Pancreas.

Even though pancreas transplant numbers have steadily declined over the past decade, new listings increased in 2014 compared with the previous year, notably for pancreas transplant alone (PTA) and simultaneous pancreas-kidney transplant. The number of new PTAs also increased over the past two years. Whether this is a sustainable trend remains to be seen. Significant events in 2014 included implementation of a new pancreas allocation system and development of a proposed uniform definition of pancreas graft failure. Meanwhile, overall pancreas transplant rates and outcomes continued to improve. Substantial decline in pancreas after kidney transplants remains a serious concern. SRTR has not published pancreas graft failure data in the program-specific reports for the past two years. While this will not change in the near future, the acceptance of a uniform definition of graft failure is a crucial first step toward resuming graft failure reporting. Continued improvements and innovation, both surgical and immunological, will be critical to keep pancreas transplant as a viable option for treatment of insulin-dependent diabetes. As alternative therapies for diabetes such as islet transplant and artificial pancreas are evolving, improved outcomes with minimizations of complications are more important than ever.

OPTN/SRTR 2013 Annual Data Report: pancreas.

Pancreas listings and transplants decreased during the past decade, most notably pancreas after kidney transplants. Center-reported outcomes of pancreas transplant across all groups, short-term and long-term, improved during the same period. Changes to the pancreas allocation system creating an efficient, uniform national system will be implemented in late 2014. Pancreas-alone and simultaneous pancreas-kidney (SPK) candidates will form a single match-run list with priority to most SPK candidates ahead of kidney-alone candidates to decrease waiting times for SPK candidates, given their higher waitlist mortality compared with nondiabetic kidney transplant candidates. The changes are expected to eliminate local variability, providing more consistent pancreas allocation nationwide. Outcomes after pancreas transplant are challenging to interpret due to lack of a uniform definition of graft failure. Consequently, SRTR has not published data on pancreas graft failure for the past 2 years. The Organ Procurement and Transplantation Network Pancreas Transplantation Committee is working on a definition that could provide greater validity for future outcomes analyses. Challenges in pancreas transplantation include high risk of technical failures, rejection (early and late), and surgical complications. Continued outcome improvement and innovation has never been more critical, as alternatives such as islet transplant and artificial pancreas move closer to clinical application.

OPTN/SRTR 2012 Annual Data Report: pancreas.

The number of pancreas transplants has decreased over the past decade, most notably numbers of pancreas after kidney (pak) and pancreas transplant alone (pta) procedures. This decrease may be mitigated in the future when changes to national pancreas allocation policy approved by the Organ Procurement and Transplantation Network Board of Directors in 2010 are implemented. The new policy will combine waiting lists for pak, pta, and simultaneous pancreas-kidney (spk) transplants), and give equal priority to candidates for all three procedures. This policy change may also eliminate geographic variation in waiting times caused by geographic differences in allocation policy. Deceased donor pancreas donation rates have been declining since 2005, and the donation rate remains low. The outcomes of pancreas grafts are difficult to describe due to lack of a uniform definition of graft failure in the transplant community. However long-term survival is better for spk versus pak and pta transplants. This may represent the difficulty of detecting rejection in the absence of a simultaneously transplanted kidney. The challenges of pancreas transplant are reflected in high rates of rehospitalization, most occurring within the first 6 months posttransplant. Pancreas transplant is associated with higher incidence of rejection compared with kidney transplant.

Optimizing recovery, utilization and transplantation outcomes for kidneys from small, ≤20 kg, pediatric donors.

The optimal balance between maximizing the number versus the outcome of transplantation utilizing kidneys from small (≤20 kg) pediatric donors remains unclear, complicated by the choice of single versus en bloc transplantation with their attendant technical risks. Using the Organ Procurement and Transplantation Network (OPTN) database, we examined kidney recovery and utilization patterns, and 1-year transplant outcomes by single kilogram weight strata. Between January 1, 2005 and June 30, 2010, 2352 kidneys from ≤20 kg donors were transplanted into 1531 recipients, 710 single kidney transplants (SKTs) and 821 en bloc kidney transplants (EBKTs). Increased donor weight was associated with higher rates of recovery, transplantation and SKT. Low donor weight (linear p < 0.001; quadratic p = 0.003), SKT versus EBKT (p = 0.008), increased cold ischemia time (p = 0.003), local versus nonlocal donor (p = 0.0044), low versus high volume center (p = 0.003) and the interaction term between center volume and donor weight (p = 0.0024) were associated with graft failure. Notably, lower donor weight exacerbated the negative impact of low center volume but did not worsen the negative impact of SKT on outcomes. Our data show that EBKT offers superior 1-year survival at the expense of accomplishing one rather than two transplants. However, SKTs yield excellent outcomes when performed at experienced centers.

OPTN/SRTR 2011 Annual Data Report: pancreas.

Numbers of pancreas transplants have been decreasing over the past decade, but outcomes continue to improve for all types: simultaneous pancreas-kidney transplant, pancreas after kidney transplant (PAK), and pancreas transplant alone (PTA). The most notable decrease occurred for PAK transplants, possibly due in part to decreases in numbers of living donor kidney transplants. The number of new candidates on the pancreas transplant waiting list has decreased steadily since 2000; only 1005 active candidates were added in 2011. Transplant rates for all pancreas transplant types reached a low in 2011 of 34.9 transplants per 100 wait-list years. Deceased donation rates have also been decreasing since 2005, but use of donation after circulatory death has been gradually increasing. The discard rate in 2011 was 27.7%, and higher for pancreata recovered from older donors. Improved outcomes during the early posttransplant period largely reflect improved donor and recipient selection and improved technical strategies. Inconsistent definitions of graft failure across reporting centers creates an ongoing challenge in the interpretation of outcome data for pancreas transplants. Rates of posttransplant re-hospitalization are high, most occurring in the first 6 months. Rejection rates are highest for PTA recipients, who also experience higher incidence of posttransplant lymphoproliferative disorder.

The isolation of carcinoembryonic antigen from tumor tissue at neutral pH.

Carcinoembryonic antigen (CEA) was purified from tumor tissue under mild conditions at neutral pH by a procedure that utilized affinity chromatography on concanvalin A. Further purification by gel filtration provided CEA in 10 to 20% yield and 10% purity. Antibody to this preparation was rendered specific for CEA by adsorption on a column of normal liver proteins bound to Sepharose. On reaction by immunodiffusion against a crude tumor extract, the adsorbed antibody produced two precipitin lines, of which one was relatively weak. These two precipitin lines fused completely with the two respective lines produced by antibody to perchloric acid-treated CEA. The major antigen found in crude tumor extracts and in CEA preparations purified at neutral pH was nearly undetectable in perchloric acid extracts of tumor homogenates. Further investigations showed that 60 to 70% of the CEA in crude tumor extracts and in preparations isolated at netural pH is destroyed and/or becomes insoluble acidic conditions.

Homogeneous substrate-labeled fluorescent immunoassay for IgG in human serum.

A homogeneous substrate-labeled fluorescent immunoassay for IgG has been developed. Purified IgG was covalently labeled with 6-(7-beta-galactosylcoumarin-3-carboxamido)-hexylamine to form a stable conjugate, GU-IgG. The galactosyl residue was hydrolyzed from GU-IgG by beta-galactosidase and the progress of the hydrolysis was monitored by the increase in fluorescence emission at 450 nm with excitation at 400 nm. Antibody to IgG diminished the activity of GU-IgG as a substrate for beta-galactosidase. Competitive binding immunoassays were conducted by allowing added IgG and GU-IgG to compete for a limited number of antibody binding sites. Hence, the fluorescence produced by enzymic hydrolysis increased with the level of added IgG. This method provides a simple and reliable immunoassay for IgG and is applicable to other proteins.

A colorimetric immunoassay based on an enzyme inhibitor method.

A competitive binding immunoassay was developed using an enzyme inhibitor for labeling the analyte. Thyroxine was labeled by covalent coupling of the alpha-amino group to the gamma-carboxyl of the glutamyl residue of methotrexate. This thyroxine-methotrexate conjugate was a potent inhibitor of dihydrofolate reductase. When antibody was bound to the thyroxine moiety, the inhibitor was inactivated. Thus, a competitive binding immunoassay for thyroxine was demonstrated based on colorimetric measurement of dihydrofolate reductase activity.

Characterization of monoclonal antibody to DNA.RNA and its application to immunodetection of hybrids.

Monoclonal antibodies were raised to a DNA.RNA heteropolymer duplex prepared by transcription of phi X174 single-stranded DNA with DNA-dependent RNA polymerase. A monoclonal antibody with the highest affinity and specificity was selected. This antibody bound the DNA.RNA heteropolymer and poly(I).poly(dC) equally but 100-fold higher levels of poly(A).poly(dT) were required to achieve a similar degree of binding in competitive binding assays using DNA.[3H]RNA. Single-stranded DNA, double-stranded DNA and RNA, and ribosomal RNA were not bound by the antibody. The observed association constant for the antibody and DNA.[3H]RNA, determined by Scatchard analysis, was 8.5 X 10(10) l/mol assuming independent antibody binding sites. The antibody and an alkaline phosphatase-labeled second antibody were used in an immunodetection method for measurement of hybrids formed between immobilized DNA probes of various lengths and 23 S ribosomal RNA. The colorimetric response of this assay increased linearly with the amount of hybrid formed.

The reversible binding of oxygen to sulfhemoglobin.

The O2 binding properties of sulfhemoglobin were studied. The oxygen tension required for half-saturation of sulfhemoglobin is more than 2 orders of magnitude higher than that for hemoglobin A. The binding of O2 exhibits an alkaline Bohr effect larger than that observed for hemoglobin, yet the Hill number is unity. From the Bohr titration curve, 0.68 proton is released during O2 binding at 0 degrees C. Sulfhemoglobin prepared from carboxypeptidase A-treated hemoglobin has an affinity for O2 which is about the same as that of sulfhemoglobin at the theoretical limit of the Bohr titration curve. Like its carboxypeptidase A-treated hemoglobin precursor, this sulfhemoglobin does not bind O2 cooperatively. Thus, sulfhemoglobin appears to be in a high affinity form at alkaline pH and a low affinity form at acid pH, similar to hemoglobin A. These results demonstrate that the magnitude of the Hill number is not always an indicator of the interaction between oxygen binding and other functions in a hemoglobin.

The source of a minor alpha 1-antitrypsin in variant serum.

An antiserum produced against human alpha1-antitrypsin gave 2 precipitin lines by immunodiffusion when tested against sera displaying MZ or MS phenotypes. Only one line (major antigen) was seen with sera displaying M phenotype, but a second line (minor antigen) became evident when the serum was concentrated 5-fold. The minor antigen appeared to be a denatured form of alpha1-antitrypsin, because the major antigen was converted to the minor one when purified alpha1-antitrypsin was incubated between pH 2.95 and 4.0. Such incubation inactivated the protein irreversibly. The purified protein was also inactivated completely within 1 hour at pH 4.95, but the activity was recovered completely by incubation for 2 to 4 hours at pH 8.0. The immunologic properties of the reactivated a1-antitrypsin were the same as those of the original untreated protein.

A homogeneous fluorescent immunoassay for human immunoglobulin M.

We describe a homogeneous substrate-labeled fluorescent immunoassay for human IgM. In this competitive-binding method we use a fluorogenic substrate for Escherichia coli beta-galactosidase, N-(6-aminohexyl)-7-beta-galactosyl-coumarin-3-carboxamide, which is covalently coupled to IgM. The fluorescence emission intensity of the labeled IgM at 450 nm (with excitation at 400 nm) is negligible, but when beta-galactosidase is added, the acetal linkage of the galactosyl moiety is hydrolyzed and the product has a greatly enhanced fluorescence. Formation of this fluorescent product is inhibited when antibody specific for IgM is bound to the labeled protein. In competitive-binding reactions, IgM from the serum competes with the labeled IgM for antibody binding sites and consequently the fluorescence produced by the enzymic reaction is related to the IgM concentration. The working range of the assay is between 0.5 and 5.0 g of IgM per liter when a 50-fold predilution of the sera is used. The assay does not cross react significantly with immunoglobulins G or A.

A solution hybridization assay for ribosomal RNA from bacteria using biotinylated DNA probes and enzyme-labeled antibody to DNA:RNA.

Rapid, convenient and non-isotopic nucleic-acid hybridization methods are needed for this technology to have practical use in clinical diagnostic tests. A method for hybridization of RNA with a DNA probe in solution followed by capture and measurement of the hybrid is described. DNA probes complementary to 23S rRNAs from Escherichia coli and Bacillus subtilis were labeled with a photoactivable biotin reagent. Hybridization of the biotinylated probes with rRNA was complete in less than 5 min. The resultant hybrids were allowed to bind simultaneously to succinylated avidin immobilized on latex and to beta-galactosidase-labeled Fab' fragments of a monoclonal antibody-specific for DNA:RNA. Finally, beta-galactosidase associated with the captured hybrids was measured colorimetrically. The hybridization method can detect less than 1000 bacteria per assay and has broad specificity to permit detection of the various genera of bacteria that infect the urinary tract.

Substrate-labeled fluorescent immunoassay for phenytoin in human serum.

A homogeneous substrate-labeled fluorescent immunoassay has been applied to the measurement of phenytoin concentrations in human serum. We coupled a fluorogenic enzyme substrate, galactosyl-umbelliferone, covalently to a derivative of phenytoin. Under assay conditions, this drug-substrate conjugate was nonfluorescent but became fluorescent upon hydrolysis catalyzed by bacterial beta-galactosidase. When antibody to phenytoin is bound to the drug-substrate conjugate, it is inactive as an enzyme substrate. Addition of phenytoin to competitive-binding reactions relieves the inactivation, and the resulting fluorescence is proportional to the phenytoin concentration. We validated the fluorescent immunoassay by comparing values for phenytoin obtained with this technique to those obtained by gas chromatography and by enzyme immunoassay (EMIT). All three methods correlated well. The major metabolite of phenytoin, 5-(p-hydroxyphenyl)-5-phenylhydantoin, and other drugs at concentrations expected in serum had no effect on the assay. The fluorescent immunoassay is rapid and simple to perform and requires only 2 microL of serum sample per test.

Homogeneous reactant-labeled fluorescent immunoassay for therapeutic drugs exemplified by gentamicin determination in human serum.

We applied a homogeneous reactant-labeled fluorescent immunoassay to the measurement of therapeutic drug concentrations in human serum, exemplified here by gentamicin. A derivative of umbelliferyl-beta-galactoside was coupled covalently to the drug and this conjugate was found to be nonfluorescent under assay conditions. The drug/dye conjugate was a substrate for bacterial beta-galactosidase and yielded a fluorescent product. When the drug/dye conjugate was bound to anti-gentamicin antibody it was inactive as an enzymatic substrate. This inactivation was relieved by the presence of gentamicin in competitive binding reactions. Hence, the rate of production of fluorescence was proportional to the gentamicin concentration. The fluorescent assay yielded values which compared favorably to a radioimmunoassay for gentamicin in clinical serum samples (r=0.94, standard error of estimate=0.66 mg/liter). The fluorescent assay requires only 1 microliter of serum and offers several advantages over existing techniques: sensitivity, specificity, simplicity, and the obviation of radioisotopes.

Evaluation of a new urease reagent strip for detection of Helicobacter pylori in gastric biopsy specimens.

OBJECTIVES: Determine sensitivity and specificity of a new urease reagent strip (URS) test for detection of Helicobacter pylori in gastric biopsy specimens. METHODS: Six paired biopsy specimens were obtained from the greater curvature of the distal antrum, the lesser curvature near the incisura, and the corpus along the greater curvature during 66 procedures on 59 patients with endoscopic findings of gastric antral mucosal erythema or erosions, or gastric or duodenal ulcers. One biopsy from each site was tested with the URS. The second was evaluated with histology. A final antral biopsy was evaluated with a urea/gel test. RESULTS: Twenty-eight of the 66 cases were histologically positive, with H. pylori observed in at least one of the three biopsy sites. The URS test correctly identified all 28. Of 193 individual biopsy specimens, 78 were positive for H. pylori. The URS correctly identified 77. Sensitivity was 0.99, specificity 0.95, positive predictive value 0.93, negative predictive value 0.99, and kappa 0.92. Average time to positive was 20 min. Twenty-seven antral biopsies were histologically positive for H. pylori. The URS test correctly identified all 27, whereas the urea/gel test correctly identified 21. For antral sites, URS sensitivity was 1.00 and specificity 0.95 versus urea/gel test sensitivity of 0.75 and specificity of 1.00. CONCLUSIONS: In this sample, the URS test is as accurate as histology in diagnosing H. pylori infections, and it provides results in less time and at a lower cost than histology.

Detection of bacteria by hybridization of rRNA with DNA-latex and immunodetection of hybrids.

A novel nucleic acid hybridization assay with a DNA probe immobilized on 1.25-micron-diameter latex particles was developed. Hybridization of the immobilized probe DNA with sample rRNA was complete in 10 to 15 min. Alkaline phosphatase-labeled anti-DNA-RNA was allowed to bind to the DNA-RNA hybrids on the latex particles. Then the latex was collected on a small glass fiber filter pad, and bound alkaline phosphatase was quantitated by reflectance rate measurement. The method detected a broad range of bacterial species and had a detection limit of 500 cells per assay. The assay was used to screen urine samples for bacteriuria and had a sensitivity of 96.2% compared with conventional culture at a decision level of greater than or equal to 10(4) CFU/ml. The hybridization method could have broad application to the detection of bacteria and viruses.