David N. Campbell

Pediatric heart transplantation after declaration of cardiocirculatory death.

In three infants awaiting orthotopic cardiac transplantation, transplantation was successfully performed with the use of organs from donors who had died from cardiocirculatory causes. The three recipients had blood group O and were in the highest-risk waiting-list category. The mean age of donors was 3.7 days, and the mean time to death after withdrawal from life support was 18.3 minutes. The 6-month survival rate was 100% for the 3 transplant recipients and 84% for 17 control infants who received transplants procured through standard organ donation. The mean number of rejection episodes among the three infants during the first 6 months after surgery was 0.3 per patient, as compared with 0.4 per patient among the controls. Echocardiographic measures of ventricular size and function at 6 months were similar among the three infants and the controls (left ventricular shortening fraction, 43.6% and 44.9%, respectively; P=0.73). No late deaths (within 3.5 years) have occurred in the three infants, and they have had functional and immunologic outcomes similar to those of controls. Mortality while awaiting a transplant is an order of magnitude higher in infants than in adults, and donors who died from cardiocirculatory causes offer an opportunity to decrease this waiting-list mortality.

A database of mass spectrometric assays for the yeast proteome

To the Editor: The current most widely used mass spectrometry (MS)-based proteomic methods sample the available proteome in a quasi-random manner. In each analysis only a subset of the proteins contained in a sample are measured, and replicate analyses sample partly overlapping proteome segments. In addition, these analyses are biased toward abundant proteins1,2. This limits the feasibility of consistently and comprehensively measuring defined sets of proteins (such as proteins constituting signaling or metabolic networks) across different samples, thus precluding the generation of complete datasets to support mathematical modeling of biological processes. To overcome these limitations we previously proposed a targeted proteomic strategy1,2 based on a MS technique called selected reaction monitoring (SRM, also referred to as multiple reaction monitoring (MRM)). This strategy can be used to generate specific, quantitative MS assays for the proteins of interest, which can be then applied for protein detection and quantification in multiple biological samples. First, for each target protein, proteotypic peptides (peptides that unambiguously represent these proteins and are preferentially detectable by MS (ref. 3)) are selected. Then precursor/fragment ion relationships are established, which identify each proteotypic peptide. These consist of pairs of massto-charge ratio (m/z) values that are selected by the two mass analyzers of a triple quadrupole (QQQ) mass spectrometer to isolate the targeted peptide ion and corresponding, diagnostic fragment ion(s). The detector then counts the analytes matching the defined relationship(s) and returns a signal intensity over the chromatographic time. These relationships, termed SRM (or MRM) transitions, therefore effectively constitute MS assays that identify a peptide and, by inference, the corresponding protein in a complex digest. The assays are quantitatively accurate, in particular if isotopically labeled standards are used. SRM measurements result in higher sensitivity (low-attomolar4) and specificity compared to other MS-based proteomic techniques. Yeast proteins spanning all ranges of abundance, from 1.3 million to <100 copies/cell can be identified and quantified by SRM in unfractionated whole yeast digests (P.P. et al., unpublished data). However, despite these favorable characteristics, SRM has not been broadly applied in proteomics, because of the effort required for establishing SRM assays for every protein. The initial selection of optimal proteotypic peptides is complicated by MS signal responses that vary greatly for different tryptic peptides of the same protein. In addition, for each proteotypic peptide, predominant fragment ions have to be selected that define the SRM transitions. As most fragment ion spectra are generated on instruments other than QQQ mass spectrometers, and operating conditions are often poorly documented, the commonly accessible fragment ion spectra can only be a starting point for establishing optimal Figure 1 | Categorization of the yeast proteins represented in MRMAtlas by function and cellular abundance. (a) Grouping of open reading frames by biological processes in S. cerevisiae is according to the Gene Ontology database nomenclature. The same ORF can map to more than one Gene Ontology biological process. (b) Yeast protein abundances in MRMAtlas are derived from a published dataset5. Organelle organization or genesis

Minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE)

One purpose of the biomedical literature is to report results in sufficient detail that the methods of data collection and analysis can be independently replicated and verified. Here we present reporting guidelines for gene expression localization experiments: the minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE). MISFISHIE is modeled after the Minimum Information About a Microarray Experiment (MIAME) specification for microarray experiments. Both guidelines define what information should be reported without dictating a format for encoding that information. MISFISHIE describes six types of information to be provided for each experiment: experimental design, biomaterials and treatments, reporters, staining, imaging data and image characterizations. This specification has benefited the consortium within which it was developed and is expected to benefit the wider research community. We welcome feedback from the scientific community to help improve our proposal.

Reoperative homograft right ventricular outflow tract reconstruction

BACKGROUND Homografts are implanted in the right ventricular outflow tract (RVOT) of children, with the knowledge that reoperation might be required. We reviewed 14 years of homograft RVOT reconstruction to assess the feasibility of homograft replacement and to determine risk factors for homograft survival. METHODS From February 1985 through March 1999, 223 children (age 5 days to 16.9 years) underwent primary RVOT reconstruction with an aortic or pulmonary homograft. Of these, 35 patients underwent homograft explant at the implanting hospital with insertion of a second homograft from 2 months to 13.3 years after the first implantation. The primary operation and reoperation patient groups were compared with regard to incidence of early death, late death, homograft-related intervention without explant, and homograft explant. RESULTS Actuarial survival and event-free curves for initial and replacement homografts were not significantly different. Univariable analysis was performed for the following risk factors: weight (p < 0.0001), age (p < 0.003), homograft diameter (p < 0.0001), homograft type (p < 0.01), surgery date (not significant [NS]), gender (NS), Blood Group match (NS), and type of distal anastomosis (NS). Multivariable analysis of significant univariable risks revealed small homograft diameter to be a significant risk factor (p < 0.001) for replacement. CONCLUSIONS The RVOT homografts eventually require replacement. Patient and homograft survival for replacement homografts is similar to primary homografts. Reoperative homograft RVOT reconstruction is possible, with reasonably low morbidity and mortality.

Modified ultrafiltration attenuates dilutional coagulopathy in pediatric open heart operations.

BACKGROUND Extreme hemodilution caused by relatively large prime volumes required for cardiopulmonary bypass in infants causes a dilutional coagulopathy, characterized by low concentrations of fibrinogen and other circulating coagulation factors. Modified ultrafiltration results in hemoconcentration and is associated with decreases in postoperative bleeding and transfusion requirements in children. This study was undertaken to quantify the effect of modified ultrafiltration on concentrations of fibrinogen, plasma proteins, and platelets in infants and small children. METHODS Twenty patients less than 15 kg were studied. Cardiopulmonary bypass circuits were primed with crystalloid solutions. Red blood cells were added during cardiopulmonary bypass for hematocrits less than 15%. Colloid solutions were not administered. Concentrations of fibrinogen, plasma proteins, and platelets, and hematocrit were measured before cardiopulmonary bypass, before modified ultrafiltration, and after modified ultrafiltration. RESULTS Modified ultrafiltration was associated with significant (p < 0.001) increases in hematocrit (19% +/- 6% to 31% +/- 9%), fibrinogen (65 +/- 29 to 101 +/- 45 mg/dL), and total plasma proteins (2.7 +/- 0.3 to 4.9 +/- 0.7 g/dL), but no change (p = 0.129) in platelet count. CONCLUSIONS We conclude that modified ultrafiltration significantly attenuates the dilutional coagulopathy associated with cardiopulmonary bypass in infants.

The St. Jude medical cardiac valve in infants and children: Role of anticoagulant therapy

The experience at the University of Colorado with the St. Jude Medical cardiac valve was reviewed to determine the feasibility of placing this prosthesis in children and the role of anticoagulation. A St. Jude Medical cardiac valve was placed in 33 patients ranging in age from 2.5 months to 17 years. Seven patients were less than 1 year of age. Nineteen valves were placed in the aortic position in patients aged 5 months to 17 years (mean 9.5 years). Five patients had valve replacement only, 13 had concomitant aortoventriculoplasty and 1 a Manouguian procedure. Indications for anulus enlarging procedures were recurrent subaortic stenosis or inability to place an adult-sized valve in the native aortic anulus, or both. There were no early or late deaths. Fourteen valves were placed in the mitral position. They were anular positioned in 6 patients aged 6 months to 16 years and supraanular positioned in 8 patients aged 2.5 months to 2 years. There were no deaths with the anular positioned replacements and seven deaths (two early and five late) with the supraanular positioned replacements. Four of the five late deaths were associated with marked pre- and postoperative left ventricular dysfunction. The follow-up time was 784 patient-months in 31 long-term survivors. Anticoagulation was achieved with warfarin, usually in combination with sulfinpyrazone, dipyridamole or aspirin. There were four episodes of thromboembolism, three occurring in patients with suboptimal anticoagulation, and one in a patient lost to follow-up.(ABSTRACT TRUNCATED AT 250 WORDS)

Outcome of Extracorporeal Membrane Oxygenation for Early Primary Graft Failure After Pediatric Heart Transplantation

OBJECTIVES We sought to analyze the indications and outcome of extracorporeal membrane oxygenation (ECMO) for early primary graft failure and determine its impact on long-term graft function and rejection risk. BACKGROUND Early post-operative graft failure requiring ECMO can complicate heart transplantation. METHODS A retrospective review of all children requiring ECMO in the early period after transplantation from 1990 to 2007 was undertaken. RESULTS Twenty-eight (9%) of 310 children who underwent transplantation for cardiomyopathy (n = 5) or congenital heart disease (n = 23) required ECMO support. The total ischemic time was significantly longer for ECMO-rescued recipients compared with our overall transplantation population (276 +/- 86 min vs. 242 +/- 70 min, p < 0.01). The indication for transplantation, for ECMO support, and the timing of cannulation had no impact on survival. Hyperacute rejection was uncommon. Fifteen children were successfully weaned off ECMO and discharged alive (54%). Mean duration of ECMO was 2.8 days for survivors (median 3 days) compared with 4.8 days for nonsurvivors (median 5 days). There was 100% 3-year survival in the ECMO survivor group, with 13 patients (46%) currently alive at a mean follow-up of 8.1 +/- 3.8 years. The graft function was preserved (shortening fraction 36 +/- 7%), despite an increased number of early rejection episodes (1.7 +/- 1.6 vs. 0.7 +/- 1.3, overall transplant population, p < 0.05) and hemodynamically comprising rejection episodes (1.3 +/- 1.9 vs. 0.7 +/- 1.3, overall transplant population, p < 0.05). CONCLUSIONS Overall survival was 54%, with all patients surviving to at least 3 years after undergoing transplantation. None of the children requiring >4 days of ECMO support survived. Despite an increased number of early and hemodynamically compromising rejections, the long-term graft function is similar to our overall transplantation population.

Left ventricular assist device as bridge to transplantation does not adversely affect one-year heart transplantation survival.

OBJECTIVE Left ventricular assist devices are increasingly used as a bridge to transplantation. It remains unclear whether the use of pretransplant left ventricular assist devices adversely affects short-term survival after cardiac transplantation. METHODS A retrospective review of 317 consecutive patients undergoing cardiac transplantation at an academic center between 1986 and 2006 was undertaken. Left ventricular assist devices were used pretransplant in 23 of these 317 patients, and 294 patients did not require left ventricular assist device support. Patients with a left ventricular assist device were supported with a Heartmate VE or Heartmate XVE (Thoratec Corp, Pleasanton, Calif). Kaplan-Meier survival estimates were compared between the left ventricular assist device group and the non-left ventricular assist device group using the log-rank test. In addition, occurrence of death was analyzed between the 2 groups with a chi-square analysis. The results are expressed as 1-year survival with 95% confidence intervals in parentheses. RESULTS The 1-year survival for all 317 patients was 0.86 (0.82-0.90). The patient survival for the group without a left ventricular assist device before cardiac transplant was 0.87 (0.83-0.90), and the survival for the group with a left ventricular assist device as bridge to transplantation was 0.83 (0.67-0.98; P = .77). For the deaths that occurred in all 317 patients, 19% of the patients without left ventricular assist devices died within 30 days of transplant, whereas 80% of the patients with left ventricular assist devices died within 30 days of transplant (P < .01). CONCLUSION When used as a bridge to transplantation, left ventricular assist devices do not compromise 1-year survival after cardiac transplantation. Of the patients who die after transplantation, patients bridged with left ventricular assist devices are at higher risk for death within 30 days of transplant. These data suggest that left ventricular assist devices as a bridge to transplantation should be considered for appropriately selected patients awaiting cardiac transplantation.

The past, present, and future of lung transplantation.

BACKGROUND The history of lung transplantation from the first human transplant performed in 1963 to the present is reviewed with particular focus on the added challenges because of the contaminated bronchus, exposure of the graft to airborne organisms, the poor blood supply to the bronchus, and the problem of reperfusion pulmonary edema. METHODS The technical aspects of single and double sequential lung transplantation are reviewed, as are the current indications for single, double sequential, and heart/lung transplantation. Criteria for lung transplant recipients, in addition to their primary disease are noted, as are absolute and relative contraindications. The standard criteria for donor selection are also reviewed. RESULTS The results of single, double sequential, and heart-lung transplantation over the past 10 years as reported by the International Society for Heart and Lung Transplantation Database are reviewed. In addition, the statistics of the lung and heart-lung transplantation program at the University of Colorado Health Sciences Center are reviewed, including the current immunosuppressive regimens and early and late monitoring for infection and rejection. This experience includes 3 early deaths in the first 53 patients for an operative mortality of 5.6%, with a 1-year actuarial survival of 90%. CONCLUSIONS During the past decade remarkable improvement in the result of single and double sequential lung transplantation have occurred. As 1-year, actuarial survival is now approaching 90% at some institutions. Living related lobar transplantation, new antirejection agents, chimerism, and xenograft transplantation are areas for continuing and future investigation. The shortage in donor organ supply continues to be a very significant factor in limiting human lung transplantation.

State of the Human Proteome in 2014/2015 As Viewed through PeptideAtlas: Enhancing Accuracy and Coverage through the AtlasProphet

The Human PeptideAtlas is a compendium of the highest quality peptide identifications from over 1000 shotgun mass spectrometry proteomics experiments collected from many different laboratories, all reanalyzed through a uniform processing pipeline. The latest 2015-03 build contains substantially more input data than past releases, is mapped to a recent version of our merged reference proteome, and uses improved informatics processing and the development of the AtlasProphet to provide the highest quality results. Within the set of ∼20,000 neXtProt primary entries, 14,070 (70%) are confidently detected in the latest build, 5% are ambiguous, 9% are redundant, leaving the total percentage of proteins for which there are no mapping detections at just 16% (3166), all derived from over 133 million peptide-spectrum matches identifying more than 1 million distinct peptides using AtlasProphet to characterize and classify the protein matches. Improved handling for detection and presentation of single amino-acid variants (SAAVs) reveals the detection of 5326 uniquely mapping SAAVs across 2794 proteins. With such a large amount of data, the control of false positives is a challenge. We present the methodology and results for maintaining rigorous quality along with a discussion of the implications of the remaining sources of errors in the build.