Journées Klotz 2015
Problems with the PTH assaysDifficultés de dosage de la PTH

https://doi.org/10.1016/j.ando.2015.03.018Get rights and content

Abstract

Even if the first assay for parathyroid hormone (PTH) was published in the early 1960s, its determination remains a challenge even today. Indeed, in the circulation, PTH is present in its active form (PTH 1–84), but many PTH fragments can also be present. These fragments accumulate when renal function declines and are recognized, at different extents, by the 2nd generation (“intact”) PTH assays that are widely used in the clinical laboratories. Some assays, called “3rd generation PTH” do not recognize these fragments, but are not available everywhere. Hence, different problems are also linked with PTH determination. Among them, one can cite the lack of a reference method, the lack of standardization of the assays and, sometimes, the lack of consistent reference range. We can also point out stability problems and a large intra-individual variation. A workgroup is working on these problems under the auspices of the IFCC and we hope that some of these problems will be resolved in the next years. In this article, we will discuss all the possible issues of PTH determination.

Résumé

Bien que le dosage de l’hormone parathyroïdienne (PTH) ait été l’un des premiers immunodosages à être publié dans les années 1960, il reste encore très problématique aujourd’hui. En effet, la PTH circule sous sa forme active (PTH 1–84) à côté de nombreux fragments qui s’accumulent dans l’insuffisance rénale et qui sont reconnus par les dosages de PTH de 2e génération (dite « intacte »), employés par la plupart des laboratoires de biologie clinique. Certains kits, dits de 3e génération, ne reconnaissent pas ces fragments, mais ils ne sont pas disponibles partout. D’autres problèmes sont également rencontrés lors du dosage de la PTH. Parmi ceux-ci, nous pouvons citer l’absence d’une méthode de référence et le manque de standardisation des trousses de dosages. Les valeurs de référence ont également souvent été établies de façon critiquable, des problèmes de stabilité sont à noter et la variation intra-individuelle de la PTH est particulièrement importante. Un groupe de travail a été constitué sous les auspices de l’IFCC pour essayer de répondre à certains de ces problèmes. Dans cet article, nous tenterons de faire la lumière sur ces difficultés évoquées.

Section snippets

The different generations of PTH assays: what they measure and what they don’t

The human parathormone (PTH) gene is located on the short arm of chromosome 11 [1]. PTH is synthesized as a large polypeptide (pre-pro PTH) containing 115 amino acids that undergoes two successive proteolytic cleavages to yield the 84 amino acids PTH, the principal form of the hormone stored in and secreted from the parathyroid glands. Its metabolization is performed in the liver, releasing different amino-truncated PTH fragments in the circulation. These fragments are generally called C

Standardisation of the PTH assays

In 1974, at its 26th meeting, the Expert Committee on Biological Standardization of the World Health Organization (ECBS, WHO), asked the National Institute for Biological Standards and Control (London), to obtain material of human origin, which could serve as an international reference preparation (IRP). In December 1978, a nominal amount of 150 μg of hPTH, stated to be more than 95% pure, was donated to WHO as a proposed IRP. Ampoules, labelled 79/500 were prepared in 1981, evaluated in a

Reference method for PTH measurement

Liquid chromatographs coupled with mass spectrometers in tandem (LCMS/MS) have recently been used for PTH quantitative analysis [28], [29]. While very sensitive, the main drawback of these techniques lies in sample preparation. Indeed, peptides as large as PTH have to undergo enzymatic digestion, followed by fragment quantification. PTH quantification is then possible, but the additional information (e.g. oxidation, new fragments, etc.) may be lost. Ideally, we need a technique selective and

Sample stability and sample type

A huge number of studies are dealing with PTH stability and a meta-analysis has recently been published on the subject [30]. The authors of this review, on behalf of the IFCC Workgroup for PTH standardization, have strongly recommended that EDTA blood samples should be taken for PTH measurement. Even if two of us (EC and JCS) are participating to this workgroup, they do not agree, for the moment, with these recommendations. Indeed, we think that the different studies on the subject present too

Problems linked with the analytical phase: heterophilic antibodies interference

Just like any other immunoassays, PTH determination is prone to heterophilic antibodies interferences. This interference, due to the presence in the serum of the patients of immunoglobulins targeted against animal proteins, can lead to falsely elevated or decreased levels of the studied parameter. These interferences can lead to significant diagnostic errors and we have shown that patients presenting human anti-goat or anti-mouse antibodies could have large discrepancies in their PTH results

Problems linked with the post-analytical phase: reference values

Establishment of reference ranges is not an easy task [34]. First, a “reference population” needs to be defined and 120 samples for each sex and/or age category have to be taken in order to establish the ranges. This task is difficult, tedious, labour intensive, time consuming and difficult to achieve in all the laboratories, reason why the ISO 15189 guidelines that rule the quality in clinical laboratories ask them to “verify” the reference ranges provided by the manufacturers. For PTH, it is

Disclosure of interest

EC is consultant at DiaSorin and IDS.

EC has received lecture fees from Shire, Amgen, Pfizer, DiaSorin, IDS, Abbott, OCD and Roche.

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