Application of the Stockholm Hierarchy to Defining the Quality of Reference Intervals and Clinical Decision Limits

The Stockholm Hierarchy is a professional consensus created to define the preferred approaches to defining analytical quality. The quality of a laboratory measurement can also be classified by the quality of the limits that the value is compared with, namely reference interval limits and clinical decision limits. At the highest level in the hierarchy would be placed clinical decision limits based on clinical outcome studies. The second level would include both formal reference interval studies (studies of intra and inter-individual variations) and clinical decision limits based on clinician survey. While these approaches are commonly used, they require a lot of resources to define accurately. Placing laboratory experts on the third level would suggest that although they can also define reference intervals by consensus, theirs aren’t as well regarded as clinician defined limits which drive clinical behaviour. Ideally both analytical and clinical considerations should be made, with clinicians and laboratorians both having important information to consider. The fourth level of reference intervals would be for those defined by survey or by regulatory authorities because of the focus on what is commonly achieved rather than what is necessarily correct. Finally, laboratorians know that adopting reference limits from kit inserts or textbook publications is problematic because both methodological issues and reference populations are often not the same as their own. This approach would rank fifth and last. When considering which so called ‘common’ or ‘harmonised reference intervals’ to adopt, both these characteristics and the quality of individual studies need to be assessed. Finally, we should also be aware that reference intervals describe health and physiology while clinical decision limits focus on disease and pathology, and unless we understand and consider the two corresponding issues of test specificity and test sensitivity, we cannot assure the quality of the limits that we report.     

Standardisation of reference intervals: an Australasian view

The development of regional databases and doctors’ desktop programs that accept pathology results from different laboratories should improve patient care by allowing easy assessment of cumulative data. This has the potential to be unnecessarily confusing unless laboratories contributing to the databases provide standardised results and common reference intervals, where this is valid. The analytical methods that produce significantly different results need to be reported in a manner that avoids inappropriate interpretation.

The process of setting reference intervals requires an organisational structure which enables appropriate intervals to be set taking all relevant factors into account, including the opinions of expert clinicians. There must also be criteria for analytical agreement between the laboratories involved based on comparison studies using patient samples.

A network of QA groups across Australasia, with leadership from the AACB and RCPA, should be formed to share the ongoing work of defining reference intervals (RIs) for common tests, and reviewing them as the testing environment changes with the introduction of new techniques and instruments.

     

Closing the Gaps in Paediatric Reference Intervals: The CALIPER Initiative

Screening, diagnosis and monitoring of paediatric diseases relies on the measurement of a spectrum of disease biomarkers in clinical laboratories to guide important clinical decisions. Physicians rely on the availability of suitable and reliable reference intervals to accurately interpret laboratory test results with data collected during medical history and physical examination. However, critical gaps currently exist in accurate and up-to-date reference intervals (normal values) for accurate interpretation of laboratory tests performed in children and adolescents. These gaps in the available paediatric laboratory reference intervals have the clear potential of contributing to erroneous diagnosis or misdiagnosis of many diseases of childhood and adolescence. Most of the available reference intervals for laboratory tests were determined over two decades ago on older instruments and technologies, and are no longer relevant considering the current testing technology used by clinical laboratories. It is thus critical and of utmost urgency that a more acceptable and comprehensive database be established. There are however many challenges when attempting to establish paediatric reference intervals. Paediatric specimen collection is a major concern for health care providers as it is frequently difficult to obtain sufficient volumes of blood or urine from paediatric patients. Common reference intervals have not been widely implemented due to lack of harmonisation of methods and differences in patient populations. Consequently, clinical laboratory accreditation organisations and licensing agencies require that each laboratory verify or establish reference intervals for each method. To provide such reference intervals requires selection criteria for suitable reference individuals, defined conditions for specimen collection and analysis, method selection to determine reference limits and validation of the reference interval. The current review will provide a brief introduction to the current approach to establishment of reference intervals, will highlight the current gaps in data available in paediatric populations, and review a recent Canadian initiative, CALIPER (Canadian Laboratory Initiative on Paediatric Reference Intervals), to establish a comprehensive database for both traditional and emerging biomarkers of paediatric disease.     

Harmonising Adult and Paediatric Reference Intervals in Australia and New Zealand: An Evidence-Based Approach for Establishing a First Panel of Chemistry Analytes

Scientific evidence supports the use of common reference intervals (RIs) for many general chemistry analytes, in particular those with sound calibration and traceability in place. Already the Nordic countries and United Kingdom have largely achieved harmonised RIs. Following a series of workshops organised by the Australasian Association of Clinical Biochemists (AACB) between 2012 and 2014 at which an evidence-based approach for determination of common intervals was developed, pathology organisations in Australia and New Zealand have reached a scientific consensus on what adult and paediatric intervals we should use across Australasia. The aim of this report is to describe the processes that the AACB and the Royal College of Pathologists of Australasia have taken towards recommending the implementation of a first panel of common RIs for use in Australasia.     

Physiology and its Importance for Reference Intervals

Reference intervals are ideally defined on apparently healthy individuals and should be distinguished from clinical decision limits that are derived from known diseased patients. Knowledge of physiological changes is a prerequisite for understanding and developing reference intervals. Reference intervals may differ for various subpopulations because of differences in their physiology, most obviously between men and women, but also in childhood, pregnancy and the elderly. Changes in laboratory measurements may be due to various physiological factors starting at birth including weaning, the active toddler, immunological learning, puberty, pregnancy, menopause and ageing. The need to partition reference intervals is required when there are significant physiological changes that need to be recognised. It is important that laboratorians are aware of these changes otherwise reference intervals that attempt to cover a widened inter-individual variability may lose their usefulness. It is virtually impossible for any laboratory to directly develop reference intervals for each of the physiological changes that are currently known, however indirect techniques can be used to develop or validate reference intervals in some difficult situations such as those for children. Physiology describes our life’s journey, and it is only when we are familiar with that journey that we can appreciate a pathological departure.     

Chlamydomonas Genome Resource for Laboratory Strains Reveals a Mosaic of Sequence Variation,Identifies True Strain Histories,and Enables Strain-Specific Studies

Chlamydomonas reinhardtii is a widely used reference organism in studies of photosynthesis, cilia, and biofuels. Most research in this field uses a few dozen standard laboratory strains that are reported to share a common ancestry, but exhibit substantial phenotypic differences. In order to facilitate ongoing Chlamydomonas research and explain the phenotypic variation, we mapped the genetic diversity within these strains using whole-genome resequencing. We identified 524,640 single nucleotide variants and 4812 structural variants among 39 commonly used laboratory strains. Nearly all (98.2%) of the total observed genetic diversity was attributable to the presence of two, previously unrecognized, alternate haplotypes that are distributed in a mosaic pattern among the extant laboratory strains. We propose that these two haplotypes are the remnants of an ancestral cross between two strains with ∼2% relative divergence. These haplotype patterns create a fingerprint for each strain that facilitates the positive identification of that strain and reveals its relatedness to other strains. The presence of these alternate haplotype regions affects phenotype scoring and gene expression measurements. Here, we present a rich set of genetic differences as a community resource to allow researchers to more accurately conduct and interpret their experiments with Chlamydomonas.     

Prerequisites for use of common reference intervals

The theory of reference values was developed more than 30 years ago, but its application in most clinical laboratories is still incomplete today. This is for several reasons, the most relevant ones being the lack of standardisation of the analytical methods, resulting in method-dependent values, and the difficulty in recruiting the proper number of reference subjects for establishment of reference intervals. With the recent progress in method standardisation the first problem is reducing while the second can be addressed optimally via multicentre collaborative studies that aim to establish common reference intervals. To be effective this approach requires the following prerequisites: 1) the existence of a reference measurement system for the analyte; 2) field methods producing results traceable to the reference system; and 3) a carefully planned multicentre reference interval study. Such a procedure will produce results traceable to the reference measurement system for a large number of reference subjects, under controlled pre-analytical conditions. It will also enable a better understanding of the various sources of population variability, if there is the need for partitioning of a reference interval or if there are any limitations to adopting the established reference intervals on a national or global scale. Once reference intervals are determined, clinical laboratories can adopt a common reference interval provided: 1) the population that the laboratory services is similar to the one studied; 2) methods producing traceable results are used; and 3) analytical quality is within defined targets of precision and bias. Moreover, some validation of the interval using a small sample of reference individuals from the laboratory's population is advisable.     

Computation of model-dependent tolerance bands for ambulatorily monitored blood pressure

The construction of time-specified reference limits requires systematic sampling in clinical health, particularly for those variables characterized by a circadian rhythm of large amplitude, as it is the case for blood pressure (BP). For the detection of false negatives, tolerance intervals (limits that will include at least a specified proportion of the population with a stated confidence) are important and should substitute when possible for prediction limits. We have previously described a nonparametric method for the computation of model-independent tolerance intervals that are constructed by first dividing the sampling range in several time spans in which no appreciable changes in population characteristics (namely, mean and variance) take place. The tolerance interval is then computed for each of the time spans. The limits thus computed, as well as results of any comparison of a given individual's profile against such tolerance intervals, are highly dependent on the sampling scheme of both the reference individuals and the test subject. To avoid this problem, we have developed an alternative method that allows the computation of model-dependent tolerance bands for hybrid time series. Assuming that a set X of longitudinal series monitored from a given group of reference individuals can be fitted with the same individual model, a population model C(X,t) can be also determined, as well as the deviation S(X,t) of each individual curve from the population model. The tolerance band will then have the form C(X,t) ± kS(X,t), where k is here estimated following a nonparametric approach based on bootstrap techniques. Alternatively, two different values of k can be estimated (for the lower and upper limits of the tolerance interval, respectively) in cases for which we cannot assume symmetry. The method is generally applicable for any population model describing the reference population (including the fit of multiple significant components, nonsinusoidal waveforms, and/or trends). The method was used to establish time-specified tolerance bands for time series of blood pressure monitored automatically in healthy individuals of both genders. Model-dependent intervals are preferred to the model-independent limits when reliance on a specified sampling rate needs to be avoided. These limits may serve for an objective and positive definition of health, for the screening and diagnosis of disease, and for gauging the subject's response to treatment. (Chronobiology International, 17(4), 567-582, 2000)     

RCPAQAP First Combined Measurement and Reference Interval Survey

Reference intervals are commonly considered to allow for between-laboratory bias. The RCPAQAP Liquid Serum Chemistry Program has collected data on laboratory measurements as well as reference intervals. This allows assessment of the between-laboratory variation in results, reference intervals and the information transmitted by the combination of these factors. For the majority of common chemistry analytes, the between-laboratory variation in reference intervals is greater than the variation in results. Additionally the reference interval variation is generally not related to bias between the results. Use of common reference intervals, either as an average of the current intervals in use, or the intervals proposed by the AACB Harmonisation Group, improved the variation seen in the information produced by different laboratories.     

COMPUTATION OF MODEL-DEPENDENT TOLERANCE BANDS FOR AMBULATORILY MONITORED BLOOD PRESSURE

The construction of time-specified reference limits requires systematic sampling in clinical health, particularly for those variables characterized by a circadian rhythm of large amplitude, as it is the case for blood pressure (BP). For the detection of false negatives, tolerance intervals (limits that will include at least a specified proportion of the population with a stated confidence) are important and should substitute when possible for prediction limits. We have previously described a nonparametric method for the computation of model-independent tolerance intervals that are constructed by first dividing the sampling range in several time spans in which no appreciable changes in population characteristics (namely, mean and variance) take place. The tolerance interval is then computed for each of the time spans. The limits thus computed, as well as results of any comparison of a given individual's profile against such tolerance intervals, are highly dependent on the sampling scheme of both the reference individuals and the test subject. To avoid this problem, we have developed an alternative method that allows the computation of model-dependent tolerance bands for hybrid time series. Assuming that a set X of longitudinal series monitored from a given group of reference individuals can be fitted with the same individual model, a population model C(X,t) can be also determined, as well as the deviation S(X,t) of each individual curve from the population model. The tolerance band will then have the form C(X,t) ± kS(X,t), where k is here estimated following a nonparametric approach based on bootstrap techniques. Alternatively, two different values of k can be estimated (for the lower and upper limits of the tolerance interval, respectively) in cases for which we cannot assume symmetry. The method is generally applicable for any population model describing the reference population (including the fit of multiple significant components, nonsinusoidal waveforms, and/or trends). The method was used to establish time-specified tolerance bands for time series of blood pressure monitored automatically in healthy individuals of both genders. Model-dependent intervals are preferred to the model-independent limits when reliance on a specified sampling rate needs to be avoided. These limits may serve for an objective and positive definition of health, for the screening and diagnosis of disease, and for gauging the subject's response to treatment. (Chronobiology International, 17(4), 567–582, 2000)     

Signal enrichment with strain-level resolution in metagenomes using topological data analysis

Background

A metagenome is a collection of genomes, usually in a micro-environment, and sequencing a metagenomic sample en masse is a powerful means for investigating the community of the constituent microorganisms. One of the challenges is in distinguishing between similar organisms due to rampant multiple possible assignments of sequencing reads, resulting in false positive identifications. We map the problem to a topological data analysis (TDA) framework that extracts information from the geometric structure of data. Here the structure is defined by multi-way relationships between the sequencing reads using a reference database.

Results

Based primarily on the patterns of co-mapping of the reads to multiple organisms in the reference database, we use two models: one a subcomplex of a Barycentric subdivision complex and the other a Čech complex. The Barycentric subcomplex allows a natural mapping of the reads along with their coverage of organisms while the Čech complex takes simply the number of reads into account to map the problem to homology computation. Using simulated genome mixtures we show not just enrichment of signal but also microbe identification with strain-level resolution.

Conclusions

In particular, in the most refractory of cases where alternative algorithms that exploit unique reads (i.e., mapped to unique organisms) fail, we show that the TDA approach continues to show consistent performance. The Čech model that uses less information is equally effective, suggesting that even partial information when augmented with the appropriate structure is quite powerful.

     

Sex differences in ischemic heart disease and heart failure biomarkers

Since 1984, each year, more women than men die of ischemic heart disease (IHD) and heart failure (HF), yet more men are diagnosed. Because biomarker assessment is often the first diagnostic employed in such patients, understanding biomarker differences in men vs. women may improve female morbidity and mortality rates.Some key examples of cardiac biomarker utility based on sex include contemporary use of “unisex” troponin reference intervals under-diagnosing myocardial necrosis in women; greater use of hsCRP in the setting of acute coronary syndrome (ACS) could lead to better stratification in women; and greater use of BNP with sex-specific thresholds in ACS could also lead to more timely risk stratification in women.Accurate diagnosis, appropriate risk management, and monitoring are key in the prevention and treatment of cardiovascular diseases; however, the assessment tools used must also be useful or at least assessed for utility in both sexes. In other words, going forward, we need to evaluate sex-specific reference intervals or cutoffs for laboratory tests used to assess cardiovascular disease to help close the diagnostic gap between men and women.     

Reconciling Hygiene and Cleanliness: A New Perspective from Human Microbiome

The term hygiene is deeply rooted with the concept of maintaining sound health and alertness towards cleanliness, while “hygiene hypothesis” depicts the protective role of microbial community exposure in development of early immunity and initial allergic and aesthetic reactions. The tug-of-war has now been pushed toward the literal term “hygiene” over the “hygiene hypothesis” and has continued with disinfection of all microbial loads from the related environments to avoid infections in humans. With the advancement in the microbiome studies, it became clear that humans possess warm, and significant relationships with diverse microbial community. With this opinion article, we have emphasized on the importance of hygiene hypothesis in immunological responses. We also propose the individual/targeted hygiene instead of application of unanimous hygiene hypothesis. This review also elaborates the common practices that should be employed to maintain hygiene along with the balanced microbiome.

     

Normal Laboratory Reference Intervals among Healthy Adults Screened for a HIV Pre-Exposure Prophylaxis Clinical Trial in Botswana

Introduction

Accurate clinical laboratory reference values derived from a local or regional population base are required to correctly interpret laboratory results. In Botswana, most reference intervals used to date are not standardized across clinical laboratories and are based on values derived from populations in the United States or Western Europe.

Methods

We measured 14 hematologic and biochemical parameters of healthy young adults screened for participation in the Botswana HIV Pre-exposure Prophylaxis Study using tenofovir disoproxil fumarate and emtricitabine (TDF/FTC) (TDF2 Study). Reference intervals were calculated using standard methods, stratified by gender, and compared with the site-derived reference values used for the TDF2 study (BOTUSA ranges), the Division of AIDS (DAIDS) Grading Table for Adverse Events, the Botswana public health laboratories, and other regional references.

Results

Out of 2533 screened participants, 1786 met eligibility criteria for participation in study and were included in the analysis. Our reference values were comparable to those of the Botswana public health system except for amylase, blood urea nitrogen (BUN), phosphate, total and direct bilirubin. Compared to our reference values, BOTUSA reference ranges would have classified participants as out of range for some analytes, with amylase (50.8%) and creatinine (32.0%) producing the highest out of range values. Applying the DAIDS toxicity grading system to the values would have resulted in 45 and 18 participants as having severe or life threatening values for amylase and hemoglobin, respectively.

Conclusion

Our reference values illustrate the differences in hematological and biochemical analyte ranges between African and Western populations. Thus, the use of western-derived reference laboratory values to screen a group of Batswana adults resulted in many healthy people being classified as having out-of-range blood analytes. The need to establish accurate local or regional reference values is apparent and we hope our results can be used to that end in Botswana.     

CLSI-Derived Hematology and Biochemistry Reference Intervals for Healthy Adults in Eastern and Southern Africa

Background

Clinical laboratory reference intervals have not been established in many African countries, and non-local intervals are commonly used in clinical trials to screen and monitor adverse events (AEs) among African participants. Using laboratory reference intervals derived from other populations excludes potential trial volunteers in Africa and makes AE assessment challenging. The objective of this study was to establish clinical laboratory reference intervals for 25 hematology, immunology and biochemistry values among healthy African adults typical of those who might join a clinical trial.

Methods and Findings

Equal proportions of men and women were invited to participate in a cross sectional study at seven clinical centers (Kigali, Rwanda; Masaka and Entebbe, Uganda; two in Nairobi and one in Kilifi, Kenya; and Lusaka, Zambia). All laboratories used hematology, immunology and biochemistry analyzers validated by an independent clinical laboratory. Clinical and Laboratory Standards Institute guidelines were followed to create study consensus intervals. For comparison, AE grading criteria published by the U.S. National Institute of Allergy and Infectious Diseases Division of AIDS (DAIDS) and other U.S. reference intervals were used. 2,990 potential volunteers were screened, and 2,105 (1,083 men and 1,022 women) were included in the analysis. While some significant gender and regional differences were observed, creating consensus African study intervals from the complete data was possible for 18 of the 25 analytes. Compared to reference intervals from the U.S., we found lower hematocrit and hemoglobin levels, particularly among women, lower white blood cell and neutrophil counts, and lower amylase. Both genders had elevated eosinophil counts, immunoglobulin G, total and direct bilirubin, lactate dehydrogenase and creatine phosphokinase, the latter being more pronounced among women. When graded against U.S.-derived DAIDS AE grading criteria, we observed 774 (35.3%) volunteers with grade one or higher results; 314 (14.9%) had elevated total bilirubin, and 201 (9.6%) had low neutrophil counts. These otherwise healthy volunteers would be excluded or would require special exemption to participate in many clinical trials.

Conclusions

To accelerate clinical trials in Africa, and to improve their scientific validity, locally appropriate reference ranges should be used. This study provides ranges that will inform inclusion criteria and evaluation of adverse events for studies in these regions of Africa.     

High-fat and obesogenic diets: current and future strategies to fight obesity and diabetes

Obesity, diabetes and their associated diseases are some of the greatest challenges that the world health care systems already face and with prospects of overburdening their capacities and funding. Due to decreased energetic expenditure and increased caloric intake, particularly in saturated fats, the number of people afflicted by said conditions is increasing by the day. Due to the failure, to this day, to effectively and ubiquity prevent and revert these diseases, the research into new compounds and therapeutic strategies is vital. In this review, we explain the most common dietary models of obesity and diabetes and the novel avenues of research we believe will be taken in the next few years in obesity and diabetes research. We primarily focus on the role of mitochondria and how the modulation of mitochondrial function and number as well as several promising therapeutic strategies involving metabolic regulators can positively affect the obese and diabetic status.     

On a habitat structure-based approach to evaluating species occurrence: cavity-nesting Hymenoptera in a secondary tropical forest remnant

Measuring bee and wasp community habitat preferences in natural systems may provide insights for biodiversity management and conservation as habitat heterogeneity can be the major factor affecting species diversity in a community. Here, we present evidence that supports the use of a vegetation structure-based approach in order to predict the occurrence of common Brazilian-remnant solitary bee and wasp species. Using trap nests, we sampled Aculeate bees and wasps in a forest remnant within an urban area of southeast Brazil. We also measured eight habitat structure components in the area. Tree trunk circumference, shrub and wood log abundance were good predictors of presence or absence of the commonest solitary bee and wasp species in the study area. We demonstrated that even on a small scale it is possible to detect significant influences of habitat structural features on species occurrences and that some of them are effective as surrogates for predicting trap-nesting Hymenoptera occurrence in a given area. Our data present evidence on the potential application of a habitat structure-based approach in conservation assessments concerning cavity-nesting Hymenoptera. We caution, however, that this habitat structure-based approach must to be taken with prudence as a detailed survey of an area’s biodiversity is always preferable.     

A phenomenological account of users' experiences of assertive community treatment

Assertive community treatment (ACT) is a widely propagated team approach to community mental health care that 'assertively' engages a subgroup of individuals with severe mental illness who continuously disengage from mental health services. It involves a number of interested parties – including clients, carers, clinicians and managers. Each operates according to perceived ethical principles related to their values, mores and principles. ACT condenses a dilemma that is common in psychiatry. ACT proffers social control whilst simultaneously holding therapeutic aspiration. The clients' perspective of this dilemma was studied in interviews with 12 clients using the 'grounded theory' approach. Results suggest that clients' disengagement is as much a historical and cultural phenomenon as a result of lack of insight. Many clients had experienced rejection of early help–seeking behaviour and all had been subject to coercive interventions. These coercive interventions were experienced as an attack on identity. All felt that their voice had not been listened to in previous interactions with psychiatric services. Consequentially the clients had an increased level of arousal around issues of power, which needs to be incorporated when examining the ethics of community psychiatry. Traditional notions of the difference between persuasion and coercion – for example – may need to be adapted for this client group. Results are compared with the provider perspective. We conclude that the perspectives differ on two key dimensions. Such an empirical approach to examining psychiatric ethics may ensure that we incorporate the subjectivities of various interested parties in the clinical decision–making process.