of ligation by an ear-bud, and subsequently its ligation 8 mm from its origin, no touch technique of the lungs during surgery, removal of air from the chest cavity prior to its closure using an in-house tubing, and deflation of the lungs before extubation. We induced MI in 24 Sprague- Dawley (SD) rats using these modifications and carried out post-MI evaluation of hemodynamic parameters, serum cardiac enzymes and histological studies upto 90 days using 13 sham operated and 3 healthy SD rats as controls. Three of the 24 rats (13%) died <24 hours of MI, but thereafter no mortality was observed till the follow-up period of 90 days. The infarct size was consistent in all the rats (21±4% of left ventricular area). This model with low early and no long-term mortality may be suitable for studying efficacy of stem cell therapy in MI, where a follow-up of at least 13 weeks is required to assess myocardial regeneration.

A prospective comparison of four techniques for diagnosis of paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder with altered expression of glycosylphosphatidylinositol (GPI)-anchored proteins, resulting in the increased susceptibility of erythrocytes to complement-mediated lysis. This study compared the available laboratory methods for detection of PNH cells and evaluated their utility in routine clinical practice. Fifty patients were evaluated by flow cytometric immunophenotyping (FCMI) using CD55 and CD59 monoclonal antibodies, PNH gel card test (GCT), Ham test and sucrose lysis test (SLT). A PNH clone was detectable in erythrocytes in 14 (28%) patients by FCMI, 13 (26%) by GCT and 10 (20%) by Ham test and SLT. The GCT and lytic tests showed 100% specificity and sensitivity was 92.8% and 71.1%, respectively. The GCT results correlated with type III cells (positive for > or =3.21% type III cells) and lytic test results correlated with CD59(-) type III cells (positive for > or =5% CD59(-) type III cells). The GCT and lytic tests were comparable in their sensitivity to detect type II cells (positive for > or =18.5% type II cells). Among the available methods, FCMI is most sensitive, can quantify and delineate PNH cells with differential expression of GPI-anchored proteins. The GCT is a useful screening tool as it is fairly sensitive, easy to perform and interpret. Well-standardized lytic tests are fairly reliable as screening tests.

Chimerism monitoring following allogeneic hematopoietic stem cell transplantation.

Information regarding the chimeric status of hematopoietic stem cell transplantation (HSCT) recipients is of great significance when comparing different conditioning and prophylactic therapies. In recent years, short tandem repeats/variable number tandem repeats (STRs/VNTRs) have emerged as the best tool for chimerism monitoring. However, the polymorphisms of STR/VNTR markers vary within and between ethnic groups. The issue is further complicated in a heterogeneous population such as occurs in the Indian subcontinent. In the present study, we attempted to devise a robust scheme to identify a set of polymorphic STRs/VNTRs most suitable for chimerism evaluation in north Indian HCST recipients. At first, we did genotyping of 11 STR and one VNTR in 1000 randomly chosen north Indian individuals to quantify different diversity parameters. Resulting data indicated that ApoB3'HVR, FES, VWA, D3S1358 and D16S310 were most polymorphic loci with the average heterozygosity being 0.756+/-0.17. Furthermore, all markers were genotyped in 77 HLA-matched donor-recipient pairs to evaluate the informativeness in differentiating donor's and recipient's cells. A panel of seven markers (ApoB3HVR-D3S1358-HUM-THO1-VWF-1-D16S310-FES-VWA) differentiated 98.70% of donor-recipient pairs. This set of markers also successfully monitored the graft status in 14 HSCT cases during multiple time points following HSCT. The results were compared to the commercially available AmpF/STR SGM Plus multiplex PCR kit (Applied Biosystems, Foster City, CA, USA). Our findings established that the panel of seven markers we identified was more cost-effective and informative.

Cellular and humoral immune responses to mycobacterial heat shock protein-65 and its human homologue in Takayasu's arteritis.

Expression of heat shock protein (HSP)-65 as well as infiltration of T-cells in arterial lesions and raised levels of circulating antibodies against mycobacterial HSP65 (mHSP65) led us to the concept that mHSP65 or its human homologue (hHSP60) might be involved in the etiopathogenesis of Takayasu's arteritis (TA). Therefore, we investigated mHSP65 and hHSP60 reactive peripheral blood T-cell subsets by BrdU incorporation assay and flow cytometry as well as investigating the different isotypes of anti-mHSP65 and hHSP60 antibodies by ELISA. Eighty-four percent (22/26) of the TA patients were observed to show T-cell proliferation to mHSP65 and hHSP60 whereas only 16% (3/18) healthy controls showed such proliferation (P <0.001). Both HSPs induced proliferation of exclusively CD4+ T-cells and not CD8+ T-cells. We also observed a significantly higher prevalence of only the IgG isotype reactive to mHSP65 and hHSP60 in TA as compared to HC (mHSP65: 92% TA versus 11% HC, P <0.0001 and hHSP60: 84% versus 22%, P <0.001). Our data show a significant correlation between mHSP65 and hHSP60 reactive T-cells (CD3+: r=0.901; CD4+: r=0.968) as well as anti-mHSP65 and anti-hHSP60 IgG antibodies (r=0.814) suggesting an infection induced autoimmunity in TA, possibly induced by molecular mimicry between mHSP65 and hHSP60 or other tissue specific antigens.

Cytokine mRNA repertoire of peripheral blood mononuclear cells in Takayasu's arteritis.

We have investigated constitutive and phytohaemagglutinin (PHA) + phorbol 12-myristate 13-acetate (PMA)-induced gene expression of tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-2, IL-3, IL-4, IL-10, IL-12 and granulocyte macrophage colony-stimulating factor (GM-CSF) in peripheral blood mononuclear cells (PBMCs) of 10 patients with Takayasu's arteritis (TA) and 10 healthy controls by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). The constitutive mRNA expression of TNF-alpha (69.0 +/- 4.0%versus 27.5 +/- 18.0%; P = 0.001) and IL-4 (60.0 +/- 10.0%versus 0%; P = 0.001) was significantly higher in patients than controls; that of IL-3 was comparable in both groups (38.0 +/- 6.0%versus 32.0 +/- 5.0%; P = 0.651) while no constitutive mRNA expression was observed for the other cytokines studied. The stimulated PBMCs of patients, as compared with the controls, had higher mRNA gene expression of TNF-alpha (127.0 +/- 16.0%versus 54.0 +/- 6.0%; P = 0.001), IFN-gamma (93.0 +/- 13.0%versus 57.0 +/- 5.0%; P = 0.032), IL-2 (109.0 +/- 13.0%versus 68.0 +/- 6.0%; P = 0.015), IL-3 (60.0 +/- 8.0%versus 21.2 +/- 3.0%; P = 0.045) and IL-4 (68.0 +/- 7.0%versus 27.0 +/- 7.2%; P = 0.01) The mRNA expression of IL-10 was lower in patients than controls (35.0 +/- 8.0%versus 75.0 +/- 12.0%; P = 0.022). The GM-CSF mRNA was similar (102.0 +/- 6.0%versus 89.0 +/- 5.0%; P = 0.475) in both groups. Stimulation of cells with PHA + PMA showed no IL-12 expression but stimulation with lipopolysaccharide induced higher IL-12 mRNA in patients than controls (83.0 +/- 14.0%versus 33.0 +/- 4.0%; P = 0.005). Our data suggest that an inflammatory cytokine signature exists in TA with a key role for TNF-alpha, IL-4, IL-10 and IL-12 in different pathological processes of the disease.

Interleukin-8 in Takayasu's arteritis: plasma levels and relationship with disease activity.

OBJECTIVE: To investigate the plasma levels of interleukin-8 (IL-8) in Takayasu's arteritis (TA) and their relationship with disease activity. METHODS: IL-8 levels were detected by quantitative enzyme-linked immunosorbent assay (ELISA) in the plasma of 53 TA patients, 25 age/sex-matched healthy controls and of 10 serially followed up active TA patients on immunosuppressive therapy. RESULTS: Significantly increased levels of IL-8 were observed in TA patients (26.32 +/- 48.96 pg/ml) compared to controls (6.0 +/- 2.45 pg/ml) (p = 0.0006) and in patients with active TA (55.0 +/- 71.43 pg/ml) compared to those with an inactive disease (8.94 +/- 6.35 pg/ml) (p = 0.0001). The increased levels of the chemokine were present in 37% (20/53) of the patients compared to 8% (2/25) of the controls (p < 0.01) and in 80% (16/20) of patients with active TA compared to 12% (4/33) of those with an inactive disease (p < 0.0001). In the follow-up study, the plasma levels of IL-8 were normalized in 6/10 of the patients and the disease in 5 of these 6 patients was also observed to undergo remission. CONCLUSION: These results suggest that IL-8 may play an important role in the pathogenesis of TA.

Anti-annexin V antibodies in Takayasu's arteritis: prevalence and relationship with disease activity.

Annexin V has an important role in the regulation of apoptosis and antibodies directed against it have been shown to lead to apoptosis of vascular endothelial cells. To evaluate the role of anti-annexin V antibodies (AA5A) in Takayasu's arteritis (TA), we investigated these antibodies in the sera of 66 TA patients, 50 healthy controls and in the follow-up sera of 12 active TA patients by enzyme-linked immunosorbent assay. The AA5A-positive patients were analysed further for the presence of anti-endothelial cell antibodies (AECA) and anticardiolipin antibodies (ACLA) to determine the relationship of AA5A with these autoantibodies. AA5A were observed in 36% (24/66) of the patients versus 6% (3/50) of the controls (P<0.001) and in 53% (19/36) of patients with active TA versus 17% (5/30) of those with inactive disease (P<0.01). Levels of AA5A were also observed to be significantly higher in patients with TA compared to controls (0.557 +/- 0.362 versus 0.259 +/- 0.069; P<0.0001) and in patients with active disease compared to those with inactive disease (0.700 +/- 0.403 versus 0.385 +/- 0.205; P<0.0001). In the follow-up study, 6/12 patients who became inactive during follow-up also showed normalization of AA5A levels. AECA and ACLA were detected in 54% (13/24) and 12% (3/24) of the AA5A-positive patients, respectively. Our results show that a significant proportion of TA patients have AA5A, which exhibit an association with AECA and because they have a correlation with disease activity thus appear to be involved in the disease pathogenesis.

Involvement of Fas and TNF pathways in the induction of apoptosis of T cells by antithymocyte globulin.

Antithymocyte globulin (ATG) is the treatment of choice for those aplastic anemia patients who are not suitable for bone marrow transplantation (BMT). ATG is also used for the treatment of rejections in organ transplantation and as a conditioning regimen in BMT. Despite the proven efficacy of ATG in these areas, its mechanism of action is not known. Profound T-cell lymphopenia observed in vivo with ATG treatment is supposed to contribute to its therapeutic effect. We have previously shown that apoptosis is one of the mechanisms responsible for ATG-induced lymphopenia. Our next objective was to investigate the effect of ATG on modulation of Fas and TNF pathways, the two main pathways of T-cell apoptosis. Maximum surface expression of Fas on T cells was observed after 24 h at an ATG dose of 100 microg/ml; at this dose 88% of cells expressed Fas as compared to 26% of untreated cells. Surface expression of FasL was found to peak after 24 h at an ATG dose of 1000 microg/ml when 34% of cells were positive for FasL as compared to 1.5% of untreated T cells. Tumor necrosis factor (TNF)-alpha production was found to be maximum after 6 h at 1000 microg/ml dose (20%) as measured by intracellular cytokine staining of T cells. TNF-alpha production was also measured by enzyme-linked immunosorbent assay (ELISA) in the supernatant of lymphocytes treated with ATG for 6 h. A dose-dependent increase in TNF-alpha production was found in these supernatants with a plateau being achieved at an ATG dose of 1000 micro g/ml. We conclude that ATG-induced apoptosis in T cells involves both Fas and TNF pathways and TNF-alpha is produced much earlier than Fas and FasL expression.

Induction of apoptosis of peripheral blood mononuclear cells by antithymocyte globulin (ATG) in aplastic anemia: an in vivo and in vitro study.

Antithymocyte globulin (ATG) is the standard therapy for aplastic anemia (AA) patients who are not eligible for allogeneic bone marrow transplantation (BMT). The exact mechanism of action of ATG is still not known. Profound lymphopenia is observed throughout the treatment period with ATG and appears to contribute to its immunomodulatory effect. One of the possible mechanisms, which could account for ATG-mediated lymphopenia, is by induction of apoptosis of peripheral blood mononuclear cells (PBMCs). However, there is no conclusive evidence to support this mechanism. We investigated whether ATG could induce an in vivo apoptosis in PBMCs of 12 AA patients undergoing ATG therapy by terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay using flow cytometry. A significant increase in the percentage of apoptosis was observed in six patients. The median percentage prior to therapy was 3 percent (range: 1-10 percent), which increased to a peak median value of 27 percent (range 17-66 percent) with therapy. ATG also induced an in vitro apoptosis of normal PBMCs as demonstrated by Annexin V and TUNEL staining using flow cytometry. Induction of apoptosis was dose dependent: 52 percent of the PBMCs exhibited Annexin V positivity after incubation with ATG at a dose of 500 microg/ml for 6 h, and 37 percent of the PBMCs exhibited DNA fragmentation after incubation with ATG at a dose of 1000 microg/ml for 24 h as demonstrated by TUNEL assay. Thus, ATG induces in vivo apoptosis in PBMCs of AA patients undergoing therapy as well as an in vitro apoptosis in normal PBMCs, and apoptosis may be an important mechanism for ATG-induced lymphopenia.

Complement and cell mediated cytotoxicity by antiendothelial cell antibodies in Takayasu's arteritis.

OBJECTIVE: To study complement and cell mediated cytotoxicity by antiendothelial cell antibodies (AECA) in Takayasu's arteritis (TA). METHODS: Complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC) of AECA positive/negative TA sera were investigated by colorimetric MTT and 51Cr release assays, respectively, using human umbilical vein endothelial cells (HUVEC) as targets. RESULTS: Seven of 12 (58%) sera positive for IgG and/or IgM AECA exhibited CDC in comparison to none of the 13 AECA negative sera (p = 0.0052). The median value of CDC of the AECA positive group was 14% (range 13-21%) and that of the AECA negative group was 1% (p = 0.0012). Interleukin 1beta (10 U/ml) treatment of HUVEC resulted in enhancement in CDC of 6 of the 7 AECA positive cytotoxic sera, the median enhancement being 17% (range 7-29%). Tumor necrosis factor-alpha (100 U/ml) treatment of the targets resulted in a median enhancement by 36% (range 25-55%) in the CDC of 3 of these 7 sera. No sera exhibited ADCC at any of the effector:target ratios tested (10:1 to 100:1). CONCLUSION: AECA in TA mediate CDC against endothelial cells and may have a pathogenic role in the perpetuation of vascular damage in this disease.

Immunophenotypic subsets in acute lymphoblastic leukemia from a single centre in North India: correlation with outcome to induction chemotherapy.

The poorer outcome of Indian patients with acute lymphoid leukemia (ALL) has been observed in earlier studies. However, little data is available on their immunophenotypic characteristics. The aim of the present study was thus to characterize the immunophenotypic subsets of Indian ALL patients and correlate with outcome at the end of induction chemotherapy. Immunophenotyping of 45 childhood and 25 adult ALL patients was performed by dual colour flow cytometry using a panel of B-lineage, T-lineage and Myeloid lineage specific monoclonal antibodies. Eighty and 17% of childhood and 92% and none of adults were B-lineage and T-lineage ALL, respectively. Aberrant myeloid marker expression was seen in 11% and 28% of childhood and adult groups, respectively. B-lineage ALL with aberrant T-lineage marker expression was observed in 4.4% and 8% of childhood and adult groups, respectively. Two each induction failures were observed in both childhood and adult groups. All of these were associated with aberrant expression of myeloid and/or T-lineage markers on B-lineage ALL. Aberrant expression of markers was associated with poorer outcome to induction chemotherapy in both childhood and adult ALL patients.