Biomedical subjects

Shealynn B Harris

Publications and source records attributed to Shealynn B Harris.

3 recordsLinked to original sources

PubMed2006

Performance characteristics of two real-time PCR assays for the quantification of Epstein-Barr virus DNA.

We compared the performance of a laboratory-developed 5'-nuclease real-time polymerase chain reaction assay and a commercial assay (LightCycler, Roche Diagnostics, Indianapolis, IN) for quantification of Epstein-Barr virus (EBV) DNA. Using standards provided by the manufacturer, the LightCycler assay was linear from 100 to 1 million copies per reaction. Based on dilution of a plasmid containing the amplicon, the laboratory-developed assay was linear from 22 to 45 million copies per reaction. Both assays detected 0.5 copies of genomic EBV DNA per reaction; both showed good reproducibility with coefficients of variation from 0.3% to 2.4% for the LightCycler and 1.8% to 5.1% for the laboratory-developed assay. For 31 whole blood specimens with measurable values in both assays, the viral load values obtained with the LightCycler averaged 2.3-fold higher than those obtained with the laboratory-developed assay. Testing 30 matched whole blood and plasma samples in the laboratory-developed assay showed whole blood viral load values averaged 10-fold higher than those for plasma. The LightCycler and laboratory-developed assays are sensitive and reproducible with broad linear ranges. Further clinical evaluation is needed to determine the viral load cutoff that is predictive of posttransplantation lymphoproliferative disorders.

DNA, Viral↗

PubMed2004

Unappreciated risk factors for transplant patients: HLA antibodies in blood components.

One of the more aggressive approaches in renal transplantation is the use of plasmapheresis (PP) and intravenous immunoglobulin to eliminate donor-directed human leukocyte antigen (HLA) alloantibodies. A potential complication of a PP protocol is iatrogenic hypocoagulability resulting from the removal of coagulation factors. To prevent bleeding, hypocoagulable patients may require transfusions with fresh frozen plasma (FFP) and/or cryoprecipitate (Cryo). Although HLA alloantibodies in these components have been linked to complications, such as transfusion-related acute lung injury (TRALI), whether they cause complications following transfusion into allograft recipients is unknown. The incidence of complications would be dependent, in part, upon the frequency of HLA alloantibodies in the various blood components. In this study, segments from 77 units of FFP, 66 units of Cryo, 106 units of packed red blood cells (RBCs), and 59 units of apheresis platelets (Plts) were tested for antibodies to HLA class I and class II antigens using FlowPRA, an HLA antigen-specific flow cytometric assay. On average, 22% of blood components tested contained HLA alloantibodies, tenfold greater than previously reported. This unappreciated frequency of HLA alloantibodies in blood components may pose a risk to transplant patients requiring transfusions by promoting allograft dysfunction or loss.

Antibodies↗

PubMed2002

Conundrums with FlowPRA beads.

In 1969, a study by Patel and Terasaki persuaded the renal transplant community that a pre-transplant cross-match should always be performed between donor and recipient to detect HLA antibodies and prevent hyperacute allograft rejection. Although the role of the cross-match among nonsensitized patients is controversial, its importance among sensitized recipients is undeniable. Over the past 30 years, more sensitive techniques, such as the flow cytometric cross-match (FCXM), were developed to identify low levels of antibodies undetectable by other approaches. The clinical relevance of a positive FCXM, however, has been hotly disputed, with some investigators maintaining that the FCXM is 'too sensitive' and rules out acceptable donor-recipient combinations. An alternative explanation is that the FCXM is non-specific, and, at least in certain situations, identifies non-HLA antibodies that are clinically irrelevant. Recently, a solid phase immunoassay utilizing purified HLA Class I or Class II molecules bound to microparticles (FlowPRA) was developed. Ideally, use of the FlowPRA for the identification of HLA antibodies in recipient sera would help ascertain whether a positive FCXM with donor cells was truly the result of an HLA-specific antibody. As shown here, this may not always be true. In this study, two unexpected serum patterns were observed. Pattern 1: FlowPRA beads were positive (with an associated HLA Class I specificity) and the FCXM with cells expressing the HLA antigen(s) to which the antibody was directed, was negative. Sequence analysis of the HLA antigens reactive with this unexpected antibody suggests that the epitope recognized resides on the floor of the groove, a site generally not expected to generate antibody activity. Pattern 2: FlowPRA beads were negative yet the FCXM was T and B cell positive. Further analysis of the FlowPRA negative/FCXM positive sera using a flow cytometric cell-based panel reactive antibody (PRA) approach revealed those sera to have specific anti-HLA Class I activity. We suspect that both types of antibodies described above have clinical relevance. Thus, a negative or positive FCXM (when the FlowPRA against donor antigens is positive or negative, respectively) is not always a straightforward interpretation.

B-Lymphocytes↗