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Biochemical Diagnosis of Acute Hepatic Porphyria: Updated Expert Recommendations for Primary Care Physicians

Open AccessPublished:April 15, 2021DOI:https://doi.org/10.1016/j.amjms.2021.03.004

      Abstract

      Acute hepatic porphyria (AHP) is a group of rare, metabolic diseases where patients can experience acute neurovisceral attacks, chronic symptoms, and long-term complications. Diagnostic biochemical testing is widely available and effective, but a substantial time from symptom onset to diagnosis often delays treatment and increases morbidity. A panel of laboratory scientists and clinical AHP specialists collaborated to produce recommendations on how to enhance biochemical diagnosis of AHP in the USA. AHP should be considered in the differential diagnosis of unexplained abdominal pain, the most common symptom, soon after excluding common causes. Measurement of porphobilinogen (PBG) and porphyrins in a random urine sample, with results normalized to creatinine, is recommended as an effective and cost-efficient initial test for AHP. Delta-aminolevulinic acid testing may be included but is not essential. The optimal time to collect a urine sample is during an attack. Substantial PBG elevation confirms an AHP diagnosis and allows for prompt treatment initiation. Additional testing can determine AHP subtype and identify at-risk family members. Increased awareness of AHP and correct diagnostic methods will reduce diagnostic delay and improve patient outcomes.

      Key Indexing Terms

      Introduction

      Porphyria is a family of rare, genetic diseases caused by altered activity of the 8 enzymes of heme biosynthesis (Supplementary Figure 1).
      • Puy H
      • Gouya L
      • Deybach JC
      Porphyrias.
      Porphyria results when transformation of porphyrins and porphyrin precursors into heme is impaired and intermediates of this pathway accumulate. The body makes heme in all tissues, but mainly in the bone marrow and liver. Acute hepatic porphyria (AHP) includes 4 of these diseases that can cause acute and debilitating neurovisceral attacks, which if not diagnosed and treated promptly can be life-threatening.
      • Anderson KE
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      Recommendations for the diagnosis and treatment of the acute porphyrias.
      ,
      • Balwani M
      • Desnick RJ
      The porphyrias: advances in diagnosis and treatment.
      Symptoms of AHP occur most commonly in women of reproductive age.
      • Gouya L
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      • Balwani M
      • et al.
      EXPLORE: a prospective, multinational, natural history study of patients with acute hepatic porphyria with recurrent attacks.
      The 4 subtypes of AHP, in order of decreasing prevalence in most countries, are acute intermittent porphyria (AIP), variegate porphyria (VP), and hereditary coproporphyria (HCP), all of which are autosomal dominant, and the ultra-rare delta-aminolevulinic acid (ALA) dehydratase deficiency porphyria (ADP), which is autosomal recessive (Supplementary Table 1).
      • Balwani M
      • Wang B
      • Anderson KE
      • et al.
      Acute hepatic porphyrias: recommendations for evaluation and long-term management.
      Upregulation of hepatic ALA synthase 1 (ALAS1), the ubiquitous form of the first and rate-limiting enzyme in the heme biosynthesis pathway, by certain stressors, drugs, hormones, and nutritional alterations, leads to the accumulation of ALA and porphobilinogen (PBG) in patients with AHP.
      • Balwani M
      • Desnick RJ
      The porphyrias: advances in diagnosis and treatment.
      ,
      • Pischik E
      • Kauppinen R
      An update of clinical management of acute intermittent porphyria.
      Substantial elevation of ALA and PBG is a distinctive biochemical feature and identifies AHP as the likely cause of acute neurovisceral attacks, which frequently require hospitalization and are associated with high morbidity.
      • Gouya L
      • Ventura P
      • Balwani M
      • et al.
      EXPLORE: a prospective, multinational, natural history study of patients with acute hepatic porphyria with recurrent attacks.
      ,
      • Bissell DM
      • Lai JC
      • Meister RK
      • Blanc PD
      Role of delta-aminolevulinic acid in the symptoms of acute porphyria.
      Clinical presentation is variable, although most attacks feature severe, diffuse abdominal pain that often occurs with a variety of other symptoms (Figure 1).
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      Acute porphyrias in the USA: features of 108 subjects from porphyrias consortium.
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      Occasionally, severe attacks lead to permanent neurologic damage or fatality.
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      • Wikberg A
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      • Lithner F
      Signs of neuropathy in the lower legs and feet of patients with acute intermittent porphyria.
      AHP has been associated with a variety of other neurologic symptoms and serious long-term complications (Figure 1).
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      • Cengia B
      • Bonkovsky HL
      Acute hepatic porphyrias: review and recent progress.
      ,
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      High prevalence of and potential mechanisms for chronic kidney disease in patients with acute intermittent porphyria.
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      Review of hepatocellular cancer, hypertension and renal impairment as late complications of acute porphyria and recommendations for patient follow-up.
      FIGURE 1
      FIGURE 1Symptoms of acute attacks and associated long-term complications in patients with acute hepatic porphyria.
      • Gouya L
      • Ventura P
      • Balwani M
      • et al.
      EXPLORE: a prospective, multinational, natural history study of patients with acute hepatic porphyria with recurrent attacks.
      In AIP, fewer than 10% of individuals with a pathogenic mutation develop disease symptoms, and the majority experience only a few attacks during their lifetime.
      • Stein PE
      • Badminton MN
      • Rees DC
      Update review of the acute porphyrias.
      ,
      • Elder G
      • Harper P
      • Badminton M
      • et al.
      The incidence of inherited porphyrias in Europe.
      ,
      • Chen B
      • Solis-Villa C
      • Hakenberg J
      • et al.
      Acute intermittent porphyria: predicted pathogenicity of HMBS variants indicates extremely low penetrance of the autosomal dominant disease.
      Research with large population databases suggests a relatively high frequency of pathogenic AIP variants in the general population (approximately 1:1600), so it is likely that disease penetrance is much lower than 10% and that a diagnosis is often not suspected and missed, particularly in patients with nonsevere or occasional attacks.
      • Pischik E
      • Kauppinen R
      An update of clinical management of acute intermittent porphyria.
      ,
      • Chen B
      • Solis-Villa C
      • Hakenberg J
      • et al.
      Acute intermittent porphyria: predicted pathogenicity of HMBS variants indicates extremely low penetrance of the autosomal dominant disease.
      Importantly, symptoms recur frequently in some individuals and may be difficult to control, resulting in a high disease burden and impaired quality of life.
      • Simon A
      • Pompilus F
      • Querbes W
      • et al.
      Patient perspective on acute intermittent porphyria with frequent attacks: a disease with intermittent and chronic manifestations.
      ,
      • Schmitt C
      • Lenglet H
      • Yu A
      • et al.
      Recurrent attacks of acute hepatic porphyria: major role of the chronic inflammatory response in the liver.
      Approximately two-thirds of these patients experience chronic symptoms between attacks, often daily, with the most common being pain, fatigue, nausea, anxiety, and trouble sleeping.
      • Gouya L
      • Ventura P
      • Balwani M
      • et al.
      EXPLORE: a prospective, multinational, natural history study of patients with acute hepatic porphyria with recurrent attacks.
      ,
      • Simon A
      • Pompilus F
      • Querbes W
      • et al.
      Patient perspective on acute intermittent porphyria with frequent attacks: a disease with intermittent and chronic manifestations.
      Management of AHP includes recognizing and removing precipitating factors that induce hepatic ALAS1 and instituting pharmacotherapies for attack treatment and/or prevention.
      • Neeleman RA
      • Wensink D
      • Wagenmakers M
      • et al.
      Diagnostic and therapeutic strategies for porphyrias.
      Attacks are treated with intravenous glucose or hemin, while prophylactic treatments include gonadotropin-releasing hormone agonists, intravenous hemin, and subcutaneous givosiran, which is the only Food and Drug Administration-approved treatment for adults with AHP to help prevent attacks.
      • Pischik E
      • Kauppinen R
      An update of clinical management of acute intermittent porphyria.
      ,
      • Yarra P
      • Faust D
      • Bennett M
      • et al.
      Benefits of prophylactic heme therapy in severe acute intermittent porphyria.
      ,
      • Balwani M
      • Sardh E
      • Ventura P
      • et al.
      Phase 3 trial of RNAi therapeutic givosiran for acute intermittent porphyria.
      Symptomatic treatments include medications for pain, nausea, and vomiting.
      • Balwani M
      • Wang B
      • Anderson KE
      • et al.
      Acute hepatic porphyrias: recommendations for evaluation and long-term management.
      ,
      • Pischik E
      • Kauppinen R
      An update of clinical management of acute intermittent porphyria.
      ,
      • Balwani M
      • Sardh E
      • Ventura P
      • et al.
      Phase 3 trial of RNAi therapeutic givosiran for acute intermittent porphyria.
      The timely diagnosis of AHP can be challenging for the following reasons:
      Tabled 1
      1. As AHP is relatively rare, it is often not considered as part of a differential diagnosis when assessing symptoms such as acute or recurrent abdominal pain.
      • Anderson KE
      • Bloomer JR
      • Bonkovsky HL
      • et al.
      Recommendations for the diagnosis and treatment of the acute porphyrias.
      ,
      • Bissell DM
      • Anderson KE
      • Bonkovsky HL
      Porphyria.
      2. Patients experiencing acute attacks often present to emergency departments where less common causes of typical presenting symptoms, such as abdominal pain, are often not considered.
      • Bissell DM
      • Anderson KE
      • Bonkovsky HL
      Porphyria.
      3. AHP has a variable presentation with no specific or definitive phenotypic manifestations, making it difficult to differentiate from other more common conditions by clinical evaluation alone.
      • Balwani M
      • Wang B
      • Anderson KE
      • et al.
      Acute hepatic porphyrias: recommendations for evaluation and long-term management.
      ,
      • Stein PE
      • Badminton MN
      • Rees DC
      Update review of the acute porphyrias.
      4. Laboratory diagnostic testing is definitive, but too often the wrong tests are ordered and results are misinterpreted or not reported rapidly, which discourages testing and delays diagnosis and treatment.
      5. As a multi-system disease, AHP has not traditionally fallen under the responsibility of a single medical specialty; hence, clinical practice guidelines for its diagnosis and treatment are seldom developed.
      These factors contribute to delays in diagnosis or to misdiagnosis, resulting in inappropriate or harmful treatments, including certain medications that can upregulate the heme biosynthesis pathway and worsen AHP symptoms.
      • Anderson KE
      • Bloomer JR
      • Bonkovsky HL
      • et al.
      Recommendations for the diagnosis and treatment of the acute porphyrias.
      ,
      • Simon A
      • Pompilus F
      • Querbes W
      • et al.
      Patient perspective on acute intermittent porphyria with frequent attacks: a disease with intermittent and chronic manifestations.
      ,
      • Stein PE
      • Badminton MN
      • Barth JH
      • et al.
      Acute intermittent porphyria: fatal complications of treatment.
      In the USA, there is a lack of awareness about which tests should be ordered for a cost-effective approach to first- and second-line testing, how samples should be collected and handled, and how results should be interpreted.
      • Anderson KE
      • Bloomer JR
      • Bonkovsky HL
      • et al.
      Recommendations for the diagnosis and treatment of the acute porphyrias.
      ,
      • Balwani M
      • Desnick RJ
      The porphyrias: advances in diagnosis and treatment.
      ,
      • Pischik E
      • Kauppinen R
      An update of clinical management of acute intermittent porphyria.
      ,
      • Woolf J
      • Marsden JT
      • Degg T
      • et al.
      Best practice guidelines on first-line laboratory testing for porphyria.
      ,
      • Aarsand AK
      • Petersen PH
      • Sandberg S
      Estimation and application of biological variation of urinary delta-aminolevulinic acid and porphobilinogen in healthy individuals and in patients with acute intermittent porphyria.
      This contrasts with other parts of the world, such as the UK, the European Union, and Australia, where testing standards and guidelines have been developed.
      • Woolf J
      • Marsden JT
      • Degg T
      • et al.
      Best practice guidelines on first-line laboratory testing for porphyria.
      ,
      • Stein P
      • Badminton M
      • Barth J
      • et al.
      Best practice guidelines on clinical management of acute attacks of porphyria and their complications.
      ,
      • Sies CW
      • Cronin V
      • Florkowski CM
      • et al.
      Regional variation in analytical techniques used in the diagnosis and monitoring of porphyria: a case for harmonisation?.
      Therefore, recommendations on diagnostic testing for AHP are needed in the USA to ensure consistency of practice by healthcare professionals, promote test availability, and provide appropriate interpretation of results. This report summarizes the recommendations of a group of clinical and laboratory specialists for biochemical diagnostic testing for AHP in the USA and is intended to reduce diagnostic delay and uncertainty and improve patient outcomes.

      Methods

      A panel consisting of laboratory directors and genetic counselors from 5 major AHP diagnostic laboratories in the USA, 1 diagnostic testing consultant, 2 clinical AHP experts, and 1 AHP patient advocate participated in a virtual meeting on April 29, 2020. A subset of this group originally convened in Bethesda, Maryland, on April 7, 2018. Representatives of the sponsoring pharmaceutical company, Alnylam Pharmaceuticals, organized and attended both meetings. The objective of the meetings was to develop practical recommendations aimed at enhancing the diagnosis of AHP in the USA.

      Material Content

      These recommendations should be reviewed and updated approximately every 2-3 years, based on evolving knowledge and changing diagnostic methods.

      Porphyria Testing

      Tests for AHP are not the same as tests for cutaneous porphyrias that present with blistering or nonblistering photosensitivity. Therefore, it is inappropriate for a healthcare professional to request a “porphyrin screen” or “porphyrin panel.” These terms do not specify which testing is needed to confirm or exclude the type of porphyria suspected and may lead to incorrect test ordering and uninformative results.
      ALA, PBG, and porphyrins are intermediates produced sequentially in the synthesis of heme. Their concentrations are markedly elevated in urine when AHP is symptomatic. However, urine porphyrin elevation occurs in many medical conditions, so is not itself specific for AHP. Substantial PBG elevation has a particularly high degree of specificity and sensitivity for diagnosis of AHP, with an elevation many multiples above normal not occurring in any other medical condition. The only exception to this is for the ultra-rare AHP subtype ADP, in which PBG levels are normal or only slightly elevated and only ALA and porphyrins are markedly increased. While ALA is the most likely causative neurotoxin in AHP attacks,
      • Stein P
      • Badminton M
      • Barth J
      • et al.
      Best practice guidelines on clinical management of acute attacks of porphyria and their complications.
      PBG elevations generally exceed those of ALA in AIP, VP, and HCP. Elevation of ALA can also occur in other conditions, including hereditary tyrosinemia type 1 and lead poisoning.
      • Bissell DM
      • Lai JC
      • Meister RK
      • Blanc PD
      Role of delta-aminolevulinic acid in the symptoms of acute porphyria.
      ,
      • Besur S
      • Hou W
      • Schmeltzer P
      • Bonkovsky HL
      Clinically important features of porphyrin and heme metabolism and the porphyrias.
      While elevations in total porphyrins are nonspecific, they may persist longer than ALA and PBG between attacks, especially in VP and HCP, and therefore their measurement can enhance sensitivity of initial testing. Urinary porphyrins include varying proportions of uroporphyrin (octacarboxylporphyrin), heptacarboxylporphyrin, hexacarboxylporphyrin, pentacarboxylporphyrin, and coproporphyrin (tetracarboxylporphyrin), but the amounts and proportions of these individual porphyrins do not reliably differentiate AHP. Of note, reddish, purple, or brown urine discoloration is common in AHP, especially during acute attacks, and urine may darken further upon exposure to light. However, these observations are nonspecific and should not be considered sufficient for diagnosis. Details of diagnostic tests used for AHP are summarized in Table 1.
      TABLE 1Summary of AHP testing.
      Test name
      Always refer to laboratory-specific procedures.
      RoleCollection timingSample typeSample requirementsTransportationCPT code
      CPT codes are used for recording medical, surgical, and diagnostic services in the USA.
      Urine PBG
      Normalized to creatinine.
      Porphyrin precursor with highest degree of specificity and sensitivity for diagnosis of AHP; potentially neurotoxicMost elevated while symptomaticRandom urine
      24-hour urine not necessary.
      ~10 mL

      Light protected
      Frozen (preferred) or refrigerated84110
      Urine ALA
      Normalized to creatinine.
      Porphyrin precursor with high degree of specificity and sensitivity for diagnosis of AHP; potentially neurotoxicMost elevated while symptomaticRandom urine
      24-hour urine not necessary.
      ~10 mL

      Light protected
      Frozen (preferred) or refrigerated82135
      Urine porphyrins
      24-hour urine not necessary.
      ,
      Porphyrins may be fractionated.
      High degree of sensitivity but not specific for diagnosis of AHPElevation may be more persistent than PBG and ALARandom urine
      24-hour urine not necessary.
      ~10 mL

      Light protected
      Frozen (preferred) or refrigerated84120
      Plasma porphyrinsHigh degree of specificity and sensitivity for diagnosis of VPElevation persistent especially in VPPlasma~2–5 mL

      Light protected
      Frozen (preferred) or refrigerated82542
      Fecal porphyrinsHigh degree of specificity and sensitivity and can differentiate VP and HCPElevation persistent especially in VP and HCPStool (random or

      24-hour)
      ~10 g

      Light protected
      Frozen (preferred) or refrigerated84126
      Genetic testing (HMBS, PPOX, CPOX, ALAD)Assessment can differentiate AHP subtypes and identify at-risk family membersCan be performed anytimeBlood
      Blood should be collected in an EDTA tube (lavender top). Abbreviations: AHP, acute hepatic porphyria; ALA, delta-aminolevulinic acid; ALAD, ALA dehydratase; CPOX, coproporphyrinogen oxidase; CPT, Current Procedural Terminology; EDTA, ethylenediaminetetraacetic acid; HCP, hereditary coproporphyria; HMBS, hydroxymethylbilane synthase; PBG, porphobilinogen; PPOX, protoporphyrinogen oxidase; VP, variegate porphyria.
      or saliva

      (if accepted)
      ~3–5 mL for blood

      Follow instructions for saliva kits
      Room temperature81479, 81405, 81406
      a Always refer to laboratory-specific procedures.
      b CPT codes are used for recording medical, surgical, and diagnostic services in the USA.
      c Normalized to creatinine.
      d 24-hour urine not necessary.
      e Porphyrins may be fractionated.
      f Blood should be collected in an EDTA tube (lavender top).Abbreviations: AHP, acute hepatic porphyria; ALA, delta-aminolevulinic acid; ALAD, ALA dehydratase; CPOX, coproporphyrinogen oxidase; CPT, Current Procedural Terminology; EDTA, ethylenediaminetetraacetic acid; HCP, hereditary coproporphyria; HMBS, hydroxymethylbilane synthase; PBG, porphobilinogen; PPOX, protoporphyrinogen oxidase; VP, variegate porphyria.
      Other porphyria types, such as porphyria cutanea tarda (PCT) and erythropoietic protoporphyria (EPP), which are due to alterations in different enzymes in the heme pathway, are characterized by photocutaneous symptoms (Supplementary Table 1). These diseases result from high levels of photosensitizing porphyrins, which are produced in the liver or bone marrow and circulate in plasma and/or erythrocytes. Notably, 2 AHPs (VP and HCP) can cause blistering skin manifestations, either with or in the absence of neurovisceral symptoms, and are often misdiagnosed as PCT. EPP is distinct in causing an acute, nonblistering type of photosensitivity.
      • Bissell DM
      • Anderson KE
      • Bonkovsky HL
      Porphyria.

      First-line (initial) testing

      For a patient with suspected AHP, simultaneous measurement of urinary PBG and total porphyrins (with or without fractionation) can be performed (Figure 2). ALA can also be measured but is never elevated if PBG and porphyrins are normal. Large elevations in PBG do not result from any medical condition other than AIP, VP, and HCP, and this high degree of specificity enables their prompt recognition and treatment. Timely results, particularly for PBG, are important for suspected acute attacks, which can be life-threatening. Therefore, a random rather than 24-hour urine specimen is preferred. Results should be normalized to creatinine measured in the same specimen, although initial reports may express results per liter. Measuring total porphyrins (with or without fractionation) adds sensitivity to first-line testing for 2 reasons. 1) It reduces the potential for missed diagnoses as PBG and ALA are often less elevated and return to normal more rapidly in VP and HCP than in AIP, whereas urine porphyrin elevations are more persistent. 2) The ultra-rare ADP (only 8 cases documented worldwide) causes little or no PBG elevation but markedly elevated urine ALA and coproporphyrin III. Expected results of first-line testing for each subtype of AHP are summarized in Table 2.
      FIGURE 2
      FIGURE 2An algorithm describing the recommended diagnostic process for first-line testing of AHP.
      aThese are the recommendations of the authors; individual laboratory guidelines may differ and should take precedence when testing is performed. Abbreviations: AHP, acute hepatic porphyria; ALA, delta-aminolevulinic acid; HCP, hereditary coproporphyria; PBG, porphobilinogen; VP, variegate porphyria.
      TABLE 2Results of recommended diagnostic biochemical tests indicating each AHP subtype.
      SymptomsFirst-line testingSecond-line testing
      AHP subtypeAcute attacksCutaneous symptoms on light-exposed skinUrinary PBGUrinary ALA
      May be used as part of second-line or follow-up biochemical testing. Abbreviations: ADP, delta-aminolevulinic acid dehydratase deficiency porphyria; AHP, acute hepatic porphyria; AIP, acute intermittent porphyria; ALA, delta-aminolevulinic acid; COPRO III, coproporphyrin III; HCP, hereditary coproporphyria; PBG, porphobilinogen; PROTO, protoporphyrin; URO, uroporphyrin; VP, variegate porphyria.
      Urinary porphyrinsPlasma porphyrins with fluorescence scanFecal porphyrins
      AIPYesNoIncreasedIncreasedIncreased URO, COPRO IIINo peak or ~619 nmNormal or slight increase
      VPYesYesIncreasedIncreasedIncreased URO, COPRO III~626 nmIncreased PROTO, COPRO III
      HCPYesYesIncreasedIncreasedIncreased URO, COPRO IIINo peak or ~619 nmIncreased COPRO III
      ADPYesNoNormal or slight increaseIncreasedIncreased COPRO IIINo peak or ~619 nmNormal or slight increase
      a May be used as part of second-line or follow-up biochemical testing.Abbreviations: ADP, delta-aminolevulinic acid dehydratase deficiency porphyria; AHP, acute hepatic porphyria; AIP, acute intermittent porphyria; ALA, delta-aminolevulinic acid; COPRO III, coproporphyrin III; HCP, hereditary coproporphyria; PBG, porphobilinogen; PROTO, protoporphyrin; URO, uroporphyrin; VP, variegate porphyria.
      First-line test results that should prompt second-line testing include the following:
      • If substantial PBG elevation is found, a diagnosis of AHP is established, and treatment can be started if clinically indicated. Subsequent second-line testing should follow to determine the AHP subtype. If treatment is required, it is advisable to refer the patient to, or seek the advice of, a physician with expertise in AHP (one resource to consider in the US is the Porphyrias Consortium: https://www.rarediseasesnetwork.org/cms/porphyrias/).
      • If total porphyrins are increased, but PBG is not, a diagnosis is not yet established, and second-line testing determines whether a type of porphyria or another medical condition explains this finding. Porphyrin elevations occur in many other medical conditions, especially those affecting the liver or bone marrow,
        • Stein PE
        • Badminton MN
        • Rees DC
        Update review of the acute porphyrias.
        ,
        • Woolf J
        • Marsden JT
        • Degg T
        • et al.
        Best practice guidelines on first-line laboratory testing for porphyria.
        and are a frequent cause for misdiagnosis of AHP. Certain medications, alcohol, hormonal changes, or diet may also elevate porphyrins. Slight elevations of individual porphyrins without elevation of total urinary porphyrins are unlikely to be diagnostically significant and are readily misinterpreted.
      • Marked elevation in ALA and coproporphyrin III without elevation of PBG suggests ADP, but further testing is needed to establish this diagnosis.
      • Minimal elevations of PBG and ALA are not diagnostic for AHP. Analytical methods and upper limits of normal can vary considerably among laboratories, but significant elevations are generally more than 3 times the upper limits of normal and exceed 10 mg/g creatinine. Additional testing may be required to determine the diagnostic significance, if any, of minimal elevations.
      First-line testing allows for cost-effective screening for AHP even if the index of suspicion is not high, as is often the case due to the nonspecific nature of the clinical presentation. If initial testing is negative, but clinical suspicion remains high, first-line testing can be repeated when symptoms recur or select second-line testing may be considered.

      Second-line (Follow-Up) Testing

      Second-line (follow-up) testing (typically indicated 5–10% of the time after first-line testing) includes additional biochemical and/or genetic testing, which may be performed in stages as guided by results. Second-line biochemical testing to differentiate the subtype of AHP or account for elevations in urine porphyrins includes measurement of plasma and fecal porphyrins and plasma fluorescence scanning.
      • Poh-Fitzpatrick MB
      A plasma porphyrin fluorescence marker for variegate porphyria.
      ,
      • Sandberg S
      • Elder GH
      Diagnosing acute porphyrias.
      Elevated porphyrins should be fractionated by high-performance liquid chromatography (HPLC), and, if not performed as part of first-line testing, urinary ALA can be measured at this time. Expected test results are summarized in Table 2. Differentiation of AIP, VP, and HCP depends greatly on analysis of porphyrins in plasma and feces, because in HCP porphyrins are more elevated in feces than urine, and in VP are more likely to be elevated in both plasma and feces than in urine.
      Measurement of enzyme activities may also contribute to biochemical differentiation of AHPs. Half-normal activity of erythrocyte porphobilinogen deaminase (PBGD, also known as hydroxymethylbilane synthase [HMBS]) can support a diagnosis of AIP. However, PBGD enzyme results are not definitive because the range in affected individuals overlaps with the lower end of the normal range. Marked deficiency of erythrocyte ALAD (ALA dehydratase) activity supports a diagnosis of ADP, but exclusion of other causes of this enzyme deficiency and molecular confirmation are required. In VP and HCP, the deficient enzymes are mitochondrial and appropriate testing assays are not currently available in the USA. For these reasons, along with increased access and convenience, genetic (DNA) testing is supplanting enzyme testing as a means of detecting latent disease in AHP family members.
      DNA testing for all porphyrias has become more widely available. Pathogenic variants associated with AHP occur in the following genes: HMBS for AIP, PPOX (protoporphyrinogen oxidase) for VP, CPOX (coproporphyrinogen oxidase) for HCP, and ALAD for ADP.
      • Balwani M
      • Desnick RJ
      The porphyrias: advances in diagnosis and treatment.
      ,
      • Whatley SD
      • Badminton MN
      Role of genetic testing in the management of patients with inherited porphyria and their families.
      Molecular genetic testing can detect ≥97% of known mutations.
      • Whatley SD
      • Mason NG
      • Woolf JR
      • et al.
      Diagnostic strategies for autosomal dominant acute porphyrias: retrospective analysis of 467 unrelated patients referred for mutational analysis of the HMBS, CPOX, or PPOX gene.
      The frequency of pathogenic HMBS variants in the general population may be as high as 1:1600, but clinical penetrance is as low as 1% in terms of diagnosed symptomatic cases.
      • Chen B
      • Solis-Villa C
      • Hakenberg J
      • et al.
      Acute intermittent porphyria: predicted pathogenicity of HMBS variants indicates extremely low penetrance of the autosomal dominant disease.
      ,
      • Lenglet H
      • Schmitt C
      • Grange T
      • et al.
      From a dominant to an oligogenic model of inheritance with environmental modifiers in acute intermittent porphyria.
      Therefore, the finding of an AHP mutation alone is insufficient to establish a diagnosis of clinically or biochemically active AHP. Disease penetrance appears to be higher in clinically affected families, suggesting the presence of unidentified modifying genes that increase the risk for symptom development.
      • Lenglet H
      • Schmitt C
      • Grange T
      • et al.
      From a dominant to an oligogenic model of inheritance with environmental modifiers in acute intermittent porphyria.
      Relatives who have inherited a familial pathogenic mutation are at risk for developing symptoms,
      • Whatley SD
      • Badminton MN
      Role of genetic testing in the management of patients with inherited porphyria and their families.
      ,
      • Whatley SD
      • Mason NG
      • Woolf JR
      • et al.
      Diagnostic strategies for autosomal dominant acute porphyrias: retrospective analysis of 467 unrelated patients referred for mutational analysis of the HMBS, CPOX, or PPOX gene.
      so genetic testing and counseling should be offered to family members. This allows for the proper diagnosis and education of at-risk mutation carriers concerning early recognition of disease symptoms and avoidance of precipitants.
      • Puy H
      • Gouya L
      • Deybach JC
      Porphyrias.
      ,
      • Chen B
      • Whatley S
      • Badminton M
      • et al.
      International Porphyria Molecular Diagnostic Collaborative: an evidence-based database of verified pathogenic and benign variants for the porphyrias.
      At-risk mutation carriers should be evaluated clinically and undergo biochemical testing to assess disease activity annually.

      Sample Requirements

      Healthcare professionals should always reference laboratory-specific instructions or contact the laboratory directly for assistance when collecting, preparing, and transporting samples.

      Collection

      The ideal time to collect urinary, plasma, or fecal samples for biochemical testing is during or shortly after a suspected acute attack, when PBG, ALA, and porphyrin levels are most likely to be elevated. Elevations in porphyrins in urine (in all AHPs), plasma (in VP) and feces (in HCP and VP) are likely to be more persistent than elevations in PBG and ALA, and genetic testing can be performed at any time regardless of the presence or absence of symptoms. Collection of a 24-hour sample has been relied upon historically for measuring PBG, ALA, and porphyrins, but is burdensome, subject to collection error, and may delay diagnosis and treatment. Therefore, a random urine sample (approximately 10 mL) is recommended.

      Handling

      Heat and light can cause oxidation and other chemical changes in PBG, ALA, and porphyrins and result in urine color change (commonly a darkening or red color).
      • Stein PE
      • Badminton MN
      • Rees DC
      Update review of the acute porphyrias.
      ,
      • Bonkovsky HL
      • Guo JT
      • Hou W
      • et al.
      Porphyrin and Heme Metabolism and the Porphyrias.
      Therefore, samples should be collected in amber, opaque, or foil-wrapped containers to protect from light exposure and frozen (preferred) or refrigerated for storage and transport. Preservatives are not required. It should be noted that minimal exposure of samples to light or ambient temperature, as occurs during collection and processing of random samples, is inconsequential, and samples do not need to be collected in total darkness or immediately refrigerated or frozen.

      Special Testing Considerations

      Certain circumstances may alter the approach to biochemical testing for suspected AHP, including: testing patients when they are asymptomatic, the need for short turnaround testing for acutely ill patients not previously diagnosed with AHP, and testing of patients with kidney disease or taking certain drugs that may interfere with testing. These considerations are summarized in Table 3.
      TABLE 3Special testing considerations.
      ConsiderationGuidance
      Testing when asymptomatic (e.g., a past medical history of suggestive symptoms or a positive family history of AHP)Testing can be initiated with typical first-line testing. If results are normal, retest if any symptoms consistent with AHP develop. Alternatively, comprehensive second-line testing including biochemical testing or genetic testing can be performed. If no abnormalities or pathogenic mutations are identified, latent AHP has not been completely excluded but is highly unlikely.
      STAT testingFor treatment to be initiated promptly, laboratories should offer expedited STAT testing for urinary PBG. Using the same sample, porphyrin and ALA levels can subsequently be performed. Healthcare professionals or local laboratories can contact the diagnostic laboratory directly to request STAT testing in urgent cases. Laboratory policies regarding maximum turnaround times should be established, especially for PBG testing, in order to limit treatment delay. If a qualitative assessment of urine PBG is used, the results should be confirmed by a quantitative method on the same sample regardless of the initial result.
      Advanced kidney diseaseThe risk of kidney disease is increased in AHP.
      • Anderson KE
      • Bloomer JR
      • Bonkovsky HL
      • et al.
      Recommendations for the diagnosis and treatment of the acute porphyrias.
      ,
      • Balwani M
      • Wang B
      • Anderson KE
      • et al.
      Acute hepatic porphyrias: recommendations for evaluation and long-term management.
      In patients with advanced renal impairment (little or no urine output), plasma PBG should be measured instead of urinary PBG.
      Drug interactionsSome drugs (e.g., methenamine) may interfere with urinary ALA and PBG measurements, so retesting patients after stopping such medications may be necessary.
      • Webber PR
      • Aarsand AK
      • Sandvik AK
      • et al.
      Acute porphyrias may be overlooked in patients taking methenamine hippurate.
      Abbreviations: AHP, acute hepatic porphyria; ALA, delta-aminolevulinic acid; PBG, porphobilinogen; STAT, short turnaround time.

      Measurement and Interpretation

      Results of analytes measured in random urine samples should be normalized to urinary creatinine (per gram or millimole creatinine rather than per liter of urine) for 2 reasons. First, the ratio to creatinine compensates for urine dilution, which may occur in patients with large fluid intakes or who are treated with diuretics. Second, this allows for more meaningful comparison with past and future results for the same patient, since creatinine excretion is relatively constant. However, in highly dilute samples (urine creatinine <2.0 mmol/L or <0.25 g/L), the accuracy of testing may be compromised, and repeat analysis using a second urine sample is recommended.
      • Woolf J
      • Marsden JT
      • Degg T
      • et al.
      Best practice guidelines on first-line laboratory testing for porphyria.
      Laboratory methods used include column chromatography followed by spectrophotometry, and mass spectrometry for PBG and ALA, and HPLC with fluorescence detection or mass spectrometry for porphyrins (Table 4). As methods differ among laboratories, reference intervals used to interpret results should be determined in-house by each laboratory. Because measurement methods vary among laboratories, efforts to compare results on shared samples would be valuable.
      TABLE 4Potential testing methods.
      Test metabolitePotential methodologies
      PBG and ALAColumn chromatography with spectrophotometry

      Tandem mass spectrometry
      Urine porphyrinsExtraction with spectrophotometry or fluorescence

      Column chromatography

      High-performance liquid chromatography

      Tandem mass spectrometry
      Plasma porphyrinsExtraction with fluorescence

      Fluorescence scan

      High-performance liquid chromatography

      Tandem mass spectrometry
      Fecal porphyrinsExtraction with spectrophotometry or fluorescence

      High-performance liquid chromatography
      Abbreviations: ALA, delta-aminolevulinic acid; PBG, porphobilinogen.
      Given the relative rarity of AHP, most healthcare professionals who consider AHP testing have limited experience in selecting tests and interpreting the results. Therefore, it is important to initially order first-line testing and follow up with second line testing if needed. If first-line testing is positive, second line testing should be initiated and the patient referred to a porphyria specialist. As already noted, substantial PBG elevation accompanied by acute attack symptoms may justify immediate treatment. Laboratory reports should provide interpretative information to aid in accurate diagnosis and to avoid overdiagnosis and unnecessary additional testing. After a diagnosis is established, treatment decisions should be based on clinical evaluation and context rather than on degree of elevation of these biochemical biomarkers alone.
      • Aarsand AK
      • Villanger JH
      • Stole E
      • et al.
      European specialist porphyria laboratories: diagnostic strategies, analytical quality, clinical interpretation, and reporting as assessed by an external quality assurance program.
      In the US, healthcare professionals can access additional information regarding AHP via the Porphyrias Consortium (https://www.rarediseasesnetwork.org/cms/porphyrias/), resources and support for healthcare professionals and patients from the American Porphyria Foundation (https://porphyriafoundation.org/). In Europe, information can be found via the European Porphyria Network (https://porphyria.eu/).

      Summary

      AHP is a group of diseases characterized by nonspecific, sometimes severe, clinical manifestations, which can have a high disease burden. Despite biochemical testing being widely available, patients with AHP often experience a delay in diagnosis of up to 15 years or longer,
      • Bonkovsky HL
      • Maddukuri VC
      • Yazici C
      • et al.
      Acute porphyrias in the USA: features of 108 subjects from porphyrias consortium.
      ,
      • Simon A
      • Pompilus F
      • Querbes W
      • et al.
      Patient perspective on acute intermittent porphyria with frequent attacks: a disease with intermittent and chronic manifestations.
      which can result in greater morbidity and even mortality. The relatively high frequency of pathogenic variants in the general population indicates low penetrance and also suggests that AHP diagnoses are often missed. Therefore, it is important to increase disease awareness and proper diagnostic testing for AHP.
      We and others
      • Woolf J
      • Marsden JT
      • Degg T
      • et al.
      Best practice guidelines on first-line laboratory testing for porphyria.
      ,
      • Stein P
      • Badminton M
      • Barth J
      • et al.
      Best practice guidelines on clinical management of acute attacks of porphyria and their complications.
      ,
      • Sies CW
      • Cronin V
      • Florkowski CM
      • et al.
      Regional variation in analytical techniques used in the diagnosis and monitoring of porphyria: a case for harmonisation?.
      recommend a cost-effective, 2-stage approach consisting of sensitive first-line testing that can be applied frequently even when clinical suspicion may be low, followed by more extensive second-line testing, which is generally performed only after positive first-line testing. As AHP is characterized by nonspecific symptoms that mimic other more common conditions, application of first-line testing is expected to yield negative results most of the time, and frequent negative results should not discourage future testing.
      Improvements in biochemical testing may also be beneficial in reducing diagnostic delays. To improve consistency of results, collaborative research is needed to establish uniform reference intervals for PBG, ALA, and porphyrins in individuals with and without AHP.
      Also, effects of other diseases, medications, environmental exposures, diet, and other stressors on these analytes are not well defined. A postanalytical biostatistical tool may aid in generating these values in selected populations and has been used in a variety of other disorders.
      • Tortorelli S
      • Eckerman JS
      • Orsini JJ
      • et al.
      Moonlighting newborn screening markers: the incidental discovery of a second-tier test for Pompe disease.
      • McHugh D
      • Cameron CA
      • Abdenur JE
      • et al.
      Clinical validation of cutoff target ranges in newborn screening of metabolic disorders by tandem mass spectrometry: a worldwide collaborative project.
      • Marquardt G
      • Currier R
      • McHugh DM
      • et al.
      Enhanced interpretation of newborn screening results without analyte cutoff values.
      Although previously commercially available, there is no longer a point-of-care (rapid) qualitative or quantitative test for urine PBG.
      • Deacon AC
      • Peters TJ
      Identification of acute porphyria: evaluation of a commercial screening test for urinary porphobilinogen.
      Advancements are underway to reintroduce such a test, which when applied would improve AHP screening.
      Educational efforts are needed to encourage healthcare professionals to include AHP testing as part of an abdominal pain work-up soon after more common causes have been excluded. Additional neuropathic symptoms are often present but their significance may not be recognized. Symptoms are most common among women of reproductive age, but may also occur in females prepuberty or postmenopause, as well as in males.

      Conclusion

      In individuals with symptoms suggestive of AHP, measurement of PBG and porphyrins in a single, random urine sample and normalized to creatinine is sufficient for first-line testing. ALA may be included but is not essential. Subsequent second-line testing will determine AHP subtype. Specimens for biochemical testing should be light protected and frozen or refrigerated. The optimal time for sample collection is during an acute attack; however, testing can be performed at any time that AHP is suspected.

      Authors’ Contribution

      All authors attended one or both of the meetings. All authors critically revised the manuscript and read and approved the final draft for submission.

      Acknowledgments

      The authors would like to acknowledge the contribution of Joseph Quill, who attended the original meeting.

      Appendix. SUPPLEMENTARY MATERIALS

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