Determine Reference Intervals Research Articles

Reference intervals for free testosterone in adult men measured using a standardized equilibrium dialysis procedure.

Free testosterone (FT) determination may be helpful in evaluating men suspected of testosterone deficiency especially in conditions with altered binding-protein concentrations. However, methods for measuring FT by equilibrium dialysis and reference intervals vary among laboratories. To determine reference intervals for FT in healthy, nonobese men by age groups as well as in healthy young men, 19-39 years, using a standardized equilibrium dialysis procedure METHODS: We measured FT in 145 healthy, nonobese men, 19 years or older, using a standardized equilibrium dialysis method performed for 16-h at 37°C using undiluted serum and dialysis buffer that mimicked the ionic composition of human plasma. FT in dialysate was measured using a CDC-certified liquid chromatography tandem mass spectrometry assay. In healthy nonobese men, the 2.5th, 10th, 50th, 90th, and 97.5th percentile values for FT were 66, 91, 141, 240, and 309 pg/ml, respectively; corresponding values for men, 19-39 years, were 120, 128, 190, 274, and 368 pg/ml, respectively. FT levels by age groups exhibit the expected age-related decline. FT levels were negatively associated with body mass index, age, and sex hormone-binding globulin (SHBG) levels. Percent FT was lower in middle-aged and older men than young men adjusting for SHBG level. Further studies are needed to determine how these reference intervals apply to the diagnosis of androgen deficiency in clinical populations and in men of different races and ethnicities in different geographic regions. Reference intervals for free FT levels (normative range 66-309 pg/ml [229-1072 pmol/L] in all men and 120-368 pg/ml [415-1274 pmol/L] in men, 19-39 years), measured using a standardized equilibrium dialysis method in healthy nonobese men, provide a rational basis for categorizing FT levels. These intervals require further validation in other populations, in relation to outcomes, and in randomized trials.

Evaluation of a novel moving threshold gating strategy for assessment of reticulated platelets in dogs using the ADVIA 2120 analyzer.

A novel method using a moving threshold (r-PLTmt) to determine canine r-PLTs (reticulated platelets) has been introduced for ADVIA 2120 software v6.11.7. We aimed to evaluate absolute (ar-PLTmt) and percent (%r-PLTmt) prior to and after visual inspection of scattergrams (ar-PLTmtv, %rPLTmtv) compared with flow cytometry (flow) and to determine reference intervals (RIs) in 120 dogs. For method comparison, 42 blood samples of healthy and thrombocytopenic dogs were included. Calculation of Spearman's rho, Bland-Altman, and Passing-Bablok analysis was performed. Coefficients of variation (CVs) were determined for three concentration levels. Moderate correlations between %r-PLTmt and %r-PLTmtv (rs 0.75-0.76) were seen compared with flow cytometry. The CV for medium %r-PLTs counts assessed with flow cytometry was 12.9%. Comparable CVs were obtained for ar-PLTmt (14.4%) and %r-PLTmt (15.7%), and ar-PLTmtv and %r-PLTmtv (10.9% and 12.9%, respectively). At low and high concentration levels, CVs for % and absolute r-PLTmt/rPLTmtv ranged between 23%-30% and 15%-20%. In patients with microcytic hypochromic erythrocytes, CVs for ar-PLTmt and %r-PLTmt were 36%-66%. Visual inspection of scattergrams resulted in a marked decrease in CV ranging between 15% and 20%. A proportional bias of 10.8% between %r-PLTmt and flow cytometry became lower (9.7%) after visual validation of scattergrams. Passing-Bablok analysis showed proportional and constant error. RIs for r-PLTmt and r-PLTmtv were 0.2%-3.8% and 0.6-10.2 × 109 /L and 0.3%-4.5% and 1.1-10.3 × 109 /L, respectively. Median values for %r-PLTmtv were higher in young adults (≤2 years) than in older dogs (P=0.03). r-PLTmt and r-PLTmtv were moderately correlated with flow cytometry. Visual inspection of scattergrams is recommended.

Ferritin and its association with anaemia in a healthy adult population in Kenya.

BackgroundIron deficiency is the commonest cause of anaemia worldwide. Serum ferritin is the most sensitive non-invasive indicator of iron stores but its utility is compromised in inflammatory states as it is an acute phase reactant. This study sought to estimate the burden of iron deficiency in a healthy adult population residing in Kenya and to determine the association between various ferritin cut-offs and anaemia in a population known to have chronic low-grade inflammation.MethodsHealthy adults aged 18–65 years were recruited from urban towns in 4 counties in Kenya at average altitudes of 1683-2099m above sea level as part of a global study conducted by the International Federation of Clinical Chemistry (IFCC) to determine reference intervals (RIs) for common laboratory tests. We analyzed complete blood count (CBC), C-reactive protein, iron, transferrin, transferrin saturation and ferritin data.ResultsWe obtained data from 528 participants. There were 254 (48.1%) males and 274 females (51.9%). Based on a ferritin cut-off of 15 μg/L and Hb cut-offs of 14.5 g/dL and 12 g/dL, the prevalence of iron deficiency anaemia was 0.8% and 7.3% in males and females respectively. The odds of having anaemia was highest if one had a ferritin value less than 15 μg/L with a sensitivity of 28.6% and specificity of 98.4% in males, and sensitivity of 83.3% and specificity of 78.0% in females.ConclusionOnly the ferritin cut-off of 15 ug/L had an association with anaemia where it can be used for ruling out iron deficiency as the cause. Sex specific ferritin cut-offs for diagnosing iron deficiency in adults in sub-Saharan Africa need to be derived by comparing ferritin levels to stainable iron in bone marrow aspirates and trephines in order to ensure that we are using appropriate clinical decision limits.

Reference Interval for Serum L-Homoarginine Determined with Enzyme-Linked Immunosorbent Assay in the Population-Based Study of Health in Pomerania.

Low levels of the endogenous amino acid L-homoarginine are a risk factor for cardiovascular morbidity and mortality. For individual risk prediction, commercially available test systems are mandatory. This study aims at formulating sex- and age-specific reference intervals of serum L-homoarginine determined with an ELISA. We determined reference intervals for serum L-homoarginine stratified by age and sex in a sample of 1285 healthy participants of the Study of Health in Pomerania (SHIP)-TREND cohort after exclusion of participants with cardiovascular diseases, diabetes mellitus, hypertension, metabolic syndrome, elevated liver enzymes, chronic kidney disease stages III or IV, or body mass index >25 kg/m2. Serum L-homoarginine was determined applying a commercially available ELISA. The reference cohort included 836 women (median age 41, 25th and 75th percentiles are 32 and 50 years) and 449 men (median age 38, 25th, and 75th percentiles are 30 and 49 years). The median serum concentration of L-homoarginine was 1.93 (25th 1.49; 75th 2.60) µmol/L in women and 2.02 (25th 1.63; 75th 2.61) µmol/L in men (P = 0.04). The reference intervals (2.5th to 97.5th percentile) were 0.89-5.29 µmol/L for women and 1.09-3.76 µmol/L for men. The L-homoarginine serum concentration declined over age decades in both sexes and a notable interaction with sex hormone intake in women was observed. The novelty of our study is that we determined reference intervals specific for the L-isomer being lower than those previously reported for homoarginine in SHIP and thus might be helpful in identifying individuals suitable for oral L-homoarginine supplementation.

Practical application of mathematical calculations and statistical methods for the routine haematology laboratory.

Statistical analyses are embedded as critical functions in the routine haematology laboratory. This educational article is aimed at providing an overview of these topics and practical application examples. Topics covered include mathematical conversion between units, maintaining a quality control (QC) system, statistical methods for reagent validation, and determining uncertainty of measurement (UoM). Additional considerations may be required when a regional laboratory program is in place, such as the harmonization of INR results and determination of therapeutic reference intervals for unfractionated heparin therapy. The coauthors of this manuscript are fortunate to be part of regional network of hospital laboratories, the Eastern Ontario Regional Laboratory Association (EORLA).

Determination of reference intervals for neutrophil granular enzymes is affected by cell isolation techniques

ObjectivesNeutrophils and their granular enzymes such as neutrophil elastase (NE) and myeloperoxidase (MPO) play important roles in inflammatory diseases, and might be utilized as biomarkers for disease severity and progression. The aim of this study was to determine reference intervals for NE and MPO activity in healthy volunteers comparing two methods of neutrophil isolation. MethodsNeutrophils were isolated using ficoll density gradient centrifugation or immunomagnetic negative selection in two separate volunteers' cohorts. Subsequently, cells were lysed and incubated with chromogens for NE and MPO activity measurements, then measured with a microplate reader at 415 or 450 nm respectively. ResultsThe enzymatic activity of NE and MPO depended on the neutrophil isolation technique. Both enzymatic activities were significantly higher (P < 0.001) after isolating neutrophils with ficoll density gradient centrifugation than using the immunomagnetic negative selection. ConclusionsWe demonstrated that neutrophil isolation is an important factor that influences the outcome of enzymatic activity measurements. Techniques based on immunomagnetic negative selection are favorable, specifically for investigations related to NE and MPO activity. When using NE and MPO activity measurements in clinical practice, care must be taken to interpret the data depending on the applied cell isolation technique.

Plasma neurofilament light chain (NfL) reference interval determination in an Age-stratified cognitively unimpaired cohort

Background and AimsNeurofilament light chain (NfL) is an emerging biomarker of neurodegenerative disease progression. As plasma NfL increases with age, characterization of NfL concentrations in an age-stratified cognitively unimpaired population was assessed. Materials and MethodsEDTA-plasma samples were measured using the Simoa® NF-light™ Advantage Kit assay. One-sided reference intervals were established from 1100 cognitive normal individuals (588 male, 512 female) aged 20 to 95 years. Of those, 927 samples were obtained from the Mayo Clinic Study of Aging cohort (age > 50 years), and the remainder (age < 50 years) were obtained from individuals without known neurological conditions. All samples were from individuals without known chronic kidney disease, stroke or myocardial infarction, and a body mass index < 30 kg/m2. ResultsThe 97.5th percentile limits for the following age ranges (in years) were (pg/mL): 20 s: ≤8.4, 30 s: ≤11.4, 40 s: ≤15.4, 50 s: ≤20.8, 60 s: ≤28.0, 70 s: ≤37.9, 80+: ≤51.2. Sex had no significant effect on reference intervals. Observed NfL concentrations increased at a rate of 3.1 % per year of age. ConclusionsCharacterization of the rate of NfL concentration increase and decade-wide reference intervals from a neurologically well-characterized patient population will aid in interpretation of NfL during the clinical evaluation of a potential neurodegenerative disease.

Soluble Isoform of Suppression of Tumorigenicity 2 (ST2) Biomarker in a Large Cohort of Healthy Pediatric Population: Determination of Reference Intervals.

Introduction: Only little data exists on ST2 reference intervals in healthy pediatric populations despite the high importance of this biomarker in adults with heart failure. The aim of the study was to assess the reference intervals of ST2 in a wide healthy pediatric cohort. Methods: We evaluated the serum concentrations of ST2 biomarker in 415 healthy pediatric subjects referred to our analysis laboratory. Subjects were categorized according to age (i.e., 0–6 (n = 79), 7–11 (n = 142) and 12–18 years (n = 191)) and sex. They were not suffering from any cardiac disorders, metabolic disorders, lung diseases, autoimmune disorders or malignancies. A written consent was obtained for each individual. No duplicate patients were included in the analysis and the presence of outliers was investigated. Reference intervals (Mean and central 95% confidence intervals) were determined. Results: Three outliers have been identified and removed from the analysis (60.0, 64.0 and 150.2 ng/mL). A total of 412 subjects were therefore included. The mean value for the whole population was 15.8 ng/mL (2.4–36.4 ng/mL). Males present a significantly higher mean concentration compared to females (17.2 versus 14.4 ng/mL, p = 0.001). A significant trend toward higher ST2 values with age was also observed, but for males only (r = 0.43, p < 0.0001). If considering age partitions, only males of 12–18 years (mean = 21.7 ng/mL) had significantly higher ST2 values compared to the other groups (ranging from 11.9 for males 0–6 years to 15.2 for females 12–18 years; p < 0.0001). Conclusions: We described age and sex-specific reference intervals for ST2 in a large healthy pediatric population. We found that ST2 values differ between sexes if considering all participants. A significant increase in ST2 with age was also observed, but only for males of 12–18 years.

Determination of reference intervals for nonesterified fatty acids in the blood serum of healthy dogs.

BackgroundNonesterified fatty acids (NEFAs) are an important energy substrate in mammals. Measurement of the NEFA concentration in blood serum is common practice and enables reliable detection of a negative energy balance in several species. This parameter can be used to detect subclinical metabolic diseases or to optimise feeding to prevent severe negative energy balance. Since no reference values for dogs have been published, the aim of this study was to establish such values.MethodsBlood serum from 85 healthy dogs was examined with a multiparameter clinical chemistry analyser. Given that NEFA values are not usually normally distributed, reference intervals (RIs) were calculated nonparametrically using bootstrapping (5000 replicates) for the 90% confidence intervals.ResultsThe examined cohort had a median age of 62.16 months (2–180 months) and a median weight of 19.2 kg (3.0–55.0 kg) and comprised 27 (31.8%) males and 58 (68.2%) females, with 32 (37.6%) neutered or spayed. The fasting time was 5.9 h (range 0–23 h). The tested confounders age, sex, neuter status, bodyweight and body condition score did not significantly affect the NEFA concentrations.ConclusionsThe NEFA RI for dogs in this study was 0.2–1.47 mmol/L. The results may be used to adjust food composition and amount in healthy dogs or to detect metabolic disorders. Further research on NEFA metabolism in dogs maintained in standardised conditions and in specific nutritional situations or with particular diseases is warranted.

Evaluation of Growing Turkey Blood Biochemistry Panel Measured Using the VetScan VS2

Point-of-care analyzers such as the Vetscan VS2 (VS2) that provide fast results are becoming an important tool in a variety of clinical and research settings. Several studies have explored the use of the VS2 to evaluate blood chemistry values in chickens; however, the VS2 analyzer has not yet been tested in turkeys. The first objective of this study was to utilize the VS2 to evaluate possible time-dependent changes in biochemical blood analytes over time, such as glucose, calcium, or sodium. Blood samples from 30 turkeys were analyzed with the VS2 at three time points (0, 15–20, and 40–60 min post collection), and it was found that the blood biochemistry values were stable when stored at 4 °C and analyzed within 60 min of collection. Next, we assessed the agreement between the VS2 portable analyzer and the reference benchtop analyzer (Cobas c501) and found that all of the blood analytes were comparable between both analyzers. Finally, we determined reference intervals using 120 blood samples collected from turkeys in the brooding period (0–7 days) and 118 samples collected from turkeys in the growing period (1–12 weeks). Although the values were compared to the published data, the understanding of any observed differences was challenging because reference intervals for the biochemical values in turkeys are limited, and the origin of these interval values could not be traced. Significant differences in the whole blood analytes were noted between brooding and growing birds. This study offers reassurance of the validity of the use of portable analyzers in the field for turkey veterinarians. We conclude that the VS2 can be used to measure biochemical analytes in turkeys and provide the first reference intervals for brooding and growing turkeys using this instrument.

Reference Intervals for Absolute and Percentage CD4+ T Lymphocytes among an Apparently Healthy Population in Addis Ababa, Ethiopia

BackgroundReference intervals for clinical laboratory parameters differ based on several factors, including age, sex, genetic variation, and geographic location. This variation influences clinical decisions and treatment monitoring. Currently, Ethiopia has used adopted reference intervals from manufacturer values derived from non-Africans. Therefore, the aim this study was to determine reference intervals for absolute and percentage CD4+ T cells for an apparently healthy population in Addis Ababa, Ethiopia.MethodsA community-based cross-sectional study was conducted on 361 apparently healthy people in four subcities in Addis Ababa from January to June 2019. Sociodemographic and clinical data were collected using a structured questionnaire after informed consent had been obtained. Blood samples were collected and CD4+ T-lymphocyte enumeration performed using a BD FACSPresto near-patient CD4 counter. Data were entered and analyzed using SPSS 20. Reference intervals were determined by a nonparametric test estimating percentiles 2.5 (lower limit) and 97.5 (upper limit) with 95% CIs. P<0.05 was considered statistically significant.ResultsA total of 337 (183 female and 154 male) healthy participants of median age 28 (IQR 17–35) years were included in the final analysis. Medians of absolute and percentage CD4+ T-cell counts (932.0 and 42.9, respectively) of female participants were significantly higher than male participants (802.5 and 38.7, respectively; P<0.05). Reference intervals for absolute CD4+ T-cell count and percentages in males were 483.8–1,310 cells/µL and 18.1–57.3 and in females 447.8–1,479.8 cells/µL and 25.6–58.9, respectively.ConclusionThe CD4+ T-count reference intervals established in this study showed some inconsistency from the manufacturer’s provided values and other studies and also revealed sex differences, necessitating sex-specific locally established reference intervals.

Indirect determination of biochemistry reference intervals using outpatient data.

The aim of this study was to determine reference intervals in an outpatient population from Vall d’Hebron laboratory using an indirect approach previously described in a Dutch population (NUMBER project). We used anonymized test results from individuals visiting general practitioners and analysed during 2018. Analytical quality was assured by EQA performance, daily average monitoring and by assessing longitudinal accuracy between 2018 and 2020 (using trueness verifiers from Dutch EQA). Per test, outliers by biochemically related tests were excluded, data were transformed to a normal distribution (if necessary) and means and standard deviations were calculated, stratified by age and sex. In addition, the reference limit estimator method was also used to calculate reference intervals using the same dataset. Finally, for standardized tests reference intervals obtained were compared with the published NUMBER results. Reference intervals were calculated using data from 509,408 clinical requests. For biochemical tests following a normal distribution, similar reference intervals were found between Vall d’Hebron and the Dutch study. For creatinine and urea, reference intervals increased with age in both populations. The upper limits of Gamma-glutamyl transferase were markedly higher in the Dutch study compared to Vall d’Hebron results. Creatine kinase and uric acid reference intervals were higher in both populations compared to conventional reference intervals. Medical test results following a normal distribution showed comparable and consistent reference intervals between studies. Therefore a simple indirect method is a feasible and cost-efficient approach for calculating reference intervals. Yet, for generating standardized calculated reference intervals that are traceable to higher order materials and methods, efforts should also focus on test standardization and bias assessment using commutable trueness verifiers.

Hematology and biochemistry of critically endangered radiated tortoises (Astrochelys radiata): Reference intervals in previously confiscated subadults and variability based on common techniques.

Madagascar’s radiated tortoises (Astrochelys radiata) are critically endangered, threatened by illegal collection, and confiscated in alarming numbers in recent years. Robust population- and technique-specific hematology and biochemistry reference intervals are valuable yet heretofore missing tools for triage, rehabilitation, and reintroduction of confiscated radiated tortoises. We determined reference intervals in 120 previously confiscated, clinically healthy subadult radiated tortoises living under human care within their native habitat at the Tortoise Conservation Center (TCC). Specific analytes measured were manual packed cell volume, total solids, white blood cell (WBC) count and differentials, and biochemistry analytes using a point of care system. To evaluate the effects of different commonly used techniques on these analytes, we compared results between two venipuncture sites (subcarapacial sinus and brachial vein) and three different WBC quantification methods (Natt and Herrick, Leukopet, and slide estimate). Reference intervals were narrower for most analytes, and sodium and potassium were qualitatively higher in the TCC population compared to previously published values from radiated tortoises housed in North American institutions. Creatine kinase, aspartate aminotransferase, glucose and inorganic phosphorus were all significantly greater in brachial samples than in subcarapacial samples. There was poor agreement and evidence of constant and/or proportional bias between all WBC quantification methods. Differences based on time of sample collection were incidentally found in some analytes. These results highlight the need for considering technique, demographic, and environmental factors in creating and applying reference intervals, and contribute foundational knowledge for improving care of radiated tortoises throughout the confiscation-to-release pathway.

Trimester-specific reference intervals for cystatin C and neutrophil gelatinase-associated lipocalin during pregnancy.

During normal pregnancy, the glomerular filtration rate (GFR) increases dramatically. Failure to obtain this physiological increase is an important risk factor for morbidity and mortality for both mother and child. The estimated GFR (eGFR) using serum creatinine levels is unsuitable for accurate measurement of renal function during pregnancy. Therefore, new biomarkers have been proposed. Elevated levels of Cystatin C (CysC) and Neutrophil Gelatinase-Associated Lipocalin (NGAL) are associated with renal failure and preeclampsia (PE). In this study, we determined reference intervals for CysC and NGAL during pregnancy. Healthy pregnant women were recruited and blood samples were collected at 9-13 weeks (T1), 27-29 weeks (T2), and 36-39 weeks (T3) of gestation and at 4-13 weeks postpartum (PP). The samples from women with uncomplicated pregnancy were analyzed to determine median values and upper reference limits (URLs, 97.5 percentiles) of creatinine, CysC, and NGAL. A total of 175 women were included. Longitudinal changes and median values of creatinine, CysC, and NGAL were determined using only complete data sets (n=59). URLs were determined using all available data. The URL at T1, T2, T3, and PP were 60, 63, 74, 93μmol/L for creatinine; 0.93, 1.04, 1.61, 1.23mg/L for CysC; and 87, 84, 88, 95ng/mL for NGAL. CysC concentrations are highly dynamic and increase during pregnancy. NGAL concentrations are less dynamic, but well below the URL specified by the manufacturer for non-pregnant women. It is therefore recommended to use trimester-specific reference values for both CysC and NGAL.

An Examination of the Sex-Specific Nature of Nutrition Assessment within the Nutrition Care Process: Considerations for Nutrition and Dietetics Practitioners Working with Transgender and Gender Diverse Clients

An Examination of the Sex-Specific Nature of Nutrition Assessment within the Nutrition Care Process: Considerations for Nutrition and Dietetics Practitioners Working with Transgender and Gender Diverse Clients

Normative values of the internal genital organs of the female pelvis in transvaginal and transabdominal ultrasound.

To conduct a systemic review of published data on reference values for both transabdominal and transvaginal ultrasound in gynecology. Literature from 1970 to 2020 of reference values for the female pelvis in healthy subjects was reviewed. According to the determination of reference intervals for laboratory values reference values are generally determined using 95%-reference intervals and their associated 90%-confidence intervals. The list of articles was supplemented with extensive crosschecking of the reference lists of all retrieved articles. A total of 33 studies were included and analyzed. The diagnostic performance of transvaginal ultrasound (TVUS) has a higher sensitivity and specificity than transabdominal ultrasound (TAUS) for high quality imaging of the uterus and the bilateral adnexa. The length of normal uterus is about 50-80 mm in fertile age. There is no consensus about the cut off value of the thickness of the endometrium in asymptomatic postmenopausal women, while a measurement of >5 mm and postmenopausal bleeding is suspect and requires further examination. The distribution of normal ovarian volumes is narrow with small volumes in postmenopausal women. Normal values are helpful in delimiting the pathological changes in the female pelvis. While sonomorphologic criteria are more important than the ovarian size for the assessment of ovarian masses and reference values of the uterus in adults have little impact on routine practice, normative values in pediatric patients are important for the detection of pathologies. Normative values of the internal genital organs in females are sufficiently validated; still further research is required to assess the role of normative values in routine clinical practice and in sonographic screening for endometrial and ovarian cancer.

Analysis of normal levels of free glycosaminoglycans in urine and plasma in adults

Plasma and urine glycosaminoglycans (GAGs) are long, linear sulfated polysaccharides that have been proposed as potential noninvasive biomarkers for several diseases. However, owing to the analytical complexity associated with the measurement of GAG concentration and disaccharide composition (the so-called GAGome), a reference study of the normal healthy GAGome is currently missing. Here, we prospectively enrolled 308 healthy adults and analyzed their free GAGomes in urine and plasma using a standardized ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry method together with comprehensive demographic and blood chemistry biomarker data. Of 25 blood chemistry biomarkers, we mainly observed weak correlations between the free GAGome and creatinine in urine and hemoglobin or erythrocyte counts in plasma. We found a higher free GAGome concentration – but not a more diverse composition - in males. Partitioned by gender, we also established reference intervals for all detectable free GAGome features in urine and plasma. Finally, we carried out a transference analysis in healthy individuals from two distinct geographical sites, including data from the Lifelines Cohort Study, which validated the reference intervals in urine. Our study is the first large-scale determination of normal free GAGomes reference intervals in plasma and urine and represents a critical resource for future physiology and biomarker research.

Determination of reference intervals of creatinine clearance in laboratory animals

Determination of reference intervals of creatinine clearance in laboratory animals

Principles of methods for indirect determination of reference intervals

Principles of methods for indirect determination of reference intervals

Establishing reference intervals for von Willebrand factor multimers.

Backgroundvon Willebrand factor (VWF) multimers (VWF:MM) methodologies are technically difficult, laborious, time consuming, non-standardized and results vary between laboratories. A new semi automated VWF:MM assay is available for routine use (Sebia). Due to lack of reference values for VWF:MM fractions, results interpretation can be challenging in some cases. The aim of this study was to determine reference intervals for low molecular weight (LMWM), intermediate molecular weight (IMWM) and high molecular weight (HMWM) multimers.MethodsBy the international cooperation initiated between 4 countries (Estonia, Latvia, France, and USA) 131 samples of relatively healthy individuals were analyzed for VWF:MM (in total 51 males and 80 non-pregnant females aged 17-69 years). Reference intervals were calculated according to CLSI C28-A3 standard.ResultsThe proposed reference intervals for VWF:MM were calculated for LMWM 10.4-22.5%, IMWM 22.6-37.6%, HMWM 45.6-66.6%. Age related differences were seen in IMWM and HMWM (p<0.001 and 0.038). There was no gender related difference observed. Geographically LMWM results of France were different from the other regions (p<0.05).ConclusionsQuantification of VWF:MM fractions, in addition to qualitative assessment of VWF:MM patterns, has the potential to aid in differential diagnosis of von Willebrand disease (VWD) subtypes. The reference values calculated in this study can be used in future research to establish clinical decision limits.