Methods to reduce lipemic interference in clinical chemistry tests: a systematic review and recommendations.

OBJECTIVES: Lipemia is the presence of abnormally high lipoprotein concentrations in serum or plasma samples that can interfere with laboratory testing. There is little guidance available from manufacturers or professional bodies on processing lipemic samples to produce clinically acceptable results. This systematic review summarizes existing literature on the effectiveness of lipid removal techniques in reducing interference in clinical chemistry tests. METHODS: A PubMed search using terms relating to lipid removal from human samples for clinical chemistry tests produced 1,558 studies published between January 2010 and July 2021. 15 articles met the criteria for further analyses. RESULTS: A total of 66 analytes were investigated amongst the 15 studies, which showed highly heterogenous study designs. High-speed centrifugation was consistently effective for 13 analytes: albumin, alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, creatine kinase (CK), creatinine (Jaffe method), gamma-glutamyl transferase (GGT), glucose (hexokinase-based method), lactate dehydrogenase (LDH), phosphate, potassium, and urea. Lipid-clearing agents were uniformly effective for seven analytes: ALT, AST, total bilirubin, CK, creatinine (Jaffe method), lipase, and urea. Mixed results were reported for the remaining analytes. CONCLUSIONS: For some analytes, high-speed centrifugation and/or lipid-clearing agents can be used in place of ultracentrifugation. Harmonized protocols and acceptability criteria are required to allow pooled data analysis and interpretation of different lipemic interference studies.

Comparative Clinical Evaluation of the Roche Elecsys and Abbott Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Serology Assays for Coronavirus Disease 2019 (COVID-19).

CONTEXT.­: The use of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serologic tests detects antibodies in the host, contributing to the identification of individuals who have been exposed to coronavirus disease 2019 (COVID-19). OBJECTIVE.­: To critically evaluate 2 commercially available SARS-CoV-2 serology tests. DESIGN.­: A total of 333 unique, nonduplicated serum samples obtained from COVID-19 patients (n = 170) and negative controls (n = 163) obtained before December 2019 were used in the study. Samples were tested on the Roche E411 and Abbott Architect i4000SR platforms, and results were correlated to reverse transcription polymerase chain reaction (PCR) results and clinical symptoms. RESULTS.­: There was a strong level of agreement in the qualitative results between both assays, with a Cohen κ value of .840, P < .001. The specificity for both Roche and Abbott were excellent at 100%. Roche exhibited marginally better performance in the 21 days or more group with a sensitivity of 90.6% (95% CI, 75.8%-96.8%) versus an Abbott sensitivity of 84.4% (95% CI, 68.3%-93.1%), as well as in the 14- to 20-day group with a sensitivity of 85.7% (95% CI, 65.4%-95.0%) versus an Abbott sensitivity of 81.0% (95% CI, 60.0%-92.3%). Less than 14 days of symptoms groups exhibited poor sensitivity at less than 50% for both assays. The areas under curve (± standard error) for Roche (0.894 ± 0.025, P < .001) and Abbott (0.884 ± 0.026, P < .001) were very similar. Potential confounders for negative serologic results include antiretroviral medication use and pauci-symptomatic patients. CONCLUSIONS.­: Specificities for high-throughput Roche and Abbott immunoassays are excellent, but users need to be cautious to interpret serologic test results after 14 days of symptoms to avoid false negatives.

Head-to-head evaluation on diagnostic accuracies of six SARS-CoV-2 serological assays.

In this study, we evaluated and compared six SARS-CoV-2 serology kits including the Abbott SARS-CoV-2 IgG assay, Beckman Access SARS-CoV-2 IgG assay, OCD Vitros OCD Anti-SARS-CoV-2 Total antibody assay, Roche Elecsys Anti SARS-CoV-2 assay, Siemens SARS-CoV-2 Total assay, and cPass surrogate viral neutralising antibody assay. A total of 336 non-duplicated residual serum samples that were obtained from COVID-19 confirmed patients (n=173) on PCR and negative controls (n=163) obtained pre-December 2019 before the COVID-19 pandemic were used for the study. These were concurrently analysed on the different immunoassay platforms and correlated with clinical characteristics. Our results showed all assays had specificity ranging from 99.3% to 100.0%. Overall sensitivity across all days of symptoms, in descending order were OCD (49.1%, 95% CI 41.8-56.5%), cPass (44.8%, 95% CI 37.5-52.3%), Roche (41.6%, 95% CI 34.5-49.0%), Siemens (39.9%, 95% CI 32.9-47.3%), Abbott (39.8%, 95% CI 32.9-47.3%) and Beckman (39.6%, 95% CI 32.5-47.3%). Testing after at least 14 days from symptom onset is required to achieve AUCs greater than 0.80. OCD and cPass performed the best in terms of sensitivity for >21 days symptoms with 93.3% (95% CI, 73.5-99.2%) and 96.7% (95% CI, 82.8-99.9%), respectively. Both also shared the greatest concordance, kappa 0.963 (95% CI 0.885-1.0), p<0.001, and had the lowest false negative rates. Serology results should be interpreted with caution in certain cases. False negatives were observed in a small number of individuals with COVID-19 on immunosuppressive therapy, pauci-symptomatic or who received antiretroviral therapy. In conclusion, all assays exhibited excellent specificity and total antibody assays with spike protein configurations generally outperformed nucleocapsid configurations and IgG assays in terms of diagnostic sensitivity.

Stromal vascular fraction promotes fibroblast migration and cellular viability in a hyperglycemic microenvironment through up-regulation of wound healing cytokines.

Diabetic wounds have impaired healing and a propensity for further morbidity, which may result in amputations. Stromal vascular fraction (SVF) is an autologous source of heterogeneous cell population obtained from adipose tissue, which is rich in stem cells and presents little immunogenicity to the host. In this study, we hypothesized that murine fibroblasts subjected to hyperglycemic conditions co-treated with SVF exhibit greater functional activity through the colorimetric MTT assay and a cell-monolayer in-vitro scratch assay. We sought to establish the underlying mechanism of action via the utility of an ELISA chemiluminescence array on the supernatant medium of the cells. Our results demonstrate that the mean percentage gap closure at 24 h in the hyperglycemia + SVF group was significantly greater at 41.1% ±â€¯1.6% compared to the hyperglycemia alone group 16.6% ±â€¯1.5% (post-hoc Bonferroni test p < 0.001, n = 3) although there was no difference between the SVF and normoglycemia group. Further, this SVF group exhibited a significantly greater 2.4 fold increase in fibroblastic cell viability as compared to the hyperglycemia alone group (p = 0.001, n = 3). The supernatant medium of the cells upon testing with ELISA indicated that early phase wound healing cytokines including platelet-derived growth factor (p = 0.012, n = 3), interleukin-1 (p = 0.003, n = 3), basic fibroblast growth factor (p = 0.003, n = 3) and interleukin-10 (p = 0.009, n = 3) were expressed in significantly greater relative luminescent units in SVF as compared to hyperglycemia alone groups (Student t-test). Taken together and for the first time, our study shows that SVF is a promising therapeutic agent for up-regulating fibroblastic activity in a hyperglycemic microenvironment, and this result can be explained in part by the stimulation of wound-healing cytokines.

A multidisciplinary approach to reducing spurious hyperkalemia in hospital outpatient clinics.

AIMS AND OBJECTIVES: To describe a multidisciplinary effort to investigate and reduce the occurence of outpatient spurious hyperkalaemia. BACKGROUND: Spurious hyperkalemia is a falsely elevated serum potassium result that does not reflect the in vivo condition of a person. A common practice of fist clenching/pumping during phlebotomy to improve vein visualisation is an under-appreciated cause of spurious hyperkalemia. DESIGN: Pre- and postinterventional study. METHOD: Objective evidence of spurious hyperkalaemia was sought by reviewing archived laboratory results. A literature review was undertaken to summarise known causes of spurious hyperkalaemia and develop a best practice in phlebotomy. Subsequently, nurses from the Urology Clinic were interviewed, observed and surveyed to understand their phlebotomy workflow and identify potential areas of improvement by comparing to the best practice in phlebotomy. Unexplained (potentially spurious) hyperkalaemia was defined as a serum potassium of >5·0 mmol/l in a patient without stage 5 chronic kidney disease or haemolysed blood sample. RESULTS AND CONCLUSION: Nurses from the Urology Clinic showed significant knowledge gap regarding causes of spurious hyperkalaemia when compared to the literature review. Direct observation revealed patients were routinely asked to clench their fists, which may cause spurious hyperkalaemia. Following these observations, several educational initiatives were administered to address the knowledge gap and stop fist clenching. The rate of unexplained hyperkalaemia at the Urology clinic reduced from a baseline of 16·0-3·8%, 58 weeks after intervention. Similar education intervention was propagated to all 18 other specialist outpatient clinic locations, which saw their rate of unexplained hyperkalaemia decrease from 5·4 to 3·7%. To ensure sustainability of the improvements, the existing phlebotomy standard operating protocol, educational and competency testing materials at variance with the best practice were revised. RELEVANCE TO CLINICAL PRACTICE: A simple intervention of avoiding fist clenching/pumping during phlebotomy produced significant reduction in the rate of spurious hyperkalemia.

Biomarkers in cardiorenal syndromes.

Cardiac and renal diseases often coexist and patients with cardiac and renal failure have high morbidity and mortality. Cardiorenal syndromes (CRSs) are disorders of the heart and kidneys whereby dysfunction in one organ may induce dysfunction in the other organ. Five subtypes of CRSs have been defined by the Acute Dialysis Quality Initiative Consensus Group. There is a need for early detection and monitoring of patients with CRSs. Biomarkers play a key role in the diagnosis and monitoring of acute myocardial infarction, chronic heart failure, and chronic kidney disease. In recent years, new biomarkers have been identified that may play a role in the early diagnosis of acute kidney injury. Herein, we review the use of serum and urine biomarkers in the diagnosis and management of CRSs. The established cardiac and renal biomarkers such as the cardiac troponins, natriuretic peptides, urine albumin, and creatinine, as well as the new renal biomarkers cystatin C and neutrophil gelatinase-associated lipocalin are reviewed in detail. The recent advances in assay methods, clinical studies, and recommendations in clinical guidelines are discussed. With advances in biomarker research, in future, perhaps a multimarker approach will become feasible to stratify the diagnosis of CRS for individualized treatment and prognosis.

Vitamin D and its relationship with markers of bone metabolism in healthy Asian women.

BACKGROUND: In this study, we aimed to determine the normal ranges of 25-hydroxy-vitamin D(3) (25-OHD(3)), parathyroid hormone (PTH), and the markers of bone turnover, procollagen type 1 N propeptide (P1NP) and C-terminal cross-linked telopeptide of type 1 collagen (CTX), in normal healthy women in Singapore, and to explore the relationship between vitamin D, PTH, and these markers of bone turnover in the women. METHODS: One hundred and ninety-seven healthy women, aged 25 to 60, were selected from a hospital staff health screening program; 68% were Chinese, 18% Malay, and 14% Indian. P1NP, CTX, and 25-OHD(3) were measured using the Roche Cobas® electrochemiluminescence immunoassay. Serum PTH was measured using the Siemens ADVIA Centaur® immunoassay. RESULTS: Sixty-five percent had 25-OHD(3) concentrations <50 nmol/l. Vitamin D insufficiency (25-OHD(3) < 50 nmol/l) was more prevalent in Malays (89%) and Indians (82%) compared to Chinese (56%). There was no correlation between vitamin D and age. PTH positively correlated with age, and Malays and Indians had higher PTH concentrations than Chinese. There was an inverse correlation between PTH and 25-OHD(3), but no threshold of 25-OHD(3) concentrations at which PTH plateaued. The bone turnover markers P1NP and CTX inversely correlated with age but were not different between ethnic groups. CTX and P1NP exhibited good correlation, however, there was no significant correlation between 25-OHD(3) or PTH concentrations and the bone turnover markers P1NP and CTX. CONCLUSIONS: Healthy women in Singapore have a high prevalence of vitamin D insufficiency. Vitamin D insufficiency was more prevalent in Malays and Indians compared to Chinese.

Comparison of the Elecsys PTH(1-84) assay with four contemporary second generation intact PTH assays and association with other biomarkers in chronic kidney disease patients.

OBJECTIVES: The COBAS Elecsys PTH(1-84) assay is a novel, electro-chemiluminescence immunoassay that exclusively measures full-length parathyroid hormone (PTH). The aim of this study is to compare the automated biointact Elecsys PTH(1-84) assay with four contemporary, iPTH assays in chronic kidney disease (CKD) patients. DESIGN AND METHODS: We compared the Elecsys PTH(1-84) assay with four iPTH assays (Siemens ADVIA Centaur, Ortho Clinical Diagnostics (OCD) VITROS, Beckman Access2, Abbott ARCHITECT) in the measurement of PTH in 83 local CKD patients. Majority of the patients (44) had CKD but were not on dialysis, 15 were on hemodialysis, 15 were on peritoneal dialysis, and 9 were post-renal transplant. The precision performance and correlation of the assays were determined. PTH(1-84) concentrations were correlated with calcium, phosphate, alkaline phosphatase, hemoglobin, HbA1c and lipid concentrations. RESULTS: The Elecsys PTH(1-84) assay showed comparable precision and good correlation with the iPTH assays. Although the four different iPTH assays correlated well with each other, there was significant discrepancy among assays. The discrepancy among assays increased with increasing PTH concentrations. The ADVIA Centaur and ARCHITECT assays measured significantly higher PTH concentrations than the VITROS and Access2 assays. PTH(1-84) showed a positive association with phosphate and alkaline phosphatase and an inverse association with HbA1c. There was no significant association with lipid concentrations. CONCLUSIONS: The third generation Elecsys PTH(1-84) assay had comparable precision performance and correlated well with second generation iPTH assays. However, significant discrepancy was found among the four iPTH assays in measuring iPTH in CKD patients.

Identification of hemoglobin variants in samples received for glycated hemoglobin testing.

BACKGROUND: In 2010 Singapore's National Health Survey reported 11.3% of the population between 18-69 years of age with diabetes, compared to 8.2% in 2006. This increasing trend reinforces the dependence on HbA(1c) for management of glycemic control. METHODS: To determine the incidence of hemoglobin variants received from our diabetic population who are attending the National University Hospital for testing of their HbA(1c) and determine whether the hemoglobin variant caused an interpretation issue, we reviewed all chromatograms from patients sending a sample for HbA(1c) analysis for a three-month period. Analysis was performed on the Variant II using the HbA(2)/HbA(1c) Dual program. RESULTS: Our sub-analysis identified 2 cases of α thalassemia, 2 cases of ß thalassemia; 5 cases of hemoglobin E variant homozygous, 1 case of hemoglobin J variant, 110 cases of hemoglobin E, 7 cases of hemoglobin S, 1 case of hemoglobin C and 1 case of hemoglobin D, all heterozygous. HbA(1c) results could be confidently reported in all cases except the homozygote variant and the hemoglobin J variant. CONCLUSIONS: Overall we obtained a prevalence of 2.3% of hemoglobin variants in our diabetic population being screened by HbA(1c) using Variant II.

Teacher led school-based surveillance can allow accurate tracking of emerging infectious diseases - evidence from serial cross-sectional surveys of febrile respiratory illness during the H1N1 2009 influenza pandemic in Singapore.

BACKGROUND: Schools are important foci of influenza transmission and potential targets for surveillance and interventions. We compared several school-based influenza monitoring systems with clinic-based influenza-like illness (ILI) surveillance, and assessed the variation in illness rates between and within schools. METHODS: During the initial wave of pandemic H1N1 (pdmH1N1) infections from June to Sept 2009 in Singapore, we collected data on nation-wide laboratory confirmed cases (Sch-LCC) and daily temperature monitoring (Sch-DTM), and teacher-led febrile respiratory illness reporting in 6 sentinel schools (Sch-FRI). Comparisons were made against age-stratified clinic-based influenza-like illness (ILI) data from 23 primary care clinics (GP-ILI) and proportions of ILI testing positive for pdmH1N1 (Lab-ILI) by computing the fraction of cumulative incidence occurring by epidemiological week 30 (when GP-ILI incidence peaked); and cumulative incidence rates between school-based indicators and sero-epidemiological pdmH1N1 incidence (estimated from changes in prevalence of A/California/7/2009 H1N1 hemagglutination inhibition titers ≥ 40 between pre-epidemic and post-epidemic sera). Variation in Sch-FRI rates in the 6 schools was also investigated through a Bayesian hierarchical model. RESULTS: By week 30, for primary and secondary school children respectively, 63% and 79% of incidence for Sch-LCC had occurred, compared with 50% and 52% for GP-ILI data, and 48% and 53% for Sch-FRI. There were 1,187 notified cases and 7,588 episodes in the Sch-LCC and Sch-DTM systems; given school enrollment of 485,723 children, this represented 0.24 cases and 1.6 episodes per 100 children respectively. Mean Sch-FRI rate was 28.8 per 100 children (95% CI: 27.7 to 29.9) in the 6 schools. We estimate from serology that 41.8% (95% CI: 30.2% to 55.9%) of primary and 43.2% (95% CI: 28.2% to 60.8%) of secondary school-aged children were infected. Sch-FRI rates were similar across the 6 schools (23 to 34 episodes per 100 children), but there was widespread variation by classrooms; in the hierarchical model, omitting age and school effects was inconsequential but neglecting classroom level effects led to highly significant reductions in goodness of fit. CONCLUSIONS: Epidemic curves from Sch-FRI were comparable to GP-ILI data, and Sch-FRI detected substantially more infections than Sch-LCC and Sch-DTM. Variability in classroom attack rates suggests localized class-room transmission.

Immunoglobulin-associated creatine kinase masquerading as macro-creatine kinase type 2 in a statin user.

Macro-creatine kinase (CK) is a cause of falsely elevated CK. Macro-CK type 1 is immunoglobulin-associated CK; type 2 is polymeric mitochondrial-CK. An elderly asymptomatic lady had an elevated CK level after receiving statin therapy. Her CK gel electrophoresis analysis demonstrated coexisting macro-CK type 1 and type 2 patterns. Further analysis by immunofixation and mixing this patient's serum with CK control material revealed an IgG-associated macro-CK that mimicked the electrophoretic pattern of macro-CK type 2. This highly unusual discovery suggests the possibility of the misinterpretation of macro-CK type 1 as macro-CK type 2. Falsely elevated CK is still common despite modern laboratory instrumentation and should be investigated.

Diagnostic testing for pandemic influenza in Singapore: a novel dual-gene quantitative real-time RT-PCR for the detection of influenza A/H1N1/2009.

With the relative global lack of immunity to the pandemic influenza A/H1N1/2009 virus that emerged in April 2009 as well as the sustained susceptibility to infection, rapid and accurate diagnostic assays are essential to detect this novel influenza A variant. Among the molecular diagnostic methods that have been developed to date, most are in tandem monoplex assays targeting either different regions of a single viral gene segment or different viral gene segments. We describe a dual-gene (duplex) quantitative real-time RT-PCR method selectively targeting pandemic influenza A/H1N1/2009. The assay design includes a primer-probe set specific to only the hemagglutinin (HA) gene of this novel influenza A variant and a second set capable of detecting the nucleoprotein (NP) gene of all swine-origin influenza A virus. In silico analysis of the specific HA oligonucleotide sequence used in the assay showed that it targeted only the swine-origin pandemic strain; there was also no cross-reactivity against a wide spectrum of noninfluenza respiratory viruses. The assay has a diagnostic sensitivity and specificity of 97.7% and 100%, respectively, a lower detection limit of 50 viral gene copies/PCR, and can be adapted to either a qualitative or quantitative mode. It was first applied to 3512 patients with influenza-like illnesses at a tertiary hospital in Singapore, during the containment phase of the pandemic (May to July 2009).

Overexpression of endoplasmic reticulum protein 29 regulates mesenchymal-epithelial transition and suppresses xenograft tumor growth of invasive breast cancer cells.

Endoplasmic reticulum protein 29 (ERp29) is a novel endoplasmic reticulum (ER) secretion factor that facilitates the transport of secretory proteins in the early secretory pathway. Recently, it was found to be overexpressed in several cancers; however, little is known regarding its function in breast cancer progression. In this study, we show that the expression of ERp29 was reduced with tumor progression in clinical specimens of breast cancer, and that overexpression of ERp29 resulted in G(0)/G(1) arrest and inhibited cell proliferation in MDA-MB-231 cells. Importantly, overexpression of ERp29 in MDA-MB-231 cells led to a phenotypic change and mesenchymal-epithelial transition (MET) characterized by cytoskeletal reorganization with loss of stress fibers, reduction of fibronectin (FN), reactivation of epithelial cell marker E-cadherin and loss of mesenchymal cell marker vimentin. Knockdown of ERp29 by shRNA in MCF-7 cells reduced E-cadherin, but increased vimentin expression. Furthermore, ERp29 overexpression in MDA-MB-231 and SKBr3 cells decreased cell migration/invasion and reduced cell transformation, whereas silencing of ERp29 in MCF-7 cells enhanced cell aggressive behavior. Significantly, expression of ERp29 in MDA-MB-231 cells suppressed tumor formation in nude mice by repressing the cell proliferative index (Ki-67 positivity). Transcriptional profiling analysis showed that ERp29 acts as a central regulator by upregulating a group of genes with tumor suppressive function, for example, E-cadherin (CDH1), cyclin-dependent kinase inhibitor (CDKN2B) and spleen tyrosine kinase (SYK), and by downregulating a group of genes that regulate cell proliferation (eg, FN, epidermal growth factor receptor (EGFR) and plasminogen activator receptor (uPAR)). It is noteworthy that ERp29 significantly attenuated the overall ERK cascade, whereas the ratio of p-ERK1 to p-ERK2 was highly increased. Taken together, our results showed that ERp29 is a novel regulator leading to cell growth arrest and cell transition from a proliferative to a quiescent state, and reprogramming molecular portraits to suppress the tumor growth of MDA--MB--231 breast cancer cells.

Natriuretic peptides in chronic kidney disease.

BACKGROUND AND OBJECTIVES: B-type natriuretic peptide (BNP) and amino-terminal pro-B-type natriuretic peptide (NT-proBNP) are biomarkers of cardiovascular disease that is common in patients with chronic kidney disease (CKD). Conflicting data on the influence of glomerular filtration rate (GFR) on BNP and NT-proBNP levels in CKD may stem from failure to account fully for the effects of coexistent cardiac disease, dysfunction, and volume overload. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Prospective head-to-head comparison of plasma BNP and NT-proBNP in ambulatory euvolemic CKD patients with normal LV ejection fraction and no manifest cardiac or vascular disease. GFR was estimated by the Modification of Diet in Renal Disease formula, BNP and NT-proBNP measured using Abbott AxSYM and Roche Elecsys assays, respectively, and cardiac morphology and function assessed by transthoracic echocardiography. RESULTS: In 142 patients (42% female) of mean age 60 +/- 11 yr, mean left ventricular ejection fraction was 71% +/- 6%, GFR 38 +/- 14 ml/min per 1.73 m(2), and median BNP and NT-proBNP level 59 and 311 pg/ml, respectively. Multivariate predictors of NT-proBNP level were GFR, beta-blocker usage, LV mass index, and hemoglobin level. Plasma BNP was independently predicted by LV mass index and beta-blocker usage but not GFR. In the 74 patients without diastolic dysfunction, there was a significant rise in NT-proBNP but not BNP as GFR declined. CONCLUSIONS: Unlike NT-proBNP, plasma BNP level is relatively independent of GFR. BNP may therefore be the more appropriate biomarker to screen for cardiac dysfunction in CKD.