Autoantibody detection is a powerful laboratory tool for clinical diagnosis in the autoimmune diseases field. Among the techniques most widely used worldwide, indirect immunfluorescence (IFA) plays a particularly important role not only in the diagnosis but in the follow up of many diseases and remains the hallmark despite the introduction of new techniques in the routine of clinical laboratories. Witness to this is the renaissance of the antinuclear antibodies (ANA) screening on HEp2 cells by this techique or the renewal of the detection of anti-endomysium antibodies on monkey esophagus as the gold standard serological test for celiac disease. Therefore, IFA is a technique in full validity and requires a level of standardization that unfortunately is far from being achieved.

by Petraki Munujos, PhD

The efforts to improve standardization of indirect immunofluorescence as a diagnostic tool are numerous worldwide. Traditionally, the players involved in standardization have been clinical laboratories, clinicians, regulators, and to a lesser degree, diagnostic reagents manufacturers. Energy has been concentrated basically in aspects like the control of laboratory procedures, unification of nomenclatures and classifications, guidelines on how to report the results, preparation of recommendations, definition of diagnostic criteria and diagnostic algorithms and development of external quality control programs. In these iniatives, laboratory staff, clinicians and regulators are mainly involved. Nevertheless, those aspects regarding the design, development and manufacturing of the reagents, which involve manufacturers, are basically ignored. And this is probably due to the fact that the evolution of the technology has led to a truncated view of the test procedure resulting in a misconception of what needs to be standardized. In other words, the execution of many procedures is nowadays being shared between the manufacturer, who actually initiates the assay, and the laboratory, where the test is finalized. In old scientific articles related to ANA, the Material and Methods section usually started with the cell culture, the preparation of the slides and the fixation among others, and the sample incubation was only one more step of the whole procedure. Currently, the Material and Methods section starts with the sample preparation and instead of describing all the preliminary steps, one can find the name and references of the manufacturer. Figure 1 illustrates what would be the whole test procedure, showing the part performed in the clinical laboratory, actually the only part which is taken into consideration when dealing with standardization. So, to ensure appropriate use of indirect immunofluorescence testing, clinicians, diagnostic laboratories, regulators and reagents manufacturers should be involved and share the tasks of identifying and managing the key points leading to proper results.

Evidences of disparity
At the level of the manufacturer, the potential variability in the performance of the kits lies in features like the reagents and materials that are purchased or manufactured to become components of the kit, the procedures and conditions of manufacturing (fixatives, temperatures, formulations), the reliability of the serum samples used to set up the calibration of the determination (basically, the sample dilution which actuallly acts as the cut-off point), and the stability of the final product (1).

When approaching the participation of the manufacturer in the standardization of antibody testing, it is observed that what basically matters for industry is the standardization of the manufacturing processes. This normally occurs in an environment of Quality System Certifications, like GMP, ISO-9001 or ISO-13485 and under the requirements of the European Directive on In Vitro Medical Devices, and it is strengthened by the manufacturer’s own interest in having robust and reliable processes. Nevertheless, despite regulatory compliant and well implemented standardized processes, there are several aspects that make final reagents differ from one manufacturer to another. Below are reviewed some examples of variation on the results depending on the manufacturer source.

Dense fine speckles 70 (DFS70) antigen
As with other fluorescence patterns, the typical DFS pattern (lens epithelium-derived growth factor) can vary depending on the manufacturer source of the HEp2 slides used. The variations consist basically in different sensitivities and even in positive and negative results for the same sample run in different slide brands. Inconsistencies are also observed when comparing fluorescence with the results obtained by means of ELISA (2,3).

Ribosomal P protein (Rib P)
In studies performed by Mahler et al. (4) to determine the sentitivity of the immunofluorescence technique to detect antibodies against ribosomal P protein, several different HEp2 slides manufacturers were used, resulting in significant differences in patterns of staining for monospecific anti-Rib-P sera. Differing patterns were observed for the same sample, from a fine speckled nucleoplasmic pattern, to a diffuse cytoplasmic staining, or a fine speckled cytoplasmic pattern.

CDC/AF Reference Human Sera
When running reference sera on HEp2 slides coming from different manufacturers, variations of unknown origin can be observed. While most brands produce the expected specific pattern, there are often differences among brands like the ones shown in Figure 2.

Labile nuclear antigens
Most of the patterns observed when analysing the presence of ANA in patients sera by IFA on HEp2 cells slides are suitably detected in most slides brands. However, there are some antigens for which expression may significantly vary from one manufacturer to another like Jo1, PCNA or SSA/Ro (5). These antigens are not always well preserved in the substrates and they can be extremely sensitive to handling, to certain fixatives and in some cases, they can be just washed out during the manufacturing process, resulting in a poor presence or a total lack of antigenic molecules available to capture the antibody being analysed.

Antineutrophil cytoplasmic antibodies (ANCA)
The neutrophil substrates used in the detection of ANCA may vary in their ability to give the typical immunofluorescence patterns described and established by consensus groups, i.e. a diffuse granular cytoplasmic staining with higher interlobular intensity (C-ANCA), a compact staining of the perinuclear zone of the cytoplasm (P-ANCA) and a broad non homogeneous perinuclear staining, eventually accompanied by a diffuse cytoplasmic pattern with no accentuation of the interlobular zone (X-ANCA). In general, substrates differ in their ability to distinguish between a C-ANCA and X-ANCA. In a study by Pollock et al. (6), it was observed that although all commercial neutrophil substrates consistently demonstrated nuclear extension of perinuclear fluorescence with sera containing P-ANCA with MPO specificity, there were more problems in P-ANCA testing than in C-ANCA, due basically to the eventual presence of additional cytoplasmic fluorescence.

Crithidia luciliae
In a similar way as observed in HEp2 cells immunofluorescence patterns, the anti-nDNA test on Crithidia luciliae slides may show significant differences among manufacturers. The variety of strains available in cell banks contribute to the heterogeneity of results. Apart from the kinetoplast, other organelles can be stained by antibodies from the sample, like the nucleus, the basal body and the flagellum. Depending on the conditions of preparation of C. luciliae substrates and on the nature of the sample analysed, different patterns of stained organelles can be observed. Nevertheless, the only specific staining to be considered as a positive result is the kinetoplast staining. In addition to anti-nDNA antibodies, there are other antibodies in the serum of lupus patients that can react with the substrate. The so called anti-nucleosome antibodies are antibodies that react with histones exposed in the nucleosome. It is well known that treating C. luciliae substrate with HCL eliminates histone from the kinetoplast (7). This could be another point of possible discrepancy among manufacturing processes if some include the histone removal procedure and some others do not. Furthermore, the cell cycle of C. luciliae may influence histone appearance in the kinetoplast. Therefore, the manufacturing process of C. luciliae slides, including culture, harvest, fixation and drying, can cause variation in the results.

Aspects providing variablity
Among the players participating in autoimmune diagnostics, there is no doubt that manufacturers hold the know-how of preparing diagnostic kits and are the true experts in the development of test methods. However, despite the standardized manufacturing processes and the CE-certifications or FDA approvals, there are several aspects that are found to be sources of variabilty. These aspects should be addressed and recommendations on key points should be created by specialized committees with the participation of laboratory experts, clinicians and manufacturers. The definition and  control of the raw materials incorporated in the kit production is a common and regulated practice in any kind of manufacturing process. But recommendations on nature, compostion or quality grades of key materials, including culture media, cell type and strain or fluorescent conjugates is still lacking. In the case of tests based on cellular substrates,  extracellular matrix (ECM) proteins are commonly used to aid the spreading and growth of cells on the slide glass surface.  Many ECM proteins contain defined amino acid sequences to which cell surface integrin receptors bind specifically. ECM, together with growth factors in the culture medium, work to produce an appropriate in vitro proliferative response, promoting cell growth and spreading. Altering cell-ECM contacts results in coordinated changes in cell, cytoskeletal, and nuclear form. Thus, the choice of the right ECM to coat the glass slides used as growing surface deserves our attention since it might have a direct effect on the fluorescent pattern finally observed (8). It is also common to use synchronization agents to achieve a greater rate of mitotic cells. Due to the fact that these compounds may be toxic for the cell, some cell disturbances may occur that can impact the morphology or the behaviour of the final cell preparation.

Diagnosis by means of tissue sections remains very important in liver autoimmune diseases like autoimmune hepatitis (AIH) or primary billiary cirrhosis (PBC). In particular, the detection of anti-smooth muscle antibodies (ASMA), antibodies to liver-kidney microsomes (LKM antibodies) and anti-mitochondrial antibodies (AMA) are considered important diagnostic tools. Only a few guidelines have been published on the obtention of tissue sections (9), while the variations in the preparation of tissue blocks regarding orientation, preservation conditions, and   sectioning keep on contributing to the heterogeneity of results, especially in the case of tissues that are not morphologically homogeneous. For instance, the LKM antibodies can only be well defined if the kidney section has the proper orientation that allows the distinction between proximal and distal renal tubules and, thus, between LKM and AMA.

Considering that the expression and topographical distribution of autoantigens is under the direct influence of the HEp-2 fixation method, some immunofluorescence patterns are not adequately expressed due to the way that the antigenic substrate is prepared. This aspect equally affects tissue and cell substrates. As for the sensitivity of the tests, differences among manufacturers are due to the use of fixatives to prolong shelf-life. The use of slides without fixation seems to be the best choice for most  autoantibody patterns. Nevertheless, there are several staining patterns that need the substrate to be fixed (figure 3), like anti-islet cells antibodies or anti-adrenal cortex antibodies.

A less frequent but significant source of variability in the immunofluorescence on tissue sections can be found in the origin of the animal used (Figure 4). Definition of suitable species and strains should be addressed in some cases in which the levels of antigen expression may differ. This affects the sensitivity of the test, especially in samples with moderate or low titers of antibody.  

Considering the complexity and diversity of manufacturing processes and subprocesses and their impact on the final test performance, it is important to combine the efforts of laboratory experts, clinicians and manufacturers in the task of standardizing those key aspects that could otherwise keep on undermining the successful harmonization of  the results obtained in the clinical laboratory.

References
1. Fritzler MJ, Wiik A, Fritzler ML, Barr SG. The use and abuse of commercial kits used to detect autoantibodies. Arthritis Res Ther 2003, 5:192-201
2. N.Bizzaro, E.Tonuttiand D.Villalta, «Recognizing the dense fine speckled/lens epithelium-derived growth factor/p75 pattern on HEP-2 cells: not an easy task! Comment on the article by Mariz et al,» Arthritis Rheum, vol. 63, no. 12, pp. 4036-4037, 2011
3. Mahler M. The clinical significance of anti-DFS70 antibodies as part of ANA testing. In: K. Conrad, E.K.L. Chan, M.J. Fritzler, R.L. Humbel, P.L. Meroni, G. Steiner, Y. Shoenfeld (Eds.). Infection, Tumors and Autoimmunity, AUTOANTIGENS, AUTOANTIBODIES, AUTOIMMUNITY, Volume 9, p.342-350. PABST, 2013.
4. Mahler M, Ngo JT, Schulte-Pelkum J, Luettich T, Fritzler MJ. Limited reliability of the indirect immunofluorescence technique for the detection of anti-Rib-P antibodies. Arthritis Research & Therapy 2008, 10:R131
5. Dellavance A, de Melo Cruvinel W, Carvalho Francescantonio PL, Pitangueira Mangueira CL, Drugowick IC, RodriguesSE; Coelho Andrade LE. Variability in the recognition of distinctive immunofluorescence patterns in different brands of HEp-2 cell slides J Bras Patol Med Lab  2013;49( 3):182-190.
6. Pollock W, Clarke K,  Gallagher K, Hall J, Luckhurst E,  McEvoy R, Melny J, Neil J, Nikoloutsopoulos A, Thompson T, Trevisin M, Savige J. Immunofluorescent patterns produced by antineutrophil cytoplasmic antibodies (ANCA) vary depending on neutrophil substrate and conjugate. J Clin Pathol 2002;55:680–683
7. Kobkitjaroen J, Jaiyen J, Kongkriengdach S, Potprasart S, Viriyataveekul R. Comparison of Three Commercial Crithidia luciliae Immunofluorescence Test (CLIFT) Kits for Anti-dsDNA Detection. Siriraj Med J 2013;65:9-11
8. (Integrin Binding and Cell Spreading on Extracellular Matrix Act at Different Points in the Cell Cycle to Promote Hepatocyte Growth  Hansen LK,. Mooney DJ, Vacanti JP, Ingber DE. Molecular Biology of the Cell 1994;5:967-975
9. Vergani D, Alvarez F, Bianchi FB, Cançado ELR, Mackay IR, Manns MP, Nishioka M, Penner E. Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group. Journal of Hepatology 2004;41: 677–683

Acute 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

Affecting 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!

Inconsistent 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.
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The authors
Emily J. Stapleton¹* BSc, Megan Hall² PhD, and Carole A. Samango-Sprouse^{1, 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