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.

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.

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.

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.

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.

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.

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.

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.

Primary Hodgkin's disease of spleen--a case report.

Primary malignant lymphoma of spleen is rare with primary Hodgkin's disease (HD) of spleen being rarer. The diagnosis of primary splenic HD is usually made on the histopathological examination of the splenectomy specimen. We report an interesting case of primary HD of spleen in a child emphasizing its rarity and the role of fine needle aspirate cytology in early diagnosis and non-surgical management of this entity.

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.

Flow cytometric analysis of CD5+ B cells: a frame of reference for minimal residual disease analysis in chronic lymphocytic leukemia.

Recent reports suggest that CD5+ B cells constitute up to 47% of the total B cells in normal peripheral blood (PB), a finding that would restrict the sensitivity of the CD5/CD19 flow cytometric assay for minimal residual disease (MRD) analysis in chronic lymphocytic leukemia (CLL). We studied 40 normal samples (PB, 20; bone marrow [BM], 20) using CD5-fluorescein isothiocyanate (FITC)/CD19-phycoerythrin (PE) immunostaining to evaluate the reference range of CD5+ B cells. The mean percentage of CD5+ B cells per total number of B cells was 12.2% (range, 3.6%-23.9%) in PB and 11.7% (range, 4.4%-19.5%) in BM. On serial dilution, this assay could detect 1 CLL cell in 1,000 leukocytes (sensitivity, 0. 1%). A distinct "bright" CD5+ B-cell subpopulation, consistent with a CLL-like-phenotype, was observed in 3 samples. Our results suggest that the CD5-FITC/CD19-PE assay has a clinically useful sensitivity for MRD analysis in CLL. The usefulness of this assay as a screening tool to identify the earliest stage of indolent CLL needs further study.

Acute basophilic leukemia with t(8;21).

Acute basophilic leukemia (ABL) is a rare form of leukemia. The diagnostic criteria have recently been described. Morphological evidence for basophilic lineage is required for its classification. However the criteria for remission status and standard therapy is not established. Here we have described an atypical case of ABL and reviewed the literature to high light issues regarding diagnosis and management, which need further discussion.

Comparison of three different staining techniques for intraoperative assessment of nodal metastasis in breast cancer.

Imprint cytology has increasingly been used for intraoperative assessment of nodal status in breast cancer. We carried out this study to compare the efficacy of Jenner Giemsa (JG), hematoxylin-eosin (H&E), and Papanicolaou (Pap) stains for intraoperative lymph node imprint cytology (IIC) in breast cancer. One hundred and seven cases of stage I-III breast cancer were studied. Overall, IIC was accurate in 95.3% cases and had a sensitivity and specificity of 98.5% and 90.0%, respectively. The accuracy of JG (95.3%) was better than that of H&E (90.6%) and Pap (94.0%), although the differences were not statistically significant. Problems encountered included cell loss and drying artifacts with H&E and Pap and the inability to distinguish between tumor cells and histiocytes confidently in tight cellular clusters that were occasionally seen. Opinion was possible in all JG cases, but not in five and four cases by H&E and Pap, respectively. Although the choice of the stain would vary depending on the experience of the pathologist, our work suggests that JG, because of fewer technical problems and superior accuracy, may be preferable over H&E and Pap.

Metastatic anaplastic oligodendroglioma simulating acute leukemia. A case report.

BACKGROUND: Anaplastic oligodendroglioma (OG) is an uncommon tumor that rarely metastasizes outside the central nervous system. Spread to the bone marrow (BM) is so rare that when it occurs in the course of follow-up of a case of OG, a disseminated second primary tumor may be a more likely possibility unless BM examination provides evidence to the contrary. Potentially misleading cytologic features of metastatic anaplastic OG can be seen in a BM touch preparation. CASE: A 50-year-old man had undergone left frontal lobectomy in September 1999 for anaplastic OG and presented seven months later with evidence, on BM scan, of focal abnormal uptake at multiple sites. Bone marrow biopsy confirmed OG secondaries, which, on the touch preparation, appeared not only in clusters but also as single cells, simulating acute leukemia. CONCLUSION: The morphology of anaplastic OG metastatic to BM simulates acute leukemia, as seen on the BM touch preparation. This is relevant particularly in the context of anaplastic OG on follow-up. This diagnostic pitfall can be heightened if a BM aspirate rather than biopsy is performed. Metastatic OG can be added to the list of tumors that metastasize to BM as single cells.

Sentinel lymph node biopsy assessment using intraoperative imprint cytology in breast cancer patients: results of a validation study.

OBJECTIVE: Sentinel lymph node biopsy (SLNB) in breast cancer patients is emerging as a promising minimally-invasive tool. There has been an exponential increase in the literature related to sentinel lymph nodes (SLN) in breast cancer patients, mainly from Western centres. This study was carried out to address issues relevant to breast cancer patients in developing countries, including the method of SLN detection, the role of imprint cytology in the assessment of SLN, and the role of SLNB in locally advanced breast cancer (LABC). METHODS: This study included 76 women with breast cancer. The blue-dye method was used to identify the sentinel node. Touch imprint smears were prepared from the sectioned node, stained using the Jenner-Geimsa technique, and examined for tumour deposits. RESULTS: Sentinel nodes were identified in 69 of 76 patients. The sensitivity, specificity and accuracy of SLNB in predicting axillary node status were 84.2%, 100% and 91.3%, respectively. The sensitivity, specificity and accuracy of intraoperative imprint cytology were 96.9%, 100% and 98.6%, respectively. CONCLUSIONS: These results prove that high levels of SLN detection can be achieved using the blue-dye method alone. Its role in LABC patients needs further evaluation. In view of promising results, imprint cytology should be used more frequently as an alternative to frozen section for the assessment of sentinel nodes.

Non hodgkin's lymphoma seven years following remission of acute lymphoblastic leukemia.

The authors describe a case of extramedullary relapse in lymph node presenting as lymphoblastic lymphoma seven years following remission of acute lymphoblastic leukemia. To the best of our knowledge, this is the first reported case of an isolated lymph node relapse with hematopoietic remission of leukemia. We have discussed cases of large cell lymphoma and other unusual areas of extramedullary relapse complicating acute lymphoblastic leukemia in hematopoietic remission.