The porphyrias are rare diseases that can be complicated to diagnose, and in most European countries the full spectrum of porphyria diagnostics is offered by national or specialized porphyria laboratories or diagnostic centres. Since 2001, many such porphyria centres have been cooperating under the EPI/EPNET, with non-European centres participating as associate members, striving to adhere to agreed quality criteria. This study shows that in 2009 there were large variations between porphyria centres with regard to the numbers and type of laboratory analyses performed, workload and dedicated personnel available. However, from 2006 through 2009 there was evidence of improvements in the analytical repertoire offered by participating centres. More importantly, increasing numbers of participating centres fulfilled the minimum criteria for being classified as an EPNET specialist porphyria centre; by 2009 94% fulfilled them.
As a part of EPNET’s WP6, the EPNET EQA scheme was established in 2008. Twice a year, biological specimens from a porphyria patient are distributed along with a case history to participating laboratories. The scheme covers diagnostic strategies, analytical laboratory performances, the clinical interpretation and reporting of results. Evaluation of the programme revealed large variations with regard to analytical and diagnostic performances . However, regular participation rapidly led to improvements in diagnostic strategies and more uniformly use of standardized units. The present study confirms that from 2006 through 2009, there was evidence of improvements in analytical laboratory services offered. The differentiation between the faecal coproporphyrin isomers I and III is of particular importance when diagnosing or excluding the rare HCP , and more centres could perform this analysis over time, and did so increasingly. Similarly, an increasing number of centres was able to distinguish between metal free and zinc-chelated protoporphyrin. This is of importance when diagnosing EPP , particularly in patients with a low erythrocyte protoporphyrin concentration and when differentiating between XLDPP caused by a mutation in ALA synthase 2 gene  and EPP caused by mutations in the ferrochelatase gene. Also, more centres offered plasma fluorescence scanning during the study period. This analysis is necessary to discriminate between both cutaneous and acute porphyrias, and of particular importance when diagnosing VP.
Having observed that more centres were able to perform essential laboratory analyses over time, we sought to investigate if centres that had introduced new analyses had in fact diagnosed and reported more cases of the relevant porphyrias. We were, however, not able to confirm any such trends (data not shown). HCP, EPP and VP are rare porphyrias of which most centres identify only a few new cases each year, some years none. Making the correct diagnosis in a symptomatic porphyria patient is a complex process, where offering the correct laboratory analyses is an essential, but not sufficient prerequisite. The clinician first has to suspect porphyria and send adequate biological samples to a diagnostic centre. After having performed and interpreted correctly the necessary laboratory analyses, the diagnostic centres can confirm or exclude the diagnosis. With such small numbers of new diagnoses reported each year, it is not surprising that an observed trend of improvement in laboratory analyses offered alone is not enough to show an improved ability to diagnose certain porphyrias. Still, we believe this to be an important step in the right direction for better care for porphyria patients.
In addition to the observed improvements in laboratory analyses offered by EPNET centres and associate members, improvement was evident from the increasing proportion of centres fulfilling the minimum criteria for being classified as a specialist porphyria centre, laboratories participating in EQA schemes and reporting to have accredited or certified facilities. The increased awareness of the porphyrias caused by the efforts of EPI/EPNET, the yearly activity feedback reports that may serve as a reminders of best practise and turn attention to areas where each centre might focus their efforts to improve their services, and the establishment of the EPNET EQA scheme in 2007  may be, at least in part, responsible for the observed improvements.
A considerable variation in numbers of individuals investigated per 100,000 inhabitants was observed between the 11 countries where participating centres reported that they provide national coverage of porphyria diagnostics, ranging from less than three to 15. The numbers of individuals investigated included both suspected new cases of porphyria as well as monitoring of known patients. The observed variation could therefore partly reflect the clinicians’ varying awareness of the porphyrias and willingness to send biological samples for testing. Also, routines for follow-up and monitoring of known patients differ between countries. For instance, where recommendations are made to analyse yearly control samples from patients in remission, as well as monitoring more frequently when symptoms occur, as in Norway, a relatively larger number of individuals investigated per 100,000 inhabitants are to be expected. Where recommendations for follow-up are not the same, not agreed upon or not widely practised, other numbers would be expected.
Large differences in the numbers of new diagnoses of AIP and PCT per 100,000 inhabitants were also evident. A certain degree of diversity in the occurrence of the porphyrias between countries is, however, to be expected. Most mutations of porphyria are private, but several founder mutations have been described, contributing to founder effects and higher occurrence of disease in geographic regions [10–13]. Also, the variation probably reflects differences in resources used to investigate healthy at-risk relatives with DNA testing. In some countries, reported new diagnoses of acute porphyrias consisted almost exclusively of presymptomatic cases, while other centres did not offer presymptomatic testing at all. In addition, in some countries or areas, intense case finding projects have already been performed, leaving fewer new cases to be diagnosed, which could also contribute to the observed differences.
In Europe, the proportion of new cases of PCT that were presymptomatic as opposed to symptomatic remained close to 10% throughout the study period, while the proportion of presymptomatic cases of AIP increased from 57% to 69%. The benefits of presymptomatic testing of healthy at-risk relatives in the acute porphyrias are obvious; knowledge about carrier status of a DNA mutation allows a person to avoid factors known to provoke acute attacks. Preventing acute attacks can improve quality of life, reduce the risk of potentially life threatening situations and reduce long term morbidity . The proportion of centres performing DNA-analyses did not increase during the study-period, suggesting perhaps a potential for identifying more at–risk relatives. For the cutaneous porphyria PCT, however, the benefits of presymptomatic DNA testing are more questionable. Although the cutaneous symptoms of PCT can be troublesome, uncomfortable and often long-lasting, they are not life-threatening and can in most cases be treated by repeated phlebotomies or low dose chloroquine treatment . In addition, a clinically indistinguishable form of non-inheritable PCT exists where the same environmental factors are necessary to cause disease (iron excess, hepatitis C infection, alcohol, oestrogens) . Thus, there is a risk of developing PCT independent of DNA mutation carrier status.
It is important to note that the reported numbers of new diagnoses in this study are hampered by uncertainties and should not be interpreted as figures of incidence. Presumably, many cases of the cutaneous porphyrias such as PCT, and to some extent EPP, are diagnosed and treated by dermatologist with little involvement from specialist porphyria centres, and thus never reported to EPNET. The reported national coverage of porphyria diagnostics is therefore thought to be more accurate for the acute porphyrias (ALA dehydratase deficiency, AIP, HCP and PV) than for the cutaneous porphyrias. Even so, a tendency towards overestimating the numbers of new acute porphyria diagnoses made may be present, as it cannot be ruled out that the same patient may occasionally be reported from more than one centre. On the other hand, centres that do not offer presymptomatic DNA testing probably report relatively lower numbers of new diagnoses. It is also likely that in certain regions and countries porphyria services could be substandard or lacking so that symptomatic patients are not diagnosed. True incidence rates of clinically overt inherited porphyrias in European countries have recently been investigated under the EPNET’s WP7 .
The minimum criteria for classification as a specialist porphyria centre set by EPNET are continuously evaluated and improved when considered necessary. For instance, participation in a relevant EQA program was introduced as a minimum criterion in 2009, and being able to differentiate between metal free and zinc-chelated protoporphyrin was added in 2010. At present, it is a concern that some centres perform only a limited amount of some or all laboratory analyses offered each year, which may compromise both the quality and the interpretation of the analyses. Therefore, some requirements regarding the numbers of yearly analyses performed may also, in the future, be included as part of the minimum criteria for classification as a specialist porphyria centre.