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European specialist porphyria laboratories: diagnostic strategies, analytical quality, clinical interpretation, and reporting as assessed by an external quality assurance program.

The porphyrias, most of which have an inherited basis, are a heterogeneous group of rare metabolic disorders that are caused by abnormal function of the enzymes of heme biosynthesis. They are usually divided into acute and cutaneous porphyrias, according to the type of symptoms (1). In symptomatic patients, the diagnosis depends on identification of specific patterns of porphyrin precursor and porphyrin accumulation in urine, feces, and blood that characterize each enzyme defect (2). As with other inborn errors of metabolism (3), however, the field of porphyrias has not yet fully benefited from the impact of evidence-based medicine, and the quality of diagnostic services is likely to vary. The European Porphyria Network (EPNET) [7] was established, initially as a 3-year project (4) funded by the European Commission through its Public Health and Consumer Protection Directorate, with the aim of setting up an effective network of European specialist porphyria laboratories and diagnostic centers. An EPNET specialist porphyria laboratory/center must be able to distinguish between all types of porphyria, requiring performance of the following analyses: urinary [delta]-aminolevulinic acid (ALA), porphobilinogen (PBG) and porphyrins (total/fractionation), fecal porphyrins (total/fractionation including coproporphyrin isomer differentiation), erythrocyte protoporphyrin (total/fractionation), and plasma fluorescence scanning. In addition, these specialist laboratories must provide interpretation of test results, offer clinical advice, and participate in an external quality assessment scheme (EQAS). In 2008, EPNET set up a clinical case-based EQAS covering preanalytical, analytical, and postanalytical aspects for all porphyria-related biochemical analyses, an area which no other publication or EQAS has previously addressed. The aim of this study was to evaluate the performance of European specialist porphyria laboratories based on results from the EPNET EQAS, with particular focus on each laboratory's selected diagnostic strategies, analytical quality, ability to establish the correct diagnoses, and reporting.

Methods

We distributed 5 sets of QC materials to specialist porphyria laboratories (5) at half-year intervals during 2008-2010 (distributions A-E). Twenty-one laboratories/centers in 15 European countries participated in 4 or more surveys and were included in this study. Each set of samples was obtained from a different patient with an unequivocally established porphyria diagnosis and was accompanied by a relevant case history (Table 1). Each laboratory received 5 mL urine, 5 g feces, 3 mL plasma, and 3 mL whole blood, sent by express service frozen on ice and light-protected. Samples were delivered within 3 days for distribution A and within 2 days for distributions B-E. Laboratories were asked to freeze the study material upon arrival and then to analyze the samples in their routine procedure for all available porphyria-related biochemical analyses. The participants reported preanalytical, analytical, and postanalytical results as described in Table 2 in a standardized Microsoft Excel worksheet within a given date. On the completion of each survey, the participants received a feedback report comparing their results with anonymous results of the other participants, including recommendations on the use and handling of sample materials, methodology, and units.

To assess metabolite stability during transportation, packed frozen study materials on ice were left at room temperature at the Norwegian Porphyria Centre for 0, 1, 2, and 3 days before being frozen, then thawed and analyzed as described in Aarsand et al. (6). If necessary, reported EQAS results were converted into units given in Table 3 before data analysis. To account for reference interval variation, results were also normalized i.e., expressed as the ratio between the reported result and the laboratories' quoted upper reference limit for each analyte (Table 4).

General statistical analysis was performed using Microsoft Excel 2003[R] and SPSS[R] 17.0 for Windows. Outliers were in general defined as values outside the overall mean [+ or -] 3 SD, but for calculation of analytical quality performance limits, the method group mean was used. Analytic quality specifications were established using data on biological variation, with desirable analytical quality being calculated as total allowable error ([TE.sub.a]): 0.25 x [square root]([CV.sup.2.sub.I] + [CV.sup.2.sub.G]) + 1.65 x (0.5 x [CV.sub.I]) (7), where [CV.sub.I] is the within-subject (intraindividual) and CVG the between-subject biological variation. By use of data from patients with stable acute porphyria (8),[TE.sub.a] was 20% for ALA and 31% for PBG. The within-subject variation corresponding to the 90th percentile was used to calculate the [TE.sub.a] of 50% for urinary total porphyrins, as the variances were not distributed homogeneously (9). With use of these values as a basis, because biological variation data were not available, [TE.sub.a] was arbitrarily set to 50% for fecal and plasma total porphyrins and 30% for erythrocyte protoporphyrin.

The laboratory reports were judged by criteria developed on the basis of the Best Practice Guidelines of the Swiss Society of Medical Genetics (10). The following 14 criteria were awarded 1 point if fulfilled/present: laboratory name, laboratory contact details, date of report, name of referring doctor/report destination, patient name and date of birth, date of sampling, date of arrival, material tested, analyses performed, quantitative or qualitative results given, units and reference intervals stated, interpretation of results given, advice on further testing given if appropriate, and name/signature of verifying/responsible laboratory personnel.

Results

More than 80% of the QC material sets were delivered within 1 day, with an additional 15% delivered within 2 days. The stability studies showed that systematic differences in metabolite levels between day 1 and 2 were <5%.

PREANALYTICAL ASPECTS: DIAGNOSTIC STRATEGIES

The number of analyses that the participants would normally have performed based on the clinical case histories ranged from 4 to 8 in A, 2 to 9 in B, 3 to 10 in C, 4 to 11 in D, and 4 to 10 in E, excluding enzyme and DNA analyses (Table 5).

ANALYTICAL ASPECTS

For all distributions, wide variations were seen in the reported results (Table 3; also see Supplemental Fig. 1, which accompanies the online version of this article at http://www.clinchem.org/content/vol57/issue11). When the reported EQA results were normalized by their corresponding upper reference limits, up to 40-fold variations were still apparent (Table 4). In the 3 patients with an acute porphyria, all laboratories reported increased excretion of PBG for distribution A, but 2 and 5 laboratories failed to detect the borderline (2- and 4-fold) increases in distributions D and E, respectively. All laboratories submitting results for erythrocyte protoporphyrin fractions found predominantly free protoporphyrin in the erythropoietic protoporphyria (EPP) patient. In the patient with porphyria cutanea tarda (PCT), a typical urinary uroporphyrin/heptaporphyrin ratio (range 2.1-4.3) was reported by all participants (6). A plasma fluorescence emission peak was found by 14 laboratories in A (n = 16), 15 in B(n = 20), 20 in C and D (n = 20 each), and 2 in E (n = 20). In distribution B, 3 of the laboratories reported peaks at 626-629 nm; in distribution D, 18 laboratories reported peaks at 624-628 nm and 2 at 623 and 629 nm. Seven of 11 and 5 of 10 results from distributions D and E, respectively, were reported as positive for qualitative/semiquantitative PBG analysis. In distribution E, 13 of the 15 reported results were belowthe lower reference limits for porphobilinogen deaminase (PBGD), and 2 of 4 for uroporphyrinogen decarboxylase (UROD).

Assessment of analytical performance in relation to quality specifications showed that most laboratories met the desirable target for urinary total porphyrins (see online Supplemental Table 1). The percentage of laboratories achieving desirable analytical quality on average for all distributions was, for ALA, 76%; for PBG, 57%; for fecal porphyrins, 63%; for erythrocyte protoporphyrin, 53%; and for plasma porphyrins, 43%. Overall, desirable analytical quality was achieved for 65% of all results.

Most laboratories used the ALA/PBG Column Test (Bio-Rad Laboratories) for ALA and PBG analysis, HPLC or spectrophotometric methods for estimation of porphyrins, and HPLC for fractionation of urinary and fecal porphyrins. During the study period, some laboratories changed to more standardized methods, set up additional porphyria-related analyses, and replaced 24-h urine collections with first morning voids as sample material for urine analyses. Methodological differences were seen for PBG and fecal total porphyrins in distributions A and C and urinary total porphyrins in B when assessed between method groups with [greater than or equal to] 5 participating laboratories.

POSTANALYTICAL ASPECTS: CLINICAL INTERPRETATION AND REPORTING

For the first 3 distributions, all the specialist porphyria laboratories reported the correct diagnoses: acute intermittent porphyria (AIP), EPP, and PCT (Table 1). In distribution D, 20 of 21 laboratories reported the correct diagnosis of variegate porphyria (VP), whereas in distribution E, 13 of 21 laboratories reported the confirmed diagnosis of AIP, 2 reported a highly likely/probable diagnosis of AIP, and 2 a probable diagnosis of an acute porphyria. The remaining 4 laboratories ruled out porphyria as a cause for the patient's symptoms, but 3 included in their evaluation a request for new samples if the patient experienced symptoms compatible with an acute attack.

With the exception of 1 laboratory in the first distribution, all would have reported the diagnosis or the exclusion of a diagnosis to the requesting physician/ward. In addition, irrespective of the type of disease, more than 80% of the laboratories provided an interpretation of the analytical results, and approximately half included clinical advice in their written response. Eighty percent of laboratory reports scored [greater than or equal to] 13 points out of a total of 14 (range 5-14). For the reporting of urinary metabolites, from 6 to 8 different quantitative units were used initially, but about 30% of the participants altered units, primarily to reporting metabolite excretion per mmol creatinine, during the study period. Some changes were also seen in reporting the other analytes, where initially 3-5 different units were used for reporting.

Discussion

Establishing a porphyria diagnosis requires the analysis of porphyrin precursors and porphyrins in appropriate samples. Moreover, information on type of symptoms and whether the samples were obtained in an active disease phase or not is necessary for choosing the correct diagnostic strategy and the adequate interpretation of results. The knowledge of porphyria diagnostics among clinicians and the availability of specialized porphyria analyses in general laboratories is limited. Thus, in many countries, laboratories and centers specializing in porphyrias have developed. As our study shows, however, there are variations with regard to diagnostic strategies, analytical quality, clinical interpretation, and reporting, even between these specialized laboratories and diagnostic centers.

PREANALYTICAL ASPECTS: DIAGNOSTIC STRATEGIES

Evaluating the laboratories' selection of analytes based on the clinical case histories showed that diverse diagnostic strategies were applied (Table 5). However, all laboratories would have analyzed PBG, the key diagnostic analysis in patients where acute porphyria is suspected (11), and all but 1 would have analyzed ALA in the 3 acute porphyria patients (distributions A, D, and E). For the diagnosis of AIP, VP, and hereditary coproporphyria (HCP), the 3 most common acute porphyrias, analysis of ALA is not strictly necessary, but the majority of laboratories used the Bio-Rad ALA/PBG Column Test in which ALA and PBG can be analyzed consecutively. In addition, ALA is required for the diagnosis of the extremely rare ALA-dehydratase deficiency that also can present with acute symptoms (12). In AIP, HCP, and VP, patients in acute attacks invariably present with increased ALA and PBG. The increased excretion normally persists in AIP (13), but in VP and HCP it may quickly normalize with the subsidence of acute symptoms (1, 14, 15). The urine samples in the 2 last distributions (D and E) were not collected during an acute attack (Table 1). Laboratories not performing specific diagnostic tests, such as plasma fluorescence scanning (16) and fecal coproporphyrin isomer (CIII:I) ratio (17) in these situations, are likely to miss or misdiagnose the type of acute porphyria. The combination of urinary PBG taken in relation to acute symptoms, plasma fluorescence scanning, and fecal CIII:I ratio is thus required to diagnose or exclude AIP, VP, and HCP (11, 12). Initially, only 8 laboratories would have performed this set of analyses. This figure increased to 16 laboratories during the study period, reflecting the implementation of improved diagnostic strategies. About a fourth of the participants would have analyzed erythrocyte protoporphyrin in the acute patients, which is of little value unless ALA-dehydratase deficiency or lead intoxication is suspected (18). The patient in distribution B was a 5-year-old boy who cried when outdoors but had no reported visible skin lesions. In this case, 40% of the laboratories would have analyzed urinary ALA and PBG, which have no diagnostic relevance. Most laboratories would, however, have performed analysis of erythrocyte free protoporphyrin, on which the diagnosis of EPP depends (18). In addition, fecal protoporphyrin and urinary coproporphyrin isomers may be of value in EPP when assessing liver function and risk of liver complications over time (19). In distribution C, a patient with erosions and skin fragility was described; thus PCT, VP, and HCP must all be considered, even if HCP rarely presents with cutaneous symptoms alone (20). About 70% of laboratories would have analyzed ALA and PBG, even when the combination of plasma fluorescence scanning and urinary and fecal porphyrin fractionation would be adequate initially (18 of 21 laboratories), and a normal plasma fluorescence scan would exclude all cutaneous porphyrias in a patient with active skin lesions (18). However, a clinically targeted cost-efficient diagnostic strategy may also carry a risk of missed diagnoses, as it depends on accurate clinical information supplied together with samples.

ANALYTICAL ASPECTS

The patients included in our EQA scheme had increased levels of the porphyrins and porphyrin precursors relevant to their diagnoses (Table 1). All participants reported increased levels of relevant markers in the first 3 distributions, where levels were substantially increased, but with wide ranges for both reported (Table 3) and normalized (Table 4) results. In distributions D and E with lower metabolite concentrations, however, some laboratories reported results within reference intervals. In a study on EQAS of 6 lysosomal enzymes, the mean interlaboratory CV decreased from 49% to 26% after normalization by the upper reference limit (21), whereas such normalization did not reduce the interlaboratory CV in our study. Thus, different reference limits cannot explain the large analytical variations, nor can the use of different methods, even if methodological differences were seen for some analytes in the first distributions. Although the stability studies showed that only minor alterations occurred (discounting transportation as a factor in poor performance), inadequate sample handling at recipient laboratories cannot be ruled out. In general, analytes measured as absolute concentrations (ALA, PBG, urinary/fecal/erythrocyte/plasma porphyrins) displayed the greatest variation. Considering the ranges and variations seen for these analytes, it is thus likely that some laboratories report false negative and/or false positive results that could lead to misdiagnosis. For the fractionations, the results were quite consistent, as the high CVs for some of the porphyrins reflect low concentrations. It is of importance, however, that some laboratories would have performed fractionation only if the concentrations of porphyrins in the relevant specimen exceeded a preset cutoff level. Thus, both an accurate calculation of the total porphyrin concentration and an adequate cutoff level for fractionation are necessary to catch a porphyria diagnosis that is dependent on the fractionation results. Positive and negative results were quite evenly distributed between the 3 qualitative/ semiquantitative PBG methods used, illustrating the well-known limitation of these screening tests, particularly in patients with borderline PBG concentrations as seen in distributions D and E.

Analytical performance in relation to quality specifications based on biological variation varied considerably between laboratories (see online Supplemental Table 1), with 3 laboratories meeting the desirable target for <50% of their results. For PBG, a key diagnostic analysis, 7 laboratories reported fewer than half of their results within given quality specifications, as was also reflected in the reduced diagnostic performance in distribution E. If applying more or less stringent analytical quality specifications (7), about 40% and 80% of all results attained "optimal" and "minimal" performance, respectively. In an EQA program of quantitative determination of amino acids, Fowler et al. (22) found that 10 amino acids were analyzed with good interlaboratory precision (CV <10%), 11 with intermediate (CV 10%-22%), and 8 with poor (CV >22%). In our EQAS, assays for all 6 constituents reported in absolute concentrations, except for ALA in 3 distributions, demonstrated poor performance according to these criteria, with a mean interlaboratory CV of 50%.

Certain biochemical findings are characteristic for the different porphyrias, such as the increase of fecal isocoproporphyrin, a specific marker for PCT (23 ). All laboratories reported increased isocoproporphyrin levels (range 13%-49%) (Table 3) in the PCT patient, but most laboratories also reported increased, but significantly lower, levels in distributions A and D (ranges 1%-7% and 0.1%-8%, respectively). A plasma fluorescence emission maximum of624-628 nm is considered diagnostic for VP, and the cut-point of 623 nm can be used to distinguish VP from AIP and HCP (11, 16). Some reported plasma fluorescence peaks in distribution D were outside this range, but were still given as consistent with VP. In the EPP patient, 3 laboratories reported peaks at 626-629 nm, indicating that unless clinical information was present and adequate diagnostic strategies were applied, misdiagnosis would be possible. The diagnosis of HCP is made on the demonstration of increased fecal CIII:I ratio after excluding VP by determination of the fluorescence emission wavelength (11, 17). The performance of the CIII:I ratio is an important indicator of laboratory expertise in porphyria diagnostic testing, and a specific criterion for designation by EPNET as a specialist porphyria laboratory. Reassuringly, the CIII:I ratio was reported as <1.5 by all participants in the 4 non-VP patients, and as moderately increased consistent with the diagnosis of VP (11) by all but 1 laboratory in distribution D. In distribution E, most laboratories used the level of PBGD activity as part of their diagnostic evaluation, and 1 laboratory diagnosed AIP based on the finding of low PBGD activity despite reporting a urinary PBG result within the reference interval in this patient. PBGD activity has, however, been shown to differentiate between patients with AIP and their healthy relatives with a likelihood ratio of 3.6 (13).

POSTANALYTICAL ASPECTS: CLINICAL INTERPRETATION AND REPORTING

Based on the results of all porphyria-related analyses available in their laboratories, the participants reported the correct diagnosis in the distributions where the levels of diagnostic markers were high (A-C). In the distribution E AIP sample, where the concentration of PBG was lower, 5 laboratories reported PBG values within reference intervals, and 4 thus missed the diagnosis. One laboratory misdiagnosed the VP patient in distribution D as HCP based on inadequate diagnostics and interpretation. Our results show that patient cases with borderline analyte levels pose a particular challenge, underlining the importance of EQA schemes that circulate cases having a wide variety of analyte concentrations. Laboratories applying inadequate diagnostic strategies are especially at risk for reduced diagnostic performance. However, overall clinical interpretation of the obtained analytical results was good.

Specialist laboratories reported the diagnosis along with the laboratory results to the requesting physician/ward, and a large percentage included both a detailed interpretation and clinical advice. In general, interpretative comments were adequate, but considerable diversity was seen. The request for new samples if the patient experienced acute symptoms, as given by 3 of the 4 laboratories that excluded an acute porphyria in distribution E, underlines the importance of relevant clinical information for adequate commenting of results. Most of the laboratory reports obtained a high score, with a few laboratories consistently obtaining the lowest scores. The most frequently missing information items were date of sampling, date of arrival, and name of referring doctor/report destination, but it is possible that such information was less diligently entered in the laboratory information system because the samples were not from an actual patient.

Conclusions

EQAS participation is essential for providing quality laboratory diagnostic services and is required for laboratory accreditation. Only a few reports on EQA in porphyria-related analyses have been published (24-26). In general, these report large variations, with CVs ranging from 6% to >200% for the small number of analytes performed in nonspecialist laboratories that were included in these schemes. There are no publications on EQA programs that address diagnostic strategies, the full spectrum of analyses relevant for porphyria diagnostics, or the ability of the laboratories to make a correct diagnosis. Standards such as the ISO 15189 (27) require that EQA programs check the entire examination process including pre- and postexamination procedures. For rare diseases with complex diagnostic testing such as porphyrias, ensuring an acceptable quality not only of the analytical components, but also of the diagnostic and clinical services offered, is essential. Schemes that assess these aspects exist for some other types of rare diseases (22, 28, 29). Our study shows that there are variations with regard to both analytical and diagnostic performance among specialist porphyria laboratories to an extent that undoubtedly can affect the quality of patient care. It is thus encouraging to see that regular participation in EQA schemes for rare diseases enhances performance (22, 28), and in our study clear improvements with regard to both standardized use of units and diagnostic strategies are already seen. The EPNET EQA scheme for porphyria will therefore continue and expand, with focus on optimization of sample preparation and handling, diagnostic strategies, methodology, analytical performance, and clinical interpretation. The scheme has been widely accepted, as indicated by an increasing number of participating specialist laboratories--at present 30--worldwide. Future plans also include standardization of methods and the development of evidence-based diagnostic strategies and criteria through international collaboration. Ensuring high analytical and diagnostic quality is essential to provide correct diagnostics and optimal care of porphyria patients.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: Cofunded by the European Commission through its Public Health and Consumer Protection Directorate (DG SANCO), PHEA Program.

Expert Testimony: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

Acknowledgments: We thank the porphyria patients for providing sample material, the staff at the Norwegian Porphyria Centre (NAPOS) and the Norwegian Quality Improvement of Primary Care Laboratories (NOKLUS) for their great organizational skills, and all the participating laboratories.

References

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Aasne K. Aarsand, [1] * Jorild H. Villanger, [1] Egil Stole, [1] Jean-Charles Deybach, [2] Joanne Marsden, [3] Jordi To-Figueras, [4] Mike Badminton, [5] George H. Elder, [5] and Sverre Sandberg [1,6]

[1] Norwegian Porphyria Centre (NAPOS), Laboratory of Clinical Biochemistry, Haukeland University Hospital, Bergen, Norway; [2] Assistance Publique-Hopitaux de Paris, Centre Francais des Porphyries, Hopital Louis Mourier, Colombes CEDEX and INSERM Unite 773, Centre de Recherche Biomedicale Bichat-Beaujon, University Paris Diderot, Paris, France; [3] Department of Clinical Biochemistry, King's College Hospital NHS Foundation Trust, Denmark Hill, London, UK; [4] Biochemistry and Molecular Genetics Unit, Hospital Clinic, IDIBAPS, University of Barcelona, Barcelona, Spain; [5] Department of Infection, Immunity and Biochemistry, School of Medicine, Cardiff University, Heath Park, Cardiff, UK; [6] Norwegian Quality Improvement of Primary Care Laboratories (NOKLUS), Section for General Practice, University of Bergen, Bergen, Norway.

* Address correspondence to this author at: Norwegian Porphyria Centre, Laboratory of Clinical Biochemistry, Haukeland University Hospital, NO-5021 Bergen, Norway. Fax +47-55973115; e-mail aasne.aarsand@helse-bergen.no.

Received June 9, 2011; accepted August 19, 2011.

Previously published online at DOI: 10.1373/clinchem.2011.170357

[7] Nonstandard abbreviations: EPNET, European Porphyria Network; ALA, [delta]-aminolevulinic acid; PBG, porphobilinogen; EQAS, external quality assessment scheme; [TE.sub.a], total allowable error; EPP, erythropoietic protoporphyria; PCT, porphyria cutanea tarda; PBGD, porphobilinogen deaminase; UROD, uroporphyrinogen decarboxylase; AIP, acute intermittent porphyria; VP, variegate porphyria; HCP, hereditary coproporphyria; CIII:I, coproporphyrin isomer III:I.
Table 1. The 5 EPNET EQAS distributions: clinical case histories,
diagnoses, and typical biochemical features (only the case histories
accompanied the distribution of sample sets to participants).

Distribution Case history Diagnosis

A Female born 12/23/1977 has had AIP
 repeated attacks of abdominal pain
 and nausea since she was 14. She
 has been hospitalized once with
 "acute abdominal pain" with no
 surgical intervention, and was
 discharged after 3 days without
 further treatment. The samples were
 obtained 14 days after her last
 attack.

B A 5-year-old boy was referred to a EPP
 pediatric clinic after having
 presented to the family physician
 on several occasions, after crying
 spells of unknown cause. According
 to the boys' parents, the crying
 spells always occurred when
 outdoors. The family doctor
 referred the patient to a pediatric
 clinic. After extensive
 investigations over a 6-month
 period, samples were sent for
 porphyrin analysis.

C A 67-year-old man with diabetes PCT
 mellitus, who had had a stinging
 rash in the face for about a year,
 consulted his [GP.sup.a] after he
 also developed ulcers and scarring
 on the hands. The GP considered
 rosacea a likely diagnosis for the
 facial symptoms, but referred the
 patient to the Dermatology Clinic
 for the hand symptoms. The
 dermatologist suspected that a
 porphyrin disease would be the most
 likely cause of the main skin
 symptoms, and sent samples for
 porphyrin analysis.

D A woman born 1957 visited her GP VP
 with complaints of depressive
 symptoms and unspecific mild
 stomach ache. As it is noted in the
 journal that the patient has
 relatives with porphyria, samples
 are sent for porphyrin analysis.

E Female, born 1962, has experienced AIP
 3 "attacks" of abdominal pain with
 additional complaints of muscle
 weakness and nausea, the first of
 which occurred during her first
 pregnancy. She has periodically
 suffered from depression. Her
 doctor sent samples for porphyrin
 analysis, obtained 2 months after
 she had recovered from the last
 attack.

 Typical biochemical features in symptomatic
 patients Deacon and Elder (18)]

Distribution Urine Plasma

A Increased PBG and ALA Increased Fluorescence
 total porphyrins Increased emission peak
 uroporphyrin at 615-620 nm

B PBG and ALA within reference Fluorescence
 interval Total porphyrins within emission peak
 reference interval at 626-634 nm

C PBG and ALA within reference Fluorescence
 interval Increased total porphyrins emission peak
 Increased uro-and heptaporphyrins at 615-620 nm

D Increased PBG and ALAb Increased Fluorescence
 total porphyrins Increased emission peak
 coproporphyrin at 624-628 nm

E Increased PBG and ALA Increased Fluorescence
 total porphyrins Increased emission peak
 uroporphyrin at 615-620 nm

 Typical biochemical features in
 symptomatic patients Deacon and
 Elder (18)]

Distribution Feces Erythrocytes

A Total Protoporphyrin
 porphyrins within
 within reference
 reference interval
 interval or
 slightly
 increased

B Increased total Increased free
 porphyrins protoporphyrin
 Increased
 protoporphyrin

C Increased total Protoporphyrin
 porphyrins within
 Increased reference
 heptaporphyrin interval
 and
 isocoproporphyrin

D Increased total Protoporphyrin
 porphyrins within
 Increased reference
 copro-and interval
 protoporphyrins
 CIII:I isomer
 ratio >2.0

E Total Protoporphyrin
 porphyrins within
 within reference
 reference interval
 interval or
 slightly
 increased

(a) GP, general practitioner.

(b) Urinary PBG and ALA are usually within reference
intervals when only skin lesions are present.

Table 2. Reporting details for the EPNET EQAS for specialist porphyria
laboratories.

 Analytical aspects:
Preanalytical aspects: laboratory analyses, with information on
diagnostic strategies methodology, units, and reference limits

 Material Analyte

Selection of Urine ALA
 analyses based on
 clinical case history

 PBG

 Qualitative/
 semiquantitative PBG (a)

 Total porphyrins

 Porphyrin fractions
 Uroporphyrin

 Total and isomers I and III

 Heptaporphyrin

 Hexaporphyrin

 Pentaporphyrin

 Coproporphyrin
 Total and isomers I and III

 Feces Percentage dry weight

 Total porphyrins

 Porphyrin fractions
 Uroporphyrin
 Total and isomers I and III

 Heptaporphyrin

 Hexaporphyrin

 Pentaporphyrin

 Coproporphyrin
 Total and isomers I and III

 Isocoproporphyrin

 Protoporphyrin

 Other dicarboxylated
 porphyrins
 Deutero- and mesoporphyrins

 Whole Erythrocyte protoporphyrin
 blood
 Erythrocyte protoporphyrin
 fractions

 Zinc protoporphyrin

 Free protoporphyrin

 Porphobilinogen deaminase (b)

 Uroporphyrinogen
 decarboxylase (b)

 Plasma Total porphyrins (c)

 Plasma fluorescence scanning

 Wavelengths for excitation
 and emission peaks

Preanalytical aspects: Postanalytical aspects:
diagnostic strategies clinical interpretation
 and reporting

Selection of The patient's diagnosis based on
 analyses based on the clinical case history and all
 clinical case history laboratory results

 Details on what would be reported
 to the requesting clinician

 Analytical results with or without
 interpretative comments

 Diagnosis

 Clinical information and advice
 for the physician and patient

 How the results would be reported
 (written and/or oral communication)

 Laboratory report forms including
 comments in English

(a) Distributions D and E.

(b) Distribution E.

(c) Distributions B, C, D, and E.

Table 3. Diagnostically important metabolites for all EQAS
distributions. (a)

 A, AIP B, EPP

 Mean CV% Mean CV%

ALA, [micro]mol/mmol 65.8 (b) 17.4 (b) 1.6 (b) 28.6 (b)
 creatinine
PBG, [micro]mol/mmol 53.7 31.0 0.6 63.6
 creatinine
Urinary total porphyrins, 651 39.3 10.2 (b) 59.1 (b)
 nmol/mmol creatinine
 Uroporphyrins (c) 85.1 5.4 29.4 33.4
 Heptaporphyrins (c) 1.2 77.2 13.1 42.5
 Coproporphyrins (c) 11.7 37.7 50.0 24.0
Fecal total porphyrins, 102 59.2 301 56.3
 nmol/g dry weight
 Coproporphyrins (c) 29.9 37.3 5.9 65.2
 CIII:I ratio 0.5 26.2 0.2 66.1
 Isocoproporphyrins (c) 3.2 90.5 0.0 211
 Dicarboxylated 36.0 44.6 92.9 5.6
 porphyrins (c)
Erythrocyte 0.7 49.6 40.2 (b) 63.0 (b)
 protoporphyrin,
 [micro]mol/L
 erythrocytes
 Zinc protoporphyrin, % 81.7 11.4 4.4 88.2
 Free protoporphyrin, % 18.3 50.9 95.6 4.0
Plasma total porphyrins, -- -- 85.9 81.4
 nmol/L

 C, PCT D, VP

 Mean CV% Mean CV%

ALA, [micro]mol/mmol 2.1 (b) 22.4 (b) 7.3 (b) 11.6 (b)
 creatinine
PBG, [micro]mol/mmol 0.3 51.7 2.4 (b) 29.2 (b)
 creatinine
Urinary total porphyrins, 1043 41.6 145 26.2
 nmol/mmol creatinine
 Uroporphyrins (c) 68.1 (b) 7.9 (b) 22.3 31.9
 Heptaporphyrins (c) 22.6 (b) 11.1 (b) 7.6 31.1
 Coproporphyrins (c) 1.9 (b) 51.1 (b) 60.8 15.1
Fecal total porphyrins, 495 55.2 1541 56.7
 nmol/g dry weight
 Coproporphyrins (c) 16.6 (b) 39.5 (b) 28.7 36.7
 CIII:I ratio 0.5 (b) 75.6 (b) 5.2 40.1
 Isocoproporphyrins (c) 26.2 (b) 40.4 (b) 1.8 140
 Dicarboxylated 12.1 (b) 66.6 (b) 66.5 19.2
 porphyrins (c)
Erythrocyte 0.9 65.5 1.4 (b) 29.6 (b)
 protoporphyrin,
 [micro]mol/L
 erythrocytes
 Zinc protoporphyrin, % 70.8 (b) 15.9 (b) 75.8 26.6
 Free protoporphyrin, % 29.2 (b) 38.6 (b) 24.2 83.2
Plasma total porphyrins, 309 61.3 59.4 (b) 151 (b)
 nmol/L

 E, AIP

 Mean CV%

ALA, [micro]mol/mmol 7.5 18.5
 creatinine
PBG, [micro]mol/mmol 6.1 64.4
 creatinine
Urinary total porphyrins, 51.1 28.1
 nmol/mmol creatinine
 Uroporphyrins (c) 23.6 37.6
 Heptaporphyrins (c) 2.5 60.1
 Coproporphyrins (c) 65.3 15.5
Fecal total porphyrins, 66.1 47.6
 nmol/g dry weight
 Coproporphyrins (c) 20.3 (d) 44.4 (d)
 CIII:I ratio 0.6d 32.6 (d)
 Isocoproporphyrins (c) 0.1d 300 (d)
 Dicarboxylated 76.7d 13.8 (d)
 porphyrins (c)
Erythrocyte 0.9 49.3
 protoporphyrin,
 [micro]mol/L
 erythrocytes
Zinc protoporphyrin, % 68.8 37.5
Free protoporphyrin, % 31.1 83.2
Plasma total porphyrins, 5.6 72.7
 nmol/L

(a) The number of results available for calculations varied from 17
to 21, except for fecal porphyrin and erythrocyte protoporphyrin
fractions and plasma porphyrins (n = 10-16).

(b) One outlier was excluded before calculations. All fractions in the
same specimen were excluded if 1 fraction was defined as an outlier.

(c) Only diagnostically most relevant porphyrin fractions are given,
as percentage of total urinary and fecal porphyrins (see Table 2).
Dicarboxylated porphyrins consisted of the sum of deutero-, meso-,
and protoporphyrin.

(d) All fractions excluded for 3 laboratories owing to outliers.

Table 4. Normalized results: ratio between the measured value and its
upper reference limit given for major metabolites in all
distributions. (a)

 A, AIP B, EPP

Urinary ALA 14.8 0.4
 (11.5, 26.0) (b) (0.1,0.8)
Urinary PBG 46.5 0.3
 (20.1,65.3) (0.04, 0.6)
Urinary total porphyrins 16.4 0.2
 (11.0, 39.8) (0.1,0.7)
Fecal total porphyrins 0.8 1.3
 (0.3, 6.8) (0.7, 4.5)
Erythrocyte protoporphyrin 0.6 25.0
 (0.1,0.8) (3.3, 68.7)
Plasma total porphyrins -- 5.3
 (0.3,12.5)

 C, PCT D, VP

Urinary ALA 0.5 1.7
 (0.4, 0.9) (b) (1.4, 2.2) (b)
Urinary PBG 0.3 2.0
 (0.04, 0.6) (1.0,3.2)
Urinary total porphyrins 30.9 4.6
 (12.7, 71.0) (b) (2.2, 7.2) (b)
Fecal total porphyrins 3.9 10.6
 (1.3, 4.6) (b) (3.3,19.3) (b)
Erythrocyte protoporphyrin 0.7 0.8
 (0.2, 1.3) (0.4,1.1) (b)
Plasma total porphyrins 17.8 4.4
 (1.8, 56.0) (0.4, 33.3)

 E, AIP

Urinary ALA 1.5
 (0.8, 2.2) (b)
Urinary PBG 4.1
 (0.4,11.4)
Urinary total porphyrins 1.4
 (0.8,2.7)
Fecal total porphyrins 0.3
 (0.2, 0.6)
Erythrocyte protoporphyrin 0.5
 (0.2, 1.1)
Plasma total porphyrins 0.5
 (0.3, 0.6)

(a) Data are medians (10th and 90th percentiles). Reference limits
available from 16-21 laboratories, except for urinary total porphyrins
(A, B), fecal total porphyrins (A-C), erythrocyte protoporphyrin (all
but C), and plasma porphyrins (all) where n = 9-15.

(b) One outlier excluded before calculations. Five of the 9 outliers
were reported from the same laboratory.

Table 5. Laboratories' selection of porphyria-related
analyses given the clinical case histories, with bold values
indicating the appropriate first-line diagnostic strategy
in each case.

 A, AIP B, EPP C, PCT

n 18 21 21
Urine
 ALA 17 8 13
 PBG 18 (a) 8 14
 Total porphyrins 13 10 16
 Porphyrin fractionation 10 6 21 (b)
Feces
 Total porphyrins 9 10 14
 Porphyrin fractionation 7 (a) 13 17 (b)
Blood
 Erythrocyte protoporphyrin 5 12 (c) 87
 Erythrocyte zinc protoporphyrin 1 17 (c) 10
 Erythrocyte free protoporphyrin 1 17 (c) 10
 Plasma total porphyrins -- 5 7
 Plasma fluorescence scanning 14 (a) 17 20 (b)
 Enzyme analyses (d) 14 7 9
 DNA analyses (e) 11 13 4

 D, VP E, AIP

n 21 21
Urine
 ALA 20 20
 PBG 21 (a) 21 (a)
 Total porphyrins 16 16
 Porphyrin fractionation 19 18
Feces
 Total porphyrins 14 15
 Porphyrin fractionation 18 (a) 17 (a)
Blood
 Erythrocyte protoporphyrin 5
 Erythrocyte zinc protoporphyrin 3 1
 Erythrocyte free protoporphyrin 3 1
 Plasma total porphyrins 6 4
 Plasma fluorescence scanning 20 (a) 19 (a)
 Enzyme analyses (d) 5/1/1/2 12
 DNA analyses (e) 1/10 11

(a) Considered necessary for the diagnosis and discrimination of
the 3 most common acute porphyrias (AIP, VP, and HCP).

(b) Considered necessary for the diagnosis and discrimination of
cutaneous porphyrias with active skin lesions (not including EPP).

(c) Two of 3 considered necessary for the diagnosis of EPP.

(d) A and E, PBGD; B, ferrochelatase; C, UROD; D, PBGD/UROD/
coproporphyrinogen oxidase/protoporphyrinogen oxidase.

(e) A and E, hydroxymethyl bilane synthase gene; B, ferrochelatase
gene; C, UROD gene; D, UROD/protoporphyrinogen oxidase genes.
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Title Annotation:Evidence-Based Medicine and Test Utilization
Author:Aarsand, Aasne K.; Villanger, Jorild H.; Stole, Egil; Deybach, Jean-Charles; Marsden, Joanne; To-Fig
Publication:Clinical Chemistry
Date:Nov 1, 2011
Words:6770
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