Combined Analysis of Anti SARS-CoV-2 IgG and IgM Responses in COVID19 Patients in India

Coronavirus disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a global health problem, India being the second most affected country. The kinetics of antibody response to SARS-CoV-2 in Indian population is not studied yet. To understand serological response in relation to age, gender, time period and severity of disease, Roche Elecsys anti-SARS-CoV-2 test was used which analysed both IgM and IgG. One hundred and three COVID-19 patients were enrolled. Seropositivity was seen in 64% of patients, with 33% at ≤ 7 days, 62% between 8 and 15 days and 81% at ≥ 16 days from the time of admission. Men (65%) showed higher antibody response than women (59%), whereas no difference was observed in seropositivity with respect to age of the patients. Dynamics of antibody responses revealed individual variations. Patients in ICU had higher antibody reactivity with 67% positivity as compared to 60% positivity in non-ICU patients. Kinetics of antibody response during COVID-19 disease varied in relation to gender, age, time period and severity and these factors might play an important role in treatment and control of COVID-19.     

Diagnosis of Major Organic Acidurias in Children: Two Years Experience at a Tertiary Care Centre

Organic acid disorders are inherited metabolic disorders in which organic acids accumulate in tissues and biological fluids of affected individuals. Classical organic acidurias include methylmalonic aciduria, propionic aciduria, isovaleric aciduria and maple syrup urine disease (MSUD). They are considered the most frequent metabolic disorders among severely ill children. Patients frequently present with acute symptoms early in life. 420 cases clinically suspected to have organic aciduria, with upper age limit of 12 years for a 2-year period (January 2007–December 2008) were enrolled into this study. Metabolic acidosis and neurological symptoms were the most common signs. Screening tests and thin layer chromatography were done for detection of organic acidurias. Identification and quantitation of organic acids in urine and quantification of amino acids in blood were done by high performance liquid chromatography. Out of 420 patients, 45 patients (10.7%) were found to have organic acidurias. 15 cases of methylmalonic aciduria, 16 cases of propionic aciduria, 13 cases of MSUD, and one case of isovaleric aciduria were diagnosed. Results demonstrate the importance of testing for organic acidurias. Since organic aciduria may cause irreversible brain damage if not treated, we recommend selective screening amongst severely ill children despite implied extra costs.     

Association of Oxidative Stress with Disease Activity and Damage in Systemic Lupus Erythematosus: A Cross Sectional Study from a Tertiary Care Centre in Southern India

To study oxidative stress in systemic lupus erythematosus (SLE) by estimating serum oxidised LDL (OxLDL), 8-hydroxy-2′-deoxyguanosine (8-OHdG), malondialdehyde (MDA), and total anti-oxidant status and to correlate with SLE disease activity and disease damage. Eighty SLE patients satisfying the Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ACR) 2012 criteria and 80 healthy controls were studied. Exclusion criteria were infections, renal insufficiency, other connective tissue diseases, drug-induced lupus, smoking, alcohol consumption. Disease activity was measured by SLE disease activity index-2 K (SLEDAI), disease damage was quantified by SLICC-Damage Index (SDI). Sera was tested for OxLDL, 8-OHdG, and total antioxidant status (TAS) by double-antibody sandwich ELISA; MDA measured by Colorimetric assay. Oxidative stress markers were compared between group1- controls, group 2-mildly active SLE (SLEDAI ≤ 5), group 3- moderate to highly active SLE (SLEDAI ≥ 6). SLE patients had significantly higher MDA, 8-OHdG and lower TAS when compared to healthy controls, while OxLDL was similar in the three groups. MDA, 8-OHdG were significantly higher, TAS lower in group 3 compared to group 2. MDA had positive correlation with SLEDAI, TAS negatively correlated with SLEDAI. SLE with neuropsychiatric manifestations, vasculitis, anti-sdDNA antibodies had higher MDA, MDA/TAS ratio. SLE patients with thrombocytopenia, and vasculitis had higher OxLDL. Only OxLDL was significantly higher in those patients who have SDI > 1. SLE patients have increased oxidative stress measured by increases in MDA, 8-OHdG, and lower total antioxidant status that was associated with disease activity and some disease manifestations. However only OxLDL was associated with damage.     

Sequential Profiling of Anti-SARS CoV-2 IgG Antibody in Post COVID-19 Patients

Upon SARS CoV-2 infection, humoral immune system triggers production of anti-SARS CoV-2 IgM and IgG antibodies. Currently, antibodies against SARS CoV-2 spike protein receptor binding domain play a central role in disease protection, making them potential target for in vitro diagnostics applications. This study determines the expression level and sustainability of anti-receptor binding domain (RBD) SARS CoV-2 IgG in post COVID-19 patients. Anti-RBD SARS CoV-2 IgG antibodies in patient serum were analysed by standardised indirect ELISA using SARS CoV-2 spike receptor binding domain protein and HRP conjugated anti-human IgG antibody (anti-h IgG). The study was conducted using 35 adult patient samples with confirmed SARS CoV-2 infection. Additionally, correlation between antibody response after each stage and disease symptoms in post COVID-19 patients were studied. Maximum antibody titre was seen at Day 40 and decreased relatively to Day 180 in antibody positive samples when compared with controls. Overall, more IgG antibody expression is observed in patients who suffered from loss of smell and taste at Day 40. 71% of the positive subjects in this study showed high SARS CoV-2 IgG antibody concentration of above 10 ng/mL and 37% showed strong antibody concentration above 20 ng/mL at the peak of seroconversion.     

Validation Protocol: First Step of a Lean-Total Quality Management Principle in a New Laboratory Set-up in a Tertiary Care Hospital in India

Method validation is pursued as the first step in establishing Lean-Total Quality Management in a new clinical laboratory, in order to eliminate error in test results. Validation of all the new tests were done (with particular reference to alkaline phosphatase) by verifying reference intervals, analytical accuracy and precision, inter-assay and intra-assay variations, analytical sensitivity, limit of detection, linearity and reportable range, i.e. (i) Analytical measurement range (AMR) and (ii) Clinically reportable range (CRR). Our obtained reference range was within that of the manufacturer’s and showed high degree of analytical accuracy between two laboratories (r ² = 0.99). Precision was comparable with the manufacturer’s claim with inter-assay variation CV 1.04% and intra-assay variation CV 1.54%. Lowest limit of detection was 1.0324 ± 0.007 with CV 0.34%. AMR was also verified with CV 1.26 and 0.69%, for level 1 and level 2 control sera, respectively. The assay was linear with different dilutions. Lean concept was also verified with high recovery percentage. Validation ensures that accurate and precise results are reported in a clinically relevant turn around time.     

Pregnancy Associated Plasma Protein-A (PAPP-A) Levels in Acute Coronary Syndrome: A Case Control Study in a Tertiary Care Centre

In recent years, an important objective of cardiovascular research has been to find new markers that would improve the risk stratification and diagnosis of patients presenting with symptoms of acute coronary syndrome (ACS). Established biomarkers for diagnosis of ACS includes troponins and creatine kinase MB (CK-MB). Pregnancy associated plasma protein A (PAPP-A) is an emerging marker which has been described as a marker of plaque instability. PAPP-A is a large metalloproteinase involved in insulin-like growth factor signaling and has been shown to be involved in pathological processes like atherosclerosis. Many studies have been published regarding release of PAPP-A in circulation during ACS. The objective of this study was to evaluate the role of PAPP-A as an early marker of ACS by comparing levels of PAPP-A in patients with acute myocardial infarction (AMI) and unstable angina (UA) with asymptomatic controls. The association of PAPP-A with markers of myocardial necrosis and the association of PAPP-A levels to the presence of risk factors for coronary artery disease was also studied. We measured PAPP-A levels in patients with AMI (30), UA (23) and asymptomatic controls (45). PAPP-A was estimated using PAPP-A US (ultra sensitive) ELISA manufactured by DRG (Germany). PAPP-A levels were significantly elevated in patients with AMI and in patients with UA (mean levels 64.26 ± 1.05 and 36.23 ± 1.05 ng/ml respectively; p < 0.001). Mean PAPP-A levels in controls were 10.68 ± 1.04 ng/ml. In UA cases PAPP-A levels were elevated when the troponin I and CK-MB levels were within the normal range. No correlation was observed between PAPP-A with markers of myocardial necrosis. PAPP-A can serve as a useful biomarker in the diagnosis of ACS, especially UA, where cardiac troponin levels and CK-MB levels are not elevated and ECG changes are inconclusive.     

Evaluation of Oxidative Stress and hsCRP in Polycystic Ovarian Syndrome in a Tertiary Care Hospital

PCOS is a heterogeneous endocrine disorder with diverse clinical presentation. Oxidative stress plays a key role in the pathophysiology of this disease. Serumhigh sensitive C-reactive protein (hsCRP), a marker of chronic low grade inflammation, is indicative of future development of cardiovascular disease. Our aim is to evaluate the oxidant status and hsCRP levels in PCOS. The study involved 61 cases and 61 controls in the age group of 18–40 years diagnosed with PCOS. Erythrocyte malondialdehyde (MDA), superoxide dismutase (SOD), serum hsCRP, gonadotrophins, thyroid stimulating hormone, prolactin, glycemic status and lipid profile were estimated. Erythrocyte MDA (p < 0.001), SOD (p = 0.007) and serum hsCRP (p < 0.001) were significantly elevated in PCOS patients than controls. Oxidative stress is present in women with PCOS along with elevated hsCRP.     

Study of Variables Affecting Critical Value Notification in a Laboratory Catering to Tertiary Care Hospital

During post-analytical phase, critical value notification to responsible caregiver in a timely manner has potential to improve patient safety which requires cooperative efforts between laboratory personnel and caregivers. It is widely accepted by hospital accreditors that ineffective notification can lead to diagnostic errors that potentially harm patients and are preventable. The objective of the study was to assess the variables affecting critical value notification, their role in affecting it’s quality and approaches to improve it. In the present study 1,187 critical values were analysed in the Clinical Chemistry Laboratory catering to tertiary care hospital for neuropsychiatric diseases. During 25 months of study period, we evaluated critical value notification with respect to clinical care area, caregiver to whom it was notified and timeliness of notification. During the study period (25 months), the laboratory obtained 1,279 critical values in clinical chemistry. The analytes most commonly notified were sodium and potassium (20.97 & 20.8 % of total critical results). Analysis of critical value notification versus area of care showed that critical value notification was high in ICU and emergency area followed by inpatients and 64.61 % critical values were notified between 30 and 120 min after receiving the samples. It was found that failure to notify the responsible caregiver in timely manner represent an important patient safety issue and may lead to diagnostic errors. The major area of concern are notification of critical value for outpatient samples, incompleteness of test requisition forms regarding illegible writing, lack of information of treating physician and location of test ordering and difficulty in contacting the responsible caregiver.     

Evaluation of Sigma-Metric and Application of Quality Tools in Clinical Laboratory of a Tertiary Care Hospital

Variability in analytical performance of some analyte indicated the need of evaluation of quality plan of our laboratory. We tried to put the same degree of effort into our quality metrics as we put into the laboratory processes themselves. Application of six sigma methodologies improve the quality by focusing on the root causes of the problems in performance and analyzing by flowcharts, fishbone diagrams and other quality tools. Sigma metric was calculated for laboratory parameters for a period of 8 months during 2018–19. The analytes with poor sigma metric were free Thyroxine (FT3, FT4), Sodium, Calcium and Magnesium. Sigma metric of free Thyroxine (FT3, FT4), Sodium, Calcium and Magnesium were below 3. A road map for process improvement was designed with DMAIC (Define-Measure-Analyze-Improve-Control) model to solve the issue. Possible causes for low analytical performance of the particular analytes were depicted in Fishbone diagram. The Fishbone analysis identified the water quality issues with electrolyte analysis while high ambient temperature was culprit for poor assay performance of free Thyroxine. Sigma metric of the analytical performance was assessed once again after root cause analysis. Sigmametric showed marked improvement in control phase. Identification of problems led to reduction in non value added work leading to adequate resource utilization by addressing the priority issue. Therefore, DMAIC tool with Fish bone model analysis can be recommended as a well suited method for troubleshooting in poor performance of laboratory parameter.     

Role of Intervention on Laboratory Performance: Evaluation of Quality Indicators in a Tertiary Care Hospital

Quality in laboratory has huge impact on diagnosis and patient management as 80–90% of all diagnosis is made on the basis of laboratory tests. Monitoring of quality indicators covering the critical areas of pre-analytical, analytical and post-analytical phases like sample misidentification, sample rejection, random and systemic errors, critical value reporting and TATs have a significant impact on performance of laboratory. This study was conducted in diagnostic laboratories receiving approximately 42,562 samples for clinical chemistry, hematology and serology. The list of quality indicators was developed for the steps of total testing process for which errors are frequent and improvements are possible. The trend was observed for all the QI before and after sensitisation of the staff over the period of 12 months. Incomplete test requisition form received in the lab was the most poor quality indicator observed (7.89%), followed by sample rejection rate (4.91%). Most significant improvement was found in pre- and post-analytical phase after sensitisation of staff but did not have much impact on analytical phase. Use of quality indicators to assess and monitor the quality system of the clinical laboratory services is extremely valuable tool in keeping the total testing process under control in a systematic and transparent way.     

Audit of the Prevalence of Noncorrelation of Immunofixation with Protein Electrophoresis and Serum Free Light Chain Assays in Multiple Myeloma in a Tertiary Cancer Care Center

Multiple myeloma (MM) is diagnosed and monitored by correlating panel of test results including serum Protein electrophoresis (SPE), Immunofixation electrophoresis (IFE), serum Free Light chain (sFLC) measurements. This audit is aimed to evaluate the prevalence of non-correlation and discrepancies amongst the three investigations (SPE/IFE/sFLC) for assessment of MM. 106 MM patients were reviewed over 16 months in a tertiary cancer care center by the availability of 3 serum test results (SPE/IFE/sFLC). Patients were divided into 2 groups: group1, newly diagnosed MM patients who were yet to receive myeloma specific treatment (n = 48); and group2, already diagnosed MM patients on treatment and followup (n = 58). Treatment modalities included stem cell transplantation and standard chemotherapy regimens. Non-correlation between the three test results (IFE/SPE/sFLC) was observed (21% in group1 and 45% in group2). Three types of discrepancies were detected as follows: (a) IFE showing less number of restriction bands as compared to SPE (8.6% patients in group2); (b) SPE/IFE negative with an abnormal sFLC ratio (12.5% patients in group1 and 13.7% in group2); (c) SPE/IFE positive but normal sFLC ratio (8% in group1 and 22% in group2). To conclude, IFE may sometimes provide information that does not always correlate with either of the SPE or sFLC results due to different sensitivities, antigen–antibody interactions, or treatment. Hence, SPE plus sFLC may be more useful particularly for patients on follow-up while IFE plus sFLC may help screen the new patients. The judicious selection of the biochemical assays can effectively reduce the treatment cost in a developing country like India.