Faculty Bibliography

Cryopreservation of parathyroid tissue (PT) provides patients undergoing parathyroidectomy with an option for delayed autologous heterotopic parathyroid transplantation. A standard protocol for quality monitoring of PT has not been established. This article describes a method for detecting the presence of bacterial contamination in PT tissue intended for autologous transplantation. PT was received in the tissue bank, processed under aseptic conditions, and placed into cryopreservation medium. Sterility testing was performed at 2 time points prior to cryopreservation. From January 2005 to October 2008, 47 PT samples were cryopreserved. The following bacteria were isolated from 11 PT specimens: Staphylococcus epidermidis, Staphylococcus capitis subspecies ureolyticus, Staphylococcus lugdunensis, Bacillus pumilus, and corynebacteria (diphtheroids). 23% of PTs were contaminated at the time of collection, predominantly with indigenous bacteria. Quality monitoring using this protocol is a useful tool to identify tissues contaminated with bacteria.

BACKGROUND:Animal models to investigate pathophysiology and xenotransplantation require complex techniques and significant animal utilization. The aim of the study was to develop a reliable ex-vivo technique to test xenotransplant interventions. MATERIAL/METHODS/METHODS:Miniature Swine being utilized for a nonsurvival study acted as donor animals. Kidneys were flushed and rapidly explanted and chilled to 4°C. Kidneys were assigned to be the control (CK) (n=3) and the mate were used as a Xenograft Kidneys (XK) (n=3). Kidneys were perfused on separate Waters RM 3 perfusion devices. Perfusion temperature was 35-37°C and pressure was 100-110/60-70 mmHg at 60 pulses per minute. CKs were reperfused with autologous blood collected at the time of organ procurement. XKs were reperfused using freshly donated whole human blood. Physical characteristics, urine output were recorded. Core needle biopsies were obtained and examined by a blinded pathologist for evidence of antibody mediated rejection. RESULTS:XK kidneys demonstrated homogenous reperfusion which rapidly became patchy at 5-7 minutes. XK kidneys had become complete black and thrombosed by 60-70 minutes. XK biopsies demonstrated peritubular capillaritis. CK kidneys demonstrated homogenous reperfusion and urine production. H&E stain of CKs only demonstrated nonspecific inflammation. CONCLUSIONS:Our ex-vivo porcine xenotransplant model shows early promise as a tool for studying Xeno- associated hyperacute rejection. This technique saves resources and animal utilization.

As the 100th anniversary of the Flexner report nears, medical student education is being reviewed at many levels. One area of concern, expressed in recent reports from some national health care organizations, is the adequacy of training in the discipline of laboratory medicine (also termed clinical pathology). The Academy of Clinical Laboratory Physicians and Scientists appointed an ad hoc committee to review this topic and to develop a suggested curriculum, which was subsequently forwarded to the entire membership for review. The proposed medical student laboratory medicine curriculum defines goals and objectives for training, provides guidelines for instructional methods, and gives examples of how outcomes can be assessed. This curriculum is presented as a potentially helpful outline for use by medical school faculty and curriculum committees.

BACKGROUND:ABO-incompatible (ABOi) and positive crossmatch (XM) renal transplants pose special immunologic challenges. It is important to compare outcomes, study resource utilization, and attempt to risk stratify patients in these higher risk transplant settings. METHODS:We compared apheresis utilization and transplant outcomes in ABOi, XM, and combined ABOi-XM renal transplants. We also analyzed multiple parameters, including patient and laboratory variables, to identify predictors of transplant outcome. RESULTS:Incidences of early (< or =30 days posttransplant) antibody-mediated rejection (AMR) and acute cellular rejection (ACR) were similar among the three incompatible groups whereas they differed in allograft rejection for late (>30 days posttransplant) AMR and ACR. Notably, there were no episodes of late AMR among ABOi patients. Patients treated with more than four pretransplant plasmapheresis/intravenous immunoglobulin (PP/IVIg) had a greater likelihood of experiencing early AMR. The median number of posttransplant PP/IVIg treatments was greater than twofold higher in ABOi-XM and XM patients compared to ABOi patients. Patients who required more than five posttransplant PP/IVIg procedures and those with one or more prior renal transplants had higher incidences of late ACR. CONCLUSIONS:Our analysis aids in defining apheresis resource utilization and helps in risk stratification of incompatible renal transplantation. It also aids in predicting allograft rejection and provides an opportunity for preemptive monitoring and treatment.

Cytokines are hypothesized to play a central role in the pathophysiology of IgG-mediated hemolytic transfusion reactions (HTRs), and deeper understanding is required for improving therapy for these events. After establishing well-defined mouse models of HTRs, we tested whether cytokines were involved. Red blood cells (RBCs) from human glycophorin A transgenic (hGPA-Tg) or wild-type (WT) mice were transfused into non-Tg recipients passively immunized with monoclonal antibodies (Mabs). Only transfusions of incompatible RBCs induced IgG-mediated HTRs, exemplified by rapid clearance and hemoglobinuria. Very high plasma levels of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6), and lower levels of tumor necrosis factor-alpha (TNF-alpha), were induced after incompatible transfusion. No significant changes in IL-10, IL-12, or interferon-gamma (IFN-gamma) levels were observed. The proinflammatory cytokines elaborated in this in vivo mouse model are also implicated in the systemic inflammatory response syndrome (SIRS) and confirm the hypothesis that cytokine storm occurs as a result of HTRs.

Wilson disease is an autosomal recessive disorder of copper metabolism that leads to the accumulation of copper mainly in the liver, cornea, brain, and kidney. Rarely, Wilson disease can present as fulminant hepatic failure with direct antiglobulin test-negative hemolytic anemia and renal failure. In the absence of liver transplantation, this disease is uniformly fatal because medical therapy is ineffective. This report describes the successful use of plasmapheresis for a patient with fulminant Wilson disease as a bridge to transplantation. Five daily therapeutic plasmapheresis procedures using fresh frozen plasma as a replacement fluid were performed over 6 days. Serum copper, urinary copper excretion, and hemolysis were significantly reduced and renal function improved. The patient's clinical status improved and she remained clinically stable until a liver transplant was possible. Plasmapheresis can be a successful medical treatment in fulminant Wilson disease and should be considered as a therapeutic measure to stabilize a patient by decreasing serum copper, reducing hemolysis, and helping to prevent renal tubular injury from copper and copper complexes until liver transplantation is possible.

Immunohistochemical staining for cytokeratin 7 (CK7), KIT, and PAX2 expression was performed on 91 renal neoplasms, 37 conventional (clear cell) renal cell carcinomas (CRCCs), 20 papillary RCCs (PRCCs), 11 chromophobe RCCs (ChCs), and 23 oncocytomas, with available karyotypes. All ChCs, 19 PRCCs, 2 CRCCs, and 1 oncocytoma were CK7+; all ChCs, 22 oncocytomas, 2 CRCCs, and no PRCCs expressed KIT; PAX2 was positive in 31 CRCCs, 17 PRCCs, 20 oncocytomas, and 1 ChC. The predominant expression profiles were as follows: CRCC, CK7-/KIT-/PAX2+ (26/37); PRCC, CK7+/KIT-/PAX2+ (17/20); ChC, CK7+/KIT+/PAX2- (10/11); and oncocytoma, CK7-/KIT+/PAX2+ (19/23). Cytogenetic analysis showed that the sole PAX2+ ChC had a retained chromosome 10, and all ChCs with chromosome 10 loss were PAX2-. These results identify specific staining patterns of the 4 major histologic subtypes of renal neoplasms and raise the question of a relationship between chromosome 10 loss and loss of PAX2 expression in ChC.

We report on the feasibility, safety, and efficacy of performing therapeutic plasmapheresis (TPE) in parallel with extracorporeal membrane oxygenation (ECMO) to alleviate antibody mediated rejection (AMR) after heart transplantation. Two pediatric and one adult patient presented with severe congestive heart failure and respiratory distress after heart transplantation and required ECMO support. TPE was initiated to treat AMR while patients remained on ECMO. Each patient received three to five procedures either every day or every other day. One equivalent total plasma volume (TPV) was processed for each procedure (patient TPV + ECMO extracorporeal TPV). A total of 13 TPE procedures were performed with 12 procedures completed without complications or adverse events; one procedure was terminated before completion because of cardiac arrhythmia. Anti-HLA antibody titers decreased after TPE in all three patients. Ventricular function improved and ECMO was discontinued in 2 of 3 patients. Performing large volume TPE with a processed volume up to 2.5 times the patient's TPV is well tolerated in both pediatric (< or = 10 kg) and adult patients. TPE in parallel with ECMO is feasible, safe, and may be measurably effective at reducing anti-HLA antibodies and should be considered as part of the treatment for patients with early AMR after heart transplantation.

Platelets, unlike red blood cells and plasma, are stored at room temperature because platelets transfused after refrigeration at 4 degrees C are rapidly cleared from the circulation. Storage at room temperature promotes bacterial proliferation, however, and transfusion-transmitted bacteremia has become an increasing problem. Traditionally, the cold storage lesion has been attributed to a change in platelet shape from disc to sphere, but Hoffmeister et al. revisited this issue and have shown that the shape change induced by cold storage does not result in poor platelet survival. Instead, they showed that poor survival results from a virtually irreversible clustering of alpha subunits of glycoprotein Ib (GPIbalpha)) on the platelet surface. In a series of elegant papers, these researchers change the way we view platelet clearance. That is, they show that exposed, terminal, beta-linked N-acetylglucosamine (beta-GlcNAc) residues on clustered GPIbalpha are recognized by the lectin domain of type 3 complement receptors on liver macrophages, leading to rapid clearance by phagocytosis. They also demonstrate that phagocytosis of chilled platelets can be inhibited--and in vivo survival prolonged--by enzymatically galactosylating the terminal beta-GlcNAc residues on GPIbalpha. Disguising the exposed beta-GlcNAc residues on the N-glycans of the clustered GPIbalpha molecules by galactosylation is a promising approach to storing platelets at 4 degrees C without affecting platelet function. Cold storage would limit bacterial proliferation and extend the duration of platelet storage, reducing the incidence of transfusion-transmitted bacteremia and improving the availability of this scarce resource.

We have observed expansions of intraepithelial lymphocytes in duodenal biopsies from patients with Helicobacter pylori gastritis. This study was undertaken to prospectively evaluate, unselected, paired gastric and duodenal biopsies from 50 patients with H. pylori gastritis and a comparison group of 30 patients with other types of gastritis (10 autoimmune and 20 reactive) to: (1) quantify duodenal intraepithelial lymphocytes, determine their distribution patterns, epithelial location, and phenotype, and (2) correlate the intraepithelial lymphocyte elevations with various features of gastric and duodenal pathology. Intraepithelial lymphocytes were analyzed with antibodies including CD3, CD8, and TIA-1. A stain for H. pylori was performed on all gastric and duodenal biopsies. Duodenal intraepithelial lymphocytes from patients with H. pylori gastritis (using CD3) ranged from 3 to 42 lymphocytes/100 epithelial cells (mean 18.5) compared to 3 to 18 lymphocytes/100 epithelial cells (mean 6.6) in the comparison group. Intraepithelial lymphocyte elevations were seen in 44% of the duodenal biopsies from patients with H. pylori gastritis (using CD3). Significant differences in the intraepithelial lymphocyte counts between patients with H. pylori gastritis and the comparison group were seen for all three T-cell antigens (P<0.001 for CD3 and CD8 and P<0.002 for TIA-1). Duodenal intraepithelial lymphocytes in the H. pylori+ cases had a latent cytotoxic phenotype, H. pylori was not visualized in any of the duodenal biopsies from patients with H. pylori gastritis, and no patient had clinical evidence of celiac disease. Our study highlights frequent duodenal intraepithelial lymphocytosis in individuals with H. pylori gastritis and the lymphocyte distribution patterns (and numbers) overlapped with those described for celiac disease patients. H. pylori gastritis must be considered as a possible explanation for duodenal intraepithelial lymphocytosis with normal villous architecture, especially when lymphocytosis is patchy, intraepithelial lymphocytes display a 'latent' cytotoxic phenotype, and the clinical findings and serologic profile does not fit celiac disease.