Processing efficacy in relation to microbial contamination of skin allografts from 723 donors

The Siena Skin Bank, established in 2000, processes skin from more than 130 cadaveric donors per year (about 400,000 cm²) and distributes it for transplants to treat burns and other types of skin loss. More than 1,500,000 cm² of homologous skin has been transplanted to date. At the Siena Skin Bank we conducted a retrospective study of our data to assess microbial contamination of skin specimens from 723 donors banked in the period 2000–2007. Our aim was to determine factors deleterious for skin quality, to optimize skin banking procedures and to reduce corrective actions. The factors analyzed were: type of donor (multi-organ, multi-tissue, live or cadaver), cause of death, time elapsing between death and procurement, different procurement centres and operator experience. Of the 723 donors considered, 26.55% (192/723) were positive for microbes, 22.68% (164) for bacteria and 5.39% (39) for mycetes. Of these 192 positives, 82 (42.70%) required corrective actions. The data obtained showed that the only variables significantly affecting microbial contamination of tissue were type of donor (live or cadaver) and type of processing (cryo- or glycerol preservation).     

Evaluation of donor skin viability: fresh and cryopreserved skin using tetrazolioum salt assay

Cell viability assessment in allograft skin is an essential step to ensure a supply of good quality allograft skin for clinical repair of wounds. It is widely recognised that ‘take’ of allografts is strongly influenced grafted by tissue viability.

The aim of this study was to set-up storage protocols that maintain high viability of the allograft after harvest, treatment and storage. In this study, the viability of post-mortem allografts (n=350) harvested from 35 different donors, was investigated using the MTT salt assay. The conditions of preparation and storage of the allograft included:

1. Fresh skin samples (about 12, 30, and 60 h after harvesting).

2. The same specimens (stored at 4 and 37 °C) tested for at least 1 month.

3. Samples after cryopreservation and thawing.

4. Thawed specimens tested daily for at least 6 days.

Parallel histomorphological analysis performed, under each of these conditions, showed a correlation between changes in structure and changes in viability as measured by the MTT quantitative assay. The viability index (VI) of skin is expressed as the ratio between the optical density (O.D.) produced in the MTT assay by the skin sample and its weight in grams. The percentage viability index is the ratio of the VI of the fresh sample (considered as 100% viability) and the value of specimens from the same harvest batch after storage or cryopreservation.

The results indicated that samples tested within 12–30 h from harvesting have an average viability index of about 75 with little variation. Samples tested within 60 h have an average viability index of 40, showing a viability decrease of about 50%. A protocol to treat skin within a maximum of 30 h was, therefore, set-up. The data suggested that skin stored at 37 °C, undergoes a viability increase during the first 2 days after harvesting. However, the viability under these conditions then decreased very quickly. After 6 days of preservation at this temperature the samples were no longer viable (PVI = 0). The tissue structure started to become damaged after 3 days. On the other hand, skin stored at 4 °C, showed a very slow viability decrease. After 15 days, viability was still almost 25% of the fresh sample. The tissue architecture showed no signs of damage under these conditions until day 7 from harvesting.

MTT analysis was performed on the specimens cryopreserved with DMSO at 10%. These measurements were compared to viability assessment of the same fresh skin samples (considered as 100%) that were analysed within 30 h from harvesting. The average PVI of thawed skin was 54% of the fresh sample. This result demonstrates that the viability of cryopreserved skin is comparable to the viability of fresh skin stored at 4 °C for 4 days.

The PVI of thawed skin samples decreased dramatically within 24 h, and had reached 0% within 6 days.     

Clinical application and viability of cryopreserved cadaveric skin allografts in severe burn: A retrospective analysis

Introduction

Cadaveric cutaneous allografts are used in burns surgery both as a temporary bio-dressing and occasionally as definitive management of partial thickness burns. Nonetheless, limitations in the understanding of the biology of these grafts have meant that their role in burns surgery continues to be controversial.

Methods

A review of all patients suffering 20% or greater total body surface area (TBSA) burns over an eight year period that received cadaveric allografts were identified. To investigate whether tissue viability plays a role in engraftment success, five samples of cryopreserved cadaveric cutaneous allograft processed at the Donor Tissue Bank of Victoria (DTBV) were submitted to our laboratory for viability analysis using two methods of Trypan Blue Exclusion and tetrazolium salt (MTT) assays.

Results

During the study period, 36 patients received cadaveric allograft at our institution. The average total burn surface area (TBSA) for this group of patients was 40% and all patients received cadaveric skin as a temporizing measure prior to definitive grafting. Cadaveric allograft was used in complicated cases such as wound contamination, where synthetic dressings had failed. Viability tests showed fewer than 30% viability in processed allografts when compared to fresh skin following the thawing process. However, the skin structure in the frozen allografts was histologically well preserved.

Conclusion

Cryopreserved cutaneous cadaveric allograft has a positive and definite role as an adjunct to conventional dressing and grafting where available, particularly in patients with large TBSA burns. The low viability of cryopreserved specimens processed at DTBV suggests that cell viability in cadaveric allograft may not be essential for its clinical function as a wound dressing or even as permanent dermal substitute.     

Short- and long-term bacterial inhibiting effect of high concentrations of glycerol used in the preservation of skin allografts

Human skin allografts are important in the treatment of severe burns. Transplantation of skin allografts can cause bacterial transmission. Glycerol in higher concentrations is an appropriate storage medium for allograft cadaver skin and has been attributed an antimicrobial effect. We investigated this effect in more detail. First, the minimal inhibitory concentration of glycerol was determined for 13 bacteria and 1 yeast. This gives an indication about an immediate (20h of incubation) antibacterial effect of glycerol. Second, effect of glycerol in the long-term was studied. Therefore, the survival time was determined for 11 different bacteria suspended in different concentrations of glycerol (50% and 85%) and incubated at three temperatures (4, 24, and 36 degrees C). The minimal inhibitory concentration exceeded 256microg/mL, thus glycerol had no direct inhibitory effect. In contrast, a long-term antimicrobial effect was present and more pronounced at higher concentrations of glycerol and higher temperatures of incubation. The mean survival time of Pseudomonas aeruginosa strains in glycerol 85% at 24 degrees C was 2.6 days, 14.7 days for the tested staphylococci and 29.6 days for three vegetative Bacillus species. In conclusion, microbial safety of glycerol-preserved skin can be increased by preserving skin allografts for some weeks at room temperature.     

神经氨酸酶预处理供体骨髓细胞延长大鼠异体移植物成活时间的观察

目的筛选神经氨酸酶(Neu)预处理供体骨髓细胞(dBMCs)的最佳浓度,使经大鼠尾静脉注射该浓度Neu后的dBMCs偏向性分布于受体肝脏,并观察Neu预处理dBMCs(Neu-dBMCs)联合环孢素A(CsA)短期应用对异体皮片成活时间的影响。方法供体为雄性SD大鼠49只,受体为雌性Wistar大鼠49只。常规制备dBMCs并留取供体皮片备用。Neu预处理浓度筛选实验：按Neu终浓度将26只受体大鼠随机分为0、0．5、1．0、2．0 U/ml 4组(各组分别为6、8、6、6只),用上述4种浓度Neu预处理dBMCs,以~(99m)Tc-SnCl_2法标记,再经受体尾静脉注入,5h后取受体各主要器官,测定其放射性活度,计算出各器官的放射性活度占全部所测器官总放射性活度的百分比,筛选出使dBMCs偏向性分布于受体肝脏的最佳Neu预处理浓度。异体皮片移植实验：将余下23只受体大鼠随机分为对照组、dBMCs组和Neu-dBMCs组,每组均行异体皮片移植,其中后两组在手术当天经尾静脉分别输注dBMCs和Neu-dBMCs(Neu为筛选出的最佳浓度),并分别于术后第2、5天腹腔注射CsA (10 mg/kg),观察各组异体皮片的成活情况。结果在Neu预处理浓度为1．0U/ml时,受体肝脏内dBMCs呈偏向性分布,其百分比为(75．3±9．8)%,明显高于其他浓度组,与0 U/ml组[(58．9±4．2)%]比较差异有统计学意义(P＜0．01)。1．0 U/ml为Neu的最佳浓度。Neu-dBMCs组的同种异体皮片成活时间较dBMCs组明显延长,两组比较差异有统计学意义(P＜0．05)。这两组异体皮片成活时间均较对照组明显延长(P＜0．01)。结论适当浓度的Neu预处理可增强dBMCs与肝脏的亲和性,使经外周静脉输注的dBMCs偏向性分布于受体肝脏内。Neu-dBMCs短期联合应用CsA可明显延长移植的供体皮片成活时间。     

Graft vasculopathy in the skin of a human hand allograft: implications for diagnosis of rejection of vascularized composite allografts

Whereas vascularized composite allografts often undergo acute rejections early in the postgraft period, rejection manifesting with severe vascular changes (graft vasculopathy) has only been observed on three occasions in humans. We report a hand‐allografted patient who developed severe rejection following discontinuation of the immunosuppressive treatment. It manifested clinically with erythematous maculopapules on the skin and pathologically with graft vasculopathy that affected both large vessels and smaller cutaneous ones. The observation that graft vasculopathy can affect skin vessels shows that it is amenable to diagnosis with usual skin biopsy as recommended for the follow‐up of these allografts. Graft vasculopathy developing in the setting of vascularized composite allografts likely represents chronic rejection due to under‐immunosuppression and, if confirmed, should be included in a future update of the Banff classification of vascularized composite allograft rejection.     

The application of glycerol-preserved skin allograft in the treatment of burn injuries: An analysis based on indications

Introduction

Glycerol-preserved skin allograft (GPA) plays a crucial role in the management of burns. Its indications include wound-bed preparation, definitive dressing and sandwich grafting technique.

Objective

We analysed the experience of using GPA and its efficacy in burn treatment in our burn centre.

Methods

All burns managed with GPA in our burn centre from October 2001 to May 2008 were analysed.

Results

Mean total body surface area (TBSA) of 43 consecutive cases was 28.7%. GPA adhered to the wound for an average of 8.4 days before rejection. The length of hospital stay of the survivors was 42.5 days. The autograft take after wound-bed preparation with GPA was 88.4%. For sandwich grafting technique, the autograft take was 74.4%. When GPA was applied for partial-thickness burn as definitive dressing, all patients achieved complete healing within an average of 19 days without further surgical intervention. Despite colonisation of burn wounds after application of skin allograft, the outcomes of autograft take and wound healing were not significantly different.

Conclusion

The selective and strategic use of the GPA in major burn patients ensures optimal benefits in the management of burns. It is versatile in various categories of burn wounds with minimal morbidity.     

Expanding donor availability in lung transplantation: A case report of 5000 miles traveled

We present the case of a 41-year-old female who underwent bilateral lung transplantation after the donor lungs were placed on a normothermic ex vivo lung perfusion and ventilation device and flown nearly 5000 miles from Honolulu, Hawaii to Durham, North Carolina. The patient experienced no primary graft dysfunction. One year after transplantation she has remained rejection-free and exhibits excellent pulmonary function. This case highlights the challenge that active organ preservation systems pose to questions of organ allocation and geographic sharing.     

四肢组织瓣移植供区继发损伤的处理方法探讨

目的探讨四肢组织瓣移植后供区创面处理的方法,为减少供区并发症提供新思路。方法在进行组织瓣移植的36例中,采用不同的处理方法来减轻或修复供区继发损伤。其中3例(足母)趾甲皮瓣和2例足背皮瓣创面采用次要部位的游离皮瓣修复;5例股前外侧皮瓣供区创面采用同侧腹股沟皮瓣逆转修复;3例(足母)趾腓侧皮瓣供区采用第一跖背动脉皮瓣逆转修复;3例足背皮瓣复合第二趾移植供区采用外踝上皮瓣逆转修复;7例肌瓣和筋膜瓣移至受区后表面植皮,供区直接缝合;13例皮神经营养血管皮瓣分离血管蒂时,保留皮神经在原位。结果所有治疗原发和继发损伤的移植皮瓣均成活,应用带蒂或游离肌瓣、筋膜瓣加表面植皮者,3例植皮完全成活,4例植皮大部成活,皮神经营养血管皮瓣供区及其以远感觉接近正常。结论四肢组织瓣移植后供区继发损伤的处理值得重视,在治疗原发损伤的同时,采用各种方法修复或减少供区损伤,是降低供区并发症的重要措施。     

Isolated vascular “v” lesions in liver allografts: How to approach this unusual finding

According to the Banff criteria for kidney allografts, isolated vascular or “v” lesions are defined as intimal inflammation, age‐inappropriate fibro‐intimal hyperplasia, or both, without the presence of associated interstitial T cell‐mediated rejection (TCMR). In general, these lesions portend a worse outcome for kidney allografts, particularly in those where the “v” lesions are identified in patients with coexistent donor specific antibodies (DSA) or later after transplantation. Although affected arteries are rarely sampled in liver allograft biopsies, we identified nine patients at a mean of 1805 days posttransplantation and compared these to matched controls. Almost half (4 of 9) of the study patient biopsies showed inflammatory arteritis associated with focal or diffuse C4d positivity, which was not observed in matched controls. One “v” lesion patient progressed to rejection‐related graft failure and two developed moderate/severe TCMR in subsequent biopsies, whereas only one rejection episode occurred in follow‐up biopsies, and no rejection‐related deaths or graft failures were detected in controls. In conclusion, patients with liver allograft isolated “v” lesions should undergo further evaluation and closer follow‐up for impending TCMR and/or underlying co‐existent chronic antibody‐mediated rejection (AMR).     

Use of anti-HBc positive allografts in adult liver transplantation: toward a safer way to expand the donor pool

The use of livers from anti-hepatitis B core (HBc) positive donors can alleviate donor shortage. Nineteen of 367 (6%) adults receiving anti-HBc positive allografts [three were hepatitis B antigen (HBsAg) negative, hepatitis B antibody (HBsAb) positive; four were HBsAg positive and 12 were not exposed to hepatitis B viral (HBV) infection] were retrospectively reviewed. In HBsAg negative recipients, immunoprophylaxis (IP) was guided by viral serology and immunohistochemistry (IH) of day 0 and day 7 liver biopsies. If IH was negative, IP was stopped. None of three HBsAg negative, HBsAb positive recipients infected; one (replicating) of four HBsAg positive recipients reinfected and seven of eight (87.5%) HBsAg, HBsAb negative recipients, who did not receive long-term IP, infected after a median time of 2 years (range 1-5); one patient died of liver failure. Four HBsAg, HBsAb negative recipients, receiving life-long IP, remained infection free. Anti-HBc positive donor livers must be directed selectively first to HBsAg positive recipients, next to recipients having HBV antibodies and finally to HBV-naive recipients. Identification of both donor and recipient risk factors for HBV infection before transplantation allows indiscriminate use of antiviral prophylaxis. The necessity for IP therapy should be guided by HBV-DNA testing of donor liver tissue and serum. IH of early liver biopsies is an unreliable marker for predicting antiviral treatment requirements.     

Prospective observational study to assess the need for postoperative antibiotics following surgical incision and drainage of skin and soft tissue abscess in pediatric patients

Background

Post-operative antibiotics are often utilized for skin and soft tissue infection (SSTI) requiring surgical incision and drainage (I&D). We propose that antibiotics are unnecessary following I&D.

Use of HCV Ab+/NAT− donors in HCV naïve renal transplant recipients to expand the kidney donor pool

Hepatitis C (HCV) disease transmission from the use of HCV antibody‐positive and HCV nucleic acid test‐negative (HCV Ab+/NAT?) kidneys have been anecdotally reported to be absent. We prospectively analyzed kidney transplant (KT) outcomes from HCV Ab+/NAT? donors to HCV naïve recipients under T‐cell depleting early steroid withdrawal immunosuppression. Allografts from 40 HCV Ab+/NAT? donors were transplanted to 52 HCV Ab? recipients between July 2016 and February 2018. Thirty‐three (82.5%) of donors met Public Health Service (PHS) increased risk criteria. De novo HCV infection was detected at 3 months post‐KT in one recipient (1.9%). This was a case of transmission from a HCV Ab+ NAT+ donor with an initial false‐negative NAT completed using sample collected on donor hospital admission (day 2). At the time of HCV diagnosis, a stored donor sample collected during procurement (day 4) was tested and resulted NAT‐positive. Subsequently, sustained virologic response (SVR) was achieved with 12 weeks of glecaprevir/pibrentasvir. One death with functioning graft at 261 days post‐KT was determined not related to HCV or donor factors. This experience provides evidence of a low transmission rate of HCV from HCV Ab+/ NAT? kidney donors, thereby arguing for increasing utilization.     

Evaluation of a new foam to increase skin hydration of the foot in type 2 diabetes: a pilot study

The aim of the present study was to evaluate the efficacy of a new product (Neuropad repair foam^®) in promoting skin hydration of the foot in type 2 diabetes. Included in this study were 20 type 2 diabetic patients (10 men, mean age 61·40 ± 2·44 years). Patients applied Neuropad repair foam^® on the plantar aspect of the right foot twice daily. No agent was applied on the left foot. Patients were examined at baseline, after 7 treatment days and after 14 treatment days. Evaluation of skin dryness was performed by means of the Multi Skin test Corneometer MC 900. In the right foot, skin capacitance was 26·55 ± 4·14 arbitrary units (a.u.) at baseline, 28·90 ± 4·53 a.u. after 7 days of treatment and 32·05 ± 4·54 a.u. after 14 days of treatment. There was a significant increase in skin capacitance from baseline to 7 days of treatment (P < 0·001), from baseline to 14 treatment days (P < 0·001), as well as from 7 to 14 days of treatment (P < 0·001). The same significant (P < 0·001) increases were observed both in men and in women. No changes were noted in the left foot. At baseline, there was no difference in skin capacitance between right and left foot (P = 0·186). However, skin capacitance was significantly higher on the right versus left foot, both after 7 days (P < 0·001) and after 14 days of treatment (P < 0·001). In conclusion, results with the new foam appear encouraging in ameliorating skin dryness in the diabetic foot and further investigation is warranted.     

The relevance of pulse repetition rate and radiant exposure to the neurophysiological effects of low-intensity laser (820 nm/pulsed wave) irradiation upon skin temperature and antidromic conduction latencies in the human median nerve

The effects of low-intensity near-infra-red laser irradiation (820 nm; 1.5 and 9.0 J cm^–2; pulsed at 12 Hz, 73 Hz and 5 kHz) upon peripheral neurophysiology and skin temperature were investigated using antidromic conduction studies in the human median nerve in vivo. Healthy human volunteers (n = 90) were recruited and allocated randomly to either a control group (n=10) or one of eight experimental groups (two radiant exposures, 1.5 J cm^–2 and 9.0 J cm^–2 at one of three pulse repetition rates, 12 Hz, 73 Hz or 5 kHz, in addition to a placebo group for each radiant exposure;n = 10 all groups). Analysis of variance (ANOVA) demonstrated a significant (p0.05) decrease in skin temperature following irradiation at the lowest radiant exposure (1.5 J cm^–2) combined with pulse repetition rates of 73 Hz and 5 kHz, with the greatest effect at 73 Hz. These changes in skin temperature were coupled with increases in negative peak latency (NPL); ie changes in NPL were inversely related to changes in skin temperature. However, in contrast to the authors' previous findings using continuous wave (CW) laser irradiation, differences in NPL were not found to be significant. These findings, therefore, provide little evidence of the neuro-physiological effects of low-intensity infra-red irradiation at the dosage levels and pulse repetition rates used here.     

应用小腿内侧带血管蒂皮瓣治疗足、踝、小腿、膝部大面积软组织缺损骨肌腱外露12例

足、踝、小腿、膝部大面积软组织缺损，骨、肌腱外露患者多见于交通事故、机器绞轧等大外力引起的损伤，治疗上的困难在于不能及时行带血管蒂皮瓣转移修复创面治疗，而直接创面植皮易感染、坏死；或待新鲜肉芽长出后再植皮造成骨、肌腱外露时间长，会引起骨肌腱干燥坏死、创面感染、病程长，而造成关节活动受限、关节强直、功能障碍，影响患者生活工作。     

Pressure by design: How to improve the consistency of pressure garments in the clinical environment and implement a simple method for gathering evidence to establish efficacy

Pressure garments are used to treat scars after major trauma including burns. However, the ideal pressure for treatment is not known. Pressures exerted are not routinely measured and garments exert a wide range of pressures. Therefore, current treatment and its efficacy are variable.Pressure Garment Design Tools were introduced in 2012 but their application in hospitals has not been reported. A Garment Dimension and Pressure Calculator was used to audit pressures delivered by 8 pressure garments made for children using the hospital department’s standard reduction factor. The tool was easy to use and showed that pressures exerted by standard garments ranged from 15 to 54 mmHg with highest pressures exerted on wrists.Results of our pilot study indicated that the Garment Dimension and Pressure Calculator was slightly quicker to use than our normal manual process for calculating garment dimensions and enabled easy auditing of past treatment. The Pressure Garment Design Tool was easy to use and calculated garments that exerted the mean target pressures of 15 mmHg and 25 mmHg, improving consistency.Pressures exerted by garments were difficult and time consuming to measure with the Picopress sensor. Pressure was not distributed evenly around the limbs and measurements were inaccurate on the smallest limbs.     

CTOTC-08: A multicenter randomized controlled trial of rituximab induction to reduce antibody development and improve outcomes in pediatric lung transplant recipients

We conducted a randomized, placebo-controlled, double-blind study of pediatric lung transplant recipients, hypothesizing that rituximab plus rabbit anti-thymocyte globulin induction would reduce de novo donor-specific human leukocyte antigen antibodies (DSA) development and improve outcomes. We serially obtained clinical data, blood, and respiratory samples for at least one year posttransplant. We analyzed peripheral blood lymphocytes by flow cytometry, serum for antibody development, and respiratory samples for viral infections using multiplex PCR. Of 45 subjects enrolled, 34 were transplanted and 27 randomized to rituximab (n = 15) or placebo (n = 12). No rituximab-treated subjects versus five placebo-treated subjects developed de novo DSA with mean fluorescence intensity >2000. There was no difference between treatment groups in time to the primary composite outcome endpoint (death, bronchiolitis obliterans syndrome [BOS] grade 0-p, obliterative bronchiolitis or listing for retransplant). A post-hoc analysis substituting more stringent chronic lung allograft dysfunction criteria for BOS 0-p showed no difference in outcome (p = .118). The incidence of adverse events including infection and rejection episodes was no different between treatment groups. Although the study was underpowered, we conclude that rituximab induction may have prevented early DSA development in pediatric lung transplant recipients without adverse effects and may improve outcomes (Clinical Trials: NCT02266888).     

Professionalization of surgical abdominal organ recovery leading to an increase in pancreatic allografts accepted for transplantation in the Netherlands: a serial analysis

Professional abdominal organ recovery with certification has been mandatory in the Netherlands since 2010. This study analyses the effects of certification (January 2010–September 2015) on pancreas transplantation and compares it to an era before certification (February 2002–May 2008) for surgical injuries and the number of pancreases transplanted. A total of 264 cases were analysed. Eighty‐four recovered pancreases (31.8%) with surgically injuries were encountered. Forty‐six of those were surgically salvaged for transplantation, resulting in a total of 226 (85.6%) being transplanted. It was found that certified surgeons recovered grafts from older donors (36.8 vs. 33.3; P = 0.021), more often from donation after circulatory death (DCD) donors (18% vs. 0%; P < 0.001) and had less surgical injuries (21.6% vs. 41.0%; P < 0.001). Certification (OR: 0.285; P < 0.001) and surgeons from a pancreas transplant centre (OR: 0.420; P = 0.002) were independent risk factors for surgical organ injury. Predictors for proceeding to the actual pancreas transplantation were a recovering surgeon from a pancreas transplantation centre (OR: 3.230; P = 0.003), certification (OR: 3.750; P = 0.004), donation after brain death (DBD) (OR: 8.313; P = 0.002) and donor body mass index (BMI) (OR: 0.851; P = 0.023). It is concluded that certification in abdominal organ recovery will limit the number of surgical injuries in pancreas grafts which will translate in more pancreases available for transplantation.