In-house standards derived from doping peptides: Enzymatic and serum stability and degradation profile of GHRP and GHRH-related peptides.

Matrix effect and sample pretreatment significantly affect the percentage recovery of peptides in biological matrices, affecting the method robustness and accuracy. To counteract this effect, an internal standard (IS) is used; however, in most cases this is not available, which limits the analytical method. It is important to identify short peptides that can be used as ISs in the quantification of peptides in biological matrices. In this study, doping peptides GHRP-4, GHRP-5, GHRP-6, Sermorelin (1-11), Sermorelin (13-20) and Sermorelin (22-29) were synthesized using solid-phase peptide synthesis. Treatment with human blood, trypsin and chymotrypsin was used to determine the stability of the peptides. Products were evaluated using the high-performance liquid chromatography-diode array detector (HPLC-DAD) method. The analytical methodology and sample pretreatment were effective for the analysis of these molecules. A unique profile related to protein binding and enzymatic stability of each peptide was established. GHRP-4, GHRP-6 and Sermorelin (22-29) can be considered as in-house ISs as they were stable to enzyme and blood treatment and can be used for the quantification of peptides in biological samples. Peptides GHRP-6 and Sermorelin (22-29) were used to analyse a dimeric peptide (26 [F] LfcinB (20-30)2 ) in four different matrices to test these peptides as in-house IS.

Establishment of dry chemistry based reference intervals of renal function test parameters for the adult population of Kaski District, Nepal.

BACKGROUND: Reference intervals (RIs) for clinical chemistry test parameters are specific to the method of measurement and population under service. However, there has been no locally available dry chemistry based RIs for the Nepalese population. Thus, the present study aimed to establish dry chemistry based RIs for sodium, potassium, urea, and creatinine specific to adult populations of Kaski districts, Nepal METHODS: This was a cross-sectional study conducted at the Manipal Teaching Hospital, Pokhara, Kaski, Nepal on 360 healthy adult participants aged 18-65 years. The test parameters under study were analyzed using a fully automated OCD Vitros 350 dry chemistry analyzer following the protocols provided by the reagent kit manufacturer. The RIs were estimated using reference limits at 2.5th and 97.5th percentiles. The normal distribution of the data was tested by Kolmogorov-Smirnov, and Shapiro-Wilk tests. The differences between males and females RIs were compared by the Mann-Whitney test while age-specific RIs for each sex was compared by One-Way-ANOVA and Dunnett's Multiple Comparisons Tests. All the data were managed and analyzed using MS Excel and SPSS version 20. RESULTS: The RIs of urea, creatinine, sodium, and potassium specific to the adult population of Kaski district, Nepal are as follows: urea: 4.20-13.70 mmol/L (males: 4.70-13.99; females: 4.20-13.23); creatinine: 44.20-106.10 µmol/L (males: 48.82-106.10; females: 35.40-83.78); sodium 135-146 mmol/L (males: 135-146; females: 135-146) and potassium 3.60-5.10 mmol/L (males: 3.54-5.0; females: 3.60-5.10). These RIs were found to be different from currently used RIs provided by the reagent manufacturer. RIs of all the test parameters were significantly influenced by the age of the study participants. However, only the RIs of urea, creatinine, and potassium were significantly influenced by sex. CONCLUSIONS: The present study has for the first time established dry chemistry based RIs for selected renal function test parameters specific to the adult population of Kaski district, Nepal. This result will aid the clinician in minimizing the errors in result interpretation and making a precise clinical decision.

Application of Sigma metrics in the quality control strategies of immunology and protein analytes.

BACKGROUND: Six Sigma (6σ) is an efficient laboratory management method. We aimed to analyze the performance of immunology and protein analytes in terms of Six Sigma. METHODS: Assays were evaluated for these 10 immunology and protein analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C-reactive protein (CRP), and Cystatin C (Cys C). The Sigma values were evaluated based on bias, four different allowable total error (TEa) and coefficient of variation (CV) at QC materials levels 1 and 2 in 2020. Sigma Method Decision Charts were established. Improvement measures of analytes with poor performance were recommended according to the quality goal index (QGI), and appropriate quality control rules were given according to the Sigma values. RESULTS: While using the TEaNCCL , 90% analytes had a world-class performance with σ>6, Cys C showed marginal performance with σ<4. While using minimum, desirable, and optimal biological variation of TEa, only three (IgG, IgM, and CRP), one (CRP), and one (CRP) analytes reached 6σ level, respectively. Based on σNCCL that is calculated from TEaNCCL , Sigma Method Decision Charts were constructed. For Cys C, five multi-rules (13s /22s /R4s /41s /6X , N = 6, R = 1, Batch length: 45) were adopted for QC management. The remaining analytes required only one QC rule (13s , N = 2, R = 1, Batch length: 1000). Cys C need to improve precision (QGI = 0.12). CONCLUSIONS: The laboratories should choose appropriate TEa goals and make judicious use of Sigma metrics as a quality improvement tool.

Establishment of reference intervals of clinical chemistry analytes for the adult population in Egypt.

BACKGROUND: This is the first Egyptian nationwide study for derivation of reference intervals (RIs) for 34 major chemistry analytes. It was conducted as a part of the global initiative by the IFCC Committee on Reference Intervals and Decision Limits (C-RIDL) for establishing country-specific RIs based on a harmonized protocol. METHODS: 691 apparently healthy volunteers aged ≥18 years were recruited from multiple regions in Egypt. Serum specimens were analyzed in two centers. The harmonization and standardization of test results were achieved by measuring value-assigned serum panel provided by C-RIDL. The RIs were calculated by parametric method. Sources of variation of reference values (RVs) were evaluated by multiple regression analysis. The need for partitioning by sex, age, and region was judged primarily by standard deviation ratio (SDR). RESULTS: Gender-specific RIs were required for six analytes including total bilirubin (TBil), aspartate and alanine aminotransferase (AST, ALT). Seven analytes required age-partitioning including glucose and low-density lipoprotein cholesterol (LDL-C). Regional differences were observed between northern and southern Egypt for direct bilirubin, glucose, and high-density-lipoprotein cholesterol (HDL-C) with all their RVs lower in southern Egypt. Compared with other collaborating countries, the features of Egyptian RVs were lower HDL-C and TBil and higher TG and C-reactive protein. In addition, BMI showed weak association with most of nutritional markers. These features were shared with two other Middle Eastern countries: Saudi Arabia and Turkey. CONCLUSION: The standardized RIs established by this study can be used as common Egyptian RI, except for a few analytes that showed regional differences. Despite high prevalence of obesity among Egyptians, their RVs of nutritional markers are less sensitive to increased BMI, compared to other collaborating countries.

The case for the repeatability intra-class correlation as a metric of precision for salivary bioscience data: Justification, assessment, application, and implications.

Best practice standards for measuring analyte levels in saliva recommend that all biospecimens be tested in replicate with mean concentrations used in statistical analyses. This approach prioritizes minimizing laboratory-based measurement error but, in the process, expends considerable resources. We explore the possibility that, due to advances in salivary assay precision, the contribution of laboratory-based measurement error in salivary analyte data is very small relative to more important and meaningful variability in analyte levels across biological replicates (i.e., between different specimens). To evaluate this possibility, we examine the utility of the repeatability intra-class correlation (rICC) as an additional index of salivary analyte data precision. Using randomly selected subsamples (Ns=200 and 60) of salivary analyte data collected as part of a larger epidemiologic study, we compute the rICCs for seven commonly assayed salivary measures in biobehavioral research - cortisol, alpha-amylase, c-reactive protein, interlekin-6, uric acid, secretory immunoglobulin A, and testosterone. We assess the sensitivity of rICC estimates to assay type and the unique distributions of the underlying analyte data. We also use simulations to examine the bias, precision, and coverage probability of rICC estimates calculated for small to large sample sizes. For each analyte, the rICCs revealed that less than 5% of variation in analyte levels was attributable to laboratory-based measurement error. rICC estimates were similar across all analytes despite differences in analyte levels, average intra-assay coefficients of variation, and in the distributional properties of the data. Guidelines for calculating rICC are provided to enable investigators and laboratory staff to apply this metric and more accurately quantify, and communicate, the magnitude of laboratory-based measurement error in their data. By helping investigators scale measurement error relative to more scientifically meaningful variability between biological replicates, the application of the rICC has the potential to influence research strategies and tactics such that resources (e.g., finances, effort, number/volume of biospecimens) are allocated more efficiently and effectively.

Evaluation of the analytical performance of endocrine analytes using sigma metrics.

BACKGROUND: (a) To evaluate the clinical performance of endocrine analytes using the sigma metrics (σ) model. (b) To redesign quality control strategies for performance improvement. METHODS: The sigma values of the analytes were initially evaluated based on the allowable total error (TEa), bias, and coefficient of variation (CV) at QC materials level 1 and 2 in March 2018. And then, the normalized QC performance decision charts, personalized QC rules, quality goal index (QGI) analysis, and root causes analysis (RCA) were performed based on the sigma values of the analytes. Finally, the sigma values were re-evaluated in September 2018 after a series of targeted corrective actions. RESULTS: Based on the initial sigma values, two analytes (FT3 and TSH) with σ > 6, only needed one QC rule (13S ) with N2 and R500 for QC management. On the other hand, seven analytes (FT4, TT4, CROT, E2, PRL, TESTO, and INS) with σ < 4 at one QC material level or both needed multiple rules (13S /22S /R4S /41S /10X ) with N6 and R10-500 depending on different sigma values for QC management. Subsequently, detailed and comprehensive RCA and timely corrective actions were performed on all the analytes base on the QGI analysis. Compared with the initial sigma values, the re-evaluated sigma metrics of all the analytes increased significantly. CONCLUSIONS: It was demonstrated that the combination of sigma metrics, QGI analysis, and RCA provided a useful evaluation system for the analytical performance of endocrine analytes.

Simultaneous determination of 30 neurologically and metabolically important molecules: A sensitive and selective way to measure tyrosine and tryptophan pathway metabolites and other biomarkers in human serum and cerebrospinal fluid.

Concurrent measurement of tyrosine, tryptophan and their metabolites, and other co-factors could help to diagnose and better understand a wide range of metabolic and neurological disorders. The two metabolic pathways are closely related to each other through co-factors, regulator molecules and enzymes. By using high performance liquid chromatography coupled to electrospray ionization triple quadrupole mass spectrometry, we present a robust, selective and comprehensive method to determine 30 molecules within 20 min using a Waters Atlantis dC18. The method was validated according to the guideline of European Medicines Agency on bioanalytical method validation. Analytical performance met all the EMA requirements and the assay covered the relevant clinical concentrations. Linear correlation coefficients were all >0.998. Intra-day and inter-day accuracy were between 80-119% and 81-117%, precision 1-19% respectively. The method was applied to measure TYR, TRP and their metabolites, and other neurologically important molecules in human serum and CSF samples. The assay can facilitate the diagnosis and is suitable for determination of reference values in clinical laboratories.

Personalized Reference Intervals in Laboratory Medicine: A New Model Based on Within-Subject Biological Variation.

BACKGROUND: The concept of personalized medicine has received widespread attention in the last decade. However, personalized medicine depends on correct diagnosis and monitoring of patients, for which personalized reference intervals for laboratory tests may be beneficial. In this study, we propose a simple model to generate personalized reference intervals based on historical, previously analyzed results, and data on analytical and within-subject biological variation. METHODS: A model using estimates of analytical and within-subject biological variation and previous test results was developed. We modeled the effect of adding an increasing number of measurement results on the estimation of the personal reference interval. We then used laboratory test results from 784 adult patients (>18 years) considered to be in a steady-state condition to calculate personalized reference intervals for 27 commonly requested clinical chemistry and hematology measurands. RESULTS: Increasing the number of measurements had little impact on the total variation around the true homeostatic set point and using ≥3 previous measurement results delivered robust personalized reference intervals. The personalized reference intervals of the study participants were different from one another and, as expected, located within the common reference interval. However, in general they made up only a small proportion of the population-based reference interval. CONCLUSIONS: Our study shows that, if using results from patients in steady state, only a few previous test results and reliable estimates of within-subject biological variation are required to calculate personalized reference intervals. This may be highly valuable for diagnosing patients as well as for follow-up and treatment.

New trimester-specific reference intervals for clinical biochemical tests in Taiwanese pregnant women-cohort of TMICS.

BACKGROUND: Because there are no published biochemical reference intervals (RI) for pregnant Taiwanese women, we used an established islandwide birth cohort, the Taiwan Maternal and Infant Cohort Study, to establish RIs for important biochemical parameters in women during their 3rd trimester in Taiwan. Additionally, we compared the differences in these biochemical parameters between early third trimester (weeks 28 to 31) and late third trimester (weeks 37 to 40) of pregnant women as well as the differences in them between the third trimester and after delivery. METHODS: Between 2012 and 2015, we recruited a total of 2,136 pregnant women from nine hospitals located in northern (n = 3), central (n = 3), southern (n = 2), and eastern Taiwan (n = 1) to receive regular prenatal health examinations during their third trimester (weeks 28 to 40). After exclusion, samples obtained from 993 eligible pregnant women were analyzed. RESULTS: There were increases in both lower and upper normal limits for blood neutrophil, thyroid profile (triiodothyronine (T3) and thyroxine (T4)), testosterone, estradiol, and progesterone and decreases for RBC, hemoglobin (Hb), alanine aminotransferase (ALT) and creatinine (Cr) during their third trimesters. Women in their late third trimester (n = 378) had higher median RBC, Hb, aspartate aminotransferase (AST), Cr, thyroid-stimulating hormone (TSH), testosterone, estradiol, and progesterone and lower median platelet and insulin, compared with those in their early third trimester (n = 490). Twenty-three of the women had both third trimester and post-pregnancy data. After delivery, the women had lower median AST, ALT, insulin, T3, T4, testosterone, estradiol, and progesterone and higher median Cr, free T4, FSH, and luteinizing hormone (LH), compared to their third trimesters. CONCLUSIONS: Gestation-related changes in important biochemical parameters should be considered when evaluating clinical laboratory values in pregnant women.

Sample preparation strategies for the determination of psychoactive substances in biological fluids.

This review focuses on the existing analytical procedures for the determination of new psychoactive substances (NPS) in biological fluids by chromatographic methods. Direct analysis of samples is scarcely employed and most proposed methodologies include a sample pre-treatment in order to remove matrix interferents and, in some cases, pre-concentrate extracts. Current extraction methods for NPS determination in plasma/serum, urine, and oral fluids have been widely discussed, such as liquid-liquid, solid-phase, and micro extraction approaches, highlighting the advantages and drawbacks of the proposed extraction methodologies. Regarding microextraction approaches, techniques like microextraction by packed sorbent, solid-phase microextraction, miniaturized solid phase extraction, and dispersive liquid-liquid extraction have been proposed for NPS determination in biological fluids with reliable analytical results.

Application and optimization of reference change values for Delta Checks in clinical laboratory.

BACKGROUND: Delta check is a patient-based QC tool for detecting errors by comparing current and previous test results of patient. Reference change value (RCV) is adopted in guidelines as method for delta check, but the performance is not verified. We applied RCV-based delta check method to patients' data and modified for application. MATERIALS AND METHODS: Reference change value were calculated using results of internal QC materials and biological variation data. Test results of 17 analytes in inpatients, outpatients, and health examination recipients were collected. The detection rates of currently used delta check method and those of RCV-based method were compared, and the methods were modified. RESULTS: Reference change value-based method had higher detection rates compared to conventional method. Applied modifications reduced detection rates. Removing the pairs of results within reference interval reduced detection rates (0.42% ~ 10.92%). When RCV was divided by time interval, the detection rates were similar to prior rates in outpatients (0.19% ~ 1.34%). Using RCV multiplied by twice the upper limit of reference value as cutoff reduced the detection rate (0.07% ~ 1.58%). CONCLUSIONS: Reference change value is a robust criterion for delta check and included in clinical laboratory practice guideline. However, RCV-based method generates high detection rates which increase workload. It needs modification for use in clinical laboratories.

A blood-based biomarker panel (NIS4) for non-invasive diagnosis of non-alcoholic steatohepatitis and liver fibrosis: a prospective derivation and global validation study.

BACKGROUND: Non-invasive tests that can identify patients with non-alcoholic steatohepatitis (NASH) at higher risk of disease progression are lacking. We report the development and validation of a blood-based diagnostic test to non-invasively rule in and rule out at-risk NASH (defined as non-alcoholic fatty liver disease [NAFLD] activity score [NAS] ≥4 and fibrosis stage ≥2). METHODS: In this prospective derivation and global validation study, blood samples, clinical data, and liver biopsy results from three independent cohorts with suspected NAFLD were used to develop and validate a non-invasive blood-based diagnostic test, called NIS4. Derivation was done in the discovery cohort, which comprised 239 prospectively recruited patients with biopsy-confirmed NASH (NAFLD NAS ≥3; fibrosis stage 0-3) from the international GOLDEN-505 phase 2b clinical trial. A complete matrix based on 23 variables selected for univariate association with the presence of at-risk NASH and avoiding high multi-collinearity was used to derive the model in a bootstrap-based process that minimised the Akaike information criterion. The overall diagnostic performance of NIS4 was externally validated in two independent cohorts: RESOLVE-IT diag and Angers. The RESOLVE-IT diag cohort comprised the first 475 patients screened for potential inclusion into the RESOLVE-IT phase 3 clinical trial. Angers was a retrospective cohort of 227 prospectively recruited patients with suspected NAFLD and clinical risk factors for NASH or fibrosis stage 2 or more according to abnormal elastography results or abnormal liver biochemistry. Both external validation cohorts were independently analysed and were combined into a pooled validation cohort (n=702) to assess clinical performance of NIS4 and other non-invasive tests. FINDINGS: The derived NIS4 algorithm comprised four independent NASH-associated biomarkers (miR-34a-5p, alpha-2 macroglobulin, YKL-40, and glycated haemoglobin; area under the receiver operating characteristics curve [AUROC] 0·80, 95% CI 0·73-0·85), and did not require adjustment for age, sex, body-mass index (BMI), or aminotransferase concentrations. Clinical cutoffs were established within the discovery cohort to optimise both rule out and rule in clinical performance while minimising indeterminate results. NIS4 was validated in the RESOLVE-IT diag cohort (AUROC 0·83, 95% CI 0·79-0·86) and the Angers cohort (0·76, 0·69-0·82). In the pooled validation cohort, patients with a NIS4 value less than 0·36 were classified as not having at-risk NASH (ruled out) with 81·5% (95% CI 76·9-85·3) sensitivity, 63·0% (57·8-68·0) specificity, and a negative predictive value of 77·9% (72·5-82·4), whereas those with a NIS4 value of more than 0·63 were classified as having at-risk NASH (ruled in) with 87·1% (83·1-90·3) specificity, 50·7% (45·3-56·1) sensitivity, and a positive predictive value of 79·2% (73·1-84·2). The diagnostic performance of NIS4 within the external validation cohorts was not influenced by age, sex, BMI, or aminotransferase concentrations. INTERPRETATION: NIS4 is a novel blood-based diagnostic that provides an effective way to non-invasively rule in or rule out at-risk NASH in patients with metabolic risk factors and suspected disease. Use of NIS4 in clinical trials or in the clinic has the potential to greatly reduce unnecessary liver biopsies in patients with lower risk of disease progression. FUNDING: Genfit.

Implementation of patient-based real-time quality control.

The quest to use patient results as quality control for routine clinical chemistry testing has long been driven by issues of the unavailability and cost of suitable quality control material and the matrix effects of synthetic material. Hematology laboratories were early adopters of average of normals techniques, primarily because of the difficulty in acquiring appropriate, stable quality control material, while in the chemistry laboratories, the perceived advantages and availability of synthetic material outweighed the disadvantages. However, the increasing volume of testing in clinical chemistry plus the capability of computer systems to deal with large and complex calculations has now made the use of patient-based quality control algorithms feasible. The desire to use patient-based quality control is also driven by increasing awareness that common quality control rules and frequency of analysis may fail to detect clinically significant assay biases. The non-commutability of quality control material has also become a problem as laboratories seek to harmonize results across regions and indeed globally. This review describes the history of patient-based quality control in clinical chemistry, summarizes the various approaches that can be implemented by laboratory professionals, and discusses how patient-based quality control can be integrated with traditional quality control techniques.

Evaluation of plasma ACTH stability using the Roche Elecsys immunoassay.

BACKGROUND: Adrenocorticotropic hormone (ACTH) has been reported to be labile in blood due to proteolytic degradation and stringent procedures are followed to prevent in vitro degradation after sample collection. OBJECTIVE: The purpose of this study was to examine the effect of time and temperature before and after separation of plasma from cells in the quantitation of plasma ACTH. METHODS: Our current protocol includes sample collection in a pre-chilled tube, transport on ice and immediate centrifugation at 4 °C. These reference conditions were compared against sample processing in tubes and centrifuge set at room-temperature; using delayed centrifugation at 4 °C. ACTH stability was evaluated at ambient and refrigerated temperatures after collection and plasma separation using the reference protocol. Plasma samples were analyzed using the Roche Elecsys ACTH immunoassay. RESULTS: Quantification of ACTH was not impacted by the use of non-chilled tubes and centrifuge and up to a 4 h delay in separation of plasma from cells. Average percent differences in plasma ACTH concentration from time 0 was <10% up to 12 h at ambient temperature. Refrigeration of plasma did not preserve ACTH stability at 12 h and longer storage resulted in significant ACTH degradation at both ambient and refrigerated temperatures. CONCLUSIONS: As supported by these data, previously recommended strict specimen collection and processing requirements are not necessary for measuring ACTH with the Roche Elecsys immunoassay.

Establishing reference intervals for major biochemical analytes for the Russian population: a research conducted as a part of the IFCC global study on reference values.

OBJECTIVES: Because reference intervals (RIs) for biochemistry analytes matched to the Russian population are not well defined, we joined the global study on reference values (RVs) coordinated by the IFCC Committee on Reference Intervals and Decision Limits (C-RIDL). METHODS: According to the C-RIDL harmonized protocol, 793 healthy volunteers were recruited in Saint-Petersburg, Moscow, and Yekaterinburg. Serum samples were tested for 34 biochemistry analytes. Sources of variation of RVs were explored using multiple regression analysis. The need for partitioning RVs by sex and age were judged using standard deviation ratio based on ANOVA. Latent abnormal values exclusion (LAVE) method was applied to reduce the influence of individuals with metabolic syndrome and/or inappropriate sampling conditions. RIs were computed by the parametric method. RESULTS: No appreciable between-city differences were observed. Partition of RVs by sex was required for 17 analytes. Age-related changes in RVs were observed in many analytes, especially in females. The trend was exaggerated in nutritional and inflammatory markers that were closely associated with body mass index (BMI), because BMI increases prominently with age. Therefore, for those analytes, volunteers with BMI > 28 kg/m2 were excluded in determining RIs for age-specific RIs. The LAVE method was effective in lowering the upper limits of the RIs for nutritional and inflammatory markers. CONCLUSION: RIs matched to the Russian population were established for 34 biochemical analytes using up-to-date methods in detailed consideration of sources of variation of RVs. The majority of Russian RIs are similar to those of Caucasian populations among the participating countries.

Establishment of reference intervals for immunoassay analytes of adult population in Saudi Arabia.

Background This is a second part of report on the IFCC global multicenter study conducted in Saudi Arabia to derive reference intervals (RIs) for 20 immunoassay analytes including five tumor makers, five reproductive, seven other hormones and three vitamins. Methods A total of 826 apparently healthy individuals aged ≥18 years were recruited in three clinical laboratories located in western, central and eastern Saudi Arabia using the protocol specified for the global study. All serum specimens were measured using Abbott, Architect analyzers. Multiple regression analysis (MRA) was performed to explore sources of variation of each analyte: age, body mass index (BMI), physical exercise and smoking. The magnitude of variation of reference values (RVs) attributable to sex, age and region was calculated by ANOVA as a standard deviation ratio (SDR). RIs were derived by the parametric (P) method. Results MRA revealed that region, smoking and exercise were not relevant sources of variation for any analyte. Based on SDR and actual between-sex differences in upper limits (ULs), we chose to partition RIs by sex for all analytes except for α-fetoprotein and parathyroid hormone (PTH). Age-specific RIs were required in females for ferritin, estradiol, progesterone, testosterone, follitropin, luteotropin and prolactin (PRL). With prominent BMI-related increase, RIs for insulin and C-peptide were derived after excluding individuals with BMI > 32 kg/m2. Individuals taking vitamin D supplements were excluded in deriving RIs for vitamin D and PTH. Conclusions RIs of major immunoassay analytes specific for Saudi Arabians were established in careful consideration of various biological sources of variation.

Preparation of In-House Quality Control Human Serum for Urea and its Use in Clinical Chemistry.

BACKGROUND: Quality control materials play a vital role in the laboratory internal and external quality assessment program. However, Ethiopia and other developing countries are challenged by the unavailability and high cost of commercial control material. Therefore, preparing in-house quality control human serum will be a cost-effective way to obtain QC material for use in poor settings in a country like Ethiopia. To prepare urea in-house control human serum and scientifically evaluate it with commercially available sera already in use in the clinical chemistry laboratory of the Ethiopian Public Health Institute. METHODS: The urea in-house quality control human serum was prepared as per ISO Guide 80, a guideline document protocol from 57 participants' normal serum specimens at the Ethiopian Public Health Institute clinical chemistry laboratory. The quality control material was analyzed on a fully automated chemistry analyzer (Cobas 6000). The initial 20 values were used for calculation of means, standard deviation (SD) and coefficient of variation (CV). Results were compared with those of commercially available lyophilized human sera. RESULTS: The average concentrations of urea were found to be near the middle of the physiological range of healthy subjects and the in-house serum could be a good substitute for the commercial serum of normal range. The prepared in-house quality control human serum is stable for about three months without any alterations in the concentration of urea. CONCLUSIONS: Well prepared in-house quality control human serum is a good substitute for commercially available control serum of normal range, especially in a developing country like Ethiopia.

Non-Parametric Combined Reference Regions and Prediction of Clinical Risk.

BACKGROUND: Many clinical decisions depend on estimating patient risk of clinical outcomes by interpreting test results relative to reference intervals, but standard application of reference intervals suffers from two major limitations that reduce the accuracy of clinical decisions: (1) each test result is assessed separately relative to a univariate reference interval, ignoring the rich pathophysiologic information in multivariate relationships, and (2) reference intervals are intended to reflect a population's biological characteristics and are not calibrated for outcome prediction. METHODS: We developed a combined reference region (CRR), derived CRRs for some pairs of complete blood count (CBC) indices (RBC, MCH, RDW, WBC, PLT), and assessed whether the CRR could enhance the univariate reference interval's prediction of a general clinical outcome, 5-year mortality risk (MR). RESULTS: The CRR significantly improved MR estimation for 21/21 patient subsets defined by current univariate reference intervals. The CRR identified individuals with >2-fold increase in MR in many cases and uniformly improved the accuracy for all five pairs of tests considered. Overall, the 95% CRR identified individuals with a >7× increase in 5-year MR. CONCLUSIONS: The CRR enhances the accuracy of the prediction of 5-year MR relative to current univariate reference intervals. The CRR generalizes to higher numbers of tests or biomarkers, as well as to clinical outcomes more specific than MR, and may provide a general way to use existing data to enhance the accuracy and precision of clinical decisions.

Clinical Application of Overlapping Confidence Intervals for Monitoring Changes in Serial Clinical Chemistry Test Results.

BACKGROUND: Interpretation of changes in serial laboratory results is necessary for both clinicians and laboratories; however, setting decision limits is not easy. Although the reference change value (RCV) has been widely used for auto-verification, it has limitations in clinical settings. We introduce the concept of overlapping confidence intervals (CIs) to determine whether the changes are statistically significant in clinical chemistry laboratory test results. METHODS: In total, 1,202,096 paired results for 33 analytes routinely tested in our clinical chemistry laboratory were analyzed. The distributions of delta% absolute values and cut-off values for certain percentiles were calculated. The CIs for each analyte were set based on biological variation, and data were analyzed at various confidence levels. Additionally, we analyzed the data using RCVs and compared their clinical utility. RESULTS: Most analytes had low indexes of individuality with large inter-individual variability. The 97.5th percentile cut-offs for each analyte were much larger than conventional RCVs. The percentages of results exceeding RCV95% and RCV99% corresponded to those with no overlap at the 83.4% and 93.2% confidence levels, respectively. CONCLUSIONS: The use of overlapping CIs in serial clinical chemistry test results can overcome the limitations of existing RCVs and replace them, especially for analytes with large intra-individual variation.