We developed an ultrasensitive bioassay using micropatterned zinc oxide nanorods (ZnO NRs) for the multiplexed detection and quantification of trace levels of cytokines implicated in acute kidney injury (AKI) directly from patient samples.  The remarkable limits of detection of the novel ZnO NR-based assay are compared directly with conventional methods.

by Manpreet Singh, Anginelle Alabanza, Lorelis E. Gonzalez, Weiwei Wang, Prof. W. Brian Reeves, and Prof. Jong-in Hahm

Introduction
Cytokines and chemokines are important immunoregulatory molecules produced by many cells such as neutrophils, monocytes, macrophages and T-cells that can serve as biomarkers of inflammatory diseases to predict and track disease pathogenesis [1–4]. Various cytokines and chemokines like interleukins (ILs) and tumour necrosis factors (TNFs) can serve as valuable clinical biomarkers of acute kidney injury (AKI), a rapidly acquired disorder associated with high morbidity and mortality that is commonly seen in hospitalized patients. As elevated levels of cytokines may reveal the activation of signalling pathways leading to inflammation and disease progression, methods enabling prompt and sensitive detection and quantification of multiple cytokines/chemokines simultaneously in a clinical setting are highly sought. Although conventional techniques such as enzyme-linked immunosorbent assays (ELISAs) are widely available and reliable, their applications may not be suitable for the rapid, multiplexed detection of weakly expressed cytokines due to their detection limits (DLs) of typically greater than tens of pg/mL, long assay times of several hours, and extensive serial workflows for detecting multiple protein analytes.

As the typical levels of important AKI-implicated cytokines and chemokines in healthy populations can often be well below the customary DLs of standard cytokine assays, which are generally around tens of pg/mL, there is great clinical interest in reducing the lower limits of detection down to the fg/mL range. In particular, the increasing need for early diagnosis and treatment in AKI and other cytokine-implicated diseases has driven the development of innovative detection schemes capable of reaching even lower DLs than have conventionally been offered. In this context, we have shown that zinc oxide nanorods (ZnO NRs) permit enhanced detection of fluorescence signals emitted by fluorophore-coupled biomolecules in the forms of custom-prepared oligonucleotide constructs and highly purified single-composition proteins in simple media [5–8]. In our most recent work, which is highlighted here, we have developed and validated an ultrasensitive fluorescence-based bioassay using micropatterned ZnO NRs as a novel optical platform for the multiplexed detection and quantification of two urinary biomarkers of AKI, tumour necrosis factor-α (TNF-α) and interleukin-8 (IL-8), in samples of patients at risk for and diagnosed with AKI [9].

In addition to the biomedical relevance of TNF-α and IL-8 in the pathophysiology of AKI, the biomarkers are ideal model cytokines and chemokines for this study due to the differences in their typical concentration levels found in human urine. The baseline expression of IL-8 in healthy populations generally ranges from tens of pg/mL to ng/mL and can be ascertained using conventional approaches, whereas TNF-α levels are typically below the DLs of traditional cytokine detection platforms. Accordingly, we performed ELISA- and ZnO NR-based assays on the same set of patient samples to first examine whether the highly expressed IL-8 levels agree between both detection methods and further demonstrated the detection capability of the ZnO NRs to reveal the ultralow protein levels of TNF-α that cannot otherwise be ascertained via ELISA.

Results and discussion
Overall approach of ZnO NRs-based fluorescence assay
Using a micropatterned array of densely grown, vertically oriented ZnO NRs synthesized using a facile, low-cost, chemical vapour-phase method, we employed a sandwich assay scheme for the multiplexed detection of both AKI-relevant biomarkers. The sequential assay steps included incubation of the ZnO NR platform with primary TNF-α and IL-8 antibodies, bovine serum albumin for surface blocking, standards for both proteins for generating calibration curves or patient urine samples for determining biomarker levels in subject individuals, and fluorophore-conjugated secondary antibodies. In Figure 1(A), representative emission data from the multiplexed assay are qualitatively presented for Alexa 488-labelled TNF-α (left) and Alexa 546-labelled IL-8 (right). As a direct comparison, the panels in Figure 1(B) display scanning electron microscope (SEM) images of the ZnO NR array platform to show the morphology and dimensions of the individual square patches of ZnO NRs. The highly crystalline ZnO NRs do not exhibit any background fluorescence, as evidenced in the inset (F) of image 1(B), and, hence, all optical signals detected for protein quantification are derived only from the surface-adsorbed fluorophore-tagged biomolecules.

Reproducibility and calibration
In Figure 1(C), exemplar fluorescence intensity plots show the different amounts of TNF-α and IL-8 simultaneously detected from selected patient urine samples as obtained by averaging the optical signal from about 550 NR square patches on different areas of the same ZnO NR detection platform. The reproducibility of the fluorescence signal on the ZnO NRs-based platform is shown in Figure 1(D) in which the same patient sample was assayed five times on the same ZnO NR platform for intra-assay variability (**) as well as on three different ZnO NR plates for inter-assay variations (*) that may arise from assay or array-to-array differences. This scheme was conducted for two patient samples, and the coefficients of variation for the intra-assay (16.5% for TNF-α and 2.5% for IL-8) and inter-assay (12% for TNF-α and 2.8% for IL-8) results were found to be below the generally accepted value of 10–20%.

In order to quantitatively compare the levels of TNF-α and IL-8 obtained via the ELISA- and ZnO NRs-based assays, calibration curves were generated using standard solutions of each cytokine. The DLs of the ELISA-based method, defined as 2 standard deviations above the mean of 20 zero concentration replicates, were determined as 5.5 and 7.5 pg/mL for TNF-α and IL-8, respectively. On the other hand, the DLs of the ZnO NR platform, assessed using the upper boundary of blank samples with a 95% accuracy goal, were found to be 4.2 and 5.5 fg/mL for TNF-α and IL-8, respectively. The unparalleled sensitivity down to the several fg/mL range enabled by the ZnO NR platform can reveal the levels of weakly expressed, disease-implicated cytokines such as TNF-α to promote early clinical diagnostics.

IL-8 testing and statistical analysis
Following calibration, the same patient samples were assayed on both the ELISA and ZnO NR platforms for quantitative comparison between both assays. When comparing the highly expressed IL-8 levels in the urine of 38 patients that ranged between several tens of pg/mL to a few ng/mL, the ZnO NRs-based assay had strong statistical agreement with the ELISA-based results allowing for direct validation of the novel bioassay. In Figure 2(A), a correlative plot displays the IL-8 readings from the same patients determined by the ELISA and ZnO NR assays on the x and y axes, respectively. The linear fit of the data points, shown in the dashed red line, lies very close to the superimposed line of y = x, shown in black, indicating excellent agreement between the two assay methods. In Figure 2(B), a histogram distribution chart reporting the differences in IL-8 readings between the two methods shows the majority of IL-8 readings from both assays fell within the range of ±2.5 pg/mL of each other. The IL-8 levels were further evaluated using the Bland-Altman analysis in Figure 2(C & D) in which the differences between the ELISA and ZnO NR readings for each patient were plotted against the mean concentration values. As shown, the data analysed over a large range of concentrations centred near the black lines, which represent the case of equivalent IL-8 readings obtained from the two different assays. The results of these comparative analyses validate the ZnO NR platform as a reliable technique to accurately quantify urinary biomarker proteins directly from patient samples.

TNF-α testing
To substantiate the applicability of the ZnO NR platforms in ultrasensitive cytokine detection using the weakly expressed biomarker of TNF-α, the protein levels for 46 patients were determined using both assay platforms. As seen in Figure 3(A), many of the patient samples exhibited values too close or below the DL of the ELISA assay (5.5 pg/mL) and are marked accordingly as grey blocks in the ELISA row. By contrast, the TNF-α values of all the patients were successfully quantified on the ZnO NR platform, well below several tens of pg/mL and into the low fg/mLrange. For the ZnO NR row in Figure 3(A), those samples that could not be measured via ELISA are shown with a magnifier sign, and their TNF-α concentrations, as determined by the ZnO NRs-based assay, are then shown in Figure 3(B & C) on two different scales for clarity. As demonstrated, the optical signal enhancement provided by the ZnO NR array platform enables the ultrasensitive detection of trace levels of proteins directly from patient samples.

Advantages of the ZnO NR-based approach and future outlook
Within the realm of biodetection, the ZnO NR-based approach can provide many direct advantages including facile platform fabrication, desirable optical properties, biocompatibility, and promising multiplexed/high-throughput integration capacities. The ZnO NR arrays are easily fabricated using a gas-phase method through well-established synthesis procedures and can be used directly after growth without any post-synthetic modifications. Further, the highly crystalline NRs exhibit many desirable optical properties including no intrinsic fluorescence (i.e. absence of autofluorescence) as well as enhancement of the optical intensity and photostability of nearby signal emitters. Since the ZnO NRs do not display any photoluminescence in the visible and near-infrared range, they do not interfere with the spectroscopic profiles of fluorophores commonly used in biology and biomedical detection. At the same time, the reduced dimensions and high shape anisotropy of the ZnO NRs enable optical enhancement and prolonged stability of the signal from fluorophore-tagged biomolecules adsorbed on their surface allowing for the ultrasensitive detection of trace levels of bioconstituents.

In addition to the demonstrated sensitivity permitted by the ZnO NR-based platform, the cytokine bioassay also has the direct advantages of rapid analysis, minimal volume requirements, and reusability. The multiplexed detection was achieved with 90 min of total assay time and only 60 μL of total bioreagent/sample volume using commonly employed fluorescence microscopy instrumentation. Further, the highly biocompatible ZnO NRs platform was found to withstand at least 25 repeated assays in complex biological and chemical reaction environments that include urine samples.

As modern automation strategies in high-throughput screening have seen great advancements in the sophistication of robotic sample delivery strategies and the detection of many analytes simultaneously via multichannel optical sensors, the ZnO NR-based platform may be able to provide much-sought detection sensitivity when integrated into these breakthrough technologies. In the microarray, each square patch of densely grown ZnO NRs with a typical dimension of 3~50 μm in side length can be configured to serve as a discrete detection element for different patient samples when coupled with appropriate sample delivery and multiplexed optical sensing/readout platforms. The demonstrated detection capabilities combined with this integration potential suggests that the ZnO NR-based approach serves as more than just an alternative or tandem detection platform to existing methods, but rather provides an advanced approach which allows the much needed, ultrasensitive detection of biomarker proteins in samples that exhibit concentration levels much lower than those which standard techniques can ascertain.

Conclusion
We successfully demonstrated a ZnO NRs-based fluorescence bioassay for the rapid, ultrasensitive, quantitative and multiplexed detection of AKI-related biomarkers in patient urine samples. We first statistically validated the ZnO NR-based approach against a conventional ELISA-based method by comparing the measurements of highly expressed levels of IL-8 that were above the DLs of ELISA. We further revealed the full detection capabilities of the ZnO NRs platform by quantifying ultratrace amounts of a weakly expressed cytokine, TNF-α, whose levels in urine are often below the DLs of conventional cytokine assays. The unparalleled detection sensitivity and other discussed advantages of the ZnO NR-based bioassay can be readily extended to advance other optical-sensing applications in biological research and clinical diagnostics.

Acknowledgement
This article is a summary of the work first presented in Singh M, Alabanza A, Gonzalez LE, Wang W, Reeves WB, Hahm J. Ultratrace level determination and quantitative analysis of kidney injury biomarkers in patient samples attained by zinc oxide nanorods. Nanoscale 2016; 8: 4613–4622 [9].

References
1. Feldmann MJ. Many cytokines are very useful therapeutic targets in disease. Clin Invest. 2008; 118: 3533–3536.
2. Fichorova RN, Richardson-Harman N, Alfano M, et al.  Biological and technical variables affecting immunoassay recovery of cytokines from human serum and simulated vaginal fluid: a multicenter study. Anal Chem. 2008; 80: 4741–4751.
3. Borish LC, Steinke JWJ. Cytokines and chemokines. Allergy Clin Immunol. 2003; 111: S460–S475.
4. Nathan C, Sporn M. Cytokines in context. J Cell Biol. 1991; 113: 981–986.
5. Adalsteinsson V, Parajuli O, Kepics S, et al. Ultrasensitive detection of cytokines enabled by nanoscale ZnO arrays. Anal Chem. 2008; 80: 6594–6601.
6. Dorfman A, Kumar N, Hahm J. Nanoscale ZnO-enhanced fluorescence detection of protein interactions. Adv. Mater. 2006; 18: 2685–2690.
7. Singh M, Song S, Hahm J. Unique temporal and spatial biomolecular emission profile on individual zinc oxide nanorods. Nanoscale 2014; 6: 308–315.
8. Singh M, Jiang R, Coia H, et al. Insight into factors affecting the presence, degree, and temporal stability of fluorescence intensification on ZnO nanorod ends. Nanoscale 2015; 7: 1424–1436.
9. Singh M, Alabanza A, Gonzalez LE, et al. Ultratrace level determination and quantitative analysis of kidney injury biomarkers in patient samples attained by zinc oxide nanorods. Nanoscale 2016; 8: 4613–4622.

The authors
Manpreet Singh¹ BS, Anginelle Alabanza¹ BS, Lorelis E. Gonzalez¹ BS, Weiwei Wang² BS, W. Brian Reeves³ MD, and Jong-in Hahm*¹ PhD
¹Department of Chemistry, Georgetown University, Washington, DC 20057, USA
²Division of Nephrology, The Penn State College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033, USA
³Department of Medicine, University of Texas Health Sciences Center at San Antonio, San Antonio, TX 78229, USA

*Corresponding author
E-mail: jh583@georgetown.edu

Porphyrias are a group of disorders of the heme biosynthetic pathway which clinically manifest with acute neurovisceral attacks and cutaneous lesions. Diagnosis of porphyrias is based on the accurate and precise measurement of various porphyrins and precursor molecules in a range of samples. In addition, molecular diagnostic assays can provide definitive diagnosis.

by Dr Vivion E. F. Crowley, Nadia Brazil, and Sarah Savage

What are porphyrias?
Porphyrias are a group of rare disorders each of which results from a deficiency of an individual enzyme within the heme biosynthetic pathway (Fig. 1) [1–3]. With the exception of an acquired form of porphyria cutanea tarda (PCT), all porphyrias are inherited as monogenic autosomal dominant, autosomal recessive or X-linked genetic disorders, with varying degrees of penetrance and expressivity and this impacts on the prevalence and incidence of clinically manifest porphyrias [4].  The biochemical consequence of each porphyria is the overproduction within the heme biosynthetic pathway of specific porphyrin intermediates and/or the porphyrin precursor molecules delta-aminolevulinic acid (ALA) and porphobilinogen (PBG) [2–3]. This in turn has implications for the clinical manifestation of these disorders, their overall classification and their diagnosis (see Table 2).

Clinical presentation
Porphyrias may present clinically with either or both of two symptom patterns. The first is the acute neurovisceral attack, which is a potentially life threatening episode related to excessive hepatic generation of ALA and PBG, and which is a feature only in acute intermittent porphyria (AIP), variegate porphyria (VP), hereditary coproporphyria (HCP) and the very rare ALA dehydratase deficiency porphyria (ADP) [5–7]. These attacks are characterized principally by autonomic dysfunction, including non-specific but severe abdominal pain, constipation, diarrhoea, nausea, vomiting, tachycardia, hypertension or occasionally postural hypotension. In addition, other features may include a predominantly motor peripheral neuropathy which, if left undiagnosed, may extend to respiratory failure reminiscent of Guillain–Barré syndrome, as well as cerebral dysfunction, which can vary from subtle alterations in mental state, to posterior reversible encephalopathy syndrome (PRES). Hyponatremia, most likely due to SIADH [syndrome of inappropriate antidiuretic hormone (ADH) secretion] may also contribute to CNS-related morbidity. The complex neuropathic manifestations appear to be primarily related to axonal degeneration due to direct neurotoxicity by ALA, which structurally resembles the neurotransmitter gamma-aminobutyric acid (GABA) [3, 5–7].

The second clinical presentation paradigm is cutaneous photosensitivity caused by the interaction of ultraviolet light with photoactive porphyrins in the skin resulting in the production of reactive oxygen species (ROS) and an associated inflammatory response [3]. In PCT, VP and HCP the skin lesions typically occur post-pubertally and consist of skin fragility, vesicles, bullae, hyperpigmentation and hypertrichosis affecting sun exposed areas, most usually the face and dorsum of hands [1–3]. In erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP), which may present in childhood, there is usually no blistering but instead erythema, edema and purpura feature in the more acute setting, with subsequent chronic skin thickening noted, whereas congenital erythropoietic porphyria (CEP) is characterized by severe cutaneous photosensitivity often occurring in early infancy with bullae and vesicles rupturing and being prone to secondary infection, with resultant scaring, bone resorption, deformation and mutilation of sun-exposed skin [1, 2, 8].

Classification
The classification of porphyrias (Table 1) has traditionally been determined either on the basis of clinical manifestations, i.e. acute or non-acute (cutaneous), or on the primary organ of porphyrin overproduction, i.e. hepatic or erythropoietic [1, 3, 8]. A combined classification has recently been proposed which takes account of both of these elements [2]. However, whichever classification is adopted there should be a realization that VP, and to a lesser extent HCP, can manifest with both acute and cutaneous features either simultaneously or separately.

Clinical and biochemical diagnosis
The clinical manifestations of porphyrias, particularly the acute hepatic porphyrias, are protean and consequently, patients with a clinically active porphyria could initially present to a relatively wide spectrum of clinical specialties including, gastroenterology, acute medicine, dermatology, neurology, endocrinology and hematology amongst others [2]. In general, cutaneous porphyrias should not pose a diagnostic difficulty for an experienced dermatologist used to investigating photosensitive skin disorders, but biochemical testing is still required to define the type of porphyria present. However, definitive diagnosis of an initial acute hepatic porphyria attack is critically dependent on biochemical testing, as symptoms are often non-specific in nature (Tables 1 & 2).

The diagnosis of an acute hepatic porphyria attack is founded on demonstrating an increase in urine PBG levels in direct temporal association with the characteristic acute symptom complex, the minimum level of increase being between 2- and 5-fold [9, 10]. The urine PBG may be measured either as a random sample, where it should be reported as urine PBG to creatinine ratio or as a 24-hour urine collection, where total PBG is reported.  The former has proven to be clinically efficacious and has the advantage of timeliness, reduced within-subject variation and convenience over the requirement for a 24 hour urine collection [9]. If the urine PBG is not elevated this effectively rules out an acute porphyria attack at the time of sampling, however, there are certain caveats to this. Thus it is important to note that if specific treatment with either heme preparations or carbohydrate loading has been instigated prior to the test these interventions could reduce the urine PBG level significantly, including normalization [3]. Furthermore, if the measurement of urine PBG is delayed or undertaken at a time removed from the actual acute clinical presentation e.g. by weeks or months, then the finding of a normal urine PBG at that later stage cannot effectively rule out acute porphyria [3]. In this authors experience another important caveat concerns patients with a previous confirmed diagnosis acute porphyria who present with symptoms suggestive of recurrent acute attack. In many instances these patients have a perpetually elevated urine PBG, even in between attacks, and therefore an elevated urine PBG cannot effectively guide diagnosis. In these situations a decision to treat as an acute attack has to be made on the basis of clinical findings.
 
Therefore, a clinically effective service for acute porphyria diagnosis requires that a timely, quality assured laboratory method for urine PBG should be available for analysis [11]. Although a qualitative method for urine PBG may suffice for the purposes of establishing a diagnosis this should be supported by the availability of a confirmatory quantitative method for urine PBG. The lack of availability of urine PBG assay is very often the basis for misdiagnosis or indeed delayed diagnosis of acute porphyria attacks [10].

In conjunction with PBG, urine ALA is often measured simultaneously and although also elevated it does not tend to reach the levels of PBG in acute porphyrias. The one exception is the extremely rare instance of autosomal recessive ADP due to defective ALA synthase 2 (ALAS2) activity, where markedly elevated urine ALA levels are reported while PBG may be normal or only slightly elevated [2, 3]. In addition, a similar pattern of urine ALA predominance relative to PBG (although not as elevated) may be observed in the context of lead poisoning, wherein patients may also present with abdominal pain and neuropathy [1, 3].

Once the diagnosis of acute porphyria has been made based on the urine PBG the next phase involves determining the type of porphyria present. This is very much dependent on the specific pattern of porphyrin overproduction observed in samples of urine, feces, plasma and erythrocytes. It is critically important that the laboratory analytical methods available extend beyond the sole measurement of total porphyrin levels [10–12]. In particular, it is essential that individual porphyrin analysis and isomer fractionation in both urine and feces is available to facilitate the identification of the porphyria-specific patterns of porphyrin overproduction [10–12]. In many instances non-porphyria disorders affecting the gastrointestinal and hepatobiliary systems or certain dietary factors may cause non-specific secondary elevations in porphyrins, e.g. coproporphyrinuria, which can be diagnostically misleading [3]. In such cases urine PBG levels will not be elevated and the pattern of porphyrins observed will not be indicative of any one of the specific porphyrias per se. Therefore, it is important to realize that a finding of elevated porphyrin levels does not automatically equate to a diagnosis of underlying porphyria. This further highlights the importance of developing specialist porphyria centres to ensure that the appropriate repertoire of quality assured testing and expert interpretation and support are available for diagnosis and management of porphyria patients [11, 13].

The diagnosis of cutaneous (non-acute) porphyrias is also very much based on the specific patterns of porphyrins observed in urine and feces. In addition, the pattern of free and zinc protoporphyrin in erythrocytes can be useful in the diagnosis of CEP, EPP and the related disorder, XLP. Moreover, the identification of the porphyria subtype, either acute or cutaneous, may also be enhanced by identifying characteristic plasma porphyrin fluorescence emission peaks, e.g. VP emission peak between 625 and 628 nm [1–3]. Finally, it is essential that all samples for porphyrin and precursor measurement are protected from light prior to analysis.

Role of genetic diagnosis
Given the heritable nature of porphyrias it is not surprising that molecular genetic analysis has also become an important diagnostic adjunct. There is an extensive allelic heterogeneity of pathogenic mutations among the implicated genes for each porphyria disorder, which means that most mutations are uniquely confined to one or at most a few kindreds. There are, however, a few exceptions to this trend, most notably in relation to founder mutations among the Swedish population and the Afrikaner population in South Africa.  The general approach in the application of genetic diagnostic strategies is firstly to characterize the causative mutation in a known affected individual (proband) using a mutation scanning approach [14]. Once a putative mutation has been identified its pathogenicity for a particular porphyria should be affirmed and then more extensive family cascade genetic screening can be organized based on the analysis of this kindred-specific mutation [14].

This approach has important implications in the diagnosis of porphyria susceptibility, particularly for the autosomal dominant acute hepatic porphyrias, where both penetrance and expressivity of the disorders is low [3, 4]. Thus the penetrance among AIP, VP and HCP is between 10 and 40%, implying that the majority of patients with an autosomal dominant acute hepatic porphyria will not manifest with an acute attack (or indeed cutaneous lesions in the case of VP and HCP) in their lifetime [3, 4]. Moreover, this lack of penetrance may also extend to the absence of subclinical biochemical abnormalities indicative of an underlying autosomal dominant acute porphyria, demonstrating the limited sensitivity of biochemical testing in identifying asymptomatic family members.

 Currently there is no clear-cut mechanism for discriminating between those who will manifest a clinical and/or biochemical phenotype and those who will not. While the role of environmental precipitating factors, e.g. porphyrinogenic medications, stress, prolonged fasting, menstruation [1–3], have long been recognized in triggering acute porphyria attacks, it is the presence of a pathogenic mutation which is still the single most important factor determining the overall susceptibility for an acute porphyria episode.  Therefore, all patients carrying a pathogenic mutation should be regarded as pre-symptomatic carriers, i.e. capable of developing an acute attack, and one of the key applications of genetic analysis in the area is in identifying pre-symptomatic carriers to allow for appropriate counselling and management advice to prevent attacks [3, 14].

In this author’s experience another useful role for molecular diagnostics in porphyrias is in relation to those patients with an historic diagnosis of acute hepatic porphyria in whom the biochemical abnormalities have subsequently normalized over years.  In such instances genetic analysis can provide a definitive diagnosis for the type of porphyria and will accommodate a more extensive family screening programme for potential pre-symptomatic carriers.

The current methods of genetic analysis vary but usually involve a confirmatory step using direct nucleotide sequencing of the putative pathogenic variants as the gold standard. However, the emergence of next generation sequencing platforms has further galvanized the diagnostic possibilities in this area. Overall, in autosomal dominant acute hepatic porphyrias, approximately 95% of mutations are identifiable [3, 14]. This sensitivity includes the application of additional methods such as ‘multiplex ligation-dependent probe amplification’ (MLPA) and gene dosage analysis for identifying complex mutations, such large gene deletions, which may not be detected using standard sequencing-based approaches [14].

In autosomal recessive porphyrias including ADP, CEP and EPP, the clinical penetrance approaches 100%. These disorders also display a level of genetic heterogeneity. In the case of EPP the presence of a relatively common low expression single nucleotide polymorphism (SNP) located in the ferrochetalase gene, FECH (IVS3-48C), appears to be essential for the clinical expression of the cutaneous phenotype in the vast majority of cases [15].

The application of molecular genetics has provided a means of establishing definitive porphyria susceptibility, however, similar to the situation for biochemical testing services any genetic diagnostic services in this area must be quality assured to a high standard and need to adopt appropriate mutation scanning assay validation protocols in accordance with international standards and best practice recommendations [11–14].

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The authors
Vivion E. F. Crowley*¹ MB MSc FRCPath FFPath(RCPI) FRCPI, Nadia Brazil² BA (Mod) FAMLS, Sarah Savage³ BSc MSc
¹Consultant Chemical Pathologist, Head of Department, Biochemistry Department, St James’s Hospital, Dublin 8, Ireland
²Porphyrin Laboratory, Biochemistry Department, St James’s Hospital, Dublin 8, Ireland
³Molecular Diagnostic Laboratory, Biochemistry Department, St James’s Hospital, Dublin 8, Ireland

*Corresponding author
E-mail: vcrowley@stjames.ie