Minder, Elisabeth

Stauch, Thomas

Vogeser, Michael

English

Universität München: Elektronischen Publikationen

Clin Chem Lab Med 2011;49(6):1059–1060  2011 by Walter de Gruyter • Berlin • Boston. DOI 10.1515/CCLM.2011.177
2010/696
Article in press - uncorrected proof
Letter to the Editor
Results of a pilot external quality assessment study on
free protoporphyrin in erythrocytes
Michael Vogeser1,*, Thomas Stauch2 and Elisabeth
Minder3
1 Institute of Clinical Chemistry, University of Munich,
Munich, Germany
2 MVZ Labor Prof. Seelig, Karlsruhe, Germany
3 Stadtspital Triemli, Zürich, Switzerland
Keywords: erythrocyte free protoporphyrin; erythropoietic
protoporphyria; interlaboratory survey.
Quantification of erythrocyte protoporphyrins (ePP) is essen-
tial to establish the diagnosis of erythropoietic protoporphy-
ria, a form of chronic porphyria that is not very rare
(prevalence roughly 1:50,000), and clinically characterized
by painful phototoxic skin reactions that usually start in early
childhood (1, 2), and X-linked dominant protoporphyria, a
recently (2008) described metabolic disorder with similar
clinical features (3). While PP with zinc chelated as the cen-
tral ion (zinc PP) is increased in iron deficiency anemia and
inefficient erythropoesis, substantial increases ()50-fold) in
metal-free ePP is pathognomonic for protoporphyria. Eryth-
rocyte protoporphyrins are measured using various methods,
and standardisation of analytic procedures has not been ad-
dressed at present.
In order to assess the comparability of free ePP results
obtained in clinical samples using different analytical meth-
ods, we performed an inter-laboratory comparison study (4).
In this trial, lyophilized aliquots of three samples (A–C) were
shipped to the participating laboratories. The samples were
based on washed red blood cells which were lysed by repeat-
ed freeze-thaw-cycles prior to dispensing and lyophilization.
Sample B was obtained from an adult male patient suffering
from protoporphyria; sample C was from a healthy volunteer;
and sample A was a 1q1 mix of samples B and C. Six
laboratories from Europe, North-America and Asia-perform-
ing ePP quantification routinely, contributed results,
*Corresponding author: Prof. Dr. med. Michael Vogeser,
Institute of Clinical Chemistry, University of Munich,
Marchioninistr. 15, 81375 München, Germany
E-mail: Michael.Vogeser@med.uni-muenchen.de
Previously published online May 17, 2011
expressed as metal-free ePP concentrations of the reconsti-
tuted sample in mmol/L. In three laboratories, HPLC meth-
ods using fluorescence detection were used, whereas the
other three laboratories used fluorometry after extraction
without chromatographic separation. Results were evaluated
according to absolute concentrations, and as the x-fold of the
respective method-specific upper limit of normal (ULN).
All participating laboratories found samples A and B to
contain free PP concentrations above the method-specific
reference ranges, and sample C was classified as normal by
all participants. However, as expressed as the x-fold of the
upper limit of the method-related reference range, very sub-
stantial between-method differences were observed: In sam-
ple A the range was from 1.2- to 123-fold of the upper limit
of the reference range, and in sample B from 2.0- to 201-
fold (Table 1). All three non-chromatographic fluorometric
methods gave substantially lower reference-range related free
ePP results when compared to HPLC methods.
Overall, our results disclose poor inter-laboratory concor-
dance of ePP measurements. In the present situation, follow-
up of patients should therefore be based on the results of a
single laboratory. HPLC methods seem to be superior to non-
chromatographic methods for the quantification of ePP with
respect to the disease-related dynamics of the results.
To improve standardization of ePP measurement, the intro-
duction of commercially available, human matrix-based, cer-
tified materials for calibration and quality control is highly
desirable. An external quality assessment scheme for ePP
measurements has very recently been initiated by the Euro-
pean porphyria network (EPNET; www.porphyria-europe.org).
Standardisation of ePP measurements aiming to stabilize
results over time is not only important for the follow-up of
patients involving results from different laboratories, but also
as a prerequisite of international clinical research on proto-
porphyria (5).
For rare diseases, laboratory investigations are often of
essential diagnostic importance. Since respective analyses are
typically performed in rather few laboratories, they are hard-
ly addressed by in-vitro diagnostic companies with respect
to standards and quality control materials. Consequently,
quality assurance of analyses related to rare disease requires
particular attention from the community of clinical chemistry
and laboratory medicine. In particular, in-vitro diagnostic
companies should be encouraged by the scientific societies
to contribute to analytical solutions related to orphan
diseases.
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1060 Vogeser et al.: EQAS of erythrocyte protoporphyrins
Article in press - uncorrected proof
Table 1 Intermethod comparison of metal-free erythrocyte protoporphyrin measurements; raw data.
Lab  Method Sample A B C Reference range
1 HPLC Metal-free ePP, mmol/L 5.9 13.0 0.06 -0.12
x-fold of ref. range 49.2 108 0.5
2 HPLC Metal-free ePP, mmol/L 11.1 18.1 0.07 -0.09
x-fold of ref. range 123 201 0.8
3 HPLC Metal-free ePP, mmol/L 8.0 15.6 0.04 -0.2
x-fold of ref. range 40.0 78.0 0.2
4 Fluorometry Metal-free ePP, mmol/L 5.2 8.9 0.7 -2.0
x-fold of ref. range 2.6 4.5 0.4
5 Fluorometry Metal-free ePP, mmol/L 1.2 2.0 0.1 -1
x-fold of ref. range 1.21 2.0 0.1
6 Fluorometry Metal-free ePP, mmol/L 12.8 21.0 0.71 -1.5
x-fold of ref. range 8.5 14.0 0.5
Conflict of interest statement
Authors’ conflict of interest disclosure: The authors stated that
there are no conflicts of interest regarding the publication of this
article. Research support played no role in the study design; in the
collection, analysis, and interpretation of data; in the writing of the
report; or in the decision to submit the report for publication.
Research funding: Hans-Fischer-Gesellschaft, München.
Employment or leadership: None declared.
Honorarium: None declared.
References
1. Puy H, Gouya L, Deyback J-C. Porphyrias. Lancet 2010;375:
924–37.
2. Holme SA, Anstey AV, Finlay AY, Elder GH, Badminton
MN. Erythropoietic protoporphyria in the UK: clinical features
and effect on quality of life. Br J Dermatol 2006;155:574–
81.
3. Whatley S, Ducamp S, Gouya L, Grandchamp B, Beaumont C,
Badminton M, et al. C-terminal deletions in the ALAS2 gene
lead to gain of function and cause X-linked dominant protopor-
phyria without anemia or iron overload. Am J Hum Genet 2008;
83:408–14.
4. Vogeser M, Müller W, Stauch Th. Inter-laboratory survey of
erythrocyte free protoporphyrin quantification – announcement
of a pilot study. Clin Chem Lab Med 2008;46:1340–1.
5. Harms J, Lautenschlager S, Minder CE, Minder EI. An alpha-
melanocyte-stimulating hormone analogue in erythropoietic pro-
toporphyria. N Engl J Med 2009;360:306–7.
Bereitgestellt von | Bayerische Staatsbibliothek
Angemeldet
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Results of a pilot external quality assessment study on free protoporphyrin in erythrocytes

Michael Vogeser

Thomas Stauch

Elisabeth Minder

Crossref

Clinical Chemistry and Laboratory Medicine (CCLM)

Open Access LMU ( Ludwig-Maximilians-Univ. München)

Clin Chem Lab Med 2011;49(6):1059–1060  2011 by Walter de Gruyter • Berlin • Boston. DOI 10.1515/CCLM.2011.1772010/696Article in press - uncorrected proofLetter to the EditorResults of a pilot external quality assessment study onfree protoporphyrin in erythrocytesMichael Vogeser1,*, Thomas Stauch2 and ElisabethMinder31 Institute of Clinical Chemistry, University of Munich,Munich, Germany2 MVZ Labor Prof. Seelig, Karlsruhe, Germany3 Stadtspital Triemli, Zürich, SwitzerlandKeywords: erythrocyte free protoporphyrin; erythropoieticprotoporphyria; interlaboratory survey.Quantification of erythrocyte protoporphyrins (ePP) is essen-tial to establish the diagnosis of erythropoietic protoporphy-ria, a form of chronic porphyria that is not very rare(prevalence roughly 1:50,000), and clinically characterizedby painful phototoxic skin reactions that usually start in earlychildhood (1, 2), and X-linked dominant protoporphyria, arecently (2008) described metabolic disorder with similarclinical features (3). While PP with zinc chelated as the cen-tral ion (zinc PP) is increased in iron deficiency anemia andinefficient erythropoesis, substantial increases ()50-fold) inmetal-free ePP is pathognomonic for protoporphyria. Eryth-rocyte protoporphyrins are measured using various methods,and standardisation of analytic procedures has not been ad-dressed at present.In order to assess the comparability of free ePP resultsobtained in clinical samples using different analytical meth-ods, we performed an inter-laboratory comparison study (4).In this trial, lyophilized aliquots of three samples (A–C) wereshipped to the participating laboratories. The samples werebased on washed red blood cells which were lysed by repeat-ed freeze-thaw-cycles prior to dispensing and lyophilization.Sample B was obtained from an adult male patient sufferingfrom protoporphyria; sample C was from a healthy volunteer;and sample A was a 1q1 mix of samples B and C. Sixlaboratories from Europe, North-America and Asia-perform-ing ePP quantification routinely, contributed results,*Corresponding author: Prof. Dr. med. Michael Vogeser,Institute of Clinical Chemistry, University of Munich,Marchioninistr. 15, 81375 München, GermanyE-mail: Michael.Vogeser@med.uni-muenchen.dePreviously published online May 17, 2011expressed as metal-free ePP concentrations of the reconsti-tuted sample in mmol/L. In three laboratories, HPLC meth-ods using fluorescence detection were used, whereas theother three laboratories used fluorometry after extractionwithout chromatographic separation. Results were evaluatedaccording to absolute concentrations, and as the x-fold of therespective method-specific upper limit of normal (ULN).All participating laboratories found samples A and B tocontain free PP concentrations above the method-specificreference ranges, and sample C was classified as normal byall participants. However, as expressed as the x-fold of theupper limit of the method-related reference range, very sub-stantial between-method differences were observed: In sam-ple A the range was from 1.2- to 123-fold of the upper limitof the reference range, and in sample B from 2.0- to 201-fold (Table 1). All three non-chromatographic fluorometricmethods gave substantially lower reference-range related freeePP results when compared to HPLC methods.Overall, our results disclose poor inter-laboratory concor-dance of ePP measurements. In the present situation, follow-up of patients should therefore be based on the results of asingle laboratory. HPLC methods seem to be superior to non-chromatographic methods for the quantification of ePP withrespect to the disease-related dynamics of the results.To improve standardization of ePP measurement, the intro-duction of commercially available, human matrix-based, cer-tified materials for calibration and quality control is highlydesirable. An external quality assessment scheme for ePPmeasurements has very recently been initiated by the Euro-pean porphyria network (EPNET; www.porphyria-europe.org).Standardisation of ePP measurements aiming to stabilizeresults over time is not only important for the follow-up ofpatients involving results from different laboratories, but alsoas a prerequisite of international clinical research on proto-porphyria (5).For rare diseases, laboratory investigations are often ofessential diagnostic importance. Since respective analyses aretypically performed in rather few laboratories, they are hard-ly addressed by in-vitro diagnostic companies with respectto standards and quality control materials. Consequently,quality assurance of analyses related to rare disease requiresparticular attention from the community of clinical chemistryand laboratory medicine. In particular, in-vitro diagnosticcompanies should be encouraged by the scientific societiesto contribute to analytical solutions related to orphandiseases.Bereitgestellt von | Bayerische StaatsbibliothekAngemeldetHeruntergeladen am | 05.11.15 11:211060 Vogeser et al.: EQAS of erythrocyte protoporphyrinsArticle in press - uncorrected proofTable 1 Intermethod comparison of metal-free erythrocyte protoporphyrin measurements; raw data.Lab  Method Sample A B C Reference range1 HPLC Metal-free ePP, mmol/L 5.9 13.0 0.06 -0.12x-fold of ref. range 49.2 108 0.52 HPLC Metal-free ePP, mmol/L 11.1 18.1 0.07 -0.09x-fold of ref. range 123 201 0.83 HPLC Metal-free ePP, mmol/L 8.0 15.6 0.04 -0.2x-fold of ref. range 40.0 78.0 0.24 Fluorometry Metal-free ePP, mmol/L 5.2 8.9 0.7 -2.0x-fold of ref. range 2.6 4.5 0.45 Fluorometry Metal-free ePP, mmol/L 1.2 2.0 0.1 -1x-fold of ref. range 1.21 2.0 0.16 Fluorometry Metal-free ePP, mmol/L 12.8 21.0 0.71 -1.5x-fold of ref. range 8.5 14.0 0.5Conflict of interest statementAuthors’ conflict of interest disclosure: The authors stated thatthere are no conflicts of interest regarding the publication of thisarticle. Research support played no role in the study design; in thecollection, analysis, and interpretation of data; in the writing of thereport; or in the decision to submit the report for publication.Research funding: Hans-Fischer-Gesellschaft, München.Employment or leadership: None declared.Honorarium: None declared.References1. Puy H, Gouya L, Deyback J-C. Porphyrias. Lancet 2010;375:924–37.2. Holme SA, Anstey AV, Finlay AY, Elder GH, BadmintonMN. Erythropoietic protoporphyria in the UK: clinical featuresand effect on quality of life. Br J Dermatol 2006;155:574–81.3. Whatley S, Ducamp S, Gouya L, Grandchamp B, Beaumont C,Badminton M, et al. C-terminal deletions in the ALAS2 genelead to gain of function and cause X-linked dominant protopor-phyria without anemia or iron overload. Am J Hum Genet 2008;83:408–14.4. Vogeser M, Müller W, Stauch Th. Inter-laboratory survey oferythrocyte free protoporphyrin quantification – announcementof a pilot study. Clin Chem Lab Med 2008;46:1340–1.5. Harms J, Lautenschlager S, Minder CE, Minder EI. An alpha-melanocyte-stimulating hormone analogue in erythropoietic pro-toporphyria. N Engl J Med 2009;360:306–7.Bereitgestellt von | Bayerische StaatsbibliothekAngemeldetHeruntergeladen am | 05.11.15 11:21

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Results of a pilot external quality assessment study on free protoporphyrin in erythrocytes

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