Quantitation of mHLA-DR and nCD64 by Flow Cytometry to Study Dysregulated Host Response: The Use of QuantiBRITE™ PE Beads and Its Stability.

Quantitation of mHLA-DR and nCD64 is useful in understanding the dysregulated host response. The down regulation of HLA-DR expression on the circulating monocytes (mHLA-DR) is associated with anti-inflammatory response, and an increased expression of CD64 on neutrophil surface (nCD64) is associated with pro-inflammatory response. Quantitation of these antigen expression using beads (QuantiBRITE™ PE) is a precision technique. These beads are reported to be stable for 24 h after reconstitution. We report the results of our investigation examining the stability of QuantiBRITE PE beads over a period of 4-week post-reconstitution. The data suggest that reconstituted QuantiBRITE PE beads, if stored in dark at 2-8 °C, can be effectively used for up to 2 weeks for determining nCD64 and mHLA-DR antibody bound per cell (ABC) values.

Monocyte HLADR and Immune Dysregulation Index as Biomarkers for COVID-19 Severity and Mortality.

Immune dysregulation in COVID-19 is the major causal factor associated with disease progression and mortality. Role of monocyte HLA-DR (mHLA-DR), neutrophil CD64 (nCD64) and Immune dysregulation index (IDI) were studied in COVID-19 patients for assessing severity and outcome. Results were compared with other laboratory parameters. Antibody bound per cell for mHLA-DR, nCD64 and IDI were measured in 100 COVID-19 patients by flow cytometry within 12 h of hospital admission. Thirty healthy controls (HC) were included. Clinical and laboratory parameters like C - reactive protein (CRP), Procalcitonin (PCT), Absolute Lymphocyte count (ALC), Absolute Neutrophil count (ANC) and Neutrophil to Lymphocyte ratio (NLR) were recorded. Patients were followed up until recovery with discharge or death. Parameters from 54 mild (MCOV-19), 46 severe (SCOV-19) and 30 HC were analysed. mHLA-DR revealed significant and graded down regulation in MCOV-19 and SCOV-19 as compared to HC whereas IDI was lowest in HC with increasing values in MCOV-19 and SCOV-19. For diagnostic discrimination of MCOV-19 and SCOV-19, IDI revealed highest AUC (0.99). All three immune parameters revealed significant difference between survivors (n = 78) and non-survivors (n = 22). mHLA-DR < 7010 and IDI > 12 had significant association with mortality. Four best performing parameters to identify patients with SCOV-19 at higher risk of mortality were IDI, NLR, ALC and PCT. mHLA-DR and IDI, in addition to NLR and ALC at admission and during hospital stay can be utilized for patient triaging, monitoring, early intervention, and mortality prediction. IDI reported for the first time in this study, appears most promising. Immune monitoring of 'in hospital' cases may provide optimized treatment options. Supplementary Information: The online version contains supplementary material available at 10.1007/s12291-022-01087-z.

Non-classical monocytes and its potential in diagnosing sepsis post cardiac surgery.

BACKGROUND: Sepsis is caused by a dysregulation of immune response to infection that results in very high mortality. Current laboratory tests and clinical criteria are inadequate to diagnose sepsis due to limited sensitivity and specificity. Circulating monocytes are important players in immune homeostasis and their altered HLA-DR expression indicate immune dysregulation. HLA-DR is an MHC Class II cell-surface receptor that can present foreign antigens to helper T cells and mount an inflammatory response. Therefore, we analyzed the variations in HLA-DR expression and the concentration of monocyte subsets for diagnosing post-surgical sepsis. METHODS: In this double-blinded prospective cohort study, we adopted immunophenotyping and quantification of antigen expression by flowcytometry to detect the changes in circulating monocyte subsets in patients undergoing cardiac surgery. Statistical analysis was performed to identify significant changes and based on the predictive potential of measured variables ROC curve analysis was done. ROC curve permitted the choice of appropriate cut-off values using which a diagnostic protocol was developed. RESULTS: We observed that the monocyte subset concentrations in circulation varied differently after surgery. There was a significant downregulation of monocytic HLA-DR on both intermediate (p = 0.0477) and non-classical monocytes (p = 0.0333) at 48 h post-surgery. The monocyte subset analysis clearly showed that the patients with reduced pre-surgical non-classical monocyte count (p = 0.0430) coupled with post-surgical down-regulation of HLA-DR expression on the same subset had a higher incidence of developing sepsis after cardiac surgery. CONCLUSIONS: Here we are reporting for the first time, the significant influence of non-classical monocytes in inducing dysregulated host response and sepsis after cardiac surgery. Using multiple biomarkers associated with this monocyte subset, we established an algorithm for the diagnosis of sepsis at 48 h post cardiac surgery with 100% sensitivity and 69.23% specificity.

Delayed Treatment of a Traumatic Lumbosacral Dislocation With Associated Injuries: A Case Report.

CASE: A 41-year-old woman sustained a degloving injury over her lumbosacral and perineal region with fractures of her right tibia and fibula. After diversion colostomy and osteosynthesis for the fractures at a primary center, a missed grade 2 lumbosacral dislocation was diagnosed at a tertiary center and the degloving injury was treated with debridement and skin grafting. After 5 months, the dislocation had progressed to grade 4 and she underwent delayed posterior lumbosacral reduction, interbody fusion, and L4-S1 fixation, with superior gluteal artery perforator flap and subsequent colostomy closure, with good outcomes (Oswestry Disability Index 10%) at the 3-year follow-up. CONCLUSION: A rare, missed, progressive traumatic L5-S1 spondylolisthesis with associated injuries is described.

Basic Multicolor Flow Cytometry.

Multicolor flow cytometry is a rapidly evolving technology that uses multiple fluorescent markers to identify and characterize cellular subpopulations of interest, allowing rapid analysis on tens of thousands of cells per second, with the possibility of isolating pure, viable populations by cell sorting for further experimentation. This unit covers the tools needed by the beginning immunologist to plan and run multicolor experiments, with information on fluorochromes and their characteristics, spectral spillover, compensation and spread, instrument and reagent variables, and the basic elements of multicolor panel design. Protocols to quantify and maximize sensitivity by titration of reagents and optimization of instrument settings, as well as basic surface and intracellular cell staining, are included. © 2017 by John Wiley & Sons, Inc.

Effect of irrigating solutions used for postspace treatment on the push-out bond strength of glass fiber posts.

AIM: To evaluate the effect of different irrigating solutions on postspace treatments on the push-out bond strength of glass fiber posts. MATERIALS AND METHODS: Thirty mandibular premolar roots were decoronated and endodontically treated. Postspaces were prepared and roots were divided into three groups: In group 1: 2.5% sodium hypochlorite irrigation (control), group 2: 17% ethylenediaminetetraacetic acid (EDTA) with hand activation, group 3: 17% EDTA irrigation with photon-induced photoacoustic streaming (PIPS) has been done to the postspaces. Scanning electron microscope (SEM) analysis has been made for two samples of each group. Fiber posts were then luted with resin cement. Each root was prepared for push-out test. Data have been statistically analyzed. RESULTS: SEM results showed clean postwalls with both group 2 and group 3, whereas group 1 showed adhesion of resin cement to intraradicular dentine. When all groups were compared, the bond strength values are higher with group 2 followed by group 3. CONCLUSION: Within the limitations of the study, clean postwalls and the highest bond strength values were obtained from 17% EDTA with hand activation and 17% EDTA with PIPS.

Ratio of neutrophilic CD64 and monocytic HLA-DR: A novel parameter in diagnosis and prognostication of neonatal sepsis.

OBJECTIVE: Approaches to monitoring of sepsis have traditionally relied upon the pro-inflammatory component of the sepsis response. This study evaluated the diagnostic and prognostic potential of the ratio of neutrophilic CD64 (nCD64) and monocytic HLA-DR (mHLA-DR) median fluorescence index in monitoring of neonatal sepsis. METHODS: Blood from 100 neonates suspected of sepsis and 29 healthy controls was collected on clinical suspicion of sepsis, and the expression of nCD64, mHLA-DR was evaluated by Flow Cytometry; thereby, a derived parameter "Sepsis index," SI = nCD64/mHLA-DR × 100 was estimated. RESULTS: At day 1, sensitivity and specificity to detect sepsis using nCD64 was 73.01% and 89.18%, respectively, while for SI it was 73.01% and 72.22%, respectively. On Kaplan-Meier analysis, neonates with SI > cut-off showed a higher 30 day-mortality than those with low SI (P = 0.096). On multivariate analysis, the factor associated with mortality in our cohort was Apgar score ≤3, while SI showed a trend toward significance. CONCLUSIONS: At day1, nCD64 is useful for the diagnosis of neonatal sepsis whereas mHLA-DR is beneficial for monitoring patients at a later time point. The SI is a marker of moderate diagnostic sensitivity and supplements the current arsenal of laboratory investigations to detect neonatal sepsis. As a marker of prognosis, a high SI shows a trend towards greater mortality. © 2015 Clinical Cytometry Society.

A Bi-Lineal Acute Leukemia (T/Myeloid, NOS) with Complex Cytogenetic Abnormalities.

We report an unusual case of bi-lineal mixed-phenotype acute leukemia (T/Myeloid, NOS) with complex cytogenetic abnormalities in a 2-year-old boy. Despite attaining complete remission with therapy, he succumbed to status epilepticus following febrile illness. Flow cytometry represents the current standard of care for the diagnosis of this malignancy and the approach adopted in our case is discussed.

Unusual presentation of an uncommon abdominal pathology.

Desmoid tumour is a non-encapsulated locally invasive tumour, originating from fibroblasts, which has ubiquitous distribution in the body. It has a high tendency for local recurrence, causing deformity in the adjacent organ and consequent organ dysfunction. A case of a 75-year-old man, presenting with high-grade fever, dull aching abdominal pain and weight loss, suspected to be due to malignancy, is reported. Contrast-enhanced computed tomography (CECT) showed a mass in the transverse colon with pericolic collection raising a suspicion of perforation. He underwent a two-stage procedure in the form of defunctioning ileostomy in the initial setting followed by colonic resection and anastomosis 6 weeks later. Histological analysis revealed mesenteric fibromatosis (desmoid tumour). The patient was completely asymptomatic one year after surgery. We report this case, as our patient had none of the predisposing factors. To the best of our knowledge, this is the first case on colonic perforation secondary to desmoid tumour.

Chronic abdominal pain--a radiological solution.

Chronic mesenteric ischaemia is not an uncommon disorder. It is associated with high morbidity and mortality. It presents with chronic abdominal pain and the diagnosis is often missed because of nonspecific clinical findings and limitations of diagnostic studies. Although surgery has been considered to be the mainstay of treatment, it is associated with significant morbidity. We report two cases of chronic mesenteric ischaemia managed effectively with endovascular therapy with no morbidity and good long term pain relief.

Renal cell carcinoma: Impact of mode of detection on its pathological characteristics.

Objective : Data correlating mode of presentation of renal cell carcinoma (RCC) with pathological prognostic factors is sparse from India. We compared RCC presenting incidentally with those presenting symptomatically with respect to pathological prognostic factors and assessed whether this could serve as a decision making resource for diagnosing small and more favorable tumors. Materials and Methods : The data were reviewed for 328 patients operated for renal tumors between January 2000 and October 2008 at our institute. The pathological factors (tumor size, stage, grade, histopathological type) in relation to the mode of presentation were analyzed according to 1997 TNM criteria. Statistical analysis was performed via the chi-square (Fisher exact) and Mann -Whitney U test. The statistical significance level utilized was P < 0.05. Results : Among the patients assessed, 93 (28.4%) had incidental diagnosis and 235 (71.6%) had symptomatic presentation. Sex and side distribution was not significantly different in the two groups. Mean tumor size was 5.75 +/- 2.73 cm in incidentally detected RCC (IRCC) and 9.32 +/- 3.70 (P < 0.001) in symptomatic RCC (SRCC). Stage I and II tumors were significantly greater in IRCC than SRCC (P < 0.001 and 0.005 respectively) whereas stage III and IV tumors were significantly less in IRCC than SRCC. There was a predominance of higher grade tumors in SRCC, 50% being higher grades (Fuhrman's grade III and IV) in SRCC than 28.1% in IRCC (P = 0.003). There were 4 tumors with collecting duct histology in SRCC and none in IRCC. Sarcomatoid differentiation was present in 14 and 1 patient in SRCC and IRCC respectively. Conclusion : Incidental detection of renal carcinoma as compared to symptomatic tumors is lower in India as compared to western world. Incidental tumors have significantly favorable pathological prognostic factors. Our results might form a basis for further studies on how to pick RCC at an earlier stage.

Flow cytometric immunophenotyping and minimal residual disease analysis in multiple myeloma.

Presence of normal plasma cells (PCs), hemodilution of bone marrow aspirate, and changes in the immunophenotype are important considerations in minimal residual disease (MRD) assessment in multiple myeloma (MM). We evaluated 124 subjects-107 with MM, 11 with Hodgkin lymphoma, and 6 allogeneic stem cell transplantation donors-for the immunophenotype of neoplastic, reactive, and normal PCs respectively. Of the patients with MM, 36 were evaluated for MRD and 23 for a change in immunophenotype after chemotherapy. The immunophenotype of normal and reactive PCs was similar and differed from that of neoplastic PCs with respect to CD19, CD45, CD56, CD52, CD20, and CD117. At least 2 antigens were aberrantly expressed in all cases and 3 in 90.7% of MM cases. A change in the immunoprofile of PCs was observed in 18 (78%) of 23 cases. By using flow cytometry, we detected MRD in all samples, and a neoplastic PC index (percentage of neoplastic PCs/total bone marrow PCs) of less than 30 could differentiate immunofixation (IFx)- from IFx+ samples (complete and partial responders, respectively).

Minimal invasive treatment of ureteropelvic junction obstruction in low volume pelvis: A comparative study of endopyelotomy and laparoscopic nondismembered pyeloplasty.

OBJECTIVE: To evaluate the role of nondismembered laparoscopic pyeloplasty and percutaneous endopyelotomy for ureteropelvic junction obstruction (UPJO) with low volume renal pelvis. MATERIAL AND METHODS: Retrospective acquired data of 34 patients of laparoscopic nondismembered pyeloplasty was compared with 26 patients of UPJO with pelvic volume less than 50 ml undergoing antegrade endopyelotomy and analyzed for clinical parameters, operative outcomes and success of procedures. All patients were followed up clinically and with diuretic renogram at regular intervals. RESULTS: Mean age, renal pelvic volume and preoperative glomerular filtration rate (GFR) was 25 years, 43.6 ml and 42.5 ml/min, respectively in endopyelotomy group and 21 years, 34.4 ml and 39.9 ml/min, respectively in laparoscopic pyeloplasty group. Mean operative time, postoperative analgesic requirement and mean hospital stay was 100min, 250 mg and 4 days, respectively in endopyelotomy group and 210 min, 300 mg and 4 days, respectively in laparoscopic pyeloplasty group. Only operative time was significantly different between two groups (P < 0.05). Mean follow-up was 36 and 39 months and success rates were 91.2% and 88.8% in laparoscopy and endopyelotomy group, respectively (P < 0.05). No significant complication was seen in endopyelotomy group while two patients had hematuria (one requiring blood transfusion) and three had increased drain output for more than 3 days in laparoscopy group. CONCLUSION: Percutaneous endopyelotomy is associated with significantly less operative time and postoperative complication rate and provides equivalent success in comparison to nondismembered laparoscopic pyeloplasty in patients with UPJO and low volume pelvis. It can be a preferred minimally invasive treatment modality for such patients.

Glanzmann's thrombasthenia in North Indians: sub classification and carrier detection by flow cytometry.

Thirty-three patients of Glanzmann's thrombasthenia (GT) and their families were assessed for the expression of alphaIIbbeta3 on platelet surface, by flow cytometry, to determine the common subtypes in North Indians as well as to assess the carrier status in family members of GT patients. GT was diagnosed in patients with bleeding manifestations accompanied by absent/reduced platelet aggregation, secondary to adenosine-di-phosphate, adrenaline, arachidonic acid and collagen. Based on alphaIIbbeta3 levels, 21 patients (64%) were classified as type I (as alphaIIbbeta3 was absent), 4 patients (12%) as type II and 8 patients (24%) as type III. Eight out of 20 fathers, 10 out of 20 mothers and 20 out of 31 siblings were found to have reduced alphaIIbbeta3 levels. Reduced alphaIIbbeta3 expression was seen in 63% of parents and 65% of siblings. It is possible that low alphaIIbbeta3 levels in family members may reflect their carrier status. It is postulated that flow cytometry estimation of alphaIIbbeta3 in parents/siblings may detect carrier status in GT. It is also revealed that type I GT is the commonest subtype in North Indians.

Analysis of bone marrow plasma cells in patients with solitary bone plasmacytoma.

Local radiotherapy is the treatment of choice for solitary bone plasmacytoma (SBP) and the role of adjuvant systemic chemotherapy in preventing progression to multiple myeloma (MM) is controversial. The purpose of this study was to examine the presence of systemic disease in the form of neoplastic plasma cells (PC) in bone marrow of patients with SBP. Flow cytometric immunophenotyping of PC was carried out on bone marrow aspirate of 7 patients using monoclonal antibodies: CD19 FITC, CD45 FITC, CD20 FITC, CD52 PE, CD117 PE, CD56 PE, CD38 PerCP-Cy5.5, CD138 APC, anti-kappa (κ) FITC and anti-lambda (λ) PE. The neoplastic as well as normal PC were identified in bone marrow aspirate of all the patients at the time of diagnosis; the neoplastic PC ranged from 0.1%to 0.7% of all BM cells and 33.5% to 89.7% of total BMPC. The κ:λ ratio was normal in all the samples ranging from 0.5% to 1.6%. The present work shows the presence of systemic disease in the form of neoplastic PC in bone marrow of patients with SBP. Prospective studies would be required to study if the levels of neoplastic PC in the bone marrow may help us identify patients who are likely to progress to overt MM and benefit from systemic chemotherapy.

Carrier detection in Glanzmann thrombasthenia: comparison of flow cytometry and Western blot with respect to DNA mutation.

We studied 20 families with Glanzmann thrombasthenia, including 20 parents and 22 siblings, for carrier detection. Carrier detection was done by phenotypic analysis (flow cytometry and Western blot) and direct gene analysis (sequencing or restriction fragment length polymorphism). Reduced expression of the glycoprotein IIb/IIIa complex was found by flow cytometry in 17 (85%) of the parents and 12 (55%) of the siblings. Western blot showed a reduced or an abnormal band in 6 (30%) of the parents and 8 (36%) of the siblings. DNA analysis in family members showed all parents and 16 (73%) of siblings to be carriers. Both techniques, flow cytometry and Western blot, were evaluated with respect to DNA analysis as a "gold standard" method. The sensitivity of flow cytometry was higher (75%) than that of Western blot (39%). We concluded that flow cytometry can effectively be used for carrier detection in Glanzmann thrombasthenia.

Omental torsion.

Omental torsion is a rare cause of acute abdominal pain, and clinically mimics acute appendicitis. A 11-year-old boy presented with symptoms and signs suggestive of appendicitis. A computed tomography of abdomen revealed findings suggestive of omental torsion. Diagnostic laparoscopy confirmed the diagnosis of torsion of a segment of the greater omentum.