David Freedom

Lifetime Alumni Member at the Univerity of Illinois.Currently with 3 PW Groups at Gift of Hope.Agent Hope Business Continuity Disaster Recovery(BCDR) PW group, Research and Development PW Group and our Laboratory PW group.

Poster: Single-antigen bead analysis: A highly accurate predictor of the outcome & strength of the flow cytometric crossmatchAndres Jaramillo · Bozena Labuda · John Turon · David Freedom ·Daniel MagasAbstract: Aim: There are conflicting reports on the accuracy of single-antigen beads (SAB) to predict the outcome & strength of the flow cytometry crossmatch (FCXM) and to identify unacceptable antigens (UA). Thus, the aim of this study was to perform a statistical analysis to determine: 1) the correlation between DSA strength and the FCXM outcome & strength, 2) the additive effect of multiple DSAs on the FCXM outcome & strength, and 3) the correlation between UAs of different strength on the overall FCXM outcome & strength. Methods: Two hundred and fifty eight (258) sensitized patients and 458 deceased donors were included in this study. The FCXMs (n=994) were performed on pronase-treated lymph node cells and expressed as the mean channel shift (MCS). All sera were analyzed by the LABScreen SAB assay (One Lambda). Then, the DSAs were grouped according to their mean flourescence intensity (MFI) as follows: 5000. The statistical analysis were performed using the Prism 5.0 Software (GraphPad). Results: There was a significant strength-dependent effect of a single HLA class I and II DSA on the T and B-cell FCXM outcome & strength, respectively (p>0.0001). In addition, There was a significant additive effect of multiple HLA class I and II DSAs on the T- and B-cell FCXM outcome & strength, respectively (p>0.0001). Moreover, there was a significant strength-dependent effect of HLA class I and II UAs on the overall T- and B-cell FCXM outcome & strength, respectively (p>0.0001). Conclusions: SABs represent a highly accurate tool to predict the outcome & strength of the FCXM and to control the positive FCXM rates using the UNOS’ UA algorithm. Show lessPoster · Nov 2009    

Poster: Significant Increase of Kidney Transplants in Highly Sensitized Patients by Implementation of the Virtual CrossmatchAndres Jaramillo · Martin D. Jendrisak · Bozena Labuda · John Turon · Sujata GaitondeAbstract: The concept of the virtual crossmatch (V -XM) has been discussed by the transplant community for more than 20 years. However, only recently, with the improved sensitivity of the antibody identification assays, has the V-XM become common practice. The solid-phase technology is now widely used to identify unacceptable antigens for the UNOS-calculated PRA (cPRA) formula. In this regard, since 2008 all transplant centers in Illinois have used solid-phase technology to identify HLA specificities. Also, all crossmatching for deceased donors is performed at the local organ procurement organization, Gift of Hope Organ & Tissue Donor Network. Because of these technological developments, the preliminary CDC crossmatch was replaced by the V-XM in January 2009 using the HLA antibody results and unacceptable antigens provided by transplant centers. Our research team assessed this policy’s impact on deceased donor kidney allocation.   

Poster · Oct 2011 Poster: The OPO Experience with (Almost) Real Time HLA Typing by Real-Time PCRSam Ho · Bozena Labuda · David Freedom · Martin D Jendrisak ·Andrés JaramilloAbstract: Purpose: Gift of Life Michigan (GOLM) and Gift of Hope Organ & Tissue Donor Network (GOH) are among the few organ procurement organizations (OPO) who possess an in-house laboratory providing full-service deceased donor testing (i.e. human leukocyte antigen (HLA) testing, blood typing and sub-typing, final crossmatch, and infectious diseases testing) for each respective OPO donation service area. Deceased donor testing is frequently performed after normal business hours with the laboratory staff constantly struggling with multitasking and time constraints. Here we report our collective experience with a new HLA typing platform recently adopted by both laboratories, which is based on the real-time polymerase chain reaction (RT-PCR) technology, and its impact on various aspects of the organ allocation process. Methods: GOLM and GOH adopted the RT-PCR HLA testing platform (Linkage Biosciences’ LinkSeq HLA-ABCDRDQDP+ 384-well typing kit) for deceased donor workup since September and December, respectively, of 2012. Prior to this new platform, GOLM used the PCR-SSP-gel-electrophoresis (SSP) method (Invitrogen SSP UniTray low- and high-resolution typing kits), whereas GOH used the PCR-rSSOP (SSO) method (Lifecodes SSO typing kits). Results: As of February of 2014, over 960 deceased donors combined have been typed by both laboratories using the RT-PCR method. The RT-PCR typing system we adopted not only types for the HLA loci required by the OPTN policies on deceased donors (HLA-A, B, C, DRB1/3/4/5 and DQB1), it also includes the typing for DPB1, DQA1 and DPA1. In addition, it meets or exceeds our accrediting agency’s (ASHI) requirements on histocompatibility testing of deceased donors (e.g. typing resolution; the identification of common null alleles). The cost of typing reagents for the RT-PCR method is $230/donor, which is comparable to that of the SSP-gel at $240/donor and the SSO method at $210/donor. On the other hand, the turn-around time for the RT-PCR method is approximately 3.5 hours, which is considerably less than the SSP method at 5.5 hours and the SSO method at 4.0 hours. To date, no repeat testing has been performed by either of the laboratories due to reaction dropout since more than half of the 245 unique reaction wells on each RT-PCR typing tray are duplicated. The drawback of implementing the RT-PCR method, however, is the need of a costly real-time PCR instrument (or two for redundancy in deceased donor workup; ~$40,000/each) which may be prohibitive for many laboratories. Conclusions: The RT-PCR HLA typing platform appears to be an excellent fit to the workflow for OPO laboratories where time-sensitive deceased donor testing is frequently performed. The additional HLA typing information provided by this system may facilitate the practice of virtual crossmatch and its accuracy; reduce the rate of organ offer refusal due to unexpected positive crossmatches and improve the post-transplant clinical management of the recipients.  

Poster · Jun 2014  ASHI Conference Paper: AUTOMATED FLOW CYTOMETRY CROSSMATCH USING THE BIOTEK ELx50 MICROPLATE WASHER David Freedom · Daniel Magas · Katarzyna Brooks · Bozena Labuda· Andres JaramilloConference Paper · Oct 2014   Poster: HLA-DQB1*03:25: A rare allele with a serological equivalency of HLA-DQ4Zeying Duorfid · Jaishree Patel · David Freedom · Brian Norton ·Andrés JaramilloAbstract: Aim HLA-DBQ1∗03:25, a rare allele of Hispanic origin identified in 2008, has not yet been assigned a serological equivalent. A recent observation in our center of a 75 year old Hispanic female patient typed as DQB1∗03:25 who also displayed HLA-DQ7, -DQ8 and -DQ9 antibodies triggered an investigation of the serological equivalency for this allele. Methods Molecular HLA typing was performed by LabType SSO (One Lambda), LinkSeq Real-Time PCR HLA typing (Linkage BioSciences) and LIFECODES HLA SSO Typing (Immucor). Serological HLA typing was performed by Lambda Monoclonal Trays (LMT) HLA class II typing tray (One Lambda). Antibody analysis was performed by LABScreen Single Antigen HLA class I and class II kits (One Lambda) and LIFECODES class I and class II ID kits (Immucor). Results The patient was initially typed by LabType SSO as HLA-A∗02, 03; B∗39, 44; C∗04, 07; DRB1∗07, 09; DQB1∗02:02, 03:25. Further DNA typing by real-time PCR SSP confirmed the same results. DQB1 typing further confirmed by LIFECODES SSO typing. Since the patient presented HLA-DQ7, -DQ8 and -DQ9 antibodies, serological HLA class II typing was subsequently performed yielding the following. Results DR7, 9; DQ2, 4. DNA and protein sequence alignments of the DQB1∗03:25 allele with other DQB1 alleles showed that its sequence is quite similar with the DQB1∗04:03 allele, but differs significantly from the DQB1∗03 alleles. The sequence difference between DQB1∗03:25 and DQB1∗04:03 is only one amino acid at codon 9 in exon 2 (Tyr vs. Phe). Thus, DQB1∗03:25 shows almost identical immunogenicity to DQB1∗04:03 in the context of their sequences. When compared with the first case of HLA-DQB1∗03:25 in IMGT/HLA database, it was noticed that both cases carry the DRB1∗09-DQB1∗03:25 haplotype. Conclusion This is the first time to confirm that the serological equivalent of DQB1∗03:25 is DQ4. In this regard, accurate antigenic entry of deceased donor HLA typing into UNET is critical since it affects multiple areas of organ allocation, particularly given the increased utilization of virtual crossmatching to determine the compatibility of a potential donor prior to organ allocation. In addition, based on our knowledge, this is the second case of the DQB1∗03:25 allele in the Hispanic population. It suggests an expanded population study needed to document this allele well.   

Poster · Oct 2015  Article: P089 Enhanced resolution in rapid HLA typing of deceased donors: A single center experience using a new Taqman-based qPCR approachJohn Lunz · Marilyn Wetmore · Ben Passey · David Freedom ·Gordon HillAbstract: Aim Organ donation is a highly orchestrated process with the initial donor testing, including donor HLA typing, needing to be completed as rapidly as possible. The need for increased donor HLA typing resolution is continually growing. However, true high-resolution typing is time and resource prohibitive for deceased donors. Real-time PCR-based HLA typing assays have improved turn-around-times and are increasingly able to provide higher-resolution typing. Here we examine the performance of a new Taqman-based qPCR approach as compared to an endpoint melt curve based assay. Methods 80 DNA samples were HLA typed by first generation QTYPE (Olerup) and LinkSeq 1575 (Linkage Biosciences) for HLA-A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1 and DPB1. Comparisons were made between assays for the number of common and well-documented (CWD) alleles (based on the IMTG/HLA 3.19.1 database) and the number of single antigen bead (SAB) specificities that could be resolved from the CWD typing. Results Average Roche Lightcycler 480 II run time was 42 min for the QTYPE assay and 75 min for LinkSeq. Compared to the endpoint melt curve-based LinkSeq assay, the TaqMan-based QTYPE assay defined CWD alleles at an equal or better resolution for HLA-A 13/18 loci, HLA-B 17/27 loci, HLA-C 13/15 loci, 17/17 HLA-DRB1, 3/3 HLA-DRB3/4/5 loci, 6/7 HLA-DQB1 loci, 4/6 HLA-DQA1 loci, 3/3 HLA-DPA1 and all HLA-DPB1. The number of SAB alleles that each CWD allele string was able to detect was determined using both Immucor and One Lambda SAB reagents. QTYPE resolved the multi-SAB antigens: A30, A34, A68, B13, Cw10, DR1, DR11, DR13, DR14, DR16, DRB3, DRB5, and DQ2 to a single SAB allele, while the LinkSeq assay detected CWD alleles that covered 1 + SAB alleles. A2, B44, DR4, DQ6 covered 1 + SAB in both tests, but QTYPE, on average, had better resolution for these SAB antigens. Other SAB were covered by 3 or more CWD alleles. All DPB1 were uniquely resolved by both assays except for DPB1∗04:02/105:01 in some LinkSeq tests. Conclusion The advantageous multiplexing capacity of the TaqMan based qPCR HLA typing permits greater resolution and, in general, resolves SAB alleles more consistently than the endpoint melt curve based assay. With increasing numbers of CWD HLA alleles, the TaqMan approach provides a greater potential for expansion and CWD allele discrimination. J. Lunz:Grant/Research Support; Company/Organization; Olerup. M. Wetmore: Employee; Company/Organization; Olerup. B. Passey: Employee; Company/Organization; Olerup SSP AB. D. Freedom: Grant/Research Support; Company/Organization; Olerup. G. Hill:Employee; Company/Organization; Olerup. · Sep 2016 · Human Immunology