Best practice guidelines on first-line laboratory testing for porphyria.

The porphyrias are disorders of haem biosynthesis which present with acute neurovisceral attacks or disorders of sun-exposed skin. Acute attacks occur mainly in adults and comprise severe abdominal pain, nausea, vomiting, autonomic disturbance, central nervous system involvement and peripheral motor neuropathy. Cutaneous porphyrias can be acute or chronic presenting at various ages. Timely diagnosis depends on clinical suspicion leading to referral of appropriate samples for screening by reliable biochemical methods. All samples should be protected from light. Investigation for an acute attack: • Porphobilinogen (PBG) quantitation in a random urine sample collected during symptoms. Urine concentration must be assessed by measuring creatinine, and a repeat requested if urine creatinine <2 mmol/L. • Urgent porphobilinogen testing should be available within 24 h of sample receipt at the local laboratory. Urine porphyrin excretion (TUP) should subsequently be measured on this urine. • Urine porphobilinogen should be measured using a validated quantitative ion-exchange resin-based method or LC-MS. • Increased urine porphobilinogen excretion requires confirmatory testing and clinical advice from the National Acute Porphyria Service. • Identification of individual acute porphyrias requires analysis of urine, plasma and faecal porphyrins. Investigation for cutaneous porphyria: • An EDTA blood sample for plasma porphyrin fluorescence emission spectroscopy and random urine sample for TUP. • Whole blood for porphyrin analysis is essential to identify protoporphyria. • Faeces need only be collected, if first-line tests are positive or if clinical symptoms persist. Investigation for latent porphyria or family history: • Contact a specialist porphyria laboratory for advice. Clinical, family details are usually required.

Audit of the Use of Regular Haem Arginate Infusions in Patients with Acute Porphyria to Prevent Recurrent Symptoms.

The National Acute Porphyria Service (NAPS) provides acute care support and clinical advice for patients in England with active acute porphyria requiring haem arginate treatment and patients with recurrent acute attacks.This audit examined the benefits and complications of regular haem arginate treatment started with prophylactic intent to reduce the frequency of recurrent acute attacks in a group of patients managed through NAPS. We included 22 patients (21 female and 1 male) and returned information on diagnosis, indications for prophylactic infusions, frequency and dose, analgesia, activity and employment and complications including thromboembolic disease and iron overload.The median age at presentation with porphyria was 21 years (range 9-44), with acute abdominal pain as the predominant symptom. Patients had a median of 12 (1-400) attacks before starting prophylaxis and had received a median of 52 (0-1,350) doses of haem arginate. The median age at starting prophylaxis was 28 years (13-58) with a median delay of 4 years (0.5-37) between presentation and prophylaxis. The frequency of prophylactic haem arginate varied from 1 to 8 per month, and 67% patients were documented as having a reduction in pain frequency on prophylaxis. Only one patient developed clinically significant iron overload and required iron chelation, but the number of venous access devices required varied from 1 to 15, with each device lasting a median of 1.2 years before requiring replacement. Six patients stopped haem arginate and in three this was because their symptoms had improved. Prophylactic haem arginate appears to be beneficial in patients with recurrent acute porphyria symptoms, but maintaining central venous access may prove challenging.

Urinary excretion of porphyrins, porphobilinogen and δ-aminolaevulinic acid following an attack of acute intermittent porphyria.

BACKGROUND AND OBJECTIVES: The porphyrias are a group of rare, mainly inherited, diseases caused by a deficiency of one of the enzymes of the haem biosynthesis pathway. The biochemical hallmark of an acute attack is an increase in urine porphobilinogen (PBG), together with an increase in urinary excretion of δ-aminolaevulinic acid (ALA) and total urine porphyrins (TUP). In patients with acute intermittent porphyria (AIP) the concentrations of the porphyrin precursors are thought to remain elevated for many years following an acute attack, although this has not been well documented. METHODS: We measured urine ALA, PBG and TUP excretion in 20 patients with AIP following an attack of acute porphyria over a time period of 3 months to 23 years after their last documented acute attack. RESULTS: We showed that urinary concentrations of all metabolites remain elevated for many years. The urinary half life of TUP was 5.3 years, ALA 7.7 years and PBG 10.6 years. Even after 20 years, PBG concentrations remained elevated above the normal range. CONCLUSIONS: Our study highlights the difficulties of using urinary analysis for diagnosing recurrent attacks, and also raises important questions about the pathophysiology of the condition.

Direct and simultaneous quantitation of 5-aminolaevulinic acid and porphobilinogen in human serum or plasma by hydrophilic interaction liquid chromatography-atmospheric pressure chemical ionization/tandem mass spectrometry.

Serum/plasma concentrations of 5-aminolaevulinic acid (ALA) and porphobilinogen (PBG) are elevated in patients with acute hepatic porphyrias, especially during acute attacks. Current assays require lengthy sample pre-treatment and derivatization steps. We report here a rapid, sensitive and specific hydrophilic interaction liquid chromatography-tandem mass spectrometry method for the direct and simultaneous quantitation of ALA and PBG in serum or plasma following simple protein precipitation with acetonitrile and centrifugation prior to injection. ALA and PBG were detected using selected reaction monitoring mode, following positive atmospheric pressure chemical ionization. Calibration was linear from 0.05 to 50 µmol/L for ALA and PBG. For both analytes, imprecision (relative standard deviation) was <13% and accuracy (percentage nominal concentrations) was between 92 and 107%. The method was successfully applied to the measurement of ALA and PBG in serum or plasma samples for the screening, biochemical diagnosis and treatment monitoring of patients with acute hepatic porphyrias.

Direct and simultaneous determination of 5-aminolaevulinic acid and porphobilinogen in urine by hydrophilic interaction liquid chromatography-electrospray ionisation/tandem mass spectrometry.

Urinary concentrations of 5-aminolaevulinic acid (ALA) and porphobilinogen (PBG) are elevated in patients with acute hepatic porphyrias, especially during acute attacks. Current assays require lengthy sample pre-treatment and derivatisation steps. We report here a rapid, sensitive and specific hydrophilic interaction liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, for the direct and simultaneous quantitation of ALA and PBG in urine following simple dilution with acetonitrile and centrifugation prior to injection. ALA and PBG were detected using selected reaction monitoring mode, following positive electrospray ionisation. Urine samples (N = 46) from active and latent mutation-confirmed acute hepatic porphyria patients and normal subjects (N = 45) were analysed and the results compared with those of a commercially available spectrophotometric method. The validated calibration range was 3-3000 µmol/L for ALA and 2-2000 µmol/L for PBG. For both analytes, imprecision (relative standard deviation) was less than 5% and accuracy (percentage nominal concentrations) was between 88 and 109%. The lower limit of quantitation was 0.1 µmol/L for both analytes. The calculated LC-MS/MS and spectrophotometric results from patient samples compared well [Pearson correlation (r²) of 0.99 and 0.95, for ALA and PBG, respectively]. The method was successfully applied to the measurement of ALA and PBG in urine samples for the screening, biochemical diagnosis and treatment monitoring of patients with acute hepatic porphyrias.

A retrospective analysis of outcome of pregnancy in patients with acute porphyria.

A survey was posted to 27 women with acute porphyria about complications and outcome of pregnancy. Fifteen women returned the completed questionnaire and the pregnancies were characterised depending on the timing of diagnosis of porphyria. Four women were diagnosed with porphyria before the first pregnancy, five during a pregnancy and six after pregnancy. Five women were diagnosed with porphyria from family studies and the remaining ten were diagnosed when they presented with acute symptoms. There were a total of 33 pregnancies and 23 live births. Four women reported symptoms associated with porphyria during pregnancy. Two women received treatment with haem arginate during pregnancy with one of them having haem arginate therapy weekly with no adverse effect either to her or the baby. One woman had acute pain and skin symptoms during pregnancy but was not diagnosed until after delivery, and another reported acute symptoms during pregnancy. There were no differences, compared to the general population, between birth weight and miscarriage rate, and there were few obstetric complications with only one patient having pre-eclampsia at 37 weeks gestation. These results show that pregnancy is typically uncomplicated in acute porphyria, and that problems are more likely if the porphyria has not been diagnosed previously. We found that administration of haem arginate during pregnancy is safe and its continuous use during pregnancy has no detrimental effect on the outcome of pregnancy.

C-terminal deletions in the ALAS2 gene lead to gain of function and cause X-linked dominant protoporphyria without anemia or iron overload.

All reported mutations in ALAS2, which encodes the rate-regulating enzyme of erythroid heme biosynthesis, cause X-linked sideroblastic anemia. We describe eight families with ALAS2 deletions, either c.1706-1709 delAGTG (p.E569GfsX24) or c.1699-1700 delAT (p.M567EfsX2), resulting in frameshifts that lead to replacement or deletion of the 19-20 C-terminal residues of the enzyme. Prokaryotic expression studies show that both mutations markedly increase ALAS2 activity. These gain-of-function mutations cause a previously unrecognized form of porphyria, X-linked dominant protoporphyria, characterized biochemically by a high proportion of zinc-protoporphyrin in erythrocytes, in which a mismatch between protoporphyrin production and the heme requirement of differentiating erythroid cells leads to overproduction of protoporphyrin in amounts sufficient to cause photosensitivity and liver disease.

Erythropoietin-- measurement and clinical applications.

Erythropoietin (EPO) is an endogenous hormone produced primarily by the kidney which controls the production of erythrocytes. The main stimulus to production is low tissue oxygen (hypoxia) and EPO triggers the formation of red blood cells by binding to a receptor on erythroid progenitor target cells. Alteration in the EPO regulatory system produces a change in circulating EPO in a variety of disease states, such as renal anaemia and polycythaemia. The availability of recombinant EPO in the 1980s transformed the treatment of anaemia, particularly anaemia of end stage renal disease, and led to the development of more sensitive and specific assays for the measurement of EPO. There are more widespread uses for EPO and preliminary studies indicate that EPO may be useful as a neuroprotective agent by reducing inflammation near the site of injury. The use of EPO to boost endurance in athletes has attracted unwanted publicity, although analytical techniques are now available that can differentiate between endogenous and recombinant EPO. Different types of erythropoietic agents have been developed with a longer plasma half-life and the ability to maintain haemoglobin levels for longer periods and reduce the need for frequent dosing with EPO.

Definition of subtypes of essential thrombocythaemia and relation to polycythaemia vera based on JAK2 V617F mutation status: a prospective study.

BACKGROUND: An acquired V617F mutation in JAK2 occurs in most patients with polycythaemia vera, but is seen in only half those with essential thrombocythaemia and idiopathic myelofibrosis. We aimed to assess whether patients with the mutation are biologically distinct from those without, and why the same mutation is associated with different disease phenotypes. METHODS: Two sensitive PCR-based methods were used to assess the JAK2 mutation status of 806 patients with essential thrombocythaemia, including 776 from the Medical Research Council's Primary Thrombocythaemia trial (MRC PT-1) and two other prospective studies. Laboratory and clinical features, response to treatment, and clinical events were compared for V617F-positive and V617F-negative patients with essential thrombocythaemia. FINDINGS: Mutation-positive patients had multiple features resembling polycythaemia vera, with significantly increased haemoglobin (mean increase 9.6 g/L, 95% CI 7.6-11.6 g/L; p<0.0001), neutrophil counts (1.1x10(9)/L, 0.7-1.5x10(9)/L; p<0.0001), bone marrow erythropoiesis and granulopoiesis, more venous thromboses, and a higher rate of polycythaemic transformation than those without the mutation. Mutation-positive patients had lower serum erythropoietin (mean decrease 13.8 U/L; 95% CI, 10.8-16.9 U/L; p<0.0001) and ferritin (n=182; median 58 vs 91 mug/L; p=0.01) concentrations than did mutation-negative patients. Mutation-negative patients did, nonetheless, show many clinical and laboratory features that were characteristic of a myeloproliferative disorder. V617F-positive individuals were more sensitive to therapy with hydroxyurea, but not anagrelide, than those without the JAK2 mutation. INTERPRETATION: Our results suggest that JAK2 V617F-positive essential thrombocythaemia and polycythaemia vera form a biological continuum, with the degree of erythrocytosis determined by physiological or genetic modifiers.

Rhabdomyolysis in a patient with acute intermittent porphyria.

There are several conditions associated with rhabdomyolysis, such as direct muscle injury, viral infections, metabolic disorders and toxic effects from ingestion of drugs such as alcohol, opiates, cocaine or heroin. We report on a patient who was admitted to the accident and emergency unit with a clinical presentation of rhabdomyolysis and acute intermittent porphyria.

Autonomic function and serum erythropoietin levels in chronic fatigue syndrome.

OBJECTIVE: Given previous findings, we wished to investigate whether there was evidence of autonomic dysfunction in patients with chronic fatigue syndrome, and whether this could be related to reduced erythropoietin levels and altered red blood cell indices. METHODS: We assessed autonomic function and analysed blood parameters (including erythropoietin) in 22 patients with chronic fatigue syndrome who were medication-free and without comorbid depression or anxiety. Results were compared to 23 iron-deficiency anaemia patients and 18 healthy individuals. RESULTS: Autonomic testing in patients with chronic fatigue syndrome yielded a significantly greater increase in heart rate together with a more pronounced systolic blood pressure fall on standing compared to healthy individuals. Heart rate beat-to-beat variation on deep breathing and responses to the Valsalva manoeuvre were normal. Two of 22 patients with chronic fatigue had mild normochromic normocytic anaemia with normal ferritin, vitamin B12 and folate levels. Serum erythropoietin levels were within reference range. CONCLUSION: Some autonomic dysfunction is present in chronic fatigue syndrome (CFS) patients; the explanation remains uncertain, but could relate to cardiovascular deconditioning. There were no major haematological, biochemical or immunological abnormalities in these patients.

Erythropoietin treatment in the neuropsychiatric porphyrias.

BACKGROUND: In a previous report, 31 patients with neuropsychiatric porphyria were studied and nine of these patients were anaemic in association with inappropriately low serum erythropoietin levels. We were also able to demonstrate that treatment with erythropoietin in non-porphyric patients (mainly diabetic patients with autonomic neuropathy) significantly reduced urinary delta-aminolaevulinic acid levels. METHODS: We treated six porphyric patients, five of whom were anaemic, with recombinant human erythropoietin (1000-2000 IU thrice weekly). They were all in clinical but not biochemical remission. Full blood count, including reticulocytes and platelets, urinary delta-aminolaevulinic acid, porphobilinogen and total porphyrins were measured monthly. Baseline serum ferritin, vitamin B(12), folate and C-reactive protein levels were all within the normal range and serum creatinine did not exceed 126 micromol/l. RESULTS: After 3 months of treatment, the average baseline haemoglobin increased significantly (p=0.01). When treatment was stopped, the haemoglobin decreased and after 3 months pre-treatment, haemoglobin levels were reached. Urinary delta-aminolaevulinic acid, porphobilinogen and porphyrin levels all tended to decrease during treatment with erythropoietin, but the difference between baseline and 3 months of erythropoietin was statistically significant only for porphobilinogen (p=0.03). The severity of porphyria attacks was reduced and the quality of life increased during treatment with erythropoietin. CONCLUSION: We conclude that in some porphyric patients treatment with erythropoietin reduces urinary delta-aminolaevulinic acid, porphobilinogen and porphyrin levels with an increase in well-being and a reduction in the severity of porphyria attacks.

Serum erythropoietin values in erythrocytoses and in primary thrombocythaemia.

Serum erythropoietin (Epo) values were estimated in samples from 125 patients with erythrocytosis to examine the specificity and sensitivity of reduced and raised values in the diagnosis of polycythaemia vera (PV) and secondary erythrocytosis (SE) respectively. Additionally, Epo values were estimated in samples from 49 patients with primary thrombocythaemia (PT) to determine whether Epo values were altered. We found high specificity (92%) and moderate sensitivity (64%) of low serum Epo values (below the reference range) in the diagnosis of PV, and also poor sensitivity (47%) of raised Epo values in the diagnosis of SE. Raised Epo values were not observed in PV patients with Hb > 14.0 g/dl and were only observed in one PV patient with a relatively low Hb recovering from a gastro-intestinal haemorrhage. Raised Epo values occurred in some patients with apparent erythrocytosis (AE) and idiopathic erythrocytosis (IE), mainly at normal (rather than raised) Hb values (< 16 g/dl). Low Epo values occurred in a few AE, IE and SE patients at higher Hb values (> 16 g/dl). Low Epo values were less specific for PV when the Hb was raised, while raised Epo values were less specific for SE when the Hb was not raised. Approximately one third of patients with PT had a low (below the reference range) Epo value, this being associated with a high normal Hb (> 14 g/dl, P < 0.001) and showing a trend towards association with absence of treatment. The high normal Hb values were in turn associated with an increased incidence of thrombotic events (P < 0.05). These findings could influence the future investigation and management of PT patients.