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Clin Biochem Rev. 2003 May; 24(2): 67–69.
PMCID: PMC1855624

Association of Clinical Pathologists [ACP] Best Practice No 165 Front line tests for the investigation of suspected Porphyria.

Reviewed by Enrico Rossi

J Clin Pathol 2001; 54: 500–7. 

Every acute hospital laboratory needs to have simple reliable methods for excluding porphyria in most of the samples tested and identifying those few patients who need more specialised investigation. Most textbook chapters on porphyria either do not describe front line tests at all or suggest methods more suitable for reference laboratories. This eight-page review article fills the gap with a complete series of practical, recommended methods, giving sufficient detail to set up the methods in any laboratory with the necessary basic equipment. The authors are AC Deacon and GH Elder, who are acknowledged experts in the field. This review will briefly address the article's contents and discuss the recommendations made.

  1. The review begins with a brief outline of the clinical features and inheritance of the main types of porphyria.
  2. Guidance is given for proper selection of front line investigations according to whether the patient presents with acute porphyria or cutaneous porphyria.

The key investigation for acute porphyria is porphobilinogen on a random urine sample. Cutaneous porphyria is more complex and is considered in two categories, acute photosensitivity without bullae and bullous porphyria, with the latter requiring collection of a random faecal sample. The advantage of this approach is that only the appropriate specimens are collected and analysed.

My comment is that the requesting clinician can be uncertain about the clinical presentation, especially if the patient is presently asymptomatic and quotes a family history of porphyria. An alternative policy is to routinely ask for collection of a random urine, random stool and EDTA or heparinised blood on every patient in whom porphyria is suspected. This avoids the problem of obtaining further specimens; however, it does lead to an increase in the numbers of unnecessary samples analysed.

Specimen collection and stability are well described. There are tables showing the different specimen requirements according to the clinical presentation and adult reference ranges. Collecting a 24-hour sample of urine offers no advantages, delays diagnosis and risks losses of porphobilinogen. The occasional random urine with a very low creatinine is unsuitable for analysis, and the authors reject specimens with creatinine below 4 mmol/litre.

It is important to choose a cut-off for random urine samples with a very low creatinine. Such urine samples often exceed the urine porphyrin reference range when corrected for creatinine excretion and may cause unnecessary alarm.

3. The largest section of the review [five pages] details the recommended methods and has been divided up according the biological matrix analysed.

♦ Porphobilinogen in urine

The authors present three alternative approaches to detecting porphobilinogen in urine. The first is a commercial semi-quantitative kit, the second a commercial quantitative kit and finally the long standing qualitative screen, the Watson-Schwartz test. Choosing between them is a perennial problem, each has pros and cons and each laboratory should carefully consider the alternatives. Briefly, the quantitative kit is time consuming and the qualitative screen is rapid, but has recently been severely criticised for low sensitivity and poor specificity. No commercial quality control material is available and whichever method is chosen, the authors r ecommend dissolving a known amount of porphobilinogen in a normal urine sample preserved with gentamicin and freezing aliquots for use as quality controls. The authors insist that positive screening tests, including those obtained with the semi-quantitative kit, should be confirmed by the quantitative method.

It is difficult to justify inclusion of the qualitative screen as a viable option, especially as the authors quote two references describing the inadequate sensitivity and specificity of qualitative screening and recommending that it be superseded. My recommendation is the commercial semi-quantitative kit [Trace Scientific] which presents the anion exchange resin in convenient small syringes and is much faster than the quantitative method. Quality control urine should always be included; this must be made in-house from solid porphobilinogen as no commercial material is available.

♦ Total porphyrin in urine

A semi-quantitative method based on spectrophotometric scanning of an acidified random urine sample, with a correction for background absorbance is recommended. Porphyrin excretion should be expressed as a ratio to creatinine concentration to correct for the urine concentration. The method is adequate for samples with raised porphyrin concentration but lacks sensitivity for results within the reference range, hence the description semi-quantitative.

This is adequate as a screening method. However, results within the reference range will occasionally be too low to detect, necessitating a report stating that total urine porphyrin is less than the detection limit [approx. 50 nmol/L]. If the laboratory possesses a fluorimeter, a similar but much more sensitive method can be performed by carrying out an excitation scan of acidified urine [Int J Biochem 1980; 12: 10512].

♦ Total porphyrin in faeces

The recommended method analyses total faecal porphyrin by first removing interfering chlorophyll pigments with diethyl ether and then scanning an acidic extract by spectrophotometry. Results are expressed on a dry weight basis, however determining faecal dry weight is troublesome. An alternative is to omit drying and express results per wet weight with a lower reference range. An increased total faecal porphyrin requires further investigation by fractionation, preferably by HPLC. Both the interpretation of raised total faecal porphyrin and subsequent faecal porphyrin fractionation can be difficult and requires experience. A valuable discussion of possible interpretations and pitfalls for the unwary is given.

Analysis of total faecal porphyrin presents many difficulties; it is an unpleasant analysis requiring accurate weighing of both wet and dry faeces. Although the drying step can be omitted, the results can be difficult to interpret and no commercial control material is available. In lieu of faecal porphyrin analysis, a simple scan of diluted plasma can exclude the porphyrias which present with either bullous lesions or acute photosensitivity symptoms. This is conditional on the availability of a scanning spectrofluorometer with sufficient sensitivity [see section on Fluorescence spectroscopy of plasma]. Thus a case could be made for abandoning faecal porphyrin analysis.

♦ Total porphyrin in erythrocytes

The recommended method involves an initial extraction of an erythrocyte suspension into diethyl ether/acetic acid followed by back-extraction into hydrochloric acid and reading fluorescence. The two major forms of erythrocyte protoporphyrin are free protoporphyrin and zinc protoporphyrin and the method does not distinguish between them. Only the free protoporphyrin can diffuse into plasma and give rise to photosensitivity.

A scanning spectrofluorometer is required for total erythrocyte porphyrin, distinguishing free protoporphyrin and zinc protoporphyrin and fluorescence spectroscopy of plasma porphyrins. Laboratories wishing to perform front line testing for porphyria will need a unit which is often dedicated to porphyrins as most other applications for stand-alone fluorimetry in clinical biochemistry laboratories have been superseded.

♦ Distinguishing free protoporphyrin and zinc protoporphyrin

If the total erythrocyte porphyrin is raised, it is necessary to perform a qualitative procedure to distinguish free protoporphyrin from zinc protoporphyrin. The recommendation is a simple extraction method using ethanol followed by recording the emission spectrum between 550 and 650 nm at an excitation wavelength of 415 nm.

A spectrofluorometer capable of scanning is required for this qualitative procedure. Identification of the free protoporphyrin spectrum is diagnostic of erythropoietic protoporphyria, provided the total erythrocyte porphyrin is significantly raised.

♦ Fluorescence spectroscopy of plasma porphyrins

This technique is very useful both for diagnosing suspected cutaneous porphyria in general and for identifying the type of cutaneous porphyria. A diluted plasma sample is scanned for emission peaks between 550 and 650 nm with an excitation wavelength of 405 nm. It is especially valuable for differentiating bullous porphyria due to porphyria cutanea tarda which is relatively common, from variegate porphyria which is rare. The scanning spectrofluorometer must be capable of detecting the small peak given by a 10 nmol/L solution of coproporphyrin in phosphate buffered saline. Such sensitivity requires a scanning spectrofluorometer fitted with a red sensitive photomultiplier; usually this is the Hamamatsu R928 photomultiplier.

This simple technique was first described in 1980 but not widely used in diagnostic laboratories, possibly as it was published in a specialist dermatology journal by a group mainly interested in research applications rather than routine diagnosis. The test, though simple, requires a scanning spectrofluorometer. The standard photomultiplier fitted to a modern unit will usually be a red sensitive type with sufficient sensitivity for this demanding application. Another advantage is that it can replace faecal porphyrin analysis in the majority of cases. Of the three types of porphyria where faecal porphyrins are routinely elevated [variegate porphyria, porphyria cutanea tarda and hereditary coproporphyria], only hereditary coproporphyria cannot readily be identified by fluorescence spectroscopy of plasma.

Concluding comments

Laboratories seeking simple reliable methods for excluding porphyria have been poorly served by textbook chapters on porphyria, which often suggest methods more suitable for reference laboratories. These aptly named Best Practice Guidelines for the investigation of suspected porphyria are an invaluable self-contained distillation of the authors' experience and the 27 well-chosen references. Highly recommended.

Footnotes

This review article can be downloaded free in PDF format from the following website: jcp.bmjjournals.com


Articles from The Clinical Biochemist Reviews are provided here courtesy of The Australian Association of Clinical Biochemists