The use of a grey zone considering measurement uncertainty in pharmacological tests. The serum growth hormone stimulation test as an example

LAZZATI JM; ZAIDMAN, V; MACEIRAS M,; BELGOROSKY A; CHALER E

CLINICAL CHEMISTRY AND LABORATORY MEDICINE

WALTER DE GRUYTER & CO

Lugar: Berlin; Año: 2016 p. 1 - 10

1434-6621

The responsibility of clinical laboratories includes adequate assay methods,measurement procedures, and the definition of the appropriate quality specifications for each mensurand as well as the identification of criteria required for obtaining the optimal interpretation and utilization of results, with reference intervals and adequate decisionlimits (1).Decision limits in pharmacological tests (PhT) are used in the study of different hormonal axes (GnRH, TRH, serum growth hormone (GH) in PhT, etc) in which an inhibitory or stimulatory factor is administered to the patient and the effects are measured at different times. Subsequently, according to a defined cut-off value, it is considered whether stimulation/inhibition was effectively achieved. The problemsbehind an evidence-based approach to laboratory diagnostics can be clearly illustrated by the components of a PhT: 1) a variety of protocols, 2) secretagogues, 3) a variable biological response to stimulation, 4) a multiplicity of assays, and finally, 5) the variabilityin clinical interpretation. The combination of these factors and the lack of standardization and other unmeasurable variables has led to a lack of confidence.Usually, the evaluation of the results obtained does not take into account that they have measurable magnitudes and are limited by the measurement system. Therefore, results have a certain measurement uncertainty, and the area around the cut-off value wherethe results are uncertain should be defined (2).The application of ISO 15189:2012 (International Organization for Standardization)to clinical laboratories requires the knowledge of the measurement uncertainty for each measurement procedure in the analysis phase used to inform the measured quantitative values of patient samples.All measurements are affected by a certain error. The measurement uncertainty tells us what size the measurement error might be. Therefore, the measurement uncertainty is an important part of the reported result.Measurement uncertainty (3) should normally be expressed as U, the combined expanded measurement uncertainty (uc), using a coverage factor k = 2, prov iding a level of confidence of approximately 95 %.U= 2. ucThe different contributions to the uc are the within-laboratory reproducibility (Rw)and the uncertainty component for bias (u(bias))uc = (u(Rw)2+ u(bias)2)1/2u(Rw) is calculated taken into account the intermediate precision (Rw) and is obtained from the internal quality control data measured for at least 6 months using different operators, reactive lots, calibrations, and storage cond itions; and using suitable material.u(bias) can be estimated by u(bias)= [(bias)2 + (Sbias/(n)½)2 + u(Cref)2]½ bias is the difference between mean measured value from a large series of test results and an accepted reference value. The most common ways of estimating the bias components are: the use of Certified Reference Material (CRM), recovery tests or participation in interlaboratory comparisons (External Quality Control).Sbias is standard deviation obtained from measurements on the CRM and n is the number of measurement on the CRM.u(Cref) Uncertainty component for the certified or nominal value.We propose to use a grey zone (GZ) based on U as it considers all sources of variation of a result attributed to the quantities in two terms: variation associated with precision and with trueness. This area consists of the GZ = cut-off ± U. Using the GZ, a ternary classification is expected; any individual result outside this zone - with its range ofuncertainty included - is guaranteed to be above or below the cut-off. (Figure).In this line, GH may be a paradigm. A diagnosis of growth hormone deficiency (GHD)implies expensive and prolonged treatment. A tremendous amount of scientific evidence regarding the physiology and physiopathology of synthesis mechanisms, secretion, and actions of GH has been published over the last years; however, in spite of these impressive advances and, deeply disappointing from a public health perspective, the real picture of diagnosis is overshadowed by widespread diagnostic inaccuracies(underdiagnosis, overdiagnosis) as well as by treatment failures generated by under- or overtreatment. The scientific, medical, and patient communities as well as decisionmakers worldwide are striving for the greatest possible health gains from available resources.The diagnostic cut-off value of serum GH in PhT for the diagnosis of GHD has been an ongoing topic of discussion. For years there was no harmonization between assays (4,5) until 2008, when a consensus (6) proposed the GH assays to measure the 22k form and use the second growth hormone-recombinant international standard IRP 98/574.Currently, there is no agreement on the cut-off point of serum GH in PhT below which we define GHD.In patients the diagnosis of GHD is based mainly on auxological criteria. The diagnosis is biochemically confirmed by the maximum peak (maxp) reached during two PhTs; if one of them is above the diagnostic cut-off value the patient is considered to have adequate GH secretion.In a recent publication (7), we defined a cut-off point for PhTs in GH of 4.7 ng/ml by chemiluminescent assay (Immulite 2000, Siemens Laboratories) using IRP 98/574 (international reference material prepared by genetic engineering).Due to a lack of laboratory or reference material Rw is traceable uniquely to the laboratory and bias is traceable uniquely to the group. We used Lyphochek Immunoassay Pluscontrol - US-Bio Rad Laboratories Irvine ? CA, with a concentration at a level = 4.5 ng/mlOver a period of 3 years, we measured GH in ng/ml in Human serum with a U =10.8%. (Rw = 5.13%, bias = 1.75%, number of result of sampling used for Rw and bias = 669, Sbias is unmeasurable and u(Cref) is unknown). If we apply the U formulation, our GZ is between 4.2 and 5.2 ng/ml.We analyzed the plotting of the results of two GH secretion PhTs (Clonidine and Arginine) in 338 patients. Using the GZ, 34.3% (n: 116) potentially had a secretory deficit (GHD group) and 57.7% (n: 195) had adequate secretion (AGH group). Finally,8% (n: 27) was found in the GZ, in which GH secretion status cannot be appropriatelydetermined (GZ group).It is widely accepted to consider insulin-like growth factor type 1 (IGF-1) as a biomarker for GH action. There are publications that defined GHD in function of IGF-1 standarddeviation score (SDS) (8). We found significant differences in IGF-1 SDS between the GHD group and the AGH group. The GZ group showed significant differences compared to the GHD group, but not compared to the AGH group. Moreover, if the GZ group is divided into the GZ group below the cut-off value (GZ low) and GZ group abovethe cut-value value (GZ high), no significant differences were found, Remarkably, theGZ low group, which may be considered as having a deficit, presented with a significant difference compared to the GHD group but not compared to the AGH group (Table).Clearly, the GZ group has different characteristics that should be assessed differently,and a single cut-off value is not sufficient to define the diagnostic limits. Savage et al (9)defined the differences in status of secretion and sensitivity as a continuum and the useof GZ would be a practical approximation to this idea.The concept of the "grey zone" is naturally used in other biochemical parameters suchas those used in serologic diagnosis; however generally it is not considered inendocrine PhTs.We strongly recommend to include an grey zone in the diagnostic cut-off valuecalculated for each analytical platform according to the U of the mensurand used.We considered GH secretion in PhTs as a paradigm, as GHD implies expensive andprolonged treatment. If the maximum peak of patients is found to be within the GZ, thespecialist should assess other features, such as family history, clinical and nutritionalstatus, and diseases to decide whether to treat or not. If this grey zone is not taken intoaccount, patients may misclassified leading to treatment errors.As the sensitivity and specificity indicate diagnostic capacity of the tests in general, theGZ indicates the analytical limitation of the mensurand that should be taken into accountfor the biochemical counseling on the interpretation of the results.