Detection of copy number variations (CNV) in the VHL gene is part of the genetic workup of VHL-related tumours. Current methods for CNV determination have complex workflows and limitations. Digital droplet PCR is a promising methodology that could be used for CNV determination. Its advantages include shorter turnaround time, decreased DNA input and superior precision.

by Dragana Milosevic, Dr Stefan K. Grebe, Dr Alicia Algeciras-Schimnich

Background
Von Hippel-Lindau (VHL) disease is an autosomal dominant cancer syndrome with an incidence of approximately 1 in 36,000 live births. It predisposes affected individuals to the development of five main types of neoplasms: retinal angioma (>90% penetrance), cerebellar hemangioblastoma (>80% penetrance), clear-cell renal cell carcinoma (~75% penetrance), spinal hemangioblastoma (~50% penetrance), and pheochromocytoma (~30% penetrance). The disease is caused by mutations or large deletions in the VHL tumour suppressor gene (VHL). The VHL gene is located on chromosome 3p25-26 and encodes a protein that is involved in ubiquitination and degradation of a variety of proteins, most notably hypoxia-inducible factor (HIF) [1]. HIF induces expression of genes that promote cell survival and angiogenesis under conditions of hypoxia. It is believed that diminished HIF degradation due to inactivation of the VHL protein causes the tumours in VHL disease. Tumours form when the remaining intact copy of the VHL gene is somatically inactivated in target tissues.

VHL patients are subdivided in two groups, based on the genotype/phenotype correlations; those at low risk of developing pheochromocytoma are designated type I, whereas those with a high risk of pheochromocytoma (with or without renal cell carcinoma) are classified as type II. Deletions in the VHL gene are more common in type II VHL syndrome [2, 3]. To date, there have been more than 300 germline mutations and large deletions identified in the VHL gene that cause loss of function [2]. Germline loss-of-function point mutations and small deletions or insertions accounts for approximately 70–80% of cases; whereas large germline deletions of one copy of the VHL gene accounts for approximately 20–30% of cases.

VHL genetic testing
The clinical diagnosis of VHL disease is suspected in individuals who present with one or several of the characteristic tumours described above. Molecular genetic testing of VHL is performed to confirm the clinical diagnosis. The genetic testing includes sequencing of the three exons of VHL gene and evaluation of copy number variations (CNV) to assess deletions of large regions of the gene. Historically, detection of these large deletions was done by Southern blot. Today, most clinical laboratories offering CNV determination use multiplex ligation probe amplification (MLPA)-based assays. MLPA is a method based on sequence specific probe hybridization, ligation and PCR amplification and detection of multiple targets with a single set of universal primers. CNVs are detected by comparison of the signal from each target region to control genes and normal control samples.

Although MLPA-based assays are of superior quality and more robust than previous technologies, technical success of MLPA assays is dependent on input of high quantities (at least 400ng of germline DNA) of high quality DNA. Although less labour intensive than Southern blotting, the MLPA work flow is still more complex than PCR-based assays and typically takes two days until completion. Finally, MLPA does not allow for absolute quantification and cannot distinguish copy numbers greater than three with high accuracy.

Digital droplet PCR
Digital droplet PCR (ddPCR) is a methodology that has gained favour as a robust alternative with improved precision to quantitative real-time PCR (qPCR) for DNA quantification. DdPCR also lends itself to exact CNV determination, detection of rare variants, translocations, and/or point mutations (SNP genotyping).

DdPCR is based on traditional PCR amplification and fluorescent probe-based detection methods, but partitions each reaction into 15 000–20 000 nanodroplets. Provided that the starting DNA concentration is not too high, some of these reactions will contain one or more target DNA molecules, whereas others will not contain any. Those with at least one target DNA molecule will yield an amplification product, while those without won’t. Quantification is based on counting the proportion of droplets that show amplification, using a microfluidic counting device. The proportion of reactions with and without amplification obeys Poisson statistics and allows back-calculation of the starting concentration based on the distribution function. When enough droplets are used, copy number ascertainment is of unprecedented accuracy and reproducibility (CVs of 2–10%) [4]. Compared to standard qPCR methods, ddPCR eliminates the need for standard curves and measures both target and reference DNA within the same well. Applications where ddPCR has been used include: rare allele detection in heterogeneous tumours, assessment of tumour burden by analysis of peripheral body fluids (mainly blood), non-invasive prenatal diagnostics, viral load detection, CNV, assays with limited sample material such as single cell gene expression and archival formalin-fixed paraffin-embedded (FFPE) samples, DNA quality control tests before sequencing, and validation of low frequency mutations identified by sequencing.

Recently, ddPCR has become commercially available in a format that allows for rapid microfluidic analysis of thousands of droplets per sample making it practical for routine use in clinical laboratories. A recent study on the analytical performance of ddPCR has shown greater precision (CVs decreased by 37–86%) and improved day-to-day reproducibility and comparable sensitivity to real-time PCR for absolute quantification of microRNAs [5]. A study that evaluated the use of ddPCR to detect BCR-ABL1 fusion transcripts demonstrated that ddPCR is able to achieve lower limit of detection and quantification than currently used in quantitative PCR methods [6].

Our group has evaluated ddPCR for VHL CNV and shown improved performance compared to MLPA [7]. The method showed 100% concordance with the MLPA method and 100% self-concordance within and between runs. The method showed reproducible results with DNA inputs as low as 10 ng, a 40-fold DNA-input reduction compared with MLPA. Because of this advantage, difficult specimen types, such as archival FFPE specimens, are now capable of being characterized for VHL CNVs, a feat previously impossible by MPLA, because of the often poor DNA quality of such samples (Fig. 1). Additionally, same-day results are available with the ddPCR method, reducing the total run-time from 48 hours for the MLPA method to 3 hours.

One limitation of current ddPCR platforms is the limited ability for multiplexing. For example the Bio-Rad’s ddPCR system can detect only two colours (FAM and HEX), limiting the number of genes that could be evaluated simultaneously in a single reaction. Development of platforms that allow greater multiplexing should, therefore, further facilitate the adaptation of this technology in clinical laboratories.

Conclusions
Improvement of current methods for VHL CNV testing is desired to obtained accurate and cost-effective results in clinical laboratories. Currently used methods are still labour intensive and not suitable for rapid turnaround time. DdPCR is an elegant adaptation of the current quantitative PCR format and has the potential to be applied widely in clinical laboratories. For VHL CNV, ddPCR provides a greatly improved turnaround time and requires only minimal nucleic acid input that does not have to be of the highest quality. With no need for standard curves or controls, ddPCR overcomes the issues associated with traditional qPCR, while increasing both robustness (superior sensitivity, specificity, and precision) and utility in other specimen types such as paraffin-embedded tissue, circulating cell-free DNA, circulating tumour cells, and microRNA detection [8–10].

References
1. Richards FM. Molecular pathology of von Hippel–Lindau disease and the VHL tumor suppressor gene. Expert Rev Mol Med. 2001; 3: 1–27. DOI: http://dx.doi.org/10.1017/S1462399401002654.
2. Maher ER, Kaelin WG Jr. von Hippel-Lindau disease. Medicine 1997; 76: 381–391.
3. Hes FJ, Höppener JW, Lips CJ. Clinical review 155: pheochromocytoma in von Hippel-Lindau disease. J Clin Endocrinol Metab. 2003; 88: 969–974.
4. Pohl G, Shih IeM. Principle and applications of digital PCR. Expert Mol Rev Diagn. 2004; 4: 41–47.
5. Hindson CM, Chevillet JR, Briggs HA, Gallichotte EN, Ruf IK, Vessella RL, Tewari M. Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods 2013; 10: 1003–1005.
6. Jennings LJ, George D, Czech J, Yu M, Joseph L. Detection and quantification of BCR-ABL1 fusion transcripts by droplet digital PCR. J Mol Diagn. 2014; 16(2): 174–179.
7. Milosevic D, Grebe SK, Algeciras-Schimnich A. Detection of Von Hippel-Lindau (VHL) gene copy number variations using digital droplet PCR. Clin Chem. 2014; 60(10S): S194.
8. Wang J, Ramakrishnan R, Tang Z, Fan W, Kluge A, Dowlati A, Jones RC, Ma PC. Quantifying EGFR alterations in the lung cancer genome with nanofluidic digital pcr arrays. Clin. Chem. 2010; 56: 623–632.
9. Lo YM, Lun FM, Chan KC, Tsui NB, Chong KC, Lau TK, Leung TY, Zee BC, Cantor CR, Chiu RW. Digital PCR for the molecular detection of fetal chromosomal aneuploidy. Proc. Natl Acad Sci U S A 2007; 104: 13116–13121.
10. Pinheiro LB, Coleman VA, Hindson CM, Herrmann J, Hindson BJ, Bhat S, Emslie KR. Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Anal Chem. 2012; 84: 1003–1011.

The authors
Dragana Milosevic MS; Stefan K. Grebe MD, PhD; Alicia Algeciras-Schimnich* PhD
Department of Laboratory Medicine and Pathology, Rochester, MN, USA

*Corresponding author
E-mail: Algeciras.Alicia@mayo.edu

Following the trend in the US, pressure is mounting on big European hospital labs to consolidate their operations and boost workflow performances while at the same time making better use of their staff. The Aptio Automation system from Siemens Healthcare Diagnostics goes a long way to fulfill these objectives. Here we take a look at three specific examples of hospitals that have integrated the system and how it has helped both their clinical and operational effectiveness

National Health Service (NHS) Tayside
NHS Tayside serves a population of 480,000 through a network of 22 hospitals/infirmaries and 69 general-practice sites that rely on two laboratories. The Blood Sciences Laboratory is located at the 900-bed Ninewells Hospital in Dundee, one of the UK’s major teaching hospitals. Here, Aptio Automation merges the three former individual labs onto a single track, providing a full complement of pre- and post-analytical sample-processing modules along with comprehensive analytics. The efficiencies gained have empowered the Ninewells Hospital laboratory to take on 73% of the testing that historically had been conducted at the 260-bed Perth Royal Infirmary (PRI), enabling the smaller PRI laboratory to focus exclusively on acute admissions and inpatient testing. Ninewells now handles 100% of the general practice testing in the entire region.
“Underpinning all of our actions is the commitment to reduce waste and variation—and most of all, to prevent harm to patients,” says Dr. Bill Bartlett, Tayside’s joint clinical director of diagnostics. “Aptio Automation is helping us enable our vision of cost-effective, patient-focused care.”
Using Aptio Automation, Tayside now processes 7,000 tubes per day—a 20% increase in the workload of its main laboratory with no additional staff. Decreased TAT across the board drove a 61% improvement in the TAT for add-on tests – all with the high-quality results derived from the consistency and standardization enabled by automation. Increased capacity has even enabled Tayside to introduce new testing protocols that improve the quality of care and can save the hospital money.
While Tayside staff had ideas about what they needed and wanted to do, Siemens gave them data-driven information to guide their decision making. “Siemens’ expertise and consultative approach was paramount to the success of this project, from beginning to end,” says Dr. Bartlett. “We relied on them to evaluate workloads from a variety of locations and to recommend the optimal mix of instruments to support peak loads. They devised the final track layout for the new space and helped optimize the use of automation to best manage the workflow.”

Carlos Haya Hospital Malaga
Rising along Spain’s Mediterranean coast, Malaga has a population of approximately 600,000 through tourism, construction, and technology services. The healthcare needs of locals and tourists are met by the 1,100-bed Universitario Carlos Haya Hospital. Part of the government-run Andalusian Public Health System, Carlos Haya is a regional institution with four hospitals: the General Hospital, Civil Hospital, CARE Joseph Estrada, and Hospital Materno Infantil. The latter, in addition to providing healthcare for mothers and infants, houses the core laboratory that serves as the reference lab for all four hospitals. The Carlos Haya General Hospital also operates a biochemistry lab and its own emergency lab.
Hospitals in Spain are classified into three tiers, depending on the complexity of the diseases they can treat. Materno Infantil holds the highest rating, Tier 3, which means it handles the most difficult cases. Its core lab provides testing across a wide spectrum of disease states, performing seven million tests a year for approximately 660,000 patients.
The great leap forward occurred in 2012, with the implementation of Siemens Aptio Automation integrated with the Siemens CentraLink Data Management System. The solution delivers extensive automation, customization, and traceability, while eliminating the need for third-party informatics software.
“Aptio Automation strengthened our preanalytical and analytical phases, giving us more capacity, more versatility, and overall, more possibility,” says Dr. Manuel Rodriguez, who is responsible for biochemistry and automation labs. “CentraLink, meanwhile, makes it much easier to manage quality. It facilitates sample follow-up and management of repetitions. With CentraLink control over instrument alarms, we gain comprehensive information on the situation of a particular sample. What’s more, the solution is simple, problem-free, and easy to use.”

Hospital Clinic de Barcelona (CDB)
In 2001, the Hospital Clinic de Barcelona in Spain was among the first healthcare providers in the world to create an automated core laboratory. Today the laboratory is gaining even greater efficiencies with Aptio Automation connecting analytical systems across all four core laboratory disciplines: clinical chemistry, immunoassay, hematology, and hemostasis.
Over the course of its 13-year journey with Siemens, the laboratory has been able to consolidate instruments, integrate and automate STAT testing, reallocate staff to higher-value responsibilities, and save upwards of €600,000 in tube costs – all while increasing clinicians’ trust in the laboratory to support excellence in patient care.
In 2000, Hospital Clinic de Barcelona established the Biomedical Diagnostic Centre (CDB) to provide high-quality, comprehensive service in all areas of laboratory medicine and to be a reference for excellence in the related specialties. The CDB uses a client-focused model to optimize the use of resources while ensuring advanced technological development in healthcare and research.
The CDB is divided into five specialty departments and an operative core laboratory where high-volume automated testing is performed. In total, the CDB is composed of 100 staff specialists from the various laboratory areas along with 300 professionals representing pathology, biochemistry, molecular genetics, hemotherapy and hemostasis, immunology, and microbiology specialties.
The CDB recently began a project to create a Molecular Biology Core Laboratory, which will integrate the most frequently tested molecular technologies.
“Economic pressure is an ongoing fact of life,” says Dr. Aurea Mira, director of the CDB. “Lab automation allows us to improve workflows, optimize human and technology resources, and save money. It also raises hospital awareness of the lab as a provider of fast, accurate testing that supports good clinical outcomes.”

Liquid chromatography-mass spectrometry (LC-MS/MS) is one of the most promising diagnostic technologies in the in-vitro diagnostics industry, but it is not yet widely adopted by mainstream laboratories. An estimated five percent of LC-MS/MS instruments reside in truly clinical diagnostic settings while the majority are deployed in research and reference laboratories.

by Dr Bori Shushan

There are three compelling reasons for clinical labs to incorporate LC-MS/MS solutions into their routine operations:

1. Quality from improved specificity
2. Workflow efficiency
3. Meeting market demand

Testing quality
Direct measurement technology is more specific and can address the limitations inherent to immunoassay testing. In particular, for small-molecule analyte testing, immunoassay results can be elevated due to the presence of metabolites from other drugs with core structures that are similar to the targeted analyte.

Workflow efficiency
As LC-MS/MS solutions are typically found in specialty laboratories, most clinical labs must outsource certain tests.  Transporting samples adds complexity, cost, and time to the testing process. In drugs of abuse testing for example, patients are initially screened using immunoassay and then confirmed using LC-MS/MS. Having this capability within the laboratory can provide quick turnaround times for faster diagnosis and treatment for patients. LC-MS/MS methods can also test for multiple analytes simultaneously where immunoassay methods require a separate test for each analyte.

Meeting market demand
The market demand that LC-MS/MS addresses arises from trends such as the growing use of opiates and increasingly more stringent regulations. On-site LC-MS/MS testing can deliver both qualitative and quantitative accuracy and precision to help clinicians understand the actual consumption of abused and or prescription drugs.

While the reasons for adopting LC-MS/MS are compelling, there are logistical and regulatory barriers to entry rooted in the current state of LC-MS/MS automation. LC-MS/MS processes are automated to some degree, but the entire process must be improved. Workflows still require many manual steps, including sample preparation and data entry into LIMS systems. This takes labour and time and can lead to errors, all of which are unacceptable to regulatory bodies and laboratory managers. The industry recognizes that innovative solutions are required to address these analytical challenges; however, only when LC-MS/MS achieves the rigorous engineering and quality developments required for regulatory approval will more labs be allowed to adopt this gold standard technology and make a meaningful advancement in diagnostic testing.