It's many years since my lab offered PTH assays (using our own reagents and assay systems, not kits) as part of its SAS menu, but in those days we recognized that "PTH" in blood represents a can of worms containing a miscellaneous collection of different molecules - intact PTH, fragments, etc. The introduction of 'two site' assays increased assay specificity, since many fragments would not react simultaneously with both antibodies. But this in itself does not guarantee that the assay system responds only the intact polypeptide, and is unaffected by fragments (some of which may interact with one or other of the antibodies used, possibly causing assay bias). In short even though PTH is an analyte of relatively simple molecular structure, and a 'right answer' to a measurement of its concentation in blood is in principle attainable , the 'right answer' will be obtained only if the assay system responds solely to the intact molecule. Also two PTH assay systems using different antibodies are likely to agree only if both respond only to the intact molecule even though both rely on the same "pure" PTH preparation as calibrant. If they don't, this is good evidence that one or other, or both, are also responding to fragments. In other words one or other or both assays constitute invalid 'analytical assays', and are therefore, in effect, 'comparative assays' as defined by Finney many years ago. Kit manufacturers faced with this sort of problem often cover up by altering the numbers on the standards so as to bring reference ranges more or less into line. (A notorious example of this was the misrepresentation of the values of FT4 standards by certain manufacturers) But this is in principle dangerous because it lulls users into a false sense of security, concealing the possibility that the various assays may give different results with certain pathological samples. Johnathan Middle is therefore right in asking "which (of two methods) is right?" if they yield different results. But I question his argument that the expression of results of measurements in SI units implies that they " must therefore be traceable back to a primary standard which contains a certified molar content of a single defined chemical entity ('PTH') and this entity must be the same as the single defined chemical entity ('PTH') in all patient samples". We can express the results of a gravimetric measurement in Kgs (a well known SI unit) , but this does not imply that that the measurement of the weight of a mixture of different substances (and the expression of the result in Kgs) is inadmissible. In other words, the expression of a result in SI units does not imply that the measurement is of the amount or concentration of a "single defined chemical entity". JM is, I think, confusing the issue of the units used to express assay results with that of whether an assay constitutes valid "analytical assay", i.e an assay that responds only to a single molecularly homogeneous substance present in standards and unknowns. In practice, many - probably most - of the proteins measured by immunoassay do not fit this description, and are of heterogeneous molecular composition. HCG is a typical example. An assay of HCG in blood is thus a "comparative assay" . It has long been known that such assays cannot be standardised. Nor do they have "right answers". But this has nothing to do with the nature of the units in which results are expressed Regrettably organisations such as NIBSC, and others have - by distributing international refence preparations whose analyte "content" is expressed in international units (claimed as constituting a "common currency") - been the originators and principal perpetuators of the myth that the results of different comparative assays (on individual samples) should be identical when expressed in such units, and that if they are not there is something wrong with the assays or their standardisation. Such organisations have stubbornly resisted the implication that the only way of dealing with this problem (other than by developing assays for each component of a heterogeneous analyte, using, eg, microarray chip technology) is by abandoning the use of either SI or international units to express assay results , and to replace them by "method dependant" units. Hopefully this would at last destroy the fiction that the results of different (comparative) immunoassays should be numerically the same or necessarily possess the same diagnostic value. -- Regards Roger Molecular Endocrinology University College London Medical School London W1N 8AA Fax +44 20 7580 2737 Phone +44 20 7679 9410 The information in this e-mail is confidential and may be legally privileged. It is intended for the exclusive attention of the addressee stated above and should not be copied or disclosed to any other without the express agreement of the sender.