LABORATORY DIAGNOSIS
 ACUTE
PORPHYRIAS
I. DIAGNOSIS OF THE
ACUTE ATTACK
Background information
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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. |
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Examination of urine for excess PBG is,
therefore, the essential investigation in
patients with a suspected attack of acute
porphyria. |
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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
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Urinary PBG is best analysed
in a fresh, random sample (10-20 mL) collected
without any preservative but protected from
light. |
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24 hour collections delay diagnosis and
increase the risk of losses during the collection
period. |
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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
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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. |
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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. |
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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). |
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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). |
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All laboratories undertaking qualitative
or quantitative determination of urinary
PBG should participate regularly in an appropriate
EQA scheme. |
Comments
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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. |
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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. |
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Interpretation
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Normal adult urine contains
< 1.5 µmol PBG/mmol creatinine
( < 10 µmol/L). |
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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. |
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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. |
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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. |
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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.
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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. |
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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
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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. |
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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. |
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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.
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1- Specimen collection and stability
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Urine: see above. |
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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. |
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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
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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).
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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). |
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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). |
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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). |
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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). |
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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
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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).
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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. |
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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. |
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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
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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. |
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Urine contains high concentrations
of coproporphyrin III in addition
to uroporphyrin (from PBG). |
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A plasma porphyrin fluorescence
emission peak at 620 nm is present
in some patients. |
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For further information on faecal
coproporphyrin isomer ratios (more
information). |
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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
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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). |
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PBG excretion may be normal or
increased up to 5-fold. |
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Erythrocyte zinc protoporphyrin
is markedly increased. |
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Faecal porphyrin concentrations
are normal. |
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B) DIAGNOSIS DURING REMISSION
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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:
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because
previous symptoms suggest
acute porphyria or |
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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. |
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Patients may
be asymptomatic or have chronic symptoms,
eg recurrent abdominal pain, suggesting
acute porphyria. |
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Diagnosis may be difficult because:
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urinary, faecal and
plasma porphyrin concentrations may
return to normal during remission
in all the autosomal dominant acute
porphyrias. |
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neither enzyme measurements nor
mutational analysis are 100% sensitive
and 100% specific. |
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Initial investigations
In all patients, determine:
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1 |
urinary PBG and ALA excretion |
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2 |
faecal protoporphyrin, coproporphyrin
and coproporphyrin III/I isomer ratio |
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3 |
plasma porphyrin fluorescence emission
spectrum |
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4 |
erythrocyte PBG deaminase activity if
routine haematology is normal. |
Interpretation and further investigation
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If
one or more of tests 1-3
is abnormal, porphyria is
confirmed
(more about
diagnostic criteria). |
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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. |
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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. |
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If all four tests are normal,
one or more of the following should be undertaken:
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the patient should
be asked to submit samples for PBG
and porphyrin analyses immediately
when symptoms recur |
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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) |
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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. |
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Comments
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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. |
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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). |
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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. |
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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. |
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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. |
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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. |
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Exclusion of autosomal dominant acute porphyria
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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. |
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When the family mutation is unknown,
it is recommended that the maximum probability
of having porphyria is estimated. |
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Estimation of the probability requires
knowledge of:
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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. |
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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. |
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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). |
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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
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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. |
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Comments
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Minimum
diagnostic criteria are indicated. |
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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. |
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In patients on chronic haemodialysis
who develop skin blisters, PCT can
be confirmed or excluded by measuring
isocoproporphyrin in faeces. |
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