Contents
1. Acute porphyrias
1.1. Diagnosis of the acute attack
1.2. Diagnosis of the type of acute porphyria
1.2.1. Diagnostic algorithms for current symptoms suggestive of an acute porphyria
1.2.2. Diagnostic algorithms for previous symptoms suggestive of an acute porphyria
2. Cutaneous porphyrias
2.1. Diagnosis of cutaneous porphyria
2.2. Diagnosis of the type of cutaneous porphyria
2.2.1. Diagnostic algorithms for cutaneous porphyria with current symptoms
2.2.2. Diagnostic algorithms for cutaneous porphyria with previous symptoms
1. Acute porphyrias
1.1. Diagnosis of the acute attack
Background information
Acute neurovisceral attacks are accompanied by increased urinary excretion
of porphobilinogen (PBG) and, usually to a lesser extent, 5-aminolevulinic
acid (ALA), except in the exceedingly rare condition, ALA Dehydrase Deficiency Porphyria (ADP), where PBG excretion is normal.
Examination of urine for excess PBG is, therefore, the essential investigation in patients with a suspected attack of acute porphyria.
Measurement of urinary porphyrin alone is unhelpful and may be misleading. In acute porphyrias, concentrations are usually increased, mainly or in part due to in vitro polymerization of PBG to uroporphyrin. However, increases, mainly of coproporphyrin, also occur in numerous other conditions such as hepatobiliary disease, alcohol abuse, infections and other common disorders.
In lead poisoning and ADP, urinary coproporphyrin III and ALA, but usually not PBG, are increased.
Specimen collection and stability
Urinary PBG is best analysed in a fresh, random sample (10-20 mL) collected without any preservative but protected from light. PBG should always be reported per mmol creatinine.
24 hour collections are cumbersome, can delay diagnosis and increase the risk of losses of in particular PBG during the collection period, and should not be used.
Data indicate that PBG is stable in urine in the dark at 4°C for up to 48 hours and for at least a month at -20°C.
Analytical procedures
The preferred method for measurement of PBG in urine is quantification of the red product formed by its reaction with 4-dimethylaminobenzaldehyde in acid (Ehrlich’s reagent) after removal of urobilinogen and other interfering substances by anion exchange chromatography. A commercial kit is available (ClinEasy® Complete Kit, Recipe, München, Germany). Results should be expressed as µmol/mmol creatinine.
Various qualitative screening tests in which the PBG-Ehrlich compound is separated from the urobilinogen-Ehrlich complex by solvent extraction have also been described. They have low sensitivity and poor specificity and should not be used.
For quality control, a normal urine sample and a quality control sample containing excess PBG should be included in every batch. Quality control materials are commercially available from Recipe.
All laboratories undertaking qualitative or quantitative determination of urinary PBG should participate regularly in an appropriate EQA scheme.
Comments
Screening tests are not recommended, due to the low sensitivity and poor specificity. If, however, used, all screening test results must be confirmed by a specific, quantitative method, preferably on the same sample of urine, in order to exclude false positive or negative results.
The identity of the PBG-Ehrlich aldehyde complex should be confirmed by determining its absorption spectrum. Very occasionally, other compounds that react with Ehrlich’s reagent to give a similar colour may co-elute with PBG.
Interpretation
Reference intervals are not harmonized between laboratories, but many laboratories apply an upper reference limit of normal for urinary PBG of around 1.5 µmol/mmol creatinine.
In most patients with an ongoing attack of acute porphyria, PBG concentrations are at least ten times the upper limit of normal within one week of the onset of symptoms. At these concentrations, urine samples may develop a brownish red colour on standing, or urine may be this colour when fresh, but this discolouration, which is produced by condensation of PBG to porphobilin, uroporphyrin and other compounds, is variable and not always observed.
ALA and PBG excretion decrease as the attack resolves. In acute intermittent porphyria (AIP), excretion usually remains increased for many months or years, but in variegate porphyria (VP) and hereditary coproporphyria (HCP) may return to normal or near normal within a week or so after the onset of symptoms.
If urinary PBG and ALA excretion is normal in a patient with current symptoms, acute porphyria is excluded as the cause of symptoms. For a patient where symptoms are subsiding or the patient is no longer symptomatic, analysis of faecal coproporphyrin III:I ratio and plasma fluorescence scanning must be performed to rule out HCP and VP.
In an individual known to have inherited one of the acute porphyrias, normal PBG/ALA concentration excludes an acute attack. However, because PBG and ALA concentrations may be persistently raised in clinically latent porphyria or during remission, and the further increase that accompanies an acute attack is often difficult to demonstrate, especially in light of the high natural biological variation of these urinary markers, attribution of symptoms to acute porphyria in such individuals depends largely on clinical assessment.
1.2. Diagnosis of the type of acute porphyria
1.2.1. Diagnostic algorithms for current symptoms suggestive of an acute
porphyria
As soon as a diagnosis of acute porphyria has been established, it is essential to identify the type of acute porphyria in order to provide appropriate advice for the patient and their family. Even when the patient comes from a family known to have a particular type of porphyria, the type of porphyria should be confirmed. Examples of two different inherited porphyrias in the same family have been reported.
Metabolite measurements (urinary PBG/ALA, faecal and plasma porphyrins) are essential for the diagnosis of clinically overt acute porphyrias because symptoms cannot be ascribed to porphyria unless specific patterns of overproduction of porphyrin precursors/porphyrins are demonstrated. Enzyme measurements are not essential and may mislead due to overlap between normal and disease ranges. Demonstration of a disease-specific mutation in the appropriate gene identifies a genetic trait for porphyria, but, by itself, gives no indication of disease activity.
This section proposes minimum diagnostic criteria that must be met in order to establish the diagnosis of the type of porphyria when symptoms due to porphyria are present. Definitive diagnosis of the porphyrias should be carried out in an appropriate porphyria expert laboratory.
- For diagnosis of porphyria in remission see section 1.2.2. Diagnostic algorithms for previous symptoms suggestive of an acute porphyria.
- For information on front line tests for non-specialist laboratories see Woolf et al, 2017.
Specimen collection and stability
Urine: Urinary PBG is best analysed in a fresh, random sample (10-20 mL) collected without any preservative but protected from light. PBG should always be reported per mmol creatinine.
About 5-10g wet weight of faeces is adequate for porphyrin analysis. Diagnostically significant changes in porphyrin concentration are unlikely to occur within 36 hours at room temperature if protected from light, allowing samples to be mailed to an expert laboratory.
Samples are stable for many months at -20° C.
For plasma porphyrin analysis, 5mL EDTA-anticoagulated blood, protected from light, is recommended. Plasma should be separated within 24 hours or as soon as practicable to avoid contamination with haemoglobin to interfere with porphyrin analysis.
Diagnostic criteria
- Acute Intermittent Porphyria (AIP)
Increased urinary PBG excretion, with a normal plasma fluorescence scan or with a maximum emission peak < 623 nm and a normal faecal coproporphyrin III:I isomer ratio.
Note : Plasma fluorescence emission spectroscopy is useful as a front-line test in all acute porphyrias because a peak at 624-627nm establishes the diagnosis of VP. However, a negative result does not rule out AIP or HCP nor does a positive result distinguish AIP from HCP; in both conditions, an emission peak around 620 nm may be present. The absence of a peak at 624-627nm makes VP very unlikely.
Comments
PBG excretion per mmol creatinine during, and for at least one week after, an acute attack is usually greater than 10 times the upper limit of normal. If it is increased less than five-fold, additional tests may be required, depending on local laboratories’ upper reference/action limits (see section 1.2.2. Diagnostic algorithms for previous symptoms suggestive of an acute porphyria).
Use of the antibiotic methenamine hippurate may cause false negative urinary ALA and PBG results.
ALA excretion is in most patients increased to a lesser extent than PBG. Its measurement is not essential to establish the diagnosis of AIP, but is often combined with that of PBG. It has a higher stability at room temperature, and it may be helpful for differentiation from other causes of abdominal pain eg lead poisoning or ADP .
Faecal coproporphyrin and dicarboxylic porphyrin concentrations are normal (total less than 200 nmol/g dry wt) or slightly elevated with coproporphyrin III:I ratios less than 2.0. Faeces may contain increased concentrations of uroporphyrin sufficient to increase the total faecal porphyrin concentration when this is measured by an acid extraction method.
Urine porphyrin concentration is usually increased, mainly due to spontaneous formation of uroporphyrin from PBG in urine, but this cannot be used diagnostically. Lower, but increased concentrations of coproporphyrin and other porphyrins may also be found.
Erythrocyte PBG deaminase measurement is not essential or recommended for the diagnosis of AIP. Activity within the reference range does not exclude the diagnosis. A strongly reduced result in a haematologically normal individual, .e.g. such as a 50% reduction of the lower limit of normal, would support it. Erythrocyte PBG deaminase activities are within the reference range in about 5-10% of haematologically normal patients with AIP because there is overlap between the AIP and reference ranges and about 3-5% of families have the non-erythroid variant.
Acute neurovisceral attack +/- skin lesions
Increased urinary PBG excretion, with a plasma porphyrin fluorescence emission peak at 624-627nm.
Skin lesions alone
A plasma porphyrin fluorescence emission peak at 624-627nm. Urinary PBG excretion is usually normal, or only borderline increased.
Note: urinary coproporphyrin III excretion is usually increased during acute and cutaneous symptomatic phases, but urinary analysis alone may not be used to diagnose or exclude VP.
Comments
An increased plasma porphyrin fluorescence emission peak at 624-627nm differentiates VP from all other porphyrias, except EPP. The fluorescence spectrometer used for its detection must be fitted with a red-sensitive photomultiplier and a reference range for normal plasma must be established for that spectrometer. Plasma from patients with EPP may show a fluorescence peak at around 628 nm if globin from haemolysed erythrocytes is present in the sample.
Faecal coproporphyrin concentrations may be increased, in combination with a coproporphyrin III:I ratio greater than 2.0. Faecal protoporphyrin concentrations are usually at least 2-fold greater than coproporphyrin. However, protoporphyrin is less fluorescent than coproporphyrin, thus HPLC peaks of similar sizes are usually observed. Calculation of relative response factors of each porphyrin using standards is therefore obligated for quantitation.
PBG excretion is initially greatly increased during an acute attack as in AIP but may decrease to near normal levels within 7 days of onset of symptoms and become normal within 3 weeks. ALA excretion is in most patients increased to a lesser extent than PBG.
- Hereditary Coproporphyria (HCP)
Increased urinary PBG excretion, with an increased total faecal porphyrin concentration (usually above 200 nmol/g dry wt) with coproporphyrin as the main component and a coproporphyrin III:I ratio greater than 2.0.
Comments
PBG excretion is initially greatly increased during an acute attack as in AIP, but may decrease to near normal levels within 7 days of onset of symptoms. ALA excretion is in most patients increased to a lesser extent than PBG.
Urine may contain high concentrations of coproporphyrin III in addition to uroporphyrin (from PBG), but in some patients, most of the porphyrins are made up of uro- and heptaporphyrins.
A plasma porphyrin fluorescence emission peak at around 620 nm is present in some patients.
- ALA Dehydrase Deficiency Porphyria (ADP)
Highly increased urinary ALA excretion, greatly in excess of PBG
excretion, and coproporphyrin III usually greater than 250 nmol/mmol. ALAD activity decreased by more than 80% and not restored by thiol reagents and lead intoxication excluded by normal lead concentrations in blood.
Comments
Less than 10 patients with ADP have been diagnosed and diagnostic data are therefore limited. Concentrations of ALA and coproporphyrin excretion are likely to be at least 8 times the upper limits of normal, but most of the identified cases have excreted much greater amounts.
PBG excretion may be normal or increased up to 5-fold.
Erythrocyte zinc protoporphyrin is markedly increased.
Faecal porphyrin concentration is normal.
1.2.2. Diagnostic algorithms for previous symptoms suggestive of an acute porphyria
This section proposes a strategy for the diagnosis or exclusion of the autosomal dominant acute porphyrias in patients, over the age of 15 years, who present for investigation:
- because previous symptoms suggest acute porphyria;
or
- with a past diagnosis of an acute porphyria for which there is no unequivocal documented evidence (ie laboratory reports) to support that diagnosis;
or
- with an inadequately documented family history of porphyria and no unequivocally affected relatives who are available for investigation.
Diagnosis may be difficult because :
- urinary, faecal and plasma porphyrin concentrations may return to normal during remission in all the autosomal dominant acute porphyrias.
- neither enzyme measurements nor mutational analysis are 100% sensitive and 100% specific.
Initial investigations
In all patients, analyse the diagnostic markers for acute porphyrias:
1 - urinary PBG and ALA
2 - faecal coproporphyrin and coproporphyrin III:I isomer ratio
3 - plasma porphyrin fluorescence emission spectrum
If one or more of these diagnostic markers are clearly abnormal, this is indicative of a porphyria; ref. diagnostic criteria in section 1.2.1. Diagnostic algorithms for current symptoms suggestive of an acute porphyria.
If all the diagnostic tests are negative, any current or recent symptoms are not caused by porphyria and an alternative cause of symptoms, if present, should be considered. For patients who are not symptomatic at the time of testing, they should be required to submit new samples when symptoms reoccur.
If all the diagnostic tests are negative, but the patient has a family history of porphyria (without a relative in whom the diagnosis can be established), further investigation by mutational analysis may be indicated. Unless the type of porphyria in the patient’s family is known, the choice of gene(s) will depend on the nature of the family history and the local relative prevalences of the different types of acute porphyria. In the absence of a family history, DNA analysis is not recommended.
Comments
In AIP, PBG and ALA excretion may become normal during remission but usually remain increased for years after cessation of symptoms.
The most sensitive metabolite tests for HCP and VP in remission are the faecal coproporphyrin isomer ratio and fluorescence emission spectroscopy of plasma, respectively. In VP, plasma porphyrin fluorescence remains abnormal for many years following remission of symptoms; current data suggest that the peak at 624-627nm may disappear in about 1% and then only after several years of remission.
Measurement of urinary porphyrin excretion is not useful for diagnosis. A small increase in coproporphyrin excretion, usually to less than 5 times the upper limit of normal, and without increased PBG or faecal porphyrin excretion, is a frequent finding in many diseases. Common causes are liver dysfunction, alcohol, various drugs, infection and miscellaneous severe illness. Coproporphyrin I usually either predominates or is present at a similar concentration to coproporphyrin III. Occasionally the latter isomer may account for greater than 80% of the total, prompting investigation for lead poisoning or HCP.
An increase in the concentration of protoporphyrin (or more usually a mixture of protoporphyrin and other dicarboxylic porphyrins) in faeces does not indicate VP. The commonest cause of this finding is increased haem in the gut, either from haemorrhage, which may be minor or trivial, or from the diet. Excess haem is converted to dicarboxylic porphyrins by gut flora but sufficient may reach the faeces to give a positive test for occult blood.
2. Cutaneous porphyrias
2.1. Diagnosis of cutaneous porphyria
Background information
Cutaneous porphyria is caused by the accumulation of phototoxic porphyrins in the skin. These can cause skin damage following sun exposure to light between 400-410nm due to photoactivation of porphyrins in the dermis.
Cutaneous porphyrias can be divided into two groups dependent on the skin symptoms.
- Those that present with acute photosensitivity (erythropoietic protoporphyria [EPP], X-linked erythropoietic protoporphyria [XLEPP]).
- Those that present mainly with bullae, fragility and or scarring (porphyria cutanea tarda [PCT], congenital erythropoietic porphyria [CEP] and VP and HCP which although they are acute porphyrias, can present with skin symptoms).
The most useful front line investigation is plasma fluorescence emission spectroscopy when carried out using a scanning fluorimeter fitted with a red sensitive photomultiplier. Active skin lesions in cutaneous porphyria are always accompanied by excess circulating porphyrins produced in either the liver or bone marrow (Schulenberg-Brand 2014). Those patients with current cutaneous symptoms would therefore be expected to have increased plasma porphyrin.
For suspected EPP, the measurement of erythrocyte protoporphyrin is essential using a sensitive fluorimetric method. It is important to determine if an increase in protoporphyrin is due to mainly metal-free protoporphyrin as in EPP or to both metal-free and zinc protoporphyrin as in XLEPP – increases in primarily zinc protoporphyrin may be due to iron deficiency or lead intoxication.
PCT can be differentiated from other bullous porphyrias by fractionation of urinary and faecal porphyrins. Uro- and heptaporphyrins predominate in urine and hepta-, penta- and isocoproporphyrins predominate in faeces.
Specimen collection and stability
Urinary porphyrins are best analysed in a fresh, random sample (10-20 mL) collected without any preservative but protected from light and should always be reported per mmol creatinine.
About 5-10g wet weight of faeces is adequate for porphyrin analysis. Diagnostically significant changes in porphyrin concentration are unlikely to occur within 36 hours at room temperature if protected from light, allowing samples to be mailed to an expert laboratory.
Samples are stable for many months at -20° C.
For plasma porphyrin analysis, 5mL EDTA-anticoagulated blood, protected from light, is recommended. Plasma should be separated within 24 hours or as soon as practicable to avoid contamination with haemoglobin to interfere with porphyrin analysis.
Analytical procedures
Erythrocyte total porphyrin The most widely used method for erythrocyte total porphyrin is based on double extraction and fluorometry.
Zinc and metal-free protoporphyrin This requires extraction with a neutral solvent followed by fluorescence spectroscopy or HPLC to distinguish metal-free protoporphyrin from zinc protoporphyrin.
Plasma porphyrin fluorescence emission spectroscopy This method determines the fluorescence emission spectrum of saline-diluted plasma excited at 405 nm.
Fractionation of porphyrins in urine and faeces Fractionation of urinary and faecal porphyrins uses sample preparation, HPLC separation, and fluorometric detection.
2.2. Diagnosis of the type of cutaneous porphyria
2.2.1. Diagnostic algorithms for cutaneous porphyria with current symptoms
Diagnostic criteria
- Erythropoietic protoporphyria (EPP)
Plasma porphyrin fluorescence emission peak greater than 623nm and an increased erythrocyte metal-free protoporphyrin.
Comments
EPP is the most frequent porphyria to present in childhood. Rarely, a form of EPP can be acquired, usually associated with myelodysplastic conditions.
DNA analysis will identify EPP patients who have biallelic FECH
mutations and may therefore have an increased risk of developing liver disease. The genetic results are also useful to determine the risk for a future child.
- X-linked Erythropoietic Protoporphyria (XLEPP)
Plasma porphyrin fluorescence emission peak greater than 623nm and increased erythrocyte metal-free and zinc protoporphyrin. DNA sequence analysis and the finding of a mutation in exon 11 of the ALAS2 gene is required to confirm the diagnosis.
Comments
Markedly increased erythrocyte total and metal-free protoporphyrin, with zinc protoporphyrin making up more than 15% of the total suggests XLEPP.
- Porphyria Cutanea Tarda (PCT)
Plasma porphyrin fluorescence emission peak equal to or less than 623nm with a predominance of uro- and hepta- porphyrins in urine and hepta-,penta- and isocoproporphyrins in faeces.
Comments
Worldwide most patients (~80%) have a sporadic form of PCT where the UROD gene is normal. Genetic family screening can be carried out in the ~20% of cases where PCT is inherited. Hepatoerythropoietic porphyria (HEP) is a rare form of PCT where UROD gene mutations are present on both alleles.
Patients with pseudoporphyria have skin manifestations that clinically and histologically imitate PCT.
- Congenital erythropoietic porphyria (CEP)
Plasma porphyrin fluorescence emission peak equal to or less than 623nm with more than 80% of the coproporphyrin in faeces being isomer I. Highly increased uroporphyrinogen I and coproporphyrinogen I in urine and erythrocytes.
Comments
This is the least common but most severe of the cutaneous porphyrias, though severity may be highly variable, and milder, late onset cases are also seen. The prevalence is less than one per million in the United Kingdom. This is an autosomal recessive disorder with homoallelic or heteroallelic for mutations in the UROS gene, or rarely, the X-linked GATA1 gene (Phillips 2007). Genetic analysis may be useful in CEP patients where prenatal diagnosis may be requested due to the serious clinical complications of this disorder.
Typical findings and biochemical differentiation of the non-acute porphyrias are shown in the Table below for symptomatic patients.
| Type | Urine ALA/PBG | Urine porphyrins | Faecal porphyrins | Erythrocyte porphyrins | Plasma fluorescence emission peak |
| EPP | Not increased | Not Increased | +/-Protoporphyrin(1) | Protoporphyrin(2) | 626-634 nm(3) |
| XLEPP | Not increased | Not increased | +/- Protoporphyrin | Zn-proto, proto(4) | 626-634 nm(3) |
| PCT | Not increased | Uro I&III
Hepta III |
Isocopro
Hepta Normal coproporphyrin III:I ratio |
Not Increased | 615-620 nm |
| CEP | Not increased | Uro I
Copro I |
Copro I | Zn-proto, proto
Copro I, uro I |
615-620 nm |
(1)Increased in about 60% of patients. (2)Total erythrocyte porphyrin typically greater than 4 µmol/L erythrocytes with more than 70% metal-free protoporphyrin. (3)Protoporphyrin bound to albumin has a fluorescence emission peak at around 636 nm whereas protoporphyrin bound to globin (if there is haemolysis in the sample) has a peak at around 628 nm. (4)Total erythrocyte porphyrin greater than 4 µmol/L erythrocytes with Zn-protoporphyrin 20 to 60% of total protoporphyrin. Analysis of the ALAS2 gene is recommended for confirmation of the diagnosis.
- Hereditary coproporphyria (HCP)
Plasma porphyrin fluorescence emission peak equal to or less than 623nm with a faecal coproporphyrin III:I isomer ratio greater than 2.0
Comments
30% of patients with this acute porphyria present with only cutaneous symptoms.
- Variegate Porphyria (VP)
Plasma porphyrin fluorescence emission peak greater than 623nm with a normal erythrocyte protoporphyrin analysis
Comments
70% of patients with this acute porphyria present with only cutaneous symptoms.
2.2.2. Diagnostic algorithms for cutaneous porphyria with previous symptoms
Background information
Confirming a diagnosis of cutaneous porphyria in an asymptomatic individual many years after the initial illness is challenging.
Diagnostic criteria
The strategy for diagnosis of a patient with previous symptoms of a cutaneous porphyria is similar to that shown above for symptomatic patients although a normal plasma porphyrin fluorescence scan would not rule out a cutaneous porphyria.
- Porphyria Cutanea Tarda (PCT)
Patients with PCT who have received adequate treatment will have normal porphyrin levels in urine, faeces and plasma when in remission, though some patients may present with slightly increased uro- and heptaporphyrin concentrations. In patients in remission reporting a past diagnosis of PCT for which there is no unequivocal documented evidence (ie laboratory reports), there are limited options to confirm such a diagnosis, as patients with sporadic PCT will have no mutation in the UROD gene.
- Hereditary coproporphyria (HCP)
The faecal coproporphyrin-isomer ratio in HCP remains abnormal for years after the onset of full clinical remission.
- Variegate Porphyria (VP)
In previously symptomatic VP patients, the plasma fluorescence peak typically persists for years after remission of clinical symptoms.
However even full analysis of blood, urine, and faeces may not result in a diagnosis, because porphyrin excretion may return to normal after long periods of remission and fresh samples would need to be taken when the patient has symptoms.
3. Suggested further reading
- Kühnel A, Gross U, Doss MO. Hereditary coproporphyria in Germany: clinical-biochemical studies in 53 patients. Clin Biochem. 2000 Aug;33(6):465-73
- Long C, Smyth SJ, Woolf J et al. The detection of latent porphyria by fluorescence emission spectroscopy of plasma. Br J Dermatol 1993; 129: 9-13
- Rossi E. Increased faecal porphyrins in acute intermittent porphyria. Clin Chem 1999;45:281-3
- Sassa S. ALAD porphyria. Seminars in Liver Disease 1998; 18: 95-101