LABORATORY DIAGNOSIS

ACUTE PORPHYRIAS

I. DIAGNOSIS OF THE ACUTE ATTACK

Background information

		Acute neurovisceral attacks are accompanied by increased urinary excretion of PBG and, to a lesser extent, ALA, except in the exceedingly rare condition, ADP (more about porphyrias), 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 by itself is unhelpful and may be misleading. Though concentrations are usually increased in acute porphyria, mainly due to in vitro polymerization of PBG to uroporphyrin, increases also occur in hepatobiliary disease, alcohol abuse, infections and other common disorders. In lead poisoning and ADP, 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.
		24 hour collections delay diagnosis and increase the risk of losses during the collection period.
		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 (more information). A commercial kit is available from BioRad Laboratories (Hemel Hempstead, Hertfordshire, UK, www.bio-rad.com). Results should be expressed as µmol/mmol creatinine.
		A more rapid semi-quantitative variation of the above method (Trace PBG kit) (more information) is available from Alpha Laboratories, Eastleigh, Hampshire, UK, for initial screening of urine for excess PBG.
		Qualitative screening tests in which the PBG-Ehrlich compound is separated from the urobilinogen-Ehrlich complex by solvent extraction have also been described (more information). Though criticised for low sensitivity and poor specificity (more information), they are rapid, cheap and still widely used (more about porphyria specialist centres).
		Whichever method is used, a normal urine sample and a quality control sample containing excess PBG should be included in every batch. Quality control material is not commercially available but can be prepared as described (more information).
		All laboratories undertaking qualitative or quantitative determination of urinary PBG should participate regularly in an appropriate EQA scheme.

Comments

		All positive screening tests should be confirmed by a specific, quantitative method (see above), preferably on the same sample of urine, in order to exclude false positives and unequivocally confirm the diagnosis of acute porphyria.
		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

		Normal adult urine contains < 1.5 µmol PBG/mmol creatinine ( < 10 µmol/L).
		In most patients with an 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, porphyrin and other compounds, is variable and not always observed.
		The detection limits for the Trace kit method and for one of the qualitative solvent-extraction methods (more information) are 25 µmol/L and about 50 µmol/L respectively. Most patients with an attack of acute porphyria excrete much more than this and their urine should give a positive screening test.
		PBG excretion decreases as the attack resolves. In AIP, excretion usually remains increased for many weeks but in VP and HCP may return to normal or near normal within a week or so after the onset of symptoms.
		If screening tests are negative and clinical suspicion of porphyria persists, it is essential to quantify urinary PBG and ALA, using a specific method (more about porphyria specialist centres) and analyse plasma and faecal porphyrins.
		If urinary PBG and ALA, plasma porphyrin concentration and faecal coproporphyrin III excretion is normal, acute porphyria is excluded as the cause of current symptoms. Enzyme measurements are not necessary for exclusion of porphyria as the cause of an acute illness and may give misleading information.
		In an individual known to have inherited one of the acute porphyrias, normal PBG/ALA excretion excludes an acute attack. However, because PBG excretion 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, attribution of symptoms to acute porphyria in such individuals depends largely on clinical assessment.

II. DIAGNOSIS OF THE TYPE OF ACUTE PORPHYRIA

A) WHEN SYMPTOMS ARE PRESENT

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 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 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.

	For diagnosis of porphyria in remission see diagnosis during remission.
	Definitive diagnosis of the porphyrias should be carried out in an appropriate reference laboratory (more about porphyria specialist centres).
	For information on front line tests for non-specialist laboratories see reference 3.

1- Specimen collection and stability

		Urine: see above.
		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, allowing samples to be mailed to a reference laboratory. Samples are stable for many months at -20° C.
		For plasma porphyrin analysis, 5mL EDTA-anticoagulated blood is recommended. Plasma should be separated within 24 hours as soon as practicable to avoid contamination with sufficient haemoglobin to interfere with porphyrin analysis. Transfer of the unseparated sample to the reference laboratory is recommended to allow additional analyses that may be required eg DNA analysis, erythrocyte porphyrin, PBG deaminase.

2– Diagnostic criteria

2.1- Acute Intermittent Porphyria (AIP)

Increased urinary PBG excretion, with normal or near normal faecal porphyrin concentration (but see comment 3 below).

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. It does not distinguish AIP from HCP; in both conditions, an emission peak at 620 nm may be present. The absence of a peak at 624-627nm excludes VP.

Comments

		PBG excretion during, and for at least one week after, an acute attack is usually greater than 10 times the upper limit of normal (more about diagnosis of acute attack). If it is increased less than five-fold, additional tests are required (more about diagnosis during remission).
		ALA excretion is 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 and may be helpful for differentiation from other causes of abdominal pain eg lead poisoning, ADP (more about 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 (more information) sufficient to increase the total faecal porphyrin concentration when this is measured by an acid extraction method (more information).
		Urine porphyrin concentration is usually increased, mainly due to formation of uroporphyrin from PBG. Lower, but increased concentrations of coproporphyrin and other porphyrins may also be found (more information).
		Erythrocyte PBG deaminase measurement is not essential for the diagnosis of AIP. Activity within the reference range does not exclude the diagnosis. Activity less than the mean – 2SD of the reference range, in a haematologically normal individual, strongly supports it (more information).

2.2– Variegate Porphyria (VP)

2.2.1- Acute neurovisceral attack +/- skin lesions

Increased urinary PBG excretion, with a plasma porphyrin fluorescence emission peak at 624-627nm. If plasma porphyrin fluorescence spectroscopy is not available or further confirmation is required, faecal porphyrin analysis shows increased protoporphyrin and, to a lesser extent, coproporphyrin concentrations with coproporphyrin III/I ratio greater than 2.0.

2.2.2- Skin lesions alone

As above, except that urinary PBG excretion is often only slightly increased or normal.

Note: urinary coproporphyrin III excretion is increased during acute and cutaneous phases but urinary analysis alone is not sufficient to establish or exclude the diagnosis of VP unequivocally.

Comments

		An increased plasma porphyrin fluorescence emission peak at 624-627 nm differentiates VP from all other porphyrias (more information). 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 628nm if globin from haemolysed erythrocytes is present in the sample (more about non-acute porphyrias).
		Faecal protoporphyrin concentrations are usually at least 2-fold greater than coproporphyrin. However, protoporphyrin is less fluorescent than coproporphyrin. For this reason, unless standards are used, HPLC traces may show coproporphyrin and protoporphyrin peaks of similar size.
		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 increased to a lesser extent than PBG.

2.3– Hereditary Coproporphyria (HCP)

Increased urinary PBG excretion, with total faecal porphyrin concentration greater than 200 nmol/g dry wt. with coproporphyrin as the main component and a coproporphyrin III/I ratio greater than 2.0.

Note: presentation with skin lesions alone is rare and often provoked by cholestasis. Such patients have markedly increased urinary coproporphyrin III excretion +/- increased PBG excretion and a plasma porphyrin fluorescence emission peak at about 620nm. Faecal coproporphrin III/I ratios are greater than 2.0 but, if cholestasis is severe, the coproporphyrin concentration may not be sufficiently increased to raise the total faecal porphyrin concentration.

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 increased to a lesser extent than PBG.
		Urine contains high concentrations of coproporphyrin III in addition to uroporphyrin (from PBG).
		A plasma porphyrin fluorescence emission peak at 620 nm is present in some patients.
		For further information on faecal coproporphyrin isomer ratios (more information).

2.4– ALA Dehydrase Deficiency Porphyria (ADP)

Urinary ALA excretion greater than 72 µmol/mmol creatinine and greatly in excess of PBG excretion. Coproporphyrin III greater than 250 nmol/mmol. Normal blood lead concentration. ALAD activity decreased by more than 80% and not restored by thiol reagents.

Comments

		Figures for ALA and coproporphyrin excretion are approximately 8 times the upper limits of normal. Most of the few reported patients have excreted much greater amounts (more information).
		PBG excretion may be normal or increased up to 5-fold.
		Erythrocyte zinc protoporphyrin is markedly increased.
		Faecal porphyrin concentrations are normal.

B) DIAGNOSIS DURING REMISSION

This section proposes a strategy for the diagnosis 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.
	Patients may be asymptomatic or have chronic symptoms, eg recurrent abdominal pain, suggesting acute porphyria.

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, determine:

	1	urinary PBG and ALA excretion
	2	faecal protoporphyrin, coproporphyrin and coproporphyrin III/I isomer ratio
	3	plasma porphyrin fluorescence emission spectrum
	4	erythrocyte PBG deaminase activity if routine haematology is normal.

Interpretation and further investigation

If one or more of tests 1-3 is abnormal, porphyria is confirmed (more about diagnostic criteria).

If metabolite tests 1-3 are normal, any current or recent symptoms are not caused by porphyria and an alternative cause of the symptoms should be sort.

If only test 4 is abnormal, mutational analysis of the HMBS gene is required. If a disease-specific mutation is identified, AIP in remission (or latent if asymptomatic with family history) is confirmed. Current data indicates that mutational analysis of the HMBS gene is 95% sensitive and 100% specific, provided disease-specific missense mutations are distinguished from rare polymorphisms.

If all four tests are normal, one or more of the following should be undertaken:

		the patient should be asked to submit samples for PBG and porphyrin analyses immediately when symptoms recur
		if there is a family history of porphyria, every attempt should be made to contact an affected relative, confirm the diagnosis and then proceed as recommended for family investigation (more about investigating the family)
		further investigation by mutational analysis to confirm or exclude latent (more about the porphyrias) acute porphyria. The main indication for mutational analysis is a family history of porphyria without a relative in whom the diagnosis can be established. 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, the likelihood of finding a disease-specific mutation is low.

Comments

		1.7 µmol/mmol is 99.9 percentile of a reference range of 0.2 – 1.2 µmol/mmol for urinary PBG. For a measurement greater than 1.7 µmol/mmol, the false positive rate is less than 1:1000.
		In AIP, PBG and ALA excretion may become normal during remission but usually remain increased for at least 6 months after cessation of symptoms. The reported sensitivities of urinary PBG and ALA measurements for detecting AIP in remission, in patients at least 15 years old, are 88% and 61% respectively (more information).
		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.
		The most sensitive metabolite tests for VP and HCP in remission are the faecal coproporphyrin isomer ratio (11) and fluorescence emission spectroscopy of plasma (more information) 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 rarely useful. 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 disease. 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 without an increase in coproporphyrin III ( either total or relative to the I isomer) does not indicate VP. The commonest cause of this finding is increased heme in the gut, either from hemorrhage, which may be minor or trivial, or from the diet. Excess heme is converted to dicarboxylic porphyrins by gut flora but sufficient may reach the faeces to give a positive test for occult blood.

Exclusion of autosomal dominant acute porphyria

There is no test or group of tests that can exclude an acute porphyria with absolute certainty unless the mutation that causes porphyria in the family of the patient under investigation is known.

When the family mutation is unknown, it is recommended that the maximum probability of having porphyria is estimated.

Estimation of the probability requires knowledge of:

		sensitivity and specificity of the relevant metabolite, enzyme and DNA analyses. For metabolite tests in particular, these are influenced by age ( before or after puberty) and clinical history (eg sensivities may be lower for patients with a family history of porphyria than for those with a previous, unsubstantiated diagnosis but no family history.
		when a family history of porphyria is present, relationship to relative suspected of having porphyria or known to have the disease but not available for mutational analysis.
		relative prevalence of AIP, VP and HCP (eg in most countries a patient with a past but unsubstantiated diagnosis of ‘acute porphyria’ is most likely to have AIP).

III. REFERENCES

1. Mauzerall D, Granick S.
The occurrence and determination of d-aminolevulinic acid and porphobilinogen in urine.
J Biol Chem 1956;219:435-46.

2. Deacon AC, Peters TJ.
Identification of acute porphyria:evaluation of a commercial screening test for urinary porphobilinogen. Ann Clin Biochem1998;35:726-32.

3. Deacon AC, Elder GH.
Front line tests for the investigation of suspected porphyria. J Clin Pathol 2001;54:500-507.

4. Buttery JE, Carrera AM, Panall PR.
Reliability of the porphobilinogen screening assay. Pathology 1990; 22:197-8.

5. Buttery JE, Carrera AM, Panall PR.
Analytical sensitivity and specificity of two screening methods for urinary porphobilinogen.
Ann Clin Biochem 1990;27:165-6.

6. Kauppinen R, Fraunberg M.
Molecular and biochemical studies of acute intermittent porphyria in 196 patients and their families. Clin Chem 2002; 48: 1891-1900.

7. Rossi E.
Increased faecal porphyrins in acute intermittent porphyria. Clin Chem 1999;45:281-3.

8. Lockwood WH, Poulos V, Rossi E et al.
Rapid procedure for faecal porphyrin assay. Clin Chem 1985; 31: 1163-7.

9. Mustajoki P, Kauppinen R, Lannfelt L et al.
Frequency of low erythrocyte porphobilinogen deaminase activity in Finland.
J Int Med 1992; 231: 389-95.

10. Nordmann Y, Puy H, Da Silva V et al.
Acute intermittent porphyria:prevalence of mutations in the porphobilinogen deaminase gene in blood donors in France. J Int Med 1997; 242: 213-217.

11. Blake D, McManus J, Cronin V et al.
Fecal coproporphyrin isomers in hereditary coproporphria. Clin Chem 1992; 38: 96-100.

12. Jacob K, Doss M.
Excretion pattern of faecal coproporphyrin isomers I-IV in human porphyrias.
Eur J Clin Chem Clin Biochem 1995; 33: 893-901.

13. Sassa S.
ALAD porphyria. Seminars in Liver Disease 1998; 18: 95-101.

14. 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.

Link to PubMed >>

NON-ACUTE PORPHYRIAS

Biochemical differentiation of the non-acute porphyrias

Type	Urine ALA/PBG	Urine porphyrins	Faecal porphyrins	Erythrocyte porphyrins	Plasma fluorescence emission peak
CEP	Not Increased	Uro I Copro I	Copro I	Zn-proto,proto Copro I, Uro I	615-620 nm
PCT	Not Increased	Uro Hepta	Isocopro Hepta	Not Increased	615-620 nm
EPP	Not Increased	Not Increased	Protoporphyrin⁽¹⁾	Protoporphyrin⁽²⁾	626-634 nm⁽³⁾
⁽¹⁾Increased in about 60% of patients. ⁽²⁾Total erythrocyte porphyrin greater than 4 µmol/L erythrocytes with greater than 70% free protoporphyrin. ⁽³⁾Protoporporhyrin bound to albumin has a fluorescence emission peak at 636 nm whereas protoporphyrin bound to globin (if there is haemolysis in the sample) has a peak at 628 nm.

Comments

		Minimum diagnostic criteria are indicated.
		Urinary porphyrin and PBG measurement is not sufficient by itself to establish the diagnosis of PCT because some patients with cutaneous VP have similar abnormalities (‘dual porphyria’). VP should be excluded by either plasma porphyrin fluorescence emission spectroscopy or faecal porphyrin analysis.
		In patients on chronic haemodialysis who develop skin blisters, PCT can be confirmed or excluded by measuring isocoproporphyrin in faeces.