Prostate specific antigen (PSA) has significantly improved the early detection of prostate cancer (PCa), reducing the related mortality rate. However, PSA has a low specificity, being affected by many benign conditions. [-2]proPSA, a PSA precursor, is a more specific and accurate biomarker indicating prostate biopsy in men at real risk of PCa.
by Dr A. Abrate, Dr M. Lazzeri and Prof. G. Guazzoni
PSA as a marker for prostate cancer
Prostate specific antigen (PSA) is a serum marker widely used for the early detection of prostate cancer (PCa). Its introduction into clinical practice in the early 1990s had an extraordinary impact on the diagnosis and management of PCa. In fact, 20 years after its introduction, the PSA-based PCa opportunistic or systematic screening has resulted in a stage migration to more organ-confined tumours at the time of diagnosis, and consequently to a consistent reduction in PCa related mortality [1, 2]. However, PSA is not a perfect marker for the detection of PCa because of its low specificity and sensitivity. Its levels may increase as a result of benign conditions, such as benign prostatic hyperplasia (BPH) and chronic prostatitis. Moreover, PSA levels are also affected by biologic variability, which may be related to differences in androgen levels, prostate manipulation or ejaculation. Finally, alterations in PSA levels may be related to sample handling, laboratory processing, or assay standardization. All these factors made it difficult to find an appropriate PSA cut-off point diagnostic for PCa (for many years considered to be 4 ng/ml).
Thus, prostate biopsy is still mandatory to confirm the diagnosis. However, this is positive in only approximately 30% of patients [3], and the European Association of Urology suggests a repeat biopsy if PSA is persistently elevated, the digital rectal examination (DRE) is suspicious, or there is a pathological diagnosis of atypical small acinar proliferation (ASAP) or high-grade prostatic intraepithelial neoplasia (HG-PIN) [4]. Finally, PCa (also high-grade cancer) is not rare (approximately 15.2%) among men with PSA levels lower than 4 ng/ml, the previously widely accepted cut-off point [5].
Considering all these observations, it is clear that PSA is an organ-specific rather than an ideal cancer-specific marker.
The introduction into clinical practice of measuring the levels of several derivatives of PSA (free PSA, percentage of free PSA, PSA density, PSA velocity) improved the accuracy of total PSA (tPSA) in detecting PCa. Recently, free PSA (fPSA) has been found to include several subforms, such as proPSA. In particular [-2]proPSA seems to be specific for PCa, opening new ways for early cancer detection.
Biological basis of proPSA
The currently measurable serum tPSA consists of either a complexed form (cPSA, 70–90%), bound by protease inhibitors (primarily alpha1-antichymotrypsin), and a non-complexed form (fPSA). Recently fPSA has been discovered to exist in at least three molecular forms: proPSA, benign PSA (BPSA), and inactive intact PSA (iPSA), covering approximately 33%, 28%, and 39% of fPSA respectively (Fig. 1) [6]. In particular, proPSA is a proenzyme (precursor) of PSA, which is associated with PCa [7].
PSA is synthesized with a 17-amino acid leader sequence (preproPSA) that is cleaved co-translationally to generate an inactive 244-amino acid precursor protein (proPSA, with seven additional amino acids compared to mature PSA). proPSA is normally secreted from the prostate luminal epithelial cells. Immediately after its release into the lumen, the pro-leader part is removed, creating the active form, by the effect of human kallikrein (hK)-2 and hK-4, which have a trypsin-like activity and are expressed predominantly by prostate secretory epithelium. Other kallikreins, localized in the prostate, such as hK216 or prostin17, are involved in the conversion and activation of proPSA. Cleavage of the N-terminal seven amino acids from proPSA generates the active enzyme, which has a mass of 33 kDa.
The partial removal of this leader sequence leads to other truncated forms of proPSA. Thus, theoretically seven isoforms of proPSA could exist, although only [-1], [-2], [-4], [-5], [-7]proPSA were found; there is still no evidence of [-3], [-6]proPSA. However, all forms of proPSA are enzymatically inactive [8]. It is possible to detect three truncated forms of proPSA in serum: [-5/-7], [-4] and [-2]proPSA, which is the most stable form (Fig. 1).
Notably, in vitro experiments showed that the [-2]proPSA form cannot be activated by either hK2 or trypsin; thus, once it is formed, [-2]proPSA is resistant to activation into the mature PSA form and consequently this is the most reliable test.
Mikolajczyk et al. [7], using a monoclonal antibody recognizing [-2]proPSA, found increased staining in the secretions from malignant prostate glands. In particular [-2]proPSA is differentially elevated in peripheral gland cancer tissue; conversely transition zone tissue contains little or no proPSA.
The increased serum tPSA concentrations in patients with PCa do not result from increased expression but rather from an increased release of PSA into the bloodstream, due to disruption of the epithelial architecture. fPSA is catalytically inactive because of internal cleavages, occurring in seminal plasma, and does not form complexes with protease inhibitors or other proteins: in PCa %fPSA is lower presumably because, consequently to an increased release of PSA into the bloodstream, a very low part is still degraded into the ducts.
In another later study [9], Mikolajczyk et al. found that [-2]proPSA was specifically higher in patients with PCa. Analysing a small number of patients with biopsy positive for PCa and tPSA between 6 and 24 ng/ml, they found that [-2]proPSA constituted a high fraction of fPSA (25% to 95%), which was greater than in patients with a negative biopsy. However, the molecular basis for the proPSA elevation in PCa is uncertain, although a decreased cleavage by hK2 could be the cause.
Clinical utility of proPSA
Sokoll et al. [10] were the first to study the role of proPSA in the early detection of PCa. The study involved archival serum from 119 men (31 PCa, 88 non-cancer), obtained before biopsy and in the tPSA range of 2.5–4.0 ng/ml. The serum levels of tPSA, fPSA, proPSA, and proPSA/fPSA ratio (%proPSA) were analysed: PSA and %fPSA values were similar between the non-cancer and PCa groups, and %proPSA was relatively higher in the PCa group (50.1±4.4%) compared to the non-cancer group (35.5±6.7%; P=0.07). Concerning the clinical utility, the area under the curve (AUC) for %proPSA was 0.688 compared to 0.567 for %fPSA. At fixed sensitivity of 75%, the specificity was significantly greater for %proPSA at 59% compared with %fPSA at 33% (P<0.0001).
Afterwards, the Prostate Health Index (PHI) has been proposed as a mathematical algorithm combining tPSA, fPSA and [-2]proPSA according to the formula: ([-2]proPSA/fPSA) × √tPSA.
A large American prospective trial [11], involving 892 men who had tPSA levels of 2–10 ng/ml and negative digital rectal examination results, showed that PHI had greater predictive accuracy for prostate biopsy outcome (AUC 0.703) than [-2]proPSA (AUC 0.557), %fPSA (AUC 0.648) and PSA (AUC 0.525), directly correlating with Gleason score (GS) (P=0.013), with an AUC of 0.724 for GS ≥4+3 disease. Moreover, men with PHI >55 had a 42% likelihood of being diagnosed with high-grade disease on biopsy compared to 26% of men with PHI 0–24.9.
Accordingly, an observational European multicenter cohort study involved 646 men with tPSA levels of 2–10 ng/ml, who had undergone prostate biopsy [12]. [-2]proPSA and PHI improved the predictive accuracy for the detection of overall PCa (and also GS ≥7 disease) compared to PSA and derivatives. In fact, at 90% sensitivity, the PHI cut-off of 27.6 could avoid 100 (15.5%) biopsies, missing 26 (9.8%) cancers (23 with GS 6, three with GS 3+4).
Moreover, a PHI based nomogram to predict PCa at extended prostate biopsy was developed and validated over 729 patients [13]. Including PHI in a multivariable logistic regression model, based on patient age, prostate volume, digital rectal examination and biopsy history, significantly increased predictive accuracy by 7% from 0.73 to 0.80 (P<0.001). Decision curve analysis showed that using the PHI based nomogram resulted in the highest net benefit.
Recently, it was demonstrated that PHI might have a role in screening patients at high risk of PCa [14]. Specifically, the study involved 158 men with a positive family history undergoing prostate biopsy within the multicentre European PROMEtheuS cohort. Similarly to previous studies in the general population, PHI outperformed tPSA and %fPSA for PCa detection on biopsy (AUC 0.73, 0.55 and 0.60, respectively). In addition, both [-2]proPSA and PHI were directly associated with GS in men with a positive family history. Overall, the authors reported that using a PHI cutoff value of 25.5 would have avoided 17.2% of biopsies while missing only two GS 7 cancers. On decision curve analysis, the addition of PHI to a base predictive model that included age, prostate volume, tPSA, fPSA and %fPSA resulted in net benefit at threshold probabilities of 35–65%. This result suggests that PHI should be incorporated into a multivariable risk assessment for high-risk patients because it offers improved performance for PCa detection.
Conclusions
[-2]proPSA and PHI are more accurate than the currently used tests (PSA and derivatives) in predicting the presence of PCa at biopsy. Their implementation in clinical practice has the potential to significantly increase physicians’ ability to detect PCa and avoid unnecessary biopsies. Further work is needed to confirm and generalize these data on wider populations.
References
1. Hoffman RM, Stone SN, Espey D, Potosky AL. Differences between men with screening-detected versus clinically diagnosed prostate cancers in the USA. BMC Cancer 2005; 5: 27.
2. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin. 2013; 63: 11–30
3. Vickers AJ, Cronin AM, Roobol MJ, et al. The relationship between prostate-specific antigen and prostate cancer risk: the Prostate Biopsy Collaborative Group. Clin Cancer Res. 2010; 16: 4374–4381.
4. Heidenreich A, Bellmunt J, Bolla M, et al. EAU guidelines on prostate cancer. Part 1: screening, diagnosis, and treatment of clinically localised disease. Eur Urol. 2011; 59: 61–71.
5. Thompson IM, Pauler DK, Goodman PJ, et al. Prevalence of prostate cancer among men with a prostate-specific antigen level < or =4.0 ng per milliliter. N Engl J Med. 2004; 350: 2239–2246.
6. Mikolajczyk SD, Rittenhouse HG. Pro PSA: a more cancer specific form of prostate specific antigen for the early detection of prostate cancer. Keio J Med. 2003; 52: 86–91.
7. Mikolajczyk SD, Millar LS, Wang TJ, et al. A precursor form of prostate-specific antigen is more highly elevated in prostate cancer compared with benign transition zone prostate tissue. Cancer Res. 2000; 60: 756–759.
8. Jansen FH, Roobol M, Jenster G, Schroder FH, Bangma CH. Screening for prostate cancer in 2008 II: the importance of molecular subforms of prostate-specific antigen and tissue kallikreins. Eur Urol. 2009; 55: 563–74.
9. Mikolajczyk SD, Marker KM, Millar LS, et al. A truncated precursor form of prostate-specific antigen is a more specific serum marker of prostate cancer. Cancer Res. 2001; 61: 6958–6963
10. Sokoll LJ, Chan DW, Mikolajczyk SD, et al. Proenzyme psa for the early detection of prostate cancer in the 2.5–4.0 ng/ml total psa range: preliminary analysis. Urology 2003; 61: 274–276.
11. Catalona WJ, Partin AW, Sanda MG, et al. A multicenter study of [-2]pro-prostate specific antigen combined with prostate specific antigen and free prostate specific antigen for prostate cancer detection in the 2.0 to 10.0 ng/ml prostate specific antigen range. J Urol. 2011; 185: 1650–1655.
12. Lazzeri M, Haese A, de la Taille A, et al. Serum isoform [-2]proPSA derivatives significantly improve prediction of prostate cancer at initial biopsy in a total PSA range of 2-10 ng/ml: a multicentric European study. Eur Urol. 2013; 63: 986–994.
13. Lughezzani G, Lazzeri M, Larcher A, et al. Development and internal validation of a Prostate Health Index based nomogram for predicting prostate cancer at extended biopsy. J Urol. 2012; 188: 1144–1150.
14. Lazzeri M, Haese A, Abrate A, et al. Clinical performance of serum prostate-specific antigen isoform [-2]proPSA (p2PSA) and its derivatives, %p2PSA and the prostate health index (PHI), in men with a family history of prostate cancer: results from a multicentre European study, the PROMEtheuS project. BJU Int. 2013; 112: 313–321.
The author
Alberto Abrate* MD; Massimo Lazzeri MD, PhD; and Giorgio Guazzoni MD
Dept of Urology, Ospedale San Raffaele Turro, San Raffaele
Scientific Institute, Milan, Italy
*Corresponding author
E-mail: alberto.abrate@gmail.com
Neurocysticercosis: can we trust serology?
, /in Featured Articles /by 3wmediaWhich is the most common parasitic disease of the nervous system, which affection is the leading cause of seizures and acquired epilepsy in the developing world but still preventable? The answer: neurocysticercosis. An orphan disease suffering from the absence of a real ‘gold standard’ diagnosis. Meanwhile, many laboratories perform immunodiagnosis but what is its real value and what can it tell us?
by Dr Jean-François Carod
What is neurocysticercosis?
Cysticercosis of the central nervous system (neurocysticercosis) is caused by the larval stage (cysticerci) of the pork tapeworm Taenia solium. When people eat undercooked pork containing viable cysticerci, they develop an intestinal tapeworm infection (Fig. 1). Humans can also become intermediate hosts, however, by directly ingesting T. solium eggs shed in the feces of human carriers of the parasite. These eggs then develop into cysticerci, which migrate mostly into muscle (causing cysticercosis) and into the central nervous system where the cysticerci can cause seizures and many other neurological symptoms, neurocysticercosis (NCC). NCC is a major cause of epilepsy in endemic countries. It is the most important neurological disease of parasitic origin in humans. The pathogenesis is unclear but symptoms seem to correlate with the stage of the cyst. Starting as a viable entity, the cyst then gradually degenerates and become calcified. Seizures seem to appear at the degenerating and calcified stage but treatment is effective on the living cysts. Human cysticercosis is endemic in the Andean area of South America, Brazil, Central America and Mexico; China, the Indian subcontinent, South-East Asia; and Sub-Saharan Africa including Madagascar.
Why do we need to diagnose it?
Diagnosing NCC is required in the event of unexplained encephalitic disorders such as first onset of seizures in countries where NCC is endemic or in patients travelling in countries where NCC is endemic and who may have been at risk of infection (e.g. exposed to NCC risk factors, such as inadequate hand and food hygiene).
How can it be diagnosed?
The diagnosis of cysticercosis of the central nervous system involves the interpretation of non-specific clinical manifestations, such as seizures, often with characteristic findings on computed tomography (CT) or magnetic resonance imaging (MRI) of the brain, and the use of specific serological tests (Fig. 2). Diagnostic criteria based on objective clinical, imaging, immunological and epidemiological data have been proposed but are not generally used in areas endemic for the disease [1].
Serology is indicated for the diagnosis of T. solium seropositivity. But from a positive serology to the assessment of NCC diagnosis, there is a huge gap. A positive T. solium serology is not predictive for a neurological localization and serology may remain positive years after the end of the infection.
No single test can lead to a definitive diagnosis of NCC. CT-scan or MRI may be performed on the presentation of clinical symptoms that could be attributed to NCC (first onset of seizure, unexplained headache…) for people who were exposed to NCC risk factors. Imaging may show typical ring lesions with or without inflammation and calcification. However, the image is not pathognomonic of NCC unless hooks (scolex) are visible inside the ring. Thus, serology may give the clue if positive. A positive serology (antibody) may be confirmed by Western-blot or electro-immuno transfer blot (EITB), which show the typical bands specific of T. solium glycoproteins. Antigen detection in the blood can also be performed. This test is specific for T. solium and does not require laboratory confirmation. Both antigen and antibody assays can be performed in the cerebrospinal fluid (CSF). The presence of antibody or antigen in the CSF may contribute towards the assessment of the neurological localization of the disease. In developing countries, the regions most affected by T. solium infection, CT-scan and, of course, MRI are unaffordable, if ever available.
What are the current laboratory tools?
The laboratory diagnosis of cysticercosis is basically the immunodiagnostic based firstly on antibody detection with ELISA (enzyme-linked immunosorbent assay) or immunoblot.
The detection of antibodies against T. solium is a common method of infection diagnosis, but presents many limitations as a single cyst carrier may not be easily detected. Commercially available tests include essentially ELISA and Western-blots. Western-blots are the ‘gold standard’ assays for the detection of specific antibodies against T. solium. The reference Western-blot assay remains the one developed at the Centers for Disease Control (CDC), Georgia, USA, by Tsang et al. [2]. It employs a specific fraction of T. solium cysts. Many of the components have been identified and cloned. The test is very specific for exposure and/or disease and to confirm the diagnosis. Both ELISA tests and Western-blot relay on antigens that have varied significantly throughout the years (Fig. 3) [3]. Historically, the first assays used crude soluble extracts, then purified proteins such as lentil lectin glycoproteins (LLGPs) Recent trends, though not yet commercialized, tend to emphasize the use of recombinant proteins. Designing recombinant antigens requires a proteinomic approach (Fig. 4) that is now frequently used in development units. Current studies propose the use of nanobodies for diagnostic purposes. These evolutions increased both the sensitivity and the specificity of the tests.
Another available technique is based on the detection of circulating parasitic antigens using monoclonal antibodies [4]. This test is capable of detecting single cyst carriers and is more specific than available antibody ELISA tests. Its main advantage is its ability to monitor the response to cysticidal therapy.
Understanding the performance assessment of T. solium detection tests
Most commercially available ELISA tests have been evaluated by poor methodology. Assessing that a performance evaluation used the proper method means ensuring that the study used a serum bank of parasitologically-defined sera to assess test sensitivity. Defined cysticercosis sera should ideally include the following sera: two or more viable cysts, single viable cysts, degenerating cysts, calcified cysts.
Each series should be initially tested separately. A parasitologically-defined sera should correspond to the Del Brutto criteria [1]. In the absence of a true ‘gold standard’ for the diagnosis of neurocysticercosis, positive sera (cases) should be taken from patients with (1) absolute diagnosis of NCC, or (2) probable NCC diagnosis.
The test specificity should be carefully evaluated using defined negative and potentially cross-reactive sera. Negative sera (control) should be taken from the same area and if possible from people exposed to the same risk factors as the positive cases, with age and sex correlation. Negative cases are usually taken from blood donors of developed countries. Those people have not been in contact with many parasitic infections and the sensitivity of the test will not be accurate/reliable for use in developing countries. This is why specificity should not only be assessed on negative samples from Western countries but also on other parasitic infections from cysticercosis-free developing countries.
What are the new trends in laboratory tests?
If only immunodiagnostic tools based on antibody or antigen detection are currently commercialized, new approaches have been developed including molecular biology (gene amplification in CSF mostly) (Fig. 5). However, so far none constitutes a ‘gold standard’. Table 1 summarizes the pros and cons of NCC diagnosis tools.
Conclusions and future
A test is reliable and useful if it contributes to a care improvement; that is to say to an appropriate therapy for all the patients. As for NCC; the decision to treat is still subject to controversy. Furthermore, even basic serologies are unaffordable or unavailable in endemic countries, not to mention imaging. The key will be in developing a reliable rapid test able to screen infected patients and correlated to neurological lesions of cysticerci.
References
1. Del Brutto OH. Diagnostic criteria for neurocysticercosis, revisited. Pathog Glob Health 2012; 106(5): 299–304.
2. Tsang VC, Brand JA, Boyer AE. An enzyme-linked immunoelectrotransfer blot assay and glycoprotein antigens for diagnosing human cysticercosis (Taenia solium). J Infec Dis. 1989; 159(1): 50–59.
3. Esquivel-Velázquez M, Ostoa-Saloma P, Morales-Montor J, Hernández-Bello R, Larralde C. Immunodiagnosis of neurocysticercosis: ways to focus on the challenge. J Biomed Biotechnol. 2011; 2011: 516042. Doi:10.1155/2011/516042.
4. Garcia HH, Harrison LJ, Parkhouse RM, Montenegro T, Martinez SM, Tsang VC, Gilman RH. A specific antigen-detection ELISA for the diagnosis of human neurocysticercosis. The Cysticercosis Working Group in Peru. Trans R Soc Trop Med Hyg. 1998; 92(4): 411–414.
The author
Jean-François Carod Pharm D, MSc
Laboratoire de Biologie Médicale, GCS de l’ARC Jurassien, Centre Hospitalier Louis Jaillon, 2 Montée de l’hôpital, 39200 Saint-Claude, France.
E-mail: jean-francois.carod@ch-stclaude.fr
Electronic alerts for acute kidney injury: the role of the laboratory
, /in Featured Articles /by 3wmediaAcute kidney injury is a common and serious complication of many hospital admissions, yet there are often delays in recognizing its development. The laboratory can play a key role in ensuring large increases in serum creatinine do not go unnoticed so that deteriorating patients receive prompt medical attention.
by Nick Flynn
Introduction
Acute kidney injury (AKI) is a sudden decline in renal function, generally occurring over hours or days. AKI is increasingly recognized as a common healthcare problem associated with poor outcomes such as increased mortality and progression of chronic kidney disease [1], prolonged hospital stay and increased healthcare costs [2]. There is also evidence that management of patients with AKI is sometimes poor: in the UK, a National Confidential Enquiry into Patient Outcomes and Death (NCEPOD) report found severe deficiencies of care in a cohort of patients who died with a primary diagnosis of AKI [3]. For example, there was often a delay in recognizing post-admission AKI. This has prompted some hospitals to implement electronic alerts (e-alerts) to systematically detect and highlight cases of AKI. As current definitions of AKI are based mainly upon changes in serum creatinine, laboratories are well placed to implement these systems (Table 1) [4]. This review will briefly discuss options for e-alerts, some considerations for their implementation, and the evidence base for their use.
AKI e-alerts
The aim of AKI e-alert systems is to improve the outcomes of patients by facilitating earlier recognition and treatment of AKI. E-alerts may be triggered by a variety of different criteria, ranging from a single threshold creatinine value to full application of AKI diagnostic criteria. This may result in an automated comment being appended to the creatinine result, a phone call, email or text message to the requesting doctor, nephrologist or critical care outreach team, or a combination of the above. The intention is for the alert to prompt medical attention for these high-risk deteriorating patients, with a resulting improvement in patient outcomes (Fig. 1). The most successful e-alert systems are therefore likely to combine the alert with a clinical protocol for AKI management, and should be developed in collaboration with clinical colleagues.
Choosing alert criteria
Although a single threshold creatinine (for example, 300 µmol/L) is the simplest approach, this lacks both sensitivity and specificity for AKI. Creatinine may need to rise significantly before reaching the threshold, so the speed at which AKI is recognized may not be improved. In addition, depending on the population served by the laboratory, a large number of elevated creatinine results are likely to be from patients with stable chronic kidney disease, rather than AKI.
Accuracy can be improved by applying a ‘delta check’ to flag an absolute or percentage increase in creatinine, for example, a 75% increase in creatinine [5]. It is usually within the realms of most modern laboratory information management systems to offer one delta check for creatinine, and it is also sometimes possible to run multiple checks with different criteria. Finally, some systems aim to fully apply current definitions, such as those recommended by KDIGO (Table 1) [4].
Accurately estimating baseline creatinine is difficult
A problem faced both by simple delta checks and e-alerts based on AKI definitions is the difficulty in reliably estimating baseline creatinine. A system employing manual estimation of baseline by clinical biochemists at the Royal Derby Hospital has been shown to have good diagnostic accuracy for detection of AKI with a false negative rate of 0.2% and a false positive rate of 1.7% [6]. However, this approach is limited to normal working hours and many laboratories do not have the resources to replicate this labour intensive system. Instead, automatic surrogate estimation methods are used, such as the lowest, most recent or median creatinine value within a certain timeframe, such as the previous three months. Laboratories should be aware of the limitations of some of these estimation methods; for example, the lowest creatinine result has been shown to be a particularly poor estimate of baseline creatinine that can lead to high rates of potential AKI misclassification [7].
Should every case fulfilling AKI criteria be highlighted?
When choosing criteria for an e-alert system, it may seem sensible to use current definitions for AKI. However, there are arguments against this approach. The KDIGO definition of AKI relies on small changes in serum creatinine based on epidemiological studies which show that even these small increases are associated with an increase in mortality risk in large populations [2]. However, in many cases an increase of 0.3 mg/dl (≥26.5 µmol/L) is within the realms of normal biological variation, particularly amongst patients with chronic kidney disease. As an illustrative example, creatinine increased by between 69% and 129% after the consumption of 300 g of animal protein in healthy volunteers, even with creatinine measurement using a specific enzymatic method [8]. The limitations of the more widely used Jaffe method for serum creatinine are well known amongst laboratory professionals, and any of a wide range of non-creatinine chromogens may cause an increased result in the absence of renal disease. When KDIGO criteria are combined with a poor method of baseline estimation (lowest previous creatinine), the proportion of creatinine results causing an AKI e-alert can approach 10%; this is unlikely to be helpful. Strict application of current AKI definitions could therefore lead to annoyance and unresponsiveness amongst clinicians alerted to minor creatinine elevations, unnecessary interventions, anxiety for patients and families, and diversion of limited healthcare resources to a large and relatively low risk group. It is therefore important for laboratories to consider both local IT and resource capabilities and the relative benefit and harm of different criteria for e-alerts before implementation.
Evidence base
A small number of studies have investigated the effect of AKI e-alerts on clinician behaviour or patient outcomes. For example, a real-time alert of worsening AKI stage through a text message sent to the clinician’s telephone was found to increase the number of early therapeutic interventions in an ICU in Belgium [9]. There was also an increase in the proportion of patients who recovered their renal function within 8 hours after an alert indicating less severe AKI, but not amongst those with more severe AKI. There was no significant effect on renal replacement therapy, ICU length of stay, mortality, maximum creatinine or maximum AKI stage. Importantly, 9 out of 10 AKI alerts were based on urine volume criteria, so the applicability of these findings to creatinine based e-alerts is questionable.
Hospitals that have already implemented AKI e-alerts have noted improved outcomes following their introduction. For example, a hospital-wide e-alert system based on changes in serum creatinine at the Royal Derby Hospital, led to a progressive reduction in 30 day mortality over consecutive 6 month periods (23.7%, 20.8%, 20.8%, 19.5%, chi-square for trend P=0.006) [10]. This improvement in survival was maintained after adjustment for age, co-morbid conditions, severity of AKI, elective/non-elective admission and baseline renal function. However, the e-alert was introduced as part of a range of educational interventions so it is difficult to determine the contribution made by the e-alert component.
The evidence base for AKI e-alerts is therefore not strong, and would benefit from further studies to demonstrate that this approach can lead to measurable improvements in patient outcomes.
Conclusions
E-alerts represent an opportunity for the laboratory to assist in the early detection of acute kidney injury. This could improve the outcomes of patients with this life threatening condition. Aside from AKI, there are undoubtedly many other opportunities for the laboratory to optimize existing resources by helping clinicians to digest the large amount of laboratory data produced on a daily basis, to highlight trends and to ensure that important changes are recognized and acted upon. The laboratory can play a key role to ensure that these systems are implemented, that they are effective in selectively capturing a high risk population, and that evidence is gathered to justify their continued use.
References
1. Coca SG, et al. Long-term risk of mortality and other adverse outcomes after acute kidney injury: a systematic review and meta-analysis. Am J Kidney Dis. 2009; 53(6): 961–973.
2. Chertow GM, et al. Acute kidney injury, mortality, length of stay, and costs in hospitalized patients. J Am Soc Nephrol. 2005; 16: 3365–3370.
3. Stewart J, et al. Adding Insult to Injury: a review of the care of patients who died in hospital with a primary diagnosis of acute kidney injury (acute renal failure). A report by the National Confidential Enquiry into Patient Outcome and Death. London: NCEPOD, 2009. www.ncepod.org.uk/2009report1/Downloads/AKI_report.pdf
4. Kidney Disease: Improving Global Outcomes (KDIGO) Acute Kidney Injury Work Group. KDIGO Clinical Practice Guideline for Acute Kidney Injury. Kidney Int. Suppl. 2012; 2: 1–138.
5. Thomas M, et al. The initial development and assessment of an automatic alert warning of acute kidney injury. Nephrol Dial Transplant 2011; 26: 2161–2168.
6. Selby N, et al. Use of electronic results reporting to diagnose and monitor aki in hospitalized patients. Clin J Am Soc Nephrol. 2012; 7: 533–540.
7. Siew ED, et al. Estimating baseline kidney function in hospitalized patients with impaired kidney function. Clin J Am Soc Nephrol. 2012; 7: 712-719.
8. Butani L, et al. Dietary protein significantly affects the serum creatinine concentration. Kidney Int. 2002; 61: 1907.
9. Colpaert K, et al. Impact of real-time electronic alerting of acute kidney injury on therapeutic intervention and progression of RIFLE class. Crit Care Med. 2012; 40: 1164–1170.
10. Kohle N, et al. Impact of a combined, hospital-wide improvement strategy on the outcomes of patients with acute kidney injury (AKI) [abstract]. Joint Congress of the British Transplantation Society & Renal Association, 2013. Bournemouth. Abstract O30. www.btsra2013.com/
The author
Nick Flynn, Pre-registration clinical scientist
Department of Clinical Biochemistry, University College London Hospitals, London, UK
E-mail: nick.flynn@nhs.net
One Company. Two World-Class Diagnostics Brands.
, /in Featured Articles /by 3wmediaSodium available in ‘enzymatic’ format
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, /in Featured Articles /by 3wmediaPre-eclampsia: the good and bad news
, /in Featured Articles /by 3wmediaAffecting around one in twenty pregnancies, pre-eclampsia is a leading cause of fetal morbidity and mortality globally. Around half a million babies die as a result of the condition annually. Severe pre-eclampsia, leading to eclampsia characterized by seizures, is also the second leading cause of maternal mortality (after hemorrhage) in most countries: an estimated 76,000 women die from it each year. A diagnosis of this multisystemic disorder has classically been made if hypertension and proteinuria are present. Pre-eclampsia can only be resolved by delivery of the placenta, thus management must weigh the severity of the condition against the risk to the fetus of an induced, premature delivery.
The launch of a rapid test measuring the plasma level of placental growth factor (PLGF), a biomarker of placental function, four years ago offered the possibility of a more timely diagnosis of pre-eclampsia and its severity that could facilitate optimal management for both mother and baby, including the administration of corticosteroids to accelerate fetal lung development prior to premature delivery. The level of PLGF normally rises during pregnancy up to 26 to 30 weeks’ gestation, and then falls until full-term, but its level is abnormally low in women with pre-term pre-eclampsia. Recently the published results of a large multicentre study using this rapid test made very encouraging reading. During the study, PLGF was measured in 625 pregnant women between 20 and 35 weeks gestation with suspected pre-eclampsia. The condition was confirmed in 55% of these women, with outcome being the delivery of the fetus within 14 days. The authors concluded that the test had high sensitivity in women presenting with suspected pre-eclampsia before 35 weeks’ gestation, and indicated need for delivery better than other diagnostic methods.
Although this research is good news for pregnant women and their babies, another aspect of pre-eclampsia has largely been ignored and is not generally known by either health-workers or women themselves, namely the subsequent increased health risk in older women who suffered from pre-eclampsia in pregnancy. A robust meta-analysis has linked the condition with a fourfold increased risk of hypertension, and a twofold increased risk of ischemic heart disease, stroke and venous thromboembolism, later in life. A recent study from Australia found that the endothelial dysfunction associated with pre-eclampsia persists, causing the increased risk. At the very least previous pre-eclampsia should be flagged as important in an older woman’s medical history!
Preimplantation genetic screening and related issues
, /in Featured Articles /by 3wmediaPreimplantation genetic screening is a diagnostic approach dedicated to patients undergoing IVF with the proper indications (advanced maternal age, recurrent implantation failure, recurrent pregnancy loss) in order to increase pregnancy rates per transfer via euploid embryo selection. This strategy, and all the associated techniques, are in constant evolution and will shed more light on unexplored aspects of embryology, such as female meiosis or chromosomal mosaicism, creating new criteria for embryo selection.
by Dr D. Cimadomo, Dr A. Capalbo, Dr L. Rienzi and Dr F. M. Ubaldi
Background
Preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS) are two diagnostic approaches increasingly exploited in recent decades within assisted reproduction facilities in the presence of specific indications. PGD is used to identify unaffected embryos in couples at high reproductive risk of a hereditary disease. Usually, these couples conceive naturally and undergo prenatal genetic testing, i.e. villocentesis or amniocentesis; procedures that are invasive and carry a high risk of subsequent miscarriage. The ultimate aim of PGD is, therefore, to prevent the conception of a fetus affected specifically and uniquely by a pathology whose causative mutations have been identified and characterized in the parental genomes before conception. Consequently, PGD depends on a preliminary ad hoc work-up for each couple approaching to an IVF cycle. PGS, instead, is meant to identify only chromosomally normal embryos, thus looking for the presence of chromosomal abnormalities. Since the development of aneuploidies is a de novo event directly linked to maternal age, this diagnostic method is independent from any specific preliminary set-up, thus being identical for each PGS cycle. The indications for this analysis are mainly advanced reproductive maternal age (more than 35 years old; AMA), recurrent implantation failure (more than three failed IVF attempts; RIF) and recurrent pregnancy loss (more than three miscarriages; RPL). From an embryological perspective there is no difference between PGD and PGS. Indeed, strategy and planning of the cycle and biopsy techniques are similar, whereas the genetic technical aspects are significantly different.
Testing for aneuploidy
Interestingly, the data collected by the ESHRE PGD consortium IX showed a constant increase in the number of the PGD cycles approached uniquely for euploid embryo selection. In particular, more than 60% of PGD cycles were actually PGS for AMA, RIF or RPL patients, and this percentage is still currently increasing. There is, in fact, a striking impact of aneuploidies on human reproduction. In particular, their incidence in newborns is around 0.3%, mostly represented by trisomies of chromosomes 13, 18 and 21 and sex chromosome aneuploidies. However, tracking backwards through the developmental stages sees this incidence sharply increase, involving other chromosomes and reaching an incidence of up to 60% in preimplantation embryos and 70% in eggs or polar bodies [1]. On the contrary, this incidence in sperm is definitely less severe, as it is never greater than 3–4%. Moreover, a significant number of spontaneous abortions are linked to aneuploidies (more than 60% of products of conception follow chromosomal abnormalities), both increase exponentially with maternal age and fertility rate collapses (Fig. 1) [2].
From a biological perspective, the origin of high trisomy rates found in clinically recognized pregnancies (which sharply increases in patients older than 35 years) resides mainly in maternal meiosis I and II [3]. Recent data obtained through array comparative genomic hybridization (aCGH) on polar bodies (PBs) showed that chromatid errors in female meiosis, such as premature separation of sister chromatids, definitely outnumber impairments involving whole chromosomes as previously thought [4, 5]. Capalbo et al. [5] performed analyses on biopsies at sequential stages of development, in particular the two PBs, a single blastomere at day 3 of embryo development and also a trophectoderm (TE) sample at the blastocyst stage (Fig. 2). This study design allowed the determination of PB analysis accuracy and the impact of male and mitotic errors as well as the evaluation of the occurrence of correction mechanisms throughout preimplantation development. It came to light that 76 out of 78 (97.4%) abnormal meiotic segregations concerned errors involving chromatids rather than whole chromosomes at meiosis I. Furthermore, it unveiled not only a false positive rate in PB biopsy analysis of 20.5%, as just 79.5% (62/78) of meiotic segregation errors identified in PB biopsies were confirmed in blastomeres, but also a false negative rate of 47.6%, as 10 out of 21 embryos showed mitotic or male-derived aneuploidies confirmed at day 3 and at the blastocyst stage of development, which are, obviously, not observable in PBs. This evidence subverts our previous scenario of chromosomal aneuploidy genesis, as well as undermining the reliability of the PB analysis strategy.
Chromosomal mosaicism
From a diagnostic perspective in PGS, post-zygotic mitotic segregation errors are definitely more troubling than meiotic ones, as, whereas the latter involve the same aberrant chromosomal layout in the whole developing embryo, the former entail the phenomenon of chromosomal mosaicism. In the last decade several publications focused on the problem of mosaicism and its influence on PGD/PGS, claiming an incidence fluctuating between 25% [6, 7] and up to more than 70% [8]. Even when these data are analysed with a critical approach, it still emerges that mosaicism is a substantial source of misdiagnosis when the embryo is biopsied at day 3 post-fertilization. This evidence encouraged a shift of the biopsy strategy toward the blastocyst stage and, to this end, different studies were conducted in order to thoroughly describe its cytogenetic constitution and the impact of biopsy itself on embryonic developmental competence. In particular, Capalbo et al. [9] published data outlining the impact of chromosomal mosaicism on a diagnosis at day 5/6 of embryo development as well as the aneuploid cells setting between inner cell mass (ICM) and TE. To this end, a novel method of ICM biopsy was conceived [as described in 9], characterized via KRT18 staining [as described in 10] and its efficiency tested. It led to the absence of TE contamination in 85.7% of the ICM biopsy products, and a low TE contamination rate (only 2% of TE cells) in the rest of them. These data attest the reliability of this biopsy procedure to test the influence of mosaicism at the blastocyst stage. The study design entailed a preliminary aCGH analysis on a TE biopsy during blastocyst-stage PGS clinical cycles, followed by FISH re-analysis of three further fragments of TE and of the ICM from those blastocysts found to be carriers of copy-number chromosomal errors as well as euploid embryos. This revealed that at the blastocyst stage of development, 79.1% of the aneuploidies were constitutional, while 20.9% of them were mosaic. However, only 4% of the blastocysts were found to be mosaic diploid/aneuploid, being at risk of misdiagnosis due to mosaicism when testing at the blastocyst stage. These data strengthen the theory that the impact of mosaicism could be critical at day 3 of embryo development, but it has definitely less influence at the blastocyst stage, thus strongly presenting the latter as the most reliable candidate biopsy stage to perform PGS. Importantly, in the same paper, Capalbo et al. demonstrated that, after excluding low grade mosaicism (<20% of aneuploid cells) and mosaicism confined to one or two TE sections, in 97.1% of cases concordance for all chromosomes re-analysed by FISH between ICM and TE was observed. On a per embryo analysis, instead, complete concordance in TE-based prediction of ICM chromosomal complement was reported (Fig. 3) [9]. Northrop et al. [11] conducted a similar analysis exploiting a single nucleotide polymorphism (SNP) array, which is a comprehensive chromosomal screening technique. This method was found to detect aneuploidy in samples possessing more than 25% aneuploidy, thus when as few as 2 of the 5 cells within a TE biopsy contain the same chromosomal error. Their data showed no preferential aneuploid cell migration to the TE layer, as aneuploidy was observed in 31% of ICM samples (15 out of 48 ICM products) and 32% of TE ones (46 of 144 TE products). Furthermore, a mosaicism rate of 24% was attested, since 12 out of 50 blastocysts screened showed more than a single diagnosis in all of the multiple sections that were re-analysed.
Does the biopsy procedure affect embryo reproductive competence?
One concern about PGS is that biopsy could affect embryo reproductive competence. To investigate this possibility, Scott et al. [12] designed a randomized and paired clinical trial. They selected two of the best quality embryos from the same patient to be transferred and randomized them, one to undergo biopsy, either at day 3 or at day 5 of embryo development, and the other as a control. The biopsy was submitted to SNP array analysis. If only one embryo implanted, buccal DNA obtained from the neonate after delivery was analysed by SNP array to determine whether the implanted embryo was the control one or not. The data collected clearly showed that conducting the biopsy at the cleavage stage affects the clinical outcome, as an absolute reduction in implantation rate of 19.6% with respect to the control was reported. On the contrary, blastocyst biopsy led to a non-significant overall reduction of implantation of 3%; thus an implantation rate equivalent to the control. It is still unclear whether this is due to a smaller proportion of the embryo’s total number of cells being removed, or to the fact that only extra-embryonic cells are involved, or to a higher stress-tolerance of the blastocyst; however, it is still additional important evidence supporting TE biopsy as the ‘gold standard’ for PGS. From a clinical perspective, the same authors also published a randomized controlled trial [13] comparing the clinical outcomes of single euploid blastocyst transfer versus double untested blastocyst transfer. Ongoing pregnancy rates per randomized patient were similar between the two groups (60.7% in the study group vs 65.1% in the control group), whereas a higher multiple pregnancy rate in the control group was recorded (54% vs 0% in the study group). Ultimately then, PGS on TE biopsy associated with a single euploid blastocyst transfer elicits the same clinical outcomes as conventional IVF, but reduces its risks.
Conclusion
In conclusion, PGS is an important diagnostic approach for patients with the proper indications (AMA, RIF or RPL), performed in order to boost implantation rate per transfer. Euploid embryo selection prevents useless and potentially detrimental embryo transfers. Consequently, further advantages of performing PGS are a lower time-to-pregnancy and a higher cost-effectiveness of each single treatment. Moreover, by adopting a biopsy strategy at day 5/6, it is possible to take advantage of a more robust genetic analysis, a high clinical predictive value, the absence of impact of the biopsy on embryo quality, a low influence of mosaicism, as well as a reduced number of embryos to analyse per cycle, as only developmentally competent ones would reach the blastocyst stage. These last aspects will help in reducing costs, thus extending the patients population that can benefit from this technology. Finally, novel comprehensive chromosomal screening techniques, i.e. aCGH, SNP array and quantitative real-time PCR (qPCR), provide us with reliable, sensible and accurate analysis methods, making of PGS also a technically solid approach.
References
1. Nagaoka SI, Hassold TJ, Hunt PA. Human aneuploidy: mechanisms and new insights into an age-old problem. Nat Rev Genet. 2012; 13(7): 493-504.
2. Heffner LJ. Advanced maternal age–how old is too old? N Engl J Med. 2004; 351(19): 1927-1929.
3. Hassold T, Hunt P. To err (meiotically) is human: the genesis of human aneuploidy. Nat Rev Genet. 2001; 2(4): 280-291.
4. Handyside AH, Montag M, Magli MC, Repping S, et al. Multiple meiotic errors caused by predivision of chromatids in women of advanced maternal age undergoing in vitro fertilisation. Eur J Hum Genet. 2012; 20(7): 742-747.
5. Capalbo A, Bono S, Spizzichino L, Biricik A, et al. Sequential comprehensive chromosome analysis on polar bodies, blastomeres and trophoblast: insights into female meiotic errors and chromosomal segregation in the preimplantation window of embryo development. Hum Reprod. 2013; 28(2): 509-518.
6. Voullaire L, Slater H, Williamson R, Wilton L. Chromosome analysis of blastomeres from human embryos by using comparative genomic hybridization. Hum Genet. 2000; 106(2): 210-217.
7. Wells D, Delhanty JD. Comprehensive chromosomal analysis of human preimplantation embryos using whole genome amplification and single cell comparative genomic hybridization. Mol Hum Reprod. 2000; 6(11): 1055-1062.
8. Mertzanidou A, Wilton L, Cheng J, Spits C, et al. Microarray analysis reveals abnormal chromosomal complements in over 70% of 14 normally developing human embryos. Hum Reprod. 2013; 28(1): 256-264.
9. Capalbo A, Wright G, Elliott T, Ubaldi FM, et al. FISH reanalysis of inner cell mass and trophectoderm samples of previously array-CGH screened blastocysts shows high accuracy of diagnosis and no major diagnostic impact of mosaicism at the blastocyst stage. Hum Reprod. 2013; 28(8): 2298-2307.
10. Cauffman G, De Rycke M, Sermon K, Liebaers I, Van de Velde H. Markers that define stemness in ESC are unable to identify the totipotent cells in human preimplantation embryos. Hum Reprod. 2009; 24(1): 63-70.
11. Northrop LE, Treff NR, Levy B, Scott RT Jr. SNP microarray-based 24 chromosome aneuploidy screening demonstrates that cleavage-stage FISH poorly predicts aneuploidy in embryos that develop to morphologically normal blastocysts. Mol Hum Reprod. 2010; 16(8): 590-600.
12. Scott RT Jr, Upham KM, Forman EJ, Zhao T, Treff NR. Cleavage-stage biopsy significantly impairs human embryonic implantation potential while blastocyst biopsy does not: a randomized and paired clinical trial. Fertil Steril. 2013; 100(3): 624-630.
13. Forman EJ, Hong KH, Ferry KM, Tao X, et al. In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial. Fertil Steril. 2013; 100(1): 100-107.
The authors
Danilo Cimadomo BSc, Antonio Capalbo* PhD, Laura Rienzi MS, Filippo Maria Ubaldi MS
G.EN.E.R.A. Centre for Reproductive
Medicine, Clinica Valle Giulia, Via G. De Notaris 2b, 00197 Rome, Italy
*Corresponding author
E-mail: capalbo@generaroma.it
NIPT: an opportunity for early detection and promising treatment for children with XXY
, /in Featured Articles /by 3wmediaInconsistent detection and false-positive rates have plagued traditional screening measures for trisomy, thus encouraging the development of less risky and invasive measures. Through the advent of single-nucleotide polymorphism-based and informatics-based non-invasive prenatal testing, accurate detection of trisomies 13, 18, 21 as well as the X and Y chromosomal aneuploidies of XXY, XYY and XXX in early in pregnancy is now possible. This technology is extremely important in ensuring infants with these disorders are identified in a timely manner so that proper care and treatment can be administered for optimal development.
by Emily J. Stapleton, Dr Megan Hall and Dr Carole A. Samango-Sprouse
Cell-free DNA-based non-invasive prenatal testing
Traditional serum- and ultrasound-based screens have high false-positive rates and less-than-ideal detection rates, resulting in unnecessary and risky invasive procedures and missed diagnoses [1]. The discovery of fetal cell-free DNA (cfDNA) in maternal circulation allowed the development of a more accurate, non-invasive approach for fetal aneuploidy screening [termed non-invasive prenatal testing (NIPT)] [2]. However, cfDNA is highly fragmented and is heavily diluted with maternal cfDNA [3]. Hence, methods to accurately detect fetal aneuploidies using cfDNA analysis had to overcome these technical limitations. Two approaches to-date have accomplished this and have been successfully commercialized. The first-generation quantitative ‘counting’ approaches amplify and sequence non-polymorphic loci and compare absolute quantities of DNA from the chromosome(s) of interest (e.g. chromosome 21) to that of reference chromosomes [4]. The second, next-generation approach specifically amplifies and sequences single-nucleotide polymorphisms (SNPs), identifying both allele identity and distribution [4].
First-generation quantitative counting methods
The most straight forward counting methods non-specifically amplify cfDNA, followed by massively parallel shotgun sequencing (MPSS) [4]. A more recent approach uses targeted amplification and sequencing, thus improving efficiency [4]. Both methods amplify non-polymorphic loci, and identify fetal aneuploidy by detecting abnormally high or low amounts of cfDNA from the chromosome(s) of interest relative to internal reference chromosomes that are presumably euploid in the fetus. If the proportion of reads associated with a particular chromosome relative to the reference chromosome(s) is found to be significantly above the expected proportion for a euploid fetus, the extra reads are presumed to have originated from an extra chromosome present in the fetal genome and fetal trisomy is inferred. Counting methods have shown remarkable improvements over serum screening and ultrasound methods, reporting >97% sensitivity for trisomies 21 and 18, and false positive rates of <0.2% for trisomy 21 [4]. However, the false positive rate can be as high as 1% for other indications [4]. Additionally, counting methods have reduced sensitivity when detecting aneuploidy of chromosomes 13 and X [4]. This is thought to be due to a combination of variable amplification efficiency due to decreased guanosine–cytosine content, as well as unusual biology specific to the X chromosome. Significantly, the requirement for a reference chromosome renders these methods unable to detect triploidy. A next-generation approach for NIPT: analysing SNPs
The next-generation PanoramaTM test is the only commercialized NIPT that incorporates genotypic information, in the form of SNPs, to accurately identify fetal chromosomal copy number from cfDNA [5, 6]. This allows a more complex and nuanced cfDNA analysis than first-generation methods that do not take into account genotypic information and only consider the number of reads. This SNP-based approach is able to identify both the allele identity and distribution, thus identifying the maternal and fetal cfDNA contribution to the sequence reads. Additionally, Panorama uses a sophisticated bioinformatics algorithm called Next-generation Aneuploidy Testing Using SNPs (NATUS) that leverages advanced Bayesian statistics.
The NATUS algorithm incorporates parental genotypic information to aid analysis of relatively noisy measurements that result from the mixture of maternal and fetal cfDNA. Specifically, NATUS considers the maternal genotype, which is obtained by measuring genomic DNA isolated from white blood cells present in the maternal blood sample, as well as the paternal genotype, if available (though not necessary); the algorithm incorporates crossover frequency data from the human genome project to bioinformatically predict all of the possible fetal genotypes that could arise from the parental genotypes. These billions of hypotheses are then compared to the actual cfDNA measurements, and a likelihood is calculated for each hypothesis. The hypothesis with the maximum likelihood indicates the actual genetic state of the fetus, thus determining the presence or absence of a chromosomal abnormality.
This approach enables the incorporation of many more quality control metrics, improving accuracy over first-generation counting approaches. First, it creates the ability to flag samples with additional abnormalities, including samples with large deletions and duplications, mosaicism, and extra parental haplotypes, which indicate undetected twins, vanishing twins, or triploidy; any of these may result in miscalls with first-generation NIPTs. Second, the algorithm can take into account a number of other indicators of accuracy in addition to fetal fraction, for example the total amount of cfDNA in the sample, and the degree of contamination. This allows the algorithm to determine when the data is insufficiently clear to make an accurate call, even if the fetal fraction is above the minimum threshold of 3.8%; this reduces the number of incorrect calls. Third, this approach does not rely on a reference chromosome, which enables highly accurate detection of abnormalities on chromosomes that do not amplify with reliable efficiency, such as chromosome 13 and the sex chromosomes, as well as the unique ability to detect triploidy [5, 6]. These advantages, therefore, overcome limitations of the first-generation approach.
This translates to a quantifiable improvement in performance [6]. Specifically, in clinical studies, the NATUS algorithm showed 100% sensitivity when detecting trisomy 21, trisomy 18, trisomy 13, fetal sex, and triploidy, and of 91.7% when detecting monosomy X (Turner syndrome) [5, 6]. Reported specificities were 100% when detecting trisomy 21, trisomy 13, triploidy, and fetal sex, and 99.9% for trisomy 18 and monosomy X [6].
Why NIPT is clinically important
With the advent of SNP-based NIPT, the increase in the number of populations that can affordably and conveniently receive prenatal testing has dramatically increased and, subsequently, so has the identification of children with genetic abnormalities. Through early identification of chromosomal aneuploidies, children can receive early intervention services that are critical to the management of the associated disorders. This is especially true regarding the X and Y chromosomal variations that the NIPT identifies, specifically 47, XXY.
The impact of prenatal testing on 47, XXY
47, XXY (Klinefelter Syndrome) is characterized by the presence of an additional X chromosome and has a frequency of occurrence of 1 in 400 to 1 in 1,000 births [7]. However, due to their mild phenotypic presentation only 25% of boys with the disorder will ever be properly diagnosed. Boys with 47, XXY present neurocognitive deficits in language-based learning disabilities, atypical social development as well as reading disorders [8]. Musculoskeletal findings consist of decreased muscle tonus with joint laxity, pectus excavatum and pescavus. MRI brain imaging in individuals with 47, XXY revealed morphological, volumetric, and gray and white matter differences that are associated with the deficits in neurodevelopmental performance [9].
Androgen insufficiency in XXY has been described in several studies and it has been posited that the androgen deficiency contributes to the neurodevelopmental challenges associated with these disorders, as small research studies report improved brain function in association with androgen replacement [10]. Additionally, recent studies on 47, XXY and 49, XXXXY showed improvement in selected aspects of neurodevelopmental outcome when treated with androgen prior to 24 months of age [11, 12]. The area of greatest difficulty in the disorder is speech and language of which early hormonal treatment (EHT) has shown the most robust improvements in select areas of the verbal domain.
Boys with 47, XXY are susceptible to atypical social interactions, social isolation, and poor self-esteem as a result of the significant language-based learning disorders [9]. Ultimately, these issues may lead to low employment rates, depression and behavioural disruptions if not treated early in life [13]. Although there is a wide variability of cognitive capabilities in 47, XXY individuals, research studies indicate that prenatally diagnosed children demonstrate higher intellectual abilities [9]. Late diagnosis and untreated learning disorders coupled with deficits in executive function may result in significant neurocognitive challenges and behavioural disruptions [13]. School failure is common in these circumstances, which is costly for society in the form of low employment and high risk for psychiatric disturbances of depression and anxiety.
The importance of prenatal diagnosis is critical for the timely implementation of targeted and syndrome-specific treatments, most importantly EHT, and ensuring an optimal developmental trajectory for the child. The development of speech, language and early neurocognitive skills is critical to the growth of later reading proficiency and academic success. These skills are the building blocks for advanced abstract thinking capabilities and as a result allow for job employment and independent living. Research suggests that without timely treatment the growth of these critical neurodevelopmental abilities would be stunted or possibly altogether halted.
Summary
Although this article highlights only one disorder that can be identified through NIPT, the studies presented throughout demonstrate that the neurodevelopmental function of a very common neurogenetic disorder may be improved through early treatment. The importance of NIPT for early identification is imperative in XXY as well as other X and Y chromosomal disorders. The ramifications of prenatal detection and early identification cannot be understated; with knowledge comes the ability to improve a child’s life as well as the family’s well being from the moment of birth onward.
References
1. Invasive prenatal testing for aneuploidy. ACOG Practice Bulletin No. 88. American College of Obstetricians and Gynecologists. Obstet Gynecol. 2007; 110: 1459–1467.
2. Noninvasive prenatal testing for fetal aneuploidy. Committee Opinion No. 545. American College of Obstetricians and Gynecologists. Obstet Gynecol. 2012; 120: 1532–1534.
3. Lo YM, Tein JS, Lau TK, Haines CJ, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for non-invasive prenatal diagnosis. Am J Hum Genet. 1998; 62: 768–775.
4. Levy B, Norwitz E. Non-invasive prenatal aneuploidy testing: technologies and clinical implication. MLO Med Lab Obs 2013; 45: 8,10,12.
5. Samango-Sprouse C, Banjevic M, Ryan A, Sigurjonsson S, et al. SNP-based non-invasive prenatal testing detects sex chromosome aneuploidies with high accuracy. Prenat Diagn. 2013; 33: 1–7.
6. Pergament E, McAdoo S, Curnow K, et al. SNP-based non-invasive prenatal aneuploidy testing of chromosomes 13, 18, 21, X, and Y in a high- and low-risk cohort. Manuscript under review.
7. Morris JK, Alberman E, Scott C, Jacobs P. Is the prevalence of Klinefelter syndrome increasing? Eur J Hum Genet. 2008; 16: 163–170.
8. Samango-Sprouse CA, Gropman AL. Introduction: Past, present, and future care of individuals with XXY. Am J Med Genet C Semin Med Genet. 2013; 163C: 1–2.
9. Lee NR, Wallace GL, Clasen LS, Lenroot RK, et al. Executive function in young males with klinefelter (XXY) syndrome with and without comorbid attention-deficit/hyperactivity disorder. J Int Neuropsychol Soc. 2011; 22: 1–9.
10. Patwardhan AJ, Eliez S, Bender B, Linden MG, Reiss AL. Brain morphology in Klinefelter syndrome: extra X chromosome and testosterone supplementation. Neurology 2000; 54(12): 2218–2223.
11. Samango-Sprouse CA, Gropman AL, Sadeghin T, Kingery M, et al. Effects of short-course androgen therapy on the neurodevelopmental profile of infants and children with 49,XXXXY syndrome. Acta Paediatrica 2011; 100(6): 861–865.
12. Samango-Sprouse CA, Sadeghin T, Mitchell FL, Dixon T, et al. Positive effects of short course androgen therapy on the neurodevelopmental outcome in boys with 47, XXY syndrome at 36 and 72 months of age. Am J Med Genet A. 2013; 161A: 501–508.
13. Simpson JL, Graham JM, Samango-Sprouse CA, Swerdloff R. 2005. Klinefelter Syndrome. In Cassidy SB, Allanson JE (editors) Management of Genetic Syndromes, pp.323–334, 2nd edn. New York: Wiley-Liss.
The authors
Emily J. Stapleton1* BSc, Megan Hall2 PhD, and Carole A. Samango-Sprouse1, 3 EdD
1The Focus Foundation, Davidsonville, MD, USA.
2Natera Inc., San Carlos, CA, USA
3George Washington University of the Health Sciences, Washington, D.C., USA
*Corresponding author
E-mail: ndckids@gmail.com
A new biomarker for prostate cancer: [-2]proPSA
, /in Featured Articles /by 3wmediaProstate specific antigen (PSA) has significantly improved the early detection of prostate cancer (PCa), reducing the related mortality rate. However, PSA has a low specificity, being affected by many benign conditions. [-2]proPSA, a PSA precursor, is a more specific and accurate biomarker indicating prostate biopsy in men at real risk of PCa.
by Dr A. Abrate, Dr M. Lazzeri and Prof. G. Guazzoni
PSA as a marker for prostate cancer
Prostate specific antigen (PSA) is a serum marker widely used for the early detection of prostate cancer (PCa). Its introduction into clinical practice in the early 1990s had an extraordinary impact on the diagnosis and management of PCa. In fact, 20 years after its introduction, the PSA-based PCa opportunistic or systematic screening has resulted in a stage migration to more organ-confined tumours at the time of diagnosis, and consequently to a consistent reduction in PCa related mortality [1, 2]. However, PSA is not a perfect marker for the detection of PCa because of its low specificity and sensitivity. Its levels may increase as a result of benign conditions, such as benign prostatic hyperplasia (BPH) and chronic prostatitis. Moreover, PSA levels are also affected by biologic variability, which may be related to differences in androgen levels, prostate manipulation or ejaculation. Finally, alterations in PSA levels may be related to sample handling, laboratory processing, or assay standardization. All these factors made it difficult to find an appropriate PSA cut-off point diagnostic for PCa (for many years considered to be 4 ng/ml).
Thus, prostate biopsy is still mandatory to confirm the diagnosis. However, this is positive in only approximately 30% of patients [3], and the European Association of Urology suggests a repeat biopsy if PSA is persistently elevated, the digital rectal examination (DRE) is suspicious, or there is a pathological diagnosis of atypical small acinar proliferation (ASAP) or high-grade prostatic intraepithelial neoplasia (HG-PIN) [4]. Finally, PCa (also high-grade cancer) is not rare (approximately 15.2%) among men with PSA levels lower than 4 ng/ml, the previously widely accepted cut-off point [5].
Considering all these observations, it is clear that PSA is an organ-specific rather than an ideal cancer-specific marker.
The introduction into clinical practice of measuring the levels of several derivatives of PSA (free PSA, percentage of free PSA, PSA density, PSA velocity) improved the accuracy of total PSA (tPSA) in detecting PCa. Recently, free PSA (fPSA) has been found to include several subforms, such as proPSA. In particular [-2]proPSA seems to be specific for PCa, opening new ways for early cancer detection.
Biological basis of proPSA
The currently measurable serum tPSA consists of either a complexed form (cPSA, 70–90%), bound by protease inhibitors (primarily alpha1-antichymotrypsin), and a non-complexed form (fPSA). Recently fPSA has been discovered to exist in at least three molecular forms: proPSA, benign PSA (BPSA), and inactive intact PSA (iPSA), covering approximately 33%, 28%, and 39% of fPSA respectively (Fig. 1) [6]. In particular, proPSA is a proenzyme (precursor) of PSA, which is associated with PCa [7].
PSA is synthesized with a 17-amino acid leader sequence (preproPSA) that is cleaved co-translationally to generate an inactive 244-amino acid precursor protein (proPSA, with seven additional amino acids compared to mature PSA). proPSA is normally secreted from the prostate luminal epithelial cells. Immediately after its release into the lumen, the pro-leader part is removed, creating the active form, by the effect of human kallikrein (hK)-2 and hK-4, which have a trypsin-like activity and are expressed predominantly by prostate secretory epithelium. Other kallikreins, localized in the prostate, such as hK216 or prostin17, are involved in the conversion and activation of proPSA. Cleavage of the N-terminal seven amino acids from proPSA generates the active enzyme, which has a mass of 33 kDa.
The partial removal of this leader sequence leads to other truncated forms of proPSA. Thus, theoretically seven isoforms of proPSA could exist, although only [-1], [-2], [-4], [-5], [-7]proPSA were found; there is still no evidence of [-3], [-6]proPSA. However, all forms of proPSA are enzymatically inactive [8]. It is possible to detect three truncated forms of proPSA in serum: [-5/-7], [-4] and [-2]proPSA, which is the most stable form (Fig. 1).
Notably, in vitro experiments showed that the [-2]proPSA form cannot be activated by either hK2 or trypsin; thus, once it is formed, [-2]proPSA is resistant to activation into the mature PSA form and consequently this is the most reliable test.
Mikolajczyk et al. [7], using a monoclonal antibody recognizing [-2]proPSA, found increased staining in the secretions from malignant prostate glands. In particular [-2]proPSA is differentially elevated in peripheral gland cancer tissue; conversely transition zone tissue contains little or no proPSA.
The increased serum tPSA concentrations in patients with PCa do not result from increased expression but rather from an increased release of PSA into the bloodstream, due to disruption of the epithelial architecture. fPSA is catalytically inactive because of internal cleavages, occurring in seminal plasma, and does not form complexes with protease inhibitors or other proteins: in PCa %fPSA is lower presumably because, consequently to an increased release of PSA into the bloodstream, a very low part is still degraded into the ducts.
In another later study [9], Mikolajczyk et al. found that [-2]proPSA was specifically higher in patients with PCa. Analysing a small number of patients with biopsy positive for PCa and tPSA between 6 and 24 ng/ml, they found that [-2]proPSA constituted a high fraction of fPSA (25% to 95%), which was greater than in patients with a negative biopsy. However, the molecular basis for the proPSA elevation in PCa is uncertain, although a decreased cleavage by hK2 could be the cause.
Clinical utility of proPSA
Sokoll et al. [10] were the first to study the role of proPSA in the early detection of PCa. The study involved archival serum from 119 men (31 PCa, 88 non-cancer), obtained before biopsy and in the tPSA range of 2.5–4.0 ng/ml. The serum levels of tPSA, fPSA, proPSA, and proPSA/fPSA ratio (%proPSA) were analysed: PSA and %fPSA values were similar between the non-cancer and PCa groups, and %proPSA was relatively higher in the PCa group (50.1±4.4%) compared to the non-cancer group (35.5±6.7%; P=0.07). Concerning the clinical utility, the area under the curve (AUC) for %proPSA was 0.688 compared to 0.567 for %fPSA. At fixed sensitivity of 75%, the specificity was significantly greater for %proPSA at 59% compared with %fPSA at 33% (P<0.0001). Afterwards, the Prostate Health Index (PHI) has been proposed as a mathematical algorithm combining tPSA, fPSA and [-2]proPSA according to the formula: ([-2]proPSA/fPSA) × √tPSA. A large American prospective trial [11], involving 892 men who had tPSA levels of 2–10 ng/ml and negative digital rectal examination results, showed that PHI had greater predictive accuracy for prostate biopsy outcome (AUC 0.703) than [-2]proPSA (AUC 0.557), %fPSA (AUC 0.648) and PSA (AUC 0.525), directly correlating with Gleason score (GS) (P=0.013), with an AUC of 0.724 for GS ≥4+3 disease. Moreover, men with PHI >55 had a 42% likelihood of being diagnosed with high-grade disease on biopsy compared to 26% of men with PHI 0–24.9.
Accordingly, an observational European multicenter cohort study involved 646 men with tPSA levels of 2–10 ng/ml, who had undergone prostate biopsy [12]. [-2]proPSA and PHI improved the predictive accuracy for the detection of overall PCa (and also GS ≥7 disease) compared to PSA and derivatives. In fact, at 90% sensitivity, the PHI cut-off of 27.6 could avoid 100 (15.5%) biopsies, missing 26 (9.8%) cancers (23 with GS 6, three with GS 3+4).
Moreover, a PHI based nomogram to predict PCa at extended prostate biopsy was developed and validated over 729 patients [13]. Including PHI in a multivariable logistic regression model, based on patient age, prostate volume, digital rectal examination and biopsy history, significantly increased predictive accuracy by 7% from 0.73 to 0.80 (P<0.001). Decision curve analysis showed that using the PHI based nomogram resulted in the highest net benefit. Recently, it was demonstrated that PHI might have a role in screening patients at high risk of PCa [14]. Specifically, the study involved 158 men with a positive family history undergoing prostate biopsy within the multicentre European PROMEtheuS cohort. Similarly to previous studies in the general population, PHI outperformed tPSA and %fPSA for PCa detection on biopsy (AUC 0.73, 0.55 and 0.60, respectively). In addition, both [-2]proPSA and PHI were directly associated with GS in men with a positive family history. Overall, the authors reported that using a PHI cutoff value of 25.5 would have avoided 17.2% of biopsies while missing only two GS 7 cancers. On decision curve analysis, the addition of PHI to a base predictive model that included age, prostate volume, tPSA, fPSA and %fPSA resulted in net benefit at threshold probabilities of 35–65%. This result suggests that PHI should be incorporated into a multivariable risk assessment for high-risk patients because it offers improved performance for PCa detection. Conclusions
[-2]proPSA and PHI are more accurate than the currently used tests (PSA and derivatives) in predicting the presence of PCa at biopsy. Their implementation in clinical practice has the potential to significantly increase physicians’ ability to detect PCa and avoid unnecessary biopsies. Further work is needed to confirm and generalize these data on wider populations.
References
1. Hoffman RM, Stone SN, Espey D, Potosky AL. Differences between men with screening-detected versus clinically diagnosed prostate cancers in the USA. BMC Cancer 2005; 5: 27.
2. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin. 2013; 63: 11–30
3. Vickers AJ, Cronin AM, Roobol MJ, et al. The relationship between prostate-specific antigen and prostate cancer risk: the Prostate Biopsy Collaborative Group. Clin Cancer Res. 2010; 16: 4374–4381.
4. Heidenreich A, Bellmunt J, Bolla M, et al. EAU guidelines on prostate cancer. Part 1: screening, diagnosis, and treatment of clinically localised disease. Eur Urol. 2011; 59: 61–71.
5. Thompson IM, Pauler DK, Goodman PJ, et al. Prevalence of prostate cancer among men with a prostate-specific antigen level < or =4.0 ng per milliliter. N Engl J Med. 2004; 350: 2239–2246.
6. Mikolajczyk SD, Rittenhouse HG. Pro PSA: a more cancer specific form of prostate specific antigen for the early detection of prostate cancer. Keio J Med. 2003; 52: 86–91.
7. Mikolajczyk SD, Millar LS, Wang TJ, et al. A precursor form of prostate-specific antigen is more highly elevated in prostate cancer compared with benign transition zone prostate tissue. Cancer Res. 2000; 60: 756–759.
8. Jansen FH, Roobol M, Jenster G, Schroder FH, Bangma CH. Screening for prostate cancer in 2008 II: the importance of molecular subforms of prostate-specific antigen and tissue kallikreins. Eur Urol. 2009; 55: 563–74.
9. Mikolajczyk SD, Marker KM, Millar LS, et al. A truncated precursor form of prostate-specific antigen is a more specific serum marker of prostate cancer. Cancer Res. 2001; 61: 6958–6963
10. Sokoll LJ, Chan DW, Mikolajczyk SD, et al. Proenzyme psa for the early detection of prostate cancer in the 2.5–4.0 ng/ml total psa range: preliminary analysis. Urology 2003; 61: 274–276.
11. Catalona WJ, Partin AW, Sanda MG, et al. A multicenter study of [-2]pro-prostate specific antigen combined with prostate specific antigen and free prostate specific antigen for prostate cancer detection in the 2.0 to 10.0 ng/ml prostate specific antigen range. J Urol. 2011; 185: 1650–1655.
12. Lazzeri M, Haese A, de la Taille A, et al. Serum isoform [-2]proPSA derivatives significantly improve prediction of prostate cancer at initial biopsy in a total PSA range of 2-10 ng/ml: a multicentric European study. Eur Urol. 2013; 63: 986–994.
13. Lughezzani G, Lazzeri M, Larcher A, et al. Development and internal validation of a Prostate Health Index based nomogram for predicting prostate cancer at extended biopsy. J Urol. 2012; 188: 1144–1150.
14. Lazzeri M, Haese A, Abrate A, et al. Clinical performance of serum prostate-specific antigen isoform [-2]proPSA (p2PSA) and its derivatives, %p2PSA and the prostate health index (PHI), in men with a family history of prostate cancer: results from a multicentre European study, the PROMEtheuS project. BJU Int. 2013; 112: 313–321.
The author
Alberto Abrate* MD; Massimo Lazzeri MD, PhD; and Giorgio Guazzoni MD
Dept of Urology, Ospedale San Raffaele Turro, San Raffaele
Scientific Institute, Milan, Italy
*Corresponding author
E-mail: alberto.abrate@gmail.com
CytoBead Assays – A state of the art combination of cell-based immunofluorescence and microparticle technology for simultaneous screening and differentiation in autoimmune diagnostics
, /in Featured Articles /by 3wmediaAutoimmune diseases affect approximately 5 % of the population of developed countries with an increasing incidence. Analysis of disease-associated autoantibodies (AAb) plays a significant role in the differential diagnosis thereof. Indirect immunofluorescence (IIF) has been established as the gold standard for AAb screening in particular for systemic rheumatic diseases. In the recommended two-tier approach for antibody serology, confirmatory testing by molecular assay techniques such as ELISA is required to confirm positive findings by screening using IIF. To cope with the constantly increasing demand for AAb testing, new efficient diagnostic approaches are required. Thus, a new generation of IIF assays have been developed to combine screening and confirmatory testing on one platform for the simultaneous detection of AAb by cell-based and bead-based assays in one reaction environment. The multiplex analysis of antineutrophil cytoplasmic antibodies (ANCA) for the differential diagnosis of vasculitides will be discussed as a first application of this novel approach.
by Dr. Christina Fritz, Mandy Sowa and Dirk Roggenbuck
ANCA-associated vasculitis
Vasculitis is an inflammation affecting blood vessel walls and resulting in their damage, fibrinoid necrosis, tissue ischemia and necrosis, and finally vessel rupture with bleeding into the surrounding tissue [1, 2]. Due to etiological factors, systemic vasculitis is differentiated into primary and secondary vasculitis. Primary systemic vasculitis of particularly small vessels often has an autoimmune pathogenesis accompanied by the occurrence of ANCA [3,5-8]. Those so called ANCA-associated systemic vasculitides (AASV) comprise microscopic polyangiitis (MPA), eosinophilic granulomatosis with polyangiitis (EGPA or Churg-Strauss syndrome) or granulomatosis with polyangiitis (GPA or Wegener’s granulomatosis)[1, 2, 4]. In contrast, secondary vasculitis occurs in 5 – 10 % of patients with rheumatoid arthritis or with other autoimmune diseases (e.g., systemic lupus erythematosus [SLE], Sjögren’s syndrome). In addition, vasculitis can occur in patients suffering from infections such as HIV or hepatitis C.
In general, an acute AASV generally requires immunosuppressive treatment with high doses of cortisone. In severe cases, cyclophosphamide is recommended. Once remission is achieved, methotrexate, azathioprin, cotrimoxazol, leflunomid or mucophenolate mofetil are used as maintenance therapy.
Diagnosis of ANCA-associated vasculitis
According to the international consensus statement for the assessment of ANCA, IIF on ethanol-fixed human neutrophils (ethN) is followed by confirmation with antigen-specific molecular immunoassays [6-8]. IIF reveals two ANCA patterns sub-classifying ANCAs into cytoplasmic ANCA (cANCA) and perinuclear ANCA (pANCA). Regarding the autoantigenic target of ANCA, c and pANCA are directed against proteinase 3 (PR3) and myeloperoxidase (MPO), respectively. A positive cANCA pattern confirmed by the presence of PR3-ANCA is pathognomonic for GPA[5], whereas a positive pANCA pattern confirmed by MPO-ANCA is decisive for MPA and EGPA. Furthermore, the corresponding ANCA titres are strongly associated with activity of disease in patients suffering from GPA and MPA.
As a matter of fact, IIF is currently the only technique to provide a single reaction environment for the combined screening and confirmation of ANCA. Simultaneous detection of c and pANCA along with PR3- and MPO-ANCA would overcome time-consuming single parameter detection by different techniques [10].
The use of multiplexing bead-based IIF assays for the simultaneous detection of single ANCA reactivities provides the ideal reaction environment to be combined with ethN-based ANCA testing. The corresponding principle is based on a covalent surface immobilization of MPO and PR3 on microbeads coded by size and fluorescence. The differentiation in size and/or intensity of a red fluorescence dye filling entirely each microbead population generates a novel reaction environment for parallel analyte analysis [11] (figure 1).
Combination of cell-based and microbead based ANCA assessment by CytoBead assay
The CytoBead assay is a unique combination of a conventional cell-based immunofluorescence assay with multiplexing microbead technology in one reaction environment. A newly designed microscopic glass slide with triple parted wells is employed to fix ethN in the middle compartment and PR3- as well as MPO-coated microbeads in the right-hand compartment of the slide (figure 2). Thus, anti-PR3 antibody positive sera show a positive cytoplasmic fluorescence on ethN and a green fluorescence halo on the surface of PR3-coated microbeads (9 µm). In contrast, anti-MPO antibody positive sera demonstrate a perinuclear fluorescence pattern on the immobilized ethN and a fluorescence halo on the surface of MPO-coated microbeads (15 µm) (figure 2). A reference microbead population (12 µm) is integrated for particle differentiation. This assay set offers the possibility of classical evaluation by a simple fluorescence microscope as well as automated analysis by interpretation systems like the AKLIDES®.
A recent clinical study with classical ANCA testing revealed a relative sensitivity and specificity of 98 % and 99.2 % for the novel CytoBead ANCA assay, respectively. Remarkably, the CytoBead ANCA assay showed a better discrimination of GPA and MPA patients in contrast to the classical anti-MPO and anti-PR3 ELISA. The detected cut-off values were determined on the basis of fluorescence intensity given in arbitrary units [AU] (personal communication).
Conclusion and future perspectives
The increasing demand for cost-effective autoimmune diagnostics requires new multiplexing technologies combining screening and confirmatory testing in one reaction environment. Thus, the novel CytoBead technology is a promising opportunity to accomplish this goal as demonstrated for the comprehensive assessment of ANCA. Automated digital immunofluorescence employed by recently established novel diagnostic interpretation system solutions such as Aklides even offers quantification and standardization of ANCA detection. The CytoBead technology provides an ideal reaction environment for the multiplexing of antinuclear antibody assessment and the simultaneous detection of celiac disease-specific antibodies.
References
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4. Waller R, Ahmed A, Patel I, Luqami R. Update on the classification of vasculitis. Best Pract Res Clin Rheumatol. 2013; 27: 3-17
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6. Jennette JC, Falk RJ, Bacon PA, Basu N, Ferrario F, Flores-Suarez LF, Gross WL, Guillevin L, Hagen EC, Hoffman GS, Jayne DR, Kallenberg CG, Lamprecht P, Langford CA, Lugmani RA, Mahr AD, Matteson EL, Merkel PA, Ozen S, Pusey CD, Rasmussen N, Rees AJ, Scott DG, Specks U, Stone JH, Takahashi K, Watts RA: 2012 revised International Chapel Hill Consensus Conference Nomenclature of Vasculitis. Arthritis Rheum. 2013, 65:1-11
7. Jennette JC, Falk RJ, Andrassy K, Bacon PA, Churg J, Gross WL, Hagen EC, Hoffman GS, Hunder GG, Kallenberg CG: Nomenclature of systemic vasculitides. Proposal of an international consensus conference. Arthritis Rheum 1994, 37:187-92
8. Savige JF, Gillis DF, Benson E, Davies DF, Esnault VF, Falk RJ, Hagen EC, Jayne D, Jennette JC, Paspaliaris B, Pollock W, Pusey C, Savage CO, Silvestrini R, van der Woude F, Wieslander J, Wiik A: International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies (ANCA). Am J Clin Pathol 1999, 111:507-13
9. Merkel PA, Polisson RP, Chang Y, Skates SJ, Niles JL: Prevalence of antineutrophil cytoplasmic antibodies in a large inception cohort of patients with connective tissue disease. Ann. Intern. Med. 1997, 126;866
10. Choi HK, Liu S, Merkel, PA, Colditz GA, Niles Jl: Diagnostic performance of antineutrophil cytoplasmic antibody tests for idiopathic vasculitides: metaanalysis with a focus on antimyeloperoxidase antibodies. J. Rheumatol. 2001, 28:1584
11. Grossmann K, Roggenbuck D, Schröder C, Conrad K, Schierack P, Sack U: Multiplex Assessment of Non-Organ-Specific Autoantibodies with a Novel Microbead-Based Immunoassay. 2011, Cytometry Part A! 79A: 118”125
Author
Dr. Christina Fritz*, Mandy Sowa and Dirk Roggenbuck
Medipan GmbH, Ludwig-Erhard-Ring 3,
15827 Dahlewitz,
Germany
*Corresponding author
E-mail: c.fritz@medipan.de