Establish Reference Intervals Research Articles

ISCEV standard pattern reversal VEP development: paediatric reference limits from 649 healthy subjects

PurposeTo establish the extent of agreement for ISCEV standard reference pattern reversal VEPs (prVEPs) acquired at three European centres, to determine any effect of sex, and to establish reference intervals from birth to adolescence.MethodsPrVEPs were recorded from healthy reference infants and children, aged 2 weeks to 16 years, from three centres using closely matched but non-identical protocols. Amplitudes and peak times were modelled with orthogonal quadratic and sigmoidal curves, respectively, and two-sided limits, 2.5th and 97.5th centiles, estimated using nonlinear quantile Bayesian regression. Data were compared by centre and by sex using median quantile confidence intervals. The ‘critical age’, i.e. age at which P100 peak time ceased to shorten, was calculated.ResultsData from the three centres were adequately comparable. Sex differences were not clinically meaningful. The pooled data showed rapid drops in P100 peak time which stabilised by 27 and by 34 weeks for large and small check widths, respectively. Post-critical-age reference limits were 87–115 ms and 96–131 ms for large and small check widths, respectively. Amplitudes varied markedly and reference limits for all ages were 5–57 μV and 3.5–56 μV for large and small check widths, respectively.ConclusionsPrVEP reference data could be combined despite some methodology differences within the tolerances of the ISCEV VEP Standard, supporting the clinical benefit of ISCEV Standards. Comparison with historical data is hampered by lack of minimum reporting guidelines. The reference data presented here could be validated or transformed for use elsewhere.

The consensus of Chinese experts on refined analysis of immune cell subsets in peripheral blood by multi-parameter flow cytometry

The detection of immune cell subsets plays a very important role in the clinical diagnosis and treatment of various benign and malignant diseases and health management. In order to better carry out in-depth research on different functional immune cell subsets, establish reference intervals for clonality related indicators, establish special reference intervals for immune aging, individualized dynamic monitoring and treatment recovery, and discover the clinical significance of immune cells other than lymphocytes, it is urgent to analyze the peripheral blood immune cell subsets in a refined way. Multiparameter flow cytometry is an important technical method to detect immune cell subsets and evaluate immune function. In order to standardize the refined detection methods and protocols of peripheral blood immune cell subsets by flow cytometry, and further promote its application in clinical diagnosis and treatment of diseases and health management, Laboratory Medicine Committee of Chinese Association of Integrative Medicine (LMC-CAIM) organized experts to formulate this expert consensus.

The effects of annual cycle, source population, and body condition on leukocyte profile and immune challenge in a basal reptile, the tuatara (Sphenodon punctatus).

Leukocyte profiles are broadly used to assess the health status of many species. Reference intervals, and an understanding of the factors that may influence these intervals, are necessary for adequate interpretation of leukograms. Using a data set that spans over three decades, we investigated variation in leukocyte profile in several populations of the evolutionarily unique reptile, the tuatara (Sphenodon punctatus). To do this, we first established reference intervals for each leukocyte type according to best practices. Next, we determined that source population and sampling date were the two most important predictors of leukocyte makeup. We found significant differences in the ratio of heterophils: lymphocytes (H:L) between populations, with tuatara on the more resource-stressed sampling island having a significantly higher ratio of H:L. Finally, we found that sampling location, sex, and life stage did not explain variation in the responses of tuatara to stimulation with Concanavalin A and lipopolysaccharide in both 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide and Griess assay experiments. Our results offer important insight into the function of leukocytes in reptiles.

Reference Intervals of Haematological Parameters for Apparently Healthy Adults in Northeast Ethiopia.

Clinical laboratory reference intervals play a vital role in evaluating overall well-being, tracking the progression of diseases, and detecting potential harmful effects and complications. Despite evidence revealing disparities, many African nations currently rely on reference intervals for blood analysis obtained mainly from Western populations. This practice increases the risk of misidentifying and misdiagnosing healthy individuals. The aim of this study was to establish common hematological parameters reference intervals for healthy adults in Northeast Ethiopia. This community-based cross-sectional study consisted of 328 individuals who were presumed to be in good health. To assess their blood-related characteristics, blood samples were collected and analyzed using the advanced Dirui BF-6500 analyzer, along with serological testing. In accordance with guidelines provided by the Clinical and Laboratory Standards Institute, the study employed a non-parametric approach to calculate the medians and 95% confidence intervals. To explore potential variations between males and females, a statistical test known as the Mann-Whitney U-test was used to compare the reference intervals. The established reference intervals were: white blood cells 3.5-11.3×109/L; red blood cells 4.0-6.1×1012/L; hemoglobin 11.2-17.5g/dL; hematocrit 35.4-52.0%; MCV 77.9-93.8fl; MCH 24.7-32.0pg; MCHC 306-349g/L; RDW-CV 12.1-13.8% and platelet 131-391×109/L. The reference values of monocytes, eosinophils, red blood cells, hemoglobin, hematocrit and RDW-CV in males were higher than females, while females had significantly higher platelet counts compared to males. The reference intervals discovered differed from the reference intervals now in use, those mentioned in earlier research in Ethiopia or other African nations, as well as those conducted on Western populations. In the adult demographic of Northeast Ethiopia, specific reference intervals for commonly observed hematological parameters were established, tailored to the local community. Consequently, these reference intervals hold the potential to enhance informed decision-making within this population, by providing valuable guidance when interpreting laboratory test outcomes.

Establishing hematologic and biochemical reference intervals for the endangered Lear's macaw (Anodorhynchus leari).

The Lear's macaw, Anodorhynchus leari (Psittaciformes, Psittacidae), is an endemic species of the Caatinga region in Brazil. With only a few free-living individuals that are constantly under threat from agricultural clearance, hunting, and trapping, A. leari is classified as "endangered" by the Red Book of Endangered Brazilian Fauna and by the International Union for Conservation of Nature. At the same time, the number of individuals in captivity has been growing due to the efforts of biologists and veterinarians in zoological parks to reproduce this species. Thus, having species-specific reference intervals for hematologic and biochemical measurands is essential to assess the health status of these captive and wild birds and guarantee their reproductive success. This study aimed to determine reference intervals for hematologic and biochemical measurands for A. leari kept in captivity in São Paulo Zoo, Brazil. Blood samples from 32 individuals were selected based on inclusion and exclusion criteria. Only samples from adults and healthy individuals were included in this study. All statistical analyses were performed according to the American Society for Veterinary Clinical Pathology guidelines and available literature. Reference values and reference intervals were determined for the captive population of Anodorhynchus leari. This is the first study to establish reference intervals for A. leari, which can aid the veterinarian in the interpretation of laboratory test results, helping in the diagnosis, treatment, and monitoring of diseases of this endangered species of Brazilian fauna.

Exploring Utilization and Establishing Reference Intervals for the Apolipoprotein B Test in the Korean Population.

We investigated the reference intervals for Apolipoprotein B (ApoB), a valuable biomarker for cardiovascular diseases, in Korean adults who had undergone health check-ups and showed normal lipid levels under traditional lipid tests, including total cholesterol, triglyceride, and high-density lipoprotein cholesterol, along with ApoB. We compared the findings with different cutoffs for ApoB from international clinical guidelines. Among a total of 264,105 traditional lipid test sets, only 464 (0.2%) included ApoB tests, indicating underutilization of this test in health check-up clinics in Korea. From these 464 samples, 334 ApoB results (164 men and 170 women) with normal traditional lipid test results were used to establish reference intervals. Using the parametric method (mean ± 2 SD), the reference intervals ranged from 46 to 134 mg/dL for men and 49 to 129 mg/dL for women. Employing the non-parametric method (central 95th percentile value), the reference intervals ranged from 50 to 131 mg/dL for men and 51 to 127 mg/dL for women. The prevalence of high ApoB did not significantly differ by sex when considering the established reference intervals for each sex and the cutoffs recommended by international clinical guidelines. This study enhances knowledge on ApoB reference intervals in the Korean population, and it will in aid test result interpretation for clinicians and laboratories.

Effect of age and sex on hematological and biochemical parameters in Chinese rhesus monkeys (Macaca mulatta) anesthetized with ketamine.

Rhesus monkeys are increasingly used in biomedical research, which makes their hematological and biochemical parameters increasingly important in preclinical research. Since age and sex can influence blood parameters, establishing reference intervals for such parameters based on age and sex becomes along with identifying the effect of age and sex on those parameters. A total of 1385 healthy Chinese rhesus monkeys (548 males and 837 females) anesthetized with ketamine were selected and segregated by age (six groups) and sex. A total of 21 hematological and 26 biochemical parameters were measured, and the effects of age and sex were analyzed. We established baseline indices for hematological and biochemical parameters based on age and sex, separately, and observed significant impacts of age, sex, and age-sex interactions on blood parameters. Among different age groups, significant differences were found in WBC, NEUT%, LYM%, EO%, LYM#, EO#, MCV, RDW-CV, PLT, MPV, PDW, PCT, TP, Alb, GLB, A/G, ALT, AST, ALP, TBIL, GGT, BUN, Cre, GLU, CK, TRIG, LDL, HCY, IL-6 FOL, Vit B12, VIT D-T, PTH, and AMH. Additionally, significant differences were observed in RBC, HGB, HCT, MPV, Alb, BUN, Cre, GLU, CHOL, TRIG, HDL, LDL, HCY, and VIT D-T between the two sexes. An age-sex interaction exerted a significant effect on WBC, NEUT#, MCV, MCHC, PDW, GLB, ALP, Cre, CHOL, TRIG, HDL, LDL, HCY, IL-6, Vit B12, VIT D-T. However, neither age, sex, and age-sex interactions exerted significant effects on MO%, MOMO#, MCH, RDW-SD, CRP, and CT. Our study investigated the blood parameters of rhesus monkeys to provide a reference basis for rhesus monkey-related scientific experimental research.

SAT015 Improving The Quality Of Disease Biomarker Measurements In Patient Care And Research-CDC's Clinical Standardization Programs

Abstract Disclosure: O. Sugahara: None. T. Buchannan: None. U. Danilenko: None. A. Ribera: None. L. Collins: None. N. Vasquez: None. K. Dahya: None. F. Pokuah: None. H. Zhou: None. L. Zhang: None. A.N. Lyle: None. H.W. Vesper: None. High-quality disease biomarker tests are critical for the correct diagnosis and treatment of patients, accurate interpretation of research data, and effective use of research findings in health care. CDC’s Clinical Standardization Programs (CDC CSP) assist researchers and laboratories with assessing and improving the analytical performance of biomarker tests performed in patient care, public health, and research. CDC CSP develops and applies reference methods, materials, and protocols to consistently calibrate biomarker tests across instruments and laboratories and to assess and improve other analytical performance characteristics, such as analytical specificity. CDC CSP works with test manufacturers and laboratories operating in-house developed tests to correctly calibrate tests. Additional programs help clinical and research laboratories monitor the accuracy and reliability of tests over time. Furthermore, CDC CSP collaborates with researchers on the development of reference intervals and on using new and emerging biomarkers in patient care and public health. CDC CSP provides comprehensive programs for traditional blood lipids, testosterone, estradiol, vitamin D, and free thyroxine. Programs for free testosterone, parathyroid hormones, apolipoproteins, and angiotensin peptides will be available soon. CDC CSP collaborated with members of the Partnership for the Accurate Testing of Hormones (PATH) and established reference intervals for testosterone in men. CDC CSP is currently working to develop reference intervals for testosterone in women and for estradiol in postmenopausal women. A CDC CSP interlaboratory study on free thyroxine measurements showed high variability in calibration accuracy among free thyroxine assays. CDC CSP is using its reference methods and materials to minimize this variability. It is anticipated that assay recalibration may change measurement values for some tests by up to 50%. CDC CSP is working with PATH and the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC) to educate stakeholders about the upcoming change in free thyroxine measurements. Presentation: Saturday, June 17, 2023

Establishment of reference intervals for free light chains and immunoglobulins in Saudi population.

Testing of serum-free light chains kappa (κ) and lambda (λ), along with ratio (FLCR) is essential for the diagnosis and management of monoclonal gammopathies. Accurate clinical diagnosis depends upon appropriate local population reference intervals (RIs). This study examined the Saudi population for serum-free light chains and other immunoglobulins to establish RIs and to explore variations in the test results by using the International Federation for Clinical Chemistry and Laboratory Medicine's global protocol for harmonized implementation of RI study. A total of 180 healthy Saudi adults were recruited. All serum samples were assayed using the Freelite reagents from the Binding Site. The variation in reference values attributable to sex, age, BMI, and region was calculated by ANOVA as a standard deviation ratio (SDR). The RIs for theFLCR were derived by the parametric method and validated by using samples from patients with hypo- and hypergammaglobulinemia. The new RIs for free κ and FLCR were shifted to a higher side from the manufacturer-adapted RIs. Based on the SDR cutoff value (>0.4), between-sex partition RIs were not required for all analytes except IgM. Validation using patients with hypo- or hypergammaglobulinemia and without multiple myeloma, was all within the new RI. BMI, smoking, and exercise were not relevant sources of variation for any analyte. Locally derived RIs for free light chains and immunoglobulins analytes specific for Saudis were established after careful consideration of various factors. These RIs were more reliable than those provided as guidance by the manufacturer, or from other countries, for appropriate classification and prediction of disease progression for Saudi patients.

Establishment of reference intervals for ultrasonographically derived caudal vena cava parameters from 110 healthy, lightly sedated cats

Objectives The present study aimed to establish reference intervals (RIs) for ultrasonographically derived caudal vena cava (CVC) parameters in healthy cats. Methods A prospective, observational, experimental single-centre study involving 110 cats was conducted. Two trained operators scanned all cats in a randomised order. Two sets of cine loops per cat were conducted by each operator from the subxiphoid view with cats in lateral recumbency. Two blinded raters measured the narrowest inspiratory (CVCinsp) and widest expiratory (CVCexp) diameter over two separate respiratory cycles for each operator (four measurements in total for each rater) at the point the CVC crosses the diaphragm. The CVC collapsibility index (CVCCI) was calculated. Reference intervals and limits were calculated for CVC parameters with outliers removed to include the central 95% of CVC values and the non-parametric 90% confidence intervals (90% CIs). The CVCCI interval included the central 95th (2.5th and 97.5th) centiles bounded by the 90% CI. Intra- and inter-rater and intra- and inter-operator reliabilities were calculated for each parameter using a two-way random-effects model. A Bland–Altman plot was used to visualise the CVCCI between the two raters. Results In total, 880 measurements were included for each diameter and 440 CVCCI values were calculated. The CVC diameters were normally distributed, whereas the CVCCI was not. The calculated RIs were 1.4–6.1 mm for CVCinsp and 2.2–6.7 mm for CVCexp. The median (range) CVCCI was 23% (11–36%) with 2.5th and 97.5th centiles of 2% and 67%, respectively. There was a moderate to good intra-rater and intra-operator reliability for CVC diameter. The intra-rater reliability for the CVCCI was moderate. The inter-rater and inter-operator reliabilities for the CVCCI were poor. Conclusions and relevance The CVC RIs determined from the subxiphoid site in cats appear to be wider than those reported in dogs, and rater and operator variability is present. Whether precise measurements of CVC parameters are suitable to guide clinical decisions remains to be determined.

P‐IS‐3 | Establishing Reference Intervals for Clinical Laboratory Tests: The Role of Blood Centers

P‐IS‐3 | Establishing Reference Intervals for Clinical Laboratory Tests: The Role of Blood Centers

PB0793 Establishing Reference Intervals of Coagulation Biomarkers in Human Immunodeficiency Virus Infection

PB0793 Establishing Reference Intervals of Coagulation Biomarkers in Human Immunodeficiency Virus Infection

A-053 Poor Clinical Utility of Free and Bioavailable Testosterone is Underscored by Overutilization

Abstract Background Symptomatic androgen deficiency has an estimate prevalence of 2%–12% in males over 40 years of age and is associated with several comorbidities such as hypertension, obesity, dyslipidemia, metabolic syndrome, diabetes, and COPD. Diagnosis relies heavily on the measurement of testosterone concentrations. While total testosterone (tT) is often sufficient to diagnose deficiency, free testosterone (fT) or bioavailable testosterone (baT; non-sex hormone binding globulin (SHBG)-bound) may be helpful in assessing patients with suspected hormone binding abnormalities. Consensus guidelines recommend relying on tT measurement and only using fT or baT testing when indicated. Despite these recommendations, requests for measured fT and baT account for a large percentage of testosterone orders in our institution. Here we examined the utilization of testosterone orders, assessed the clinical utility of measured fT and baT relative to tT alone, and devised a reflex algorithm to optimize fT and baT utilization. Methods Testosterone data was collected over a 12 month period, September 2020–August 2021. Only data from adult males with established reference intervals was included in the final cohort; ≥20 and 20–69 years of age for fT and baT, respectively. Logistic regression was used to determine the probability of low fT and baT based on tT concentration. An algorithm based on initial tT concentration was established to reflex to fT/baT when most likely to provide additional benefit. Results Of 9754 testosterone tests performed on male patients during the study period, 4514 (46%) included a measured fT and/or baT. After excluding data without established reference intervals, the final cohort consisted of 1851 fT and 2514 baT measurements. Overall concordance with tT was 70.0% and 74.8% for fT and baT, respectively. However, only 0.2% (3/1479) were found to have low fT and 8.4% (166/1988) low baT when tT was greater than the reference interval lower limit (i.e., normal or high). The probability of a patient having low fT or baT with normal tT peaked at 0.01 and 0.21, respectively, and was greatest at the tT reference interval lower limit (240 ng/dL). An algorithm was devised in which only samples with tT concentrations of 240–500 ng/dL would reflex to baT testing. In this cohort, 90% of discordant (low baT with normal tT) samples would have triggered the baT reflex while reducing the overall utilization of baT by 55%. Applying the same reflex algorithm would have also reflexed 100% of discordant fT samples. Conclusions Despite being performed with nearly half of all testosterone orders, baT, and especially fT, provided little additional clinical utility to tT alone. By determining the probability of a low baT or fT when Tt is above the reference interval lower limit, a reflex algorithm can be devised to help optimize baT and fT utilization. While this algorithmic approach has been proposed by others, the reflex values in our cohort differ significantly from previously published values suggesting that laboratories should determine limits based on their individual testosterone assays. The logistic regression method utilized here provides one such example laboratories can follow for undertaking such an analysis.

A-048 Accurate Measurement of Free Testosterone in Serum using Equilibrium Dialysis Coupled with ID-UHPLC-MS/MS

Abstract Background Assessment of free testosterone (FT) has been recommended in recent clinical practice guidelines as a biomarker for diagnosis and management of testosterone related disorders such as male testosterone deficiency due to hypogonadism and female androgen excess due to polycystic ovary syndrome (PCOS). FT is currently measured using direct commercial immunoassays, assays using equilibrium dialysis (ED) or other separation techniques. Though the ED approach is recognized as the recommended approach, it is laborious and technically challenging for routine clinical use. The CDC Clinical Standardization Program is developing a high throughput automated method using the ED procedure coupled with isotope dilution ultra-high-performance liquid chromatography tandem mass spectrometry (ID-UHPLC-MS/MS) that is suitable for routine patient care and large-scale epidemiologic studies. Methods Serum samples are dialyzed in a custom-designed multi-well plate against a protein-free HEPES buffer (pH 7.4) at 37 °C until equilibrium. After isolating endogenous FT from protein-bound testosterone by ED, isotope-labeled internal standard (13C3-testosterone) was added to the dialysate for quantification. Certified pure primary reference material (National Measurement Institute-M914) was used to prepare calibrators, enabling traceability and ensuring measurement trueness. FT was further isolated from the dialysate matrix using supported liquid extraction and a chromatographic separation from interfering compounds and quantitation by tandem MS. Results The measurement ranges covered 0.2–1000 ng/dL for testosterone, with the bias within ±5% and precision less than 10% CV. A total of 45 samples with a wide range of total testosterone (TT, 21–912 ng/dL) and SHBG (11–129 nmol/L) were analyzed and showed the suitability of this assay to measure the serum free testosterone levels in normal, hypogonadal males as well as in the majority of normal females and females with androgen excess. The application of 48-well format plate and automated liquid handler system significantly improves the throughput of sample preparation. In addition, comparison of the TT results measured with the presented TT method with calculated TT concentrations, suggest that on average calculation overestimates TT concentration by 34%. Conclusion The described high throughput method for FT allows for sufficiently accurate and precise measurement for routine applications including large epidemiologic studies to help with establishing reference intervals.

B-165 Establishment of a Labile Bound Copper and Labile Bound Copper Ratio Reference Interval in a Healthy Adult Population

Abstract Background While clinical tests are available to aid in the diagnosis of Wilson’s Disease (WD), some such as total copper determination lack sensitivity and specificity, while others such as liver tissue copper assessment are costly and invasive. WD affects copper metabolism and is known to cause low serum copper concentrations. In addition, an elevated percentage of total serum copper exists as non-protein bound or labile bound copper (LBC). Previous studies have shown that measuring the ratio of labile bound copper to total serum copper may aid in the diagnosis of WD. To support the utility of a laboratory developed test using serum LBC ratios to aid in the diagnosis of WD, serum samples from healthy donors were used to establish a reference range for LBC and the ratio of LBC to total copper in adults. Methods Serum samples were collected in trace metal free tubes from 214 donors (n = 110 male, n = 104 female) between the ages of 19–80 years old, selected against pre-determined exclusion criteria including acute hepatitis, chronic hepatitis, renal failure, biliary cirrhosis, celiac disease, and leukemia. Prior to analysis, samples went through a centrifugation-based preparation process to isolate the LBC fraction using molecular weight size exclusion filters and EDTA chelation. Samples were then analyzed via ICP-MS in kinetic energy discrimination (KED) mode to directly measure the LBC and total copper concentrations. LBC ratio values were determined from the LBC/total copper concentrations. Results Preliminary reference ranges were developed for both LBC and the LBC ratio based on the 2.5th and 97.5th percentiles with 95% confidence intervals. Established sex specific reference intervals for total copper were deemed acceptable per CLSI guidelines in document EP28-A3c, with ≤10% of results from this sample set falling outside of the established intervals. No statistically significant relationship was found in the mid-95th percentile between LBC concentration or ratio with age or sex. The reference intervals for LBC concentration were calculated as 13.41–104.66 and 11.69–106.50 ng/mL for females and males respectively. The reference intervals for LBC ratio were calculated as 1.04–8.12 and 1.17–10.48 for females and males respectively. Conclusions A reference range of LBC and LBC ratio has now been determined for the healthy adult population. Further testing can now be done on disease state populations to determine the sensitivity and specificity of LBC ratio in the detection, monitoring, or diagnosis of WD.

A-335 Pediatric Cortisol Reference Intervals (RI): Impact of Age-Specific RI on the Rate of Abnormal “Low” Cortisol Results

Abstract Background Serum cortisol, measured by automated immunoassay, is a rapid test used for the initial assessment of adrenal dysfunction. Physiologic serum cortisol concentrations are influenced by many factors including diurnal variation, stress, and patient age. In the absence of established pediatric reference intervals (RIs), our laboratory has utilized adult RIs to flag pediatric cortisol results as abnormally high or low in the electronic health record. However, pediatric cortisol RIs recently defined by the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) are considerably lower than the adult RIs currently in use. This could cause pediatric cortisol results to be incorrectly flagged as “low” when they are within the age-specific RI and lead to unnecessary follow-up testing. The aim of this study was to determine the impact of implementing CALIPER pediatric cortisol RIs on the rate of “low” result flagging for pediatric patients. Methods AM cortisol results for patients age <18 years reported between 10/2012- 09/2022 were obtained from the Mayo Clinic data repository. These results contained values measured by both Beckman Access (n = 3 818) and Roche Cobas (n = 625) immunoassays. Results classified as abnormal “low” by current adult RIs (Beckman: 7–25 mcg/dL, Roche: 4.8–20 mcg/dL) were compared to results which would flag low according to the appropriate method- and age-specific RIs defined by CALIPER (Beckman: 1-<13 yrs 2.19–12.8 mcg/dL, 13-<16 yrs 3.03–17.1 mcg/dL, 16-<19 yrs 3.78–19.4 mcg/dL; Roche: <1 yr 0.58–17.2 mcg/dL, 1-<12 yrs 2.39–14.9 mcg/dL, 12-<19 yrs 3.63–17.4 mcg/dL). The proportion of results flagging low by adult versus pediatric RI were compared by chi-squared test, with P < 0.05 considered significant. Results For Beckman, the percentages of pediatric patient cortisol results flagging low using the adult RI versus (vs) the pediatric age-specific RI were: 35% vs 7% for 1–12 years of age (P < 0.001, n = 1866), 22% vs 5% for 13–15 years (P < 0.001, n = 966), and 16% vs 5% for 16–18 years (P < 0.001, n = 986). For Roche, the same analysis yielded: 52% vs 9% for 0–1 years (P = 0.001, n = 23), 14% vs 8% for 1–11 years (P = 0.03, n = 238), and 9% vs 6% for 12–18 years (P = 0.16, n = 364). Conclusion The implementation of pediatric RIs for serum cortisol will have a significant impact on the number of “low” result flags for patient results on both Beckman and Roche platforms. Statistically significant decreases in the percentage of low result flags were observed on both platforms for all age ranges except for the age bracket of 12–18 years by Roche. Furthermore, pediatric RIs appeared to produce more consistent rates of abnormal results across methods and age ranges, with all groups flagging low at a rate of 5–9%, whereas the same age ranges with adult RIs flagged at rates ranging from 9–52%. Lowering the RIs for pediatric cortisol to more appropriate ranges as identified by CALIPER will decrease the number of “low” flag results. This has the potential to decrease unnecessary follow-up, including repeat testing and further diagnostic procedures such as ACTH stimulation testing. Implementation of pediatric RIs has potential benefits regarding stress and cost for patients and parents, alarm fatigue for physicians, and utilization of laboratory resources.

B-380 Drift of Freelite on Optilite

Abstract Background Serum free light chains (sFLC) which include kappa (KFLC), lambda (LFLC) and their ratio (K/L) are used for diagnosis and monitoring of plasma cell dyscrasias (PCDs). In 2002, the Binding Site Freelite assay established reference intervals (RIs) for KFLC, LFLC and K/L. Since that time, renal RIs have been proposed and drift for the Freelite assays has been reported. The objective of this study was to evaluate healthy and renal insufficiency population RIs and examine retrospective data to ascertain the reported assay drift. Methods 260 serum samples were measured using the Freelite assay on the Optilite. Creatinine was measured on the Abbott Alinity and eGFR was calculated. Binding Site’s RIs for KFLC, LFLC and K/L were compared to samples with ≥60 eGFR and ≤59 eGFR samples were compared to the hypergammaglobulinemia population from a previous report in 2002. IFE was used to exclude samples with detectable monoclonal proteins. Retrospective data from University Health Network (UHN) was examined. Results The comparison results for the samples are in the tables below: The data suggest that Freelite has drifted up approximately 300% for KFLC, 150% for LFLC and 200% for K/L. Using 220 000 data points from UHN and applying these recalibration factors, the normal KFLC vs LFLC bivariate distribution appears centered to the well-known ‘0.26–1.65’ K/L 100% diagnostic range. Conclusions Drift of Freelite is confirmed with this study and recalibration factors for Freelite are proposed which are lower KFLC values by 300% and lower LFLC values by 150% for K/L to be centered to 0.26–1.65. Institutions should review their data with Freelite on Optilite and any clinical data to confirm this finding. Drift of this magnitude has the potential to cause false positives for kappa and false negatives for lambda in the diagnosis and monitoring of PCDs.

B-123 Utilization of Five Data Mining Algorithms Combined with Simplified Preprocessing to Establish Reference Intervals of Thyroid Related Hormones for Nonelderly Adults

Abstract Background Despite the extensive research on data mining algorithms, there is still a lack of a standard protocol to evaluate the performance of the existing algorithms. Therefore, the study aims to provide a novel procedure that combines data mining algorithms and simplified preprocessing to establish reference intervals (RIs), with the performance of five algorithms assessed objectively as well. Methods The Test data set and the Reference data set are the two data sets derived from the population undergoing a physical examination. After the thyroid-related hormone including thyroid stimulating hormone (TSH), free triiodo-thyronine (FT3), total triiodo-thyronine (TT3), free thyroxine(FT4), and total thyroxine (TT4) were measured by an ADVIA Centaur XP chemiluminescence immunoassay analyzer, five data algorithms were used to calculated RIs. Hoffmann, Bhattacharya, Expectation Maximum (EM), kosmic, and refineR algorithms combined with two-step data preprocessing respectively were implemented in the Test data set to establish RIs for thyroid-related hormones. The first step is to conduct a random sampling strategy to balance the ratio of sex and age, and the second step is to identify the outliers of variables in each subgroup by the Tukey method. Algorithm-calculated RIs were compared with the standard RIs calculated by transformed parametric method from the Reference data set in which reference individuals were selected following strict inclusion and exclusion criteria. RIs partition were comprehensively determined by the multiple linear regression and variance component analysis. Objective assessment of the methods is implemented by the bias ratio (BR) matrix, of which the BR threshold was set to 0.375. Results The levels of the all five thyroid-related hormones are significantly different in sex, with the male having lower TSH and higher FT3, FT4, TT3, and TT4 compared to the female. Further analysis indicates the establishment of sex-specific RIs for FT3 and FT4. Standard RIs derived from the Reference data set by transformed parametric method are 0.801–4.221 μIU/L for TSH, 2.58–3.82 pg/mL for FT3, 0.98–1.53 ng/dL for FT4, 0.80–1.38 ng/mL for TT3, 5.46–10.05 g/dL for TT4, respectively. There is a high consistency between TSH RIs established by the EM algorithm and the standard TSH RIs (BR = 0.063), although EM algorithms seems to perform poor on other hormones with the BR higher than 0.375. RIs calculated by Hoffmann, Bhattacharya, and refineR methods for free and total triiodo-thyronine, free and total thyroxine respectively are close and matched the standard RIs. Conclusion An effective approach for objectively evaluating the performance of the algorithm based on the BR matrix is established. EM algorithm combined with simplified preprocessing can handle data with significant skewness, but its performance is limited in other scenarios. The other four algorithms perform well for data with Gaussian or near-Gaussian distribution. Using the appropriate algorithm based on the data distribution characteristics is recommended.

Reporting and Establishment of Reference Intervals for Antiphospholipid Antibody Immunoassays: A Survey of Participants in the College of American Pathologists Proficiency Testing Program.

Misdiagnosis of antiphospholipid syndrome can occur owing to the wide diversity of antiphospholipid (aPL) assays and a lack of international calibrators and harmonized reference intervals. To assess laboratory practices regarding reporting and establishing reference intervals for immunoglobulin (Ig) G/IgM anti-cardiolipin (aCL) and anti-beta-2 glycoprotein I (anti-β2GPI) assays. Supplemental questions related to reporting and establishing reference ranges for aPL assays were sent as part of the Antiphospholipid Antibody (ACL)-B 2019 College of American Pathologists (CAP) proficiency testing survey. The response rate and methods assessment details were determined, as well as qualitative and quantitative results for 3 test samples. The number of participants reporting results for IgG aCL (n = 489), IgM aCL (n = 476), IgG anti-β2GPI (n = 354), and IgM anti-β2GPI (n = 331) varied by antibody type. The enzyme-linked immunosorbent assay (ELISA) (up to 58.6%, 260 of 444) was the most used method; others included multiplex (from 18.9% to 23.9%), fluorescence enzyme immunoassay (14.4%-17.6%), and chemiluminescence immunoassay (6.5%-9.0%). More respondents reported quantitative than qualitative results, and manufacturer cutoff ranges were used by 92.9% and 94.2% of respondents for aCL and anti-β2GPI, respectively. Despite variation in the use of semiquantitative ranges, qualitative negative/positive reporting of the test samples achieved almost 100% consensus. Qualitative consensus was met in contrast to the wide range of quantitative results obtained for each analyte across different kits. ELISA remains the most used method for detecting aPL antibodies, with most laboratories reporting quantitative results based on manufacturers' suggested reference ranges. The categorization of quantitative results as equivocal, weak positive, or positive for responders using kits from the same manufacturer was variable.

Determination of Coagulation Parameters by Whole Blood Dynamic Viscoelastic Coagulometry in Strigiformes.

No reference values are available in Strigiformes to evaluate blood coagulation using dynamic viscoelastic coagulometry (DVC) with the Sonoclot (Sienco, Boulder, CO, USA) analyzer. The objectives of this study were 1) to assess the feasibility of DVC in Strigiformes, 2) to calculate the index of individuality of each coagulation parameter, and 3) to assess interspecies variability and establish reference intervals, if relevant, based on the index of individuality. Fresh whole blood samples were obtained from healthy Strigiformes, including 13 barred owls (Strix varia), 10 great horned owls (Bubo virginianus), 6 snowy owls (Bubo scandiacus), and 7 eastern screech owls (Megascops asio), and analyzed with DVC with glass bead (gb) and kaolin clay (k) coagulation activators. Activated clotting time (ACT), clot rate (CR), and platelet function were determined immediately after collection using fresh native whole blood. Intraindividual variability was assessed with a second fresh native whole blood sample from 5 barred owls. Interindividual variability was assessed using a Kruskall-Wallis test. For the parameters gbACT (n = 35), gbCR (n = 34), and kACT (n = 27), no significant differences were detected between species (all P ≥ 0.05). Based on low index of individuality, global Strigiformes reference intervals were determined for gbACT (32.3-852.5 seconds; n = 35), gbCR (0-20.1 units/min; n = 29), and kACT (0-1570.3 seconds; n = 27). In conclusion, DVC can be used in Strigiformes and the gb coagulation activator would be more appropriate when basal individual values are not available in a tested individual.