Su Kah Goh

Probe-Free Digital PCR Quantitative Methodology to Measure Donor-Specific Cell-Free DNA after Solid-Organ Transplantation

BACKGROUND Donor-specific cell-free DNA (dscfDNA) is increasingly being considered as a noninvasive biomarker to monitor graft health and diagnose graft rejection after solid-organ transplantation. However, current approaches used to measure dscfDNA can be costly and/or laborious. A probe-free droplet digital PCR (ddPCR) methodology using small deletion/insertion polymorphisms (DIPs) was developed to circumvent these limitations without compromising the quantification of dscfDNA. This method was called PHABRE-PCR (Primer to Hybridize across an Allelic BREakpoint-PCR). The strategic placement of one primer to hybridize across an allelic breakpoint ensured highly specific PCR amplification, which then enabled the absolute quantification of donor-specific alleles by probe-free ddPCR. METHODS dscfDNA was serially measured in 3 liver transplant recipients. Donor and recipient genomic DNA was first genotyped against a panel of DIPs to identify donor-specific alleles. Alleles that differentiated donor-specific from recipient-specific DNA were then selected to quantify dscfDNA in the recipient plasma. RESULTS Lack of amplification of nontargeted alleles confirmed that PHABRE-PCR was highly specific. In recipients who underwent transplantation, dscfDNA was increased at day 3, but decreased and plateaued at a low concentration by 2 weeks in the 2 recipients who did not develop any complications. In the third transplant recipient, a marked increase of dscfDNA coincided with an episode of graft rejection. CONCLUSIONS PHABRE-PCR was able to quantify dscfDNA with high analytical specificity and sensitivity. The implementation of a DIP-based approach permits surveillance of dscfDNA as a potential measure of graft health after solid-organ transplantation.

Preoperative left hepatic lobe devascularisation to minimize perioperative bleeding in a Jehovah’s Witness undergoing left hepatectomy

Highlights • Liver resection in a Jehovah’s Witness patient requires multimodal blood minimization strategies to improve patient centred outcomes.• Combination portal vein embolization and hepatic lobe revascularization for total vascular inflow occlusion can allow a bloodless resection.• Preoperative angio-embolization should be researched in a larger patient cohort to reduce blood loss and blood transfusion

Serum carbohydrate antigen 19-9 in pancreatic adenocarcinoma: a mini review for surgeons

The optimal management of oncological conditions is reflected by the careful interpretation of investigations for screening, diagnosis, staging, prognostication and surveillance. Serum tumour markers are examples of commonly requested tests in conjunction with other imaging and endoscopic tests that are used to help clinicians to stratify therapeutic decisions. Serum carbohydrate antigen 19‐9 (CA19‐9) is a key biomarker for pancreatic cancers. Although this biomarker is considered clinically useful and informative, clinicians are often challenged by the accurate interpretation of elevated serum CA19‐9 levels. Recognizing the pitfalls of normal and abnormal serum CA19‐9 concentrations will facilitate its appropriate use. In this review, we appraised the biomarker, serum CA19‐9, and highlighted the clinical utility and limitations of serum CA19‐9 in the investigation and management of pancreatic cancers.

Donor-Specific Cell-Free DNA as an Emerging Biomarker of Organ Rejection after Liver Transplantation

Introduction There is an increasing interest in the use of donor-specific cell-free DNA (dscfDNA) as a non-invasive biomarker of organ health after transplantation. We have developed a PCR-based approach that readily measures dscfDNA. Using this approach, we evaluated the utility of dscfDNA in two separate cohorts of recipients. Methods Deletion/insertion polymorphisms (DIP) were used to distinguish donor-specific DNA and recipient-specific DNA. Post-transplant dscfDNA was measured using a simple and novel probe-free droplet digital PCR (ddPCR) methodology. In the longitudinal cohort, dscfDNA was serially measured at days 3, 7, 14, 28 and 42 in 20 recipients. In the cross-sectional cohort, dscfDNA was quantified in 16 recipients (>3 months post-transplant) undergoing liver biopsies.\ Results DscfDNA levels were reflective of organ health after liver transplantation. In the recipients who underwent uncomplicated transplantation, dscfDNA markedly reduced at day 7 and remained at a low median level of 76.45 (IQR: 50.60-108.90) dscfDNA copies/mL from day 14 onwards. Furthermore, dscfDNA was consistently lower in recipients who did not have any evidence of rejection compared to those who developed biopsy-proven organ rejection (281.50 dscfDNA copies/mL vs 1,661.00 dscfDNA copies/mL respectively, p<0.05). The area under the receiver operator characteristic curve of dscfDNA was higher compared to routine serum liver biochemistry (dscfDNA: 0.818, ALT: 0.745, GGT: 0.627, ALP: 0.436). Conclusion In this study, we demonstrated a readily performed methodology to measure dscfDNA. We also highlighted the utility of dscfDNA as a promising non-invasive biomarker for the surveillance of organ health and the diagnosis of organ rejection after liver transplantation.

Malnutrition is Associated with Poor Outcomes after Liver Transplantation: A Promising Target for Pre-Transplantation Nutritional Intervention

Introduction Malnutrition is prevalent in recipients undergoing liver transplantation. However, the implication of nutritional status on morbidity and mortality remains unclear. This study aims to evaluate the impact of malnutrition on recipient outcomes. Methods A retrospective review of 390 adult recipients who underwent liver transplantation between January 2009 and June 2016 was performed. Recipients with fulminant liver failure or those requiring re-transplantation were excluded. A total of 321 recipients was analysed. Nutritional status was determined by the subjective global assessment (SGA) at wait-listing and at transplantation. Outcome measures comprised the development of infection within the index admission, rejection within 90-days, and biliary anastomotic strictures within 90-days, as well as readmission within 90-days, intensive care unit (ICU) length of stay (LOS), hospital LOS, and overall survival. Results Malnutrition (SGA-B & SGA-C) was identified in 67% of the recipients at wait-listing. Progressive decline in nutritional status was observed at transplantation (wait-list median 4.4-months), where 77% of the recipients were malnourished. Of these, 18% (n=58) were severely malnourished (SGA-C). Severe malnutrition at transplantation was associated with longer ICU LOS (147-hours vs 89-hours, p<0.01), longer hospital LOS (40-days vs 16-days, p<0.01) and increased incidences of infection (55.2% vs 33.8%, p<0.01) when compared to the well-nourished recipients (SGA-A). Other outcome measures were not associated with nutritional status. Conclusion This large study demonstrates the impact of malnutrition on early recipient morbidity. Nutritional interventions prior to transplantation may potentially improve outcomes and reduce the economic burden on our healthcare system.

Laparoscopic treatment of a patent ductus venosus and the use of indocyanine green to monitor perioperative hepatic function.

Abstract Patent ductus venosus (PDV) is an uncommon but important congenital portocaval shunt that can lead to numerous complications if untreated. This case describes the successful management of a 17-year-old male with symptomatic PDV. Doppler ultrasonography and contrast-enhanced computed tomography (CT) confirmed a large communication between the left portal vein and the inferior vena cava. Angiography demonstrated a large and high flow PDV which precluded its therapeutic embolization. Based on these findings, laparoscopic closure of the PDV was elected and successfully performed. Perioperative indocyanine green (ICG) clearance was performed and marked improvement was observed following the occlusion of the PDV. The patient showed immediate resolution of symptoms post-operatively and remains asymptomatic 2 years after his surgery. Laparoscopic approach to the management of PDV is feasible. ICG clearance, for the first time, was demonstrated in this setting to be a useful and rapid bedside test for the real-time assessment of liver function.

Adapting an Established Clinical Chemistry Quality Control Measure for Droplet Generation Performance in Digital PCR

To the Editor: Droplet digital PCR (ddPCR)1 is an emerging platform that is being increasingly adopted by clinical laboratories. The partitioning of a PCR reaction into discrete droplets enables the accurate detection and quantification of molecular targets. Single molecule analysis by this manner is accurate, cost-effective, and readily performed compared with next-generation sequencing and mass spectrometry. The typical ddPCR workflow comprises 4 key steps: ( a ) preparation of the PCR reaction, ( b ) droplet generation (DG), ( c ) endpoint PCR, and ( d ), droplet analysis. The process of DG is a critical step of the workflow because this step is considered to be most prone to droplet loss (1). It is thus important to recognize that a loss of droplets during DG will affect the number of droplets recoverable for droplet analysis in an optimized assay. Consequently, lower droplet numbers can lead to a loss of analytical sensitivity and may confound the evaluation of samples for molecular targets that are present in very low concentrations. To date, informative measures to evaluate DG performance within the ddPCR workflow remain unaddressed. The Levey–Jennings chart in conjunction with the “Westgard rules” is the staple of quality management in clinical chemistry measurement systems. Here, we outline an approach that uses the Levey–Jennings chart and a set of adapted Westgard rules to monitor DG performance in …

Fresh Frozen Plasma Transfusion Can Confound the Analysis of Circulating Cell-Free DNA.

To the Editor: Advances in DNA detection methodologies have enabled the accurate assessment of circulating cell-free DNA (cfDNA).1 In transplantation, the quantification of donor-specific cfDNA (dscfDNA) can guide the noninvasive diagnosis of immune-mediated rejection episodes (1). Similarly, in oncology, quantification of circulating tumor DNA enables the surveillance of minimal residual disease, the selection of targeted therapies, and prognostication (2). As the adoption of cfDNA-based diagnostic assays broadens, it is important to standardize preanalytical procedures that can facilitate the reliable assessment of cfDNA. Procedures such as blood collection, blood processing, cfDNA extraction, and, potentially, other unknown factors can impact the final amount of cfDNA and, thus, downstream analysis. Furthermore, there is increasing evidence that patient factors such as recent exercise and trauma are important contributors to the variation (3). Here, we describe the observation that the transfusion of fresh frozen plasma is an unrecognized clinical variable that can confound cfDNA analysis. We previously reported a deletion/insertion polymorphism (DIP)-based approach to quantify dscfDNA in recipients after transplantation (4). The first step of the approach involves the genotyping of pretransplant blood samples derived from potential recipients and the corresponding organ donors using a panel …

Salmonella typhimurium: a rare cause of mesh-related infection

Abstract The use of mesh in the management of abdominal wall hernias has significantly reduced the incidences of hernia recurrences. The placement of synthetic meshes to reinforce the abdominal wall is not without caveats. Synthetic meshes are associated with a risk of infection. Common causative microorganisms for mesh-related infection range from a diversity of gram positive, gram negative and anaerobic bacteria. However, non-typhoidal Salmonella spp. mesh-related infection remains poorly described in the literature. In this case, we report the management of an immunocompromised patient who developed Salmonella typhimurium mesh-related infection that was complicated by abscess formation.