Pitfalls When Determining HNA-1 Genotypes and Finding Novel Alleles.

Genetic variation in the FCGR3B gene is responsible for different variants of human neutrophil antigen 1 (HNA-1). Laboratory techniques currently utilized for routine HNA-1 genotyping, predominantly PCR-sequence-specific primer (PCR-SSP) and PCR-sequence-based typing (PCR-SBT), lack specificity for FCGR3B. This study compares the capabilities and limitations of existing technologies including an in-house TaqMan PCR, a commercial PCR-SSP test, PCR-SBT and multiplex ligation-dependent probe amplification (MLPA) with those of a long-read nanopore sequencing assay. Testing was performed with both related and unrelated Danish samples with different copy numbers and/or rare alleles. Long-read nanopore sequencing was validated by blind testing of ten English samples. The results showed that FCGR3B copy numbers correlate with a dose-dependent distribution of alleles that complicates genotyping by TaqMan PCR, PCR-SSP and PCR-SBT, due to co-amplification of the homologous FCGR3A gene. MLPA can correctly quantify the dose-dependent distribution but not detect novel variants. Long-read nanopore sequencing showed high specificity for FCGR3B and was able to detect dosage-dependent distribution, and rare and novel variants that were previously not described. Current HNA-1 genotyping methods cannot produce unambiguous allele-level results, whereas long-read nanopore sequencing has shown the potential to resolve observed ambiguities, identify new HNA-1 variants and allow definitive allele assignment.

Human neutrophil antigen 2 sequence-based typing: Joining the hunt for the CD177 answer.

BACKGROUND AND OBJECTIVES: Isoantibodies to human neutrophil antigen 2 (CD177) have been associated with several clinical conditions but to date the molecular basis for altered or non-expression has not been determined. Reliance on phenotyping and crossmatch to investigate these neutropenic clinical cases are inconvenient for the patients and demanding of resources within the laboratory. Therefore, a molecular approach has been introduced to address both issues. MATERIALS AND METHODS: A DNA panel of 100 randomly selected blood donors were collected and supplemented with 18 DNA samples from blood donors previously shown to be CD177 null. All DNA samples were sequence-based typed for all exons and observed polymorphisms recorded. The DNA from two families previously investigated for neonatal alloimmune neutropenia due to CD177 isoantibodies were also analysed. RESULTS: The incidence of CD177 null could be associated with a known exon 7 single-nucleotide polymorphism in 16/21 known CD177 null samples, which is consistent with previously published findings. Two additional mutations that may lead to null expression were also identified, of which one may be novel. In both family investigations, this same mutation could also be observed in the maternal DNA sample. CONCLUSION: Based on these observations, introduction of CD177 genotyping into routine use would identify null expression in over 75% (16/21) of associated cases. In turn, this could significantly reduce the need for supplementary testing and associated inconvenience to patients while permitting increased efficiency of laboratory testing. An added benefit would potentially elucidate other clinically relevant mutations and associated antigenic targets.

Abdominal Aortic Neck Wrap for Refractory Type 1a Endoleak: A Case Series and a Novel Intraoperative Assessment Technique.

Objective: Refractory type 1a endoleak after endovascular aneurysm repair (EVAR) can pose a significant challenge to surgeons and interventional radiologists. Continuous sac expansion results in aneurysm rupture and mortality. In such circumstances, an external infrarenal aortic wrap could serve as an essential and alternative solution. Methods: We assessed the application of an infrarenal aortic neck wrap for the treatment of refractory type 1a endoleak in n=6 consecutive patients along with the introduction of a novel assessment technique in order to assure their intraoperative success with no radiation exposure and contrast use. Results: The median sac expansion was 8.5 mm (interquartile range [IQR], 5-20 mm). The median neck diameter and length of the aortic neck were 23 mm (IQR, 18-25 mm) and 21 mm (IQR, 18-25 mm), respectively. The median length of follow-up post wrap is 24 months (IQR, 14-34 months). There was no associated mortality or morbidity and requirement for any further interventions. Conclusion: The study demonstrates that aortic wrapping for the treatment of refractory type 1a endoleak for any given neck diameter and length is safe, effective, and long lasting. The suggested novel intraoperative assessment technique contributes to the safety of the procedure by diminishing the need for intraoperative radiation exposure, contrast, and shorter operative time.

Human neutrophil antigens: Nature, clinical significance and detection.

Granulocytes are an essential part of both the innate and adaptive immune systems. Human neutrophil antigens (HNAs) are a family of epitopes that are located on glycoproteins that are mostly expressed on human granulocytes. Antibodies that recognize these epitopes have been associated with neutropenia, transfusion complications, haematopoietic stem cell transplant nonengraftment and renal transplant rejection. Currently, there are fourteen recognized HNA alleles across five antigen systems (HNA-1 through HNA-5), the molecular basis of which are located on the genes FCGR3B, CD177, SLC44A2, ITGAM and ITGAL, respectively. Elucidation of the associated genes has permitted the development of testing strategies for HNA typing and aided understanding of the associated epitopes. This review will outline the associated clinical conditions that require HNA investigation and how these are performed in specialized laboratories. Investigations provided are both reactive for patients with a variety of existing or suspected neutropenias and proactive in the testing of blood component donors in order to reduce the potential risk to patients who require transfusion.

Novel Assessment (BlueDop) Device for Detection of Lower Limb Arterial Disease: A Prospective Comparative Study.

According to National Institute of Clinical Excellence guidelines, the ankle-brachial pressure index coupled with a full clinical evaluation has been the mainstay of detecting peripheral arterial disease on its suspicion. However, this technique is not free of its own limitations in calcified arteries, ulcerative and diabetic patients. We introduce a new, novel, and effective assessment device (BlueDop) with a minimal learning curve that could overcome such barriers and serve as a valid replacement in perihospital settings.

The Clinical Implication of Blood Product Transfusion on Morbidity and Mortality of Ruptured Abdominal Aortic Aneurysm.

BACKGROUND: Transfusion of blood products occurs frequently in ruptured abdominal aortic aneurysm surgery (rAAA). The aim of this study is to establish the impact of packed red blood cell (PRBC), fresh frozen plasma (FFP), and platelet (PLT) transfusion on the 30-day mortality and morbidity (thrombotic versus non thrombotic complications) of rAAAs. METHODS: A retrospective study of 90 consecutive rAAAs from November 2007 to June 2015 was conducted. Multivariable regression models were produced to determine blood products associated with 30-day morbidity and mortality post-rAAA. RESULTS: The overall mortality was 14.6%. At multivariable analyses, transfusion with FFP (>3 units) was independently associated with an increased risk of mortality (odds ratio [OR]: 11.27, 95% confidence interval [CI]: 1.13-96.72, P = .027). The overall morbidity was 26.8%, and subgroup analysis (thrombotic vs nonthrombotic complications) demonstrated transfusion of PLTs (>1 pool) to be independently associated with thrombotic events (OR: 4.3, 95% CI: 1.37-13.6, P = .012). Thrombotic events were responsible for 50% (n = 11 of 22) of all morbidities and mortalities (n = 6 of 12). CONCLUSION: Transfusion of FFP and PLTs may be associated with an increased risk of postoperative morbidity and mortality. The use of these blood components should be considered in the context of the patient's clinical and laboratory data as opposed to a fixed ratio to PRBCs. This may result in the reduction in thrombotic complications emerging in rAAA cohort.

Neutrophil to lymphocyte ratio (NLR) of five predicts 30-day morbidity in ruptured abdominal aortic aneurysms (rAAA): a retrospective cohort study.

BACKGROUND: In recent years the role of neutrophil to lymphocyte ratio (NLR) as an independent predictor of mortality and morbidity has gained significant attention in colorectal, upper gastrointestinal and cancer surgery. To date, no study has examined this in ruptured abdominal aortic aneurysms (rAAAs). This study aims to assess the role of NLR as a prognostic marker of 30-day (30d) morbidity and mortality in patients undergoing repair of rAAAs. METHODS: Data from 80 consecutive patients with a diagnosis of rAAAs from November 2007 to June 2014 were included. Receiver operating characteristic curve analysis was used to identify the optimal value for NLR in relation to 30d mortality and morbidity. Univariate and multivariate logistic regression analysis were used to determine the role of NLR after stratification by several clinical factors. RESULTS: 25 patients (31.2%) had a low NLR (LNLR) and 55 patients (68.8%) had a high NLR (HNLR). Elevated NLR was significantly associated with low Hemoglobin and it was not associated with gender, age, AAA Size, history of HTN, COPD, smoking and renal failure. Patients with HNLR had higher 30d morbidity compared with the LNLR group (35 vs. 6 p = 0.001) but no difference in intraoperative blood loss, length of hospital, ITU stay and 30d mortality. High NLR through multivariate analysis was an independent prognostic factor for 30d morbidity [OR = 4.28, 95% (1.27-14.42), p = 0.02]. CONCLUSION: A preoperative NLR >5 is an independent predictive marker of 30d morbidity in rAAAs. This appears to be in line with earlier literature demonstrating similar outcome in the elective group of abdominal aortic aneurysm.

Late partial failure of a free ALT flap.

Late failure of microsurgical flaps is a rare event and it has been reported as a consequence of compression of the vascular pedicle or late infection. We report a case of late partial failure occurring 3 weeks post-operatively which was shown by vascular imaging to be caused by a previously unidentified complete occlusion of the right external iliac artery. After successful vascular bypass surgery, the suffering flap developed granulation tissue and was skin grafted. In patients carrying multiple risk factors for peripheral vascular disease, the risk of proximal vessel occlusion as a cause of flap failure, should be kept in mind.

Balloon control of the saphenofemoral junction during foam sclerotherapy: proposed innovation.

Venous obliteration using foam sclerosant is a relatively new procedure that holds great promise and may prove to be as effective as conventional surgical treatments in obliteration of the great saphenous vein. Foam sclerotherapy can have minor and major complications, most of which occur as a result of gas or sclerosant leakage into the systemic venous system. Conventionally, the saphenofemoral junction is occluded by digital pressure to prevent escape of sclerosant foam into the deep venous system. We propose balloon control of the saphenofemoral junction to prevent sclerosant leakage. This also promotes foam contact with the endothelium in a prolonged, controlled fashion. This method requires no anesthesia, is suitable for ambulatory care, and has been safe and successful in all five cases. Duplex examinations at 1 week and 3 months have shown complete obliteration of the great saphenous vein using this technique.

Effect of postharvest storage on the expression of the apple allergen Mal d 1.

Consumption of fresh apples can cause allergy in susceptible individuals. A competitive enzyme-linked immunosorbent assay (ELISA) has been developed to determine Mal d 1 levels in apple pulp using a monoclonal antibody (BIP-1). The ELISA was able to rank ten cultivars according to their Mal d 1 content (between 3.8 and 72.5 mug/g pulp). For the first time, it has been demonstrated that growing conditions and postharvest storage, using three different treatments over a 5 month period in 2 consecutive years, increase Mal d 1 expression at a translational and transcriptional level (3.5- and 8.5-fold under controlled atmosphere storage). Expression of three major Mal d 1 isoforms was observed by real-time polymerase chain reaction over the 5 month storage period, and Mal d 1.02 was the most highly expressed isoform. In conclusion, Mal d 1 gene expression was significantly increased during modified atmosphere storage. Individuals suffering from birch pollen-apple allergy syndrome might experience fewer problems consuming freshly picked apples.

Catheter occlusion technique to facilitate distal anastomoses using vein grafts.

Several different modes of achieving a bloodless operating field by proximal and distal occlusion of blood vessels have been described. Vascular clamps, vessel loops, intraluminal occluding devices, and tourniquets have been used to control the tibial and pedal vessels but may cause injury or fail to control heavily calcified arteries. We describe an alternative way to control proximal and distal small arteries by using small, umbilical vein, feeding catheters. The catheters are introduced in the proximal and distal arterial lumen and in the vein graft lumen, achieving hemostasis, technically facilitating the suturing, and providing a means for the administration of vasodilator or thrombolytic agents as well as for a completion angiography.