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Detection of antibody directed against human leukocyte antigens (HLA) using a combination of flow cytometric crossmatch (FCXM) and antibody tests, is an important responsibility of Histocompatibility laboratories. Proficiency testing surveys utilize the results of these assays to assess concordance across multiple laboratories. In this study, we reviewed the ASHI Proficiency Testing (PT) antibody and crossmatching (AC) survey results obtained over a 6-year period, to evaluate the degree and nature of inter-laboratory FCXM and antibody assay variability. National and international laboratories representing 22 countries produced >10,000 T cell and >10,000 B cell FCXM results. Based on the 80% consensus threshold established for FCXM surveys, 92.5% T cell FCXM and 91.7% B cell FCXM results reached positive or negative consensus and were respectively consistent with the presence or absence of donor specific HLA antibodies (DSA) that reached a 90% consensus. The 7.5% of T cell and 8.3% of B cell FCXM results that did not reach consensus were associated with a combination of consensus and non-consensus DSA. This analysis shows that despite differences in testing protocols and algorithms, there is good consensus for the FCXM assay among laboratories. The data show correlation between FCXM and bead-based assays and support the use of both for reliable information when assessing immunological risk. [ABSTRACT FROM AUTHOR]

Since the first allogeneic hematopoietic stem cell transplantation (HCT) was performed by Dr. E. Donnall Thomas in 1957, the field has advanced with new stem cell sources, immune suppressive regimens, and transplant protocols. Stem cells may be collected from bone marrow, peripheral or cord blood from an identical twin, a sibling, or a related or unrelated donor, which can be human leukocyte antigen (HLA) matched, mismatched, or haploidentical. Although HLA matching is one of the most important criteria for successful allogeneic HCT (allo-HCT) to minimize graft vs host disease (GVHD), prevent relapse, and improve overall survival, the novel immunosuppressive protocols for GVHD prophylaxis offered improved outcomes in haploidentical HCT (haplo-HCT), expanding donor availability for the majority of HCT candidates. These immunosuppressive protocols are currently being tested with the HLA-matched and mismatched donors to improve HCT outcomes further. In addition, fine-tuning the DPB1 mismatching and discovering the B leader genotype and mismatching may offer further optimization of donor selection and transplant outcomes. While the decision about a donor type largely depends on the patient's characteristics, disease status, and the transplant protocols utilized by an individual transplant center, there are general approaches to donor selection dictated by donor-recipient histocompatibility and the urgency for HCT. This review highlights recent advances in understanding critical factors in donor selection strategies for allo-HCT. It uses clinical vignettes to demonstrate the importance of making timely decisions for HCT candidates. [ABSTRACT FROM AUTHOR]

Phosphorylation of signal transducer and activator of transcription 3 (STAT3) on a single tyrosine residue in response to growth factors, cytokines, interferons, and oncogenes activates its dimerization, translocation to the nucleus, binding to the interferon γ (gamma)-activated sequence (GAS) DNA-binding site and activation of transcription of target genes. STAT3 is constitutively phosphorylated in various cancers and drives gene expression from GAS-containing promoters to promote tumorigenesis. Recently, roles for unphosphorylated STAT3 (U-STAT3) have been described in response to cytokine stimulation, in cancers, and in maintenance of heterochromatin stability. However, the mechanisms underlying U-STAT3 binding to DNA has not been fully investigated. Here, we explore STAT3-DNA interactions by atomic force microscopy (AFM) imaging. We observed that U-STAT3 molecules bind to theGAS DNA-binding site as dimers and monomers. In addition, we observed that U-STAT3 binds to AT-rich DNA sequence sites and recognizes specific DNA structures, such as 4-way junctions and DNA nodes, within negatively supercoiled plasmid DNA. These structures are important for chromatin organization and our data suggest a role for U-STAT3 as a chromatin/genome organizer. Unexpectedly, we found that a C-terminal truncated 67.5-kDa STAT3 isoform recognizes single-stranded spacers within cruciform structures that also have a role in chromatin organization and gene expression. This isoform appears to be abundant in the nuclei of cancer cells and, therefore, may have a role in regulation of gene expression. Taken together, our data highlight novel mechanisms by which U-STAT3 binds to DNA and supports U-STAT3 function as a transcriptional activator and a chromatin/genomic organizer. [ABSTRACT FROM AUTHOR]

Abstract: Although developmental exposures of rats to low levels of the organophosphate pesticides (OPs), chlorpyrifos (CPF) or diazinon (DZN), both cause persistent neurobehavioral effects, there are important differences in their neurotoxicity. The current study extended investigation to parathion (PTN), an OP that has higher systemic toxicity than either CPF or DZN. We gave PTN on postnatal days (PND) 1–4 at doses spanning the threshold for systemic toxicity (0, 0.1 or 0.2mg/kg/day, s.c.) and performed a battery of emotional and cognitive behavioral tests in adolescence through adulthood. The higher PTN dose increased time spent on the open arms and the number of center crossings in the plus maze, indicating greater risk-taking and overall activity. This group also showed a decrease in tactile startle response without altering prepulse inhibition, indicating a blunted acute sensorimotor reaction without alteration in sensorimotor plasticity. T-maze spontaneous alternation, novelty-suppressed feeding, preference for sweetened chocolate milk, and locomotor activity were not significantly affected by neonatal PTN exposure. During radial-arm maze acquisition, rats given the lower PTN dose committed fewer errors compared to controls and displayed lower sensitivity to the amnestic effects of the NMDA receptor blocker, dizocilpine. No PTN effects were observed with regard to the sensitivity to blockade of muscarinic and nicotinic cholinergic receptors, or serotonin 5HT2 receptors. This study shows that neonatal PTN exposure evokes long-term changes in behavior, but the effects are less severe, and in some incidences opposite in nature, to those seen earlier for CPF or DZN, findings consistent with our neurochemical studies showing different patterns of effects and less neurotoxic damage with PTN. Our results reinforce the conclusion that low dose exposure to different OPs can have quite different neurotoxic effects, obviously unconnected to their shared property as cholinesterase inhibitors. [Copyright &y& Elsevier]

Abstract: The susceptibility to hepatocellular carcinoma (HCC) varies greatly within human populations in response to environmental risk agents. The mechanisms underlying differential susceptibility are still largely unknown and need to be clarified to improve HCC chemoprevention and therapeutic treatment. Inbred rodent strains with established predispositions for hepatocarcinogenesis offer the opportunity to identify intrinsic susceptibility and resistance factors. Previously, we have characterized mouse strains showing differential susceptibility to o-aminoazotoluene (OAT) and established that susceptibility does not result from OAT metabolism or genotoxicity in the livers of resistant and susceptible mice. In this study we have found that OAT differently affects hepatocyte proliferation in mice after partial hepatectomy (PH). OAT inhibited hepatocyte proliferation by 60–80% in the livers of susceptible mice, whereas resistant mice showed less than 15% inhibition. The inhibition resulted in significant delay of hepatic mass recovery in susceptible mice. OAT induced p53 stabilization and transcriptional activation in response to carcinogen treatment to the same degree in both, susceptible and resistant mice. Taken together, our data support inhibition of hepatocyte proliferation as a major cause for increased mouse susceptibility to hepatocarcinogenesis, and acceleration of functional liver recovery may offer a way to increase resistance to hepatic neoplasms. These results may have relevance to clinical observations of HCCs and implications for HCC chemoprevention and treatment. [Copyright &y& Elsevier]

Abstract: Rationale: Alpha2-adrenoreceptor (α2-AR) antagonists have been shown to improve, while α2-AR agonists impair cognitive function in subjects with functioning NMDA receptors (NMDAR). In subjects with inhibited NMDAR (a model of schizophrenia) α2-AR agonists attenuate the cognitive impairments. The effect with α2-AR antagonists remains unclear. Objectives: We investigated the effects of the α2-AR antagonist idazoxan on memory function in rats treated/not treated with NMDAR antagonist dizocilpine or a combination of dizocilpine and nicotine to clarify noradrenergic/cholinergic regulation of memory function. Methods: Female Sprague–Dawley rats (n =12) were trained for food reward on the radial maze. Working and reference memory errors and response latency were assessed after injections of idazoxan (0.5, 1.0 mg/kg), dizocilpine (0.05 mg/kg), nicotine (0.2, 0.4 mg/kg) or vehicle, alone or in combination. Results: Dizocilpine potently impaired memory. Nicotine (0.4 mg/kg) reversed this impairment. Idazoxan at the doses tested did not affect performance when given alone or with dizocilpine, but it did block the nicotine reversal of the dizocilpine-induced memory impairment. Three rats after 10–12 drug treatments developed limbic seizures. Our findings suggest that combination of drugs which block α2-AR with nicotinic agonists in schizophrenia may prevent therapeutic effect of nicotinic agonists and increase risk for convulsive activity with repeated administration. [Copyright &y& Elsevier]

Abstract: Developmental exposure to the organophosphorus pesticides chlorpyrifos and diazinon (DZN) alters serotonergic synaptic function at doses below the threshold for cholinesterase inhibition, however there are some indications that the two agents may differ in several important attributes. Previously, we found that low-dose chlorpyrifos exposure in neonatal rats causes lasting changes in emotional response and in the current study we did a comparable evaluation for DZN. Male and female Sprague–Dawley rat pups (N =10–12 of each sex per treatment group) were given 0, 0.5 or 2mg/(kgday) of DZN s.c. daily on postnatal days (PND) 1–4. These doses bracket the threshold for barely-detectable cholinesterase inhibition. Starting on PND 52, these rats began a battery of tests to assess emotional reactivity. In the elevated plus maze, there was a slight decrease in the time spent in the open arms for DZN-exposed males, while DZN-exposed females were not different from control females. In the novelty-suppressed feeding test, DZN-exposed males had significantly shorter latencies to begin eating than did control males, reducing the values to those normally seen in females. DZN-exposed rats of either sex showed reduced preference for chocolate milk in the anhedonia test that compared the consumption of chocolate milk to water. These findings show that neonatal exposures to DZN at a dose range below the threshold for cholinesterase inhibition nevertheless evokes specific, later alterations in emotional behaviors, particularly in males. The effects show not only some similarities to those of chlorpyrifos but also some differences, in keeping with neurochemical findings comparing the two agents. [Copyright &y& Elsevier]

Abstract: Background: Developmental neurotoxicity of organophosphorous insecticides (OPs) involves multiple mechanisms in addition to cholinesterase inhibition. We have found persisting effects of developmental chlorpyrifos (CPF) and diazinon (DZN) on cholinergic and serotonergic neurotransmitter systems and gene expression as well as behavioral function. Both molecular/neurochemical and behavioral effects of developmental OP exposure have been seen at doses below those which cause appreciable cholinesterase inhibition. Objectives: We sought to determine if developmental DZN exposure at doses which do not produce significant acetylcholinesterase inhibition cause persisting cognitive deficits. Methods: Rats were exposed to DZN on postnatal days 1–4 at doses (0.5 and 2 mg/kg/d) that span the threshold for cholinesterase inhibition. They were later examined with a cognitive battery tests similar to that used with CPF. Results: In the T-maze DZN caused significant hyperactivity in the initial trials of the session, but not later. In a longer assessment of locomotor activity no DZN-induced changes were seen over a 1-hour session. Prepulse inhibition was reduced by DZN exposure selectively in males vs. females; DZN eliminated the sex difference present in controls. In the radial maze, the lower but not higher DZN dose significantly impaired spatial learning. This type of nonmonotonic dose-effect function has previously been seen with CPF as well. The lower dose DZN group also showed significantly greater sensitivity to the memory-impairing effects of scopolamine a muscarinic acetylcholine antagonist. Conclusions: Neonatal DZN exposure below the threshold for appreciable cholinesterase inhibition caused persisting neurocognitive deficits in adulthood. The addition of some inhibition of AChE with a higher dose reversed the cognitive impairment. This non-monotonic dose–effect function has also been seen with neurochemical effects. Some of the DZN effects on cognition resemble those seen earlier for CPF, some differ. Our data suggest that DZN and CPF affect transmitter systems supporting memory function, differently, implying participation of mechanisms other than their common inhibition of cholinesterase. [Copyright &y& Elsevier]

Abstract: Molecular oxygen is involved in hydroxylation and subsequent degradation of HIF-1α, a subunit of HIF-1 transcription factor; therefore oxygen shortage (hypoxia) stabilizes this protein. However, HIF-1α can also be stabilized by transition metal ions in the presence of oxygen, suggesting that a different mechanism is involved in metal-induced hypoxic stress. Recently, we showed that the depletion of intracellular ascorbate by metals may lead to the inhibition of hydroxylases. Because nickel(II) has similarity to iron(II), an alternative hypothesis suggests that iron substitution for nickel in the enzyme inhibits hydroxylase activity. Here we investigated the induction of HIF-1 by another metal, chromium, which cannot replace iron in the enzyme. We show that chromium(VI), but not chromium(III), can oxidize ascorbate both in cells and in a cell-free system. In agreement with these data chromium(VI) stabilizes HIF-1α protein in cells only until it is reduced to chromium(III). In contrast, nickel(II) was found to be a catalyst, which facilitated continuous oxidation of ascorbate by ambient oxygen. These data correlate with extended stabilization of HIF-1α after acute exposure to nickel(II). The HIF-1-dependent reporter assays revealed that 20–24 h was required to fully develop the HIF-1 transcriptional response, and the acute exposure to nickel(II), but not chromium(VI), meets this requirement. However, repeated (chronic) exposure to chromium(VI) can also lead to extended stabilization of HIF-1α. Thus, the obtained data emphasize the important role of ascorbate in regulation of HIF-1 transcriptional activity in metal-exposed human lung cells. [Copyright &y& Elsevier]

Exposure to organophosphates (OP) has been associated with sleep disorders such as insomnia and “excessive dreaming.” The central mechanisms of these effects are not well understood. OPs inhibit acetylcholinesterase (AChE) activity, leading to a hyperactivity of the brain cholinergic systems that are involved in sleep regulation. We studied alterations in the EEG, behavioral states, motor activity and core temperature in rats orally administered with 10 or 40 mg/kg of the OP insecticide chlorpyrifos (CHP). Occipital EEG, motor activity and core temperature were recorded with telemetric transmitters. Behavioral sleep–wake states were visually scored. Both doses of CHP produced alterations of the EEG (decrease in power of σ/β and increase in slow θ and fast γ bands) characteristic of arousal. EEG alterations were consistent with behavioral changes such as an increase in wakefulness and a decrease in sleep. Waking immobility was a prevalent behavior. We did not detect any overt signs of CHP toxicity, such as an abnormal posture or gait, suggesting that reduced locomotion can be a result of central effects of CHP (such as activation of cholinergic motor inhibitory system) rather than peripheral (such as an impairment of neuromuscular function). Changes in the EEG and behavior occurred independently of the decrease in core temperature. Increased wakefulness together with reduced motor activity after exposure to CHP seems to be a result of hyperactivity in brain cholinergic neuronal networks. [Copyright &y& Elsevier]

Highlights from the article: Antibody-mediated rejection (AMR) caused by HLA donor-specific antibodies (DSA) is an important cause of graft failure after lung transplantation. To test whether high level DSAs could be treated with more aggressive protocol, three patients with strong DSA who needed AMR treatment underwent 4 double TPE sessions with bortezomib and IVIG as previously described by Patel et al. for pre-transplant desensitization.

Highlights from the article: A Transplant window was needed for a 53 year old, female Raynaud's lung pretransplant patient. By Single Antigen Bead (SAB) and FlowPRA testing she had a UNOS UA (Unacceptable donor HLA Antigen) A2 (13,000 MFI, Median Fluorescence Intensity) and Bw6 antibodies, 1100-3600 MFI. A concern was how strong the 1100-3600 MFI Bw6 antibodies actually were, as a shared epitope the MFI could be much stronger when spread over many HLA Bw6 determinants.

Aim Virtual crossmatch (VXM) has increased non-local organ utilization and improved clinical outcomes for sensitized lung patients. However, the concordance between VXM and cell-based crossmatch is not absolute because Single Antigen Bead (SAB) assay is prone to both false-negative and false-positive reactions. Since false-positive antibody specificities may unnecessarily deny an organ based on VXM, the goal of this study was to evaluate what additional testing may be warranted to recognize such reactions and to improve virtual crossmatch accuracy. Methods All sera were pre-treated with EDTA to inactivate complement in order to avoid prozon-like effect and were analyzed by SAB. A positive VXM was defined as the presence of donor specific antibodies based on SAB that would result in unacceptably positive Flow Crossmatch (FCXM). FCXM was done using 3-color analysis on a 1024 channel scale. All sera was retrospectively tested by FlowPRA Screen and LSPRA (phenotype bead) assays to rule out antibodies against denatured HLA epitopes detected by SAB. Results Of 58 consecutive VXM performed during July-December 2016 for lung candidates with CPRA > 10%, 28 had no DSAs or had acceptably weak DSAs and were predicted to result in negative or acceptably low positive FCXM. Other 30 VXM had one or more strong DSAs and were predicted to result in unacceptably positive FCXM and the organ offers were refused. Additional antibody testing showed that only 23 out of 30 VMXs should have been called positive. The other 7 VXM were called unacceptably positive due to the presence of antibody against denatured antigens. Three patients had antibodies against class I denatured antigens (MFI ranging 2500–3500) and four patients had antibodies against class II denatured antigens (MFI ranging 2000–14,000). After using LSPRA and FlowPRA Screen assays, the antibody against denatured antigens were removed from the list of UA and 5 out of 7 patients were successfully transplanted. Conclusions We found that at our center 12% VXM are false-positive due to the presence of antibodies against denatured antigens detected by SAB. Multiple antibody testing platforms, including SAB, LSPRA, and FlowPRA Screen, can be used for antibody characterization to avoid erroneous assignment of UA that may lead to an unnecessary organ offer refusal. [ABSTRACT FROM AUTHOR]

Aim: Reagent receipt and quality control is very important in each laboratory, our aim was to implement an easy and efficient system that would document the receipt of each reagent and automatically trigger QC testing prior to use. Methods: The following steps were implemented: 1) One key technologist was identified to oversee the process. 2) An electronic Excel-based reagent tracking spreadsheet was developed. The information recorded on the tracking spreadsheet includes: reagent name, lot number, order date, receipt date, date of QC, technologist performing QC, date of QC review, date the reagent was put in-use, and, last date the reagent was in-use. The spreadsheet is simple to complete and a filtering option was added for easy searching. 3) A procedure was written and competency was documented. 4) QC testing to be completed within one week of reagent receipt was assigned by the key technologist to a specific workbench. 5) A simplified reagent QC guideline was developed. 6) QC data were reviewed for acceptability by the key technologist. 7) All QC data were then approved by the supervisor. 8) An audit of the tracking system was performed monthly. Results: In January of 2013, after six months of implementing the system, we performed an audit of thirty random patient charts focusing on the completeness and documentation of reagent QC. The results of this audit revealed that all reagents used were documented and QC testing was complete. This represented a significant improvement over audit findings prior to the implementation of the tracking system. Conclusions: Streamlining reagent receiving and quality control testing is extremely important in every laboratory. Its successful implementation requires everyone’s participation. We have found that our restructured systematic process is not only easily monitored, but also 1)easily adhered to by all personnel, 2)saves time and facilitates inventory control, 3)avoids midnight reagent crises, and (best of all), 4)we are always ready for inspection. [Copyright &y& Elsevier]

Aim: Preliminary clinical data suggest that the C1q assay is a promising noninvasive technique for prediction of antibody mediated rejection (AMR). However, conflicting reports exist suggesting that assay performance and sensitivity vary between laboratories. Our aim was to evaluate a modified protocol for the commercially available C1qScreen™ assay to achieve increased sensitivity. We hypothesized that sensitivity of the assay could be increased by preincubation of serum with LABScreen beads prior to incubation with C1q. The rationale for this modification was that individual heads on a C1q molecule bind to the Fc region with weak affinity. Antibody aggregation on an antigenic surface facilitates binding of several heads significantly enhancing C1q affinity to low-MFI antibody. Methods: The standard and modified C1q assays were performed in parallel. The difference between protocols was that in the standard C1qScreen™ protocol, heat-inactivated serum was spiked with human C1q and incubated with LABScreen beads for 20 min at RT. In the modified protocol, heat-inactivated serum was first incubated with LABScreen beads for 20 minutes followed by a 20 minute incubation with C1q. The rest of the assay was performed as described in the standard protocol. Results: Of 30 samples tested, 17 samples that were positive for class I and 19 samples that were positive for class II C1q-binding antibody by both protocols. Some specificities that were weak or negative for C1q-binding using the standard protocol were strongly positive using the modified protocol. Also, of 13 ClassI and 11 ClassII samples that were negative for C1q-binding antibody by the standard protocol, 5 and 2 samples, respectively, were positive by the modified protocol. Conclusions: Preincubation of serum with LABScreen beads prior to addition of human C1q facilitates increased sensitivity for detection of C1q-binding antibody. Further investigation will reveal whether increase in sensitivity offers better prediction of AMR. [Copyright &y& Elsevier]

Abstract: In the epileptic brain, hippocampal dentate granule cells become synaptically interconnected through the sprouting of mossy fibers. This new circuitry is expected to facilitate epileptiform discharge. Prolonged seizures induce the long-lasting neoexpression of neuropeptide Y (NPY) in mossy fibers. NPY is released spontaneously from recurrent mossy fiber terminals, reduces glutamate release from those terminals by activating presynaptic Y2 receptors, and depresses granule cell epileptiform activity dependent on the recurrent pathway. These effects are much greater in rats than in C57BL/6 mice, despite apparently equivalent mossy fiber sprouting and neoexpression of NPY. This species difference can be explained by contrasting changes in the expression of mossy fiber Y2 receptors; seizures upregulate Y2 receptors in rats but downregulate them in mice. The recurrent mossy fiber pathway may synchronize granule cell discharge more effectively in humans and mice than in rats, due to its lower expression of either NPY (humans) or Y2 receptors (mice). [Copyright &y& Elsevier]

Highlights from the article: Next Generation Sequencing (NGS) has given HLA Laboratories the opportunity to obtain high-resolution HLA genotyping with minimal ambiguities at a reduced cost. During the sample selection a representation of our sample population with a mix of homozygous samples as well as rare typings were considered, leading to a collection of 150 DNA samples. This included 50 BMT samples that were previously typed by SBT/SSP for 6 loci, 50 deceased donors and SOT patients previously typed by rSSO/SSP, and 30 proficiency samples from ASHI and CAP.

Abstract: We have shown previously that activation of STAT1 contributes to the pathogenesis of Wilms tumor. This neoplasm caricatures metanephric development and is believed to originate from embryonic renal mesenchymal progenitors that lose their ability to undergo mesenchymal–epithelial transition (MET). Therefore, we hypothesized that STAT1 is also activated and functional during metanephric development. Here we have demonstrated that both STAT1 and STAT3 are activated during normal development of the embryonic kidney. Furthermore, activation of STAT1 stimulated the proliferation of metanephric mesenchymal cells, but it prevented MET and tubulogenesis induced by leukemia inhibitory factor, which preferentially activates STAT3. Consistent with its negative regulation of metanephric mesenchymal differentiation, inhibition of STAT1 activation with protein kinase CK2 inhibitor TBB or RNAi-mediated knockdown of STAT1 promoted differentiation of metanephric progenitors and abolished the effect of cytokine-induced STAT1 activation in these cells. Additionally, a cell-permeable peptide that inhibits STAT1-mediated transactivation by targeting the STAT1 N-domain also blocked cytokine-induced STAT1-dependent proliferation in metanephric progenitors and promoted LIF-induced MET and tubulogenesis. Finally, the STAT1 peptide inhibitor caused the down regulation of survival/anti-apoptotic factors, Mcl-1 and Hsp-27, and induced apoptosis in renal tumor cells with constitutively active STAT1, indicating that STAT1 is required for these cells to survive. These findings show that both metanephric progenitors and renal tumor cells utilize a STAT1-dependent mechanism for growth or survival. [ABSTRACT FROM AUTHOR]

Abstract: The objective of this study was to investigate the CYP1A1 and CYP1A2 mRNAs and enzyme activities in mouse liver during induction with o-aminoazotoluene (OAT) as well as the capability of the hepatic S9-fraction from OAT-treated mice to induce its own activation to mutagens in the Ames test using S. typhymurium strain TA98. The data obtained indicate that when used at appropriate doses, OAT is a PAH-type inducer of mouse hepatic microsomal monooxygenases, which activity is not less than that of the known inducer 3,4-benzo[α]pyrene. In the absence of S9-fraction enzymes no OAT-mediated mutagenicity was observed in the Ames test. In the presence of the S9-fraction from OAT-pretreated mice, OAT induced as high revertant numbers, as it did in the presence of the S9 fraction from the liver of Aroclor 1254-treated mice. Thus, OAT does induce the enzymes of its own mutagenic activation in mouse liver. [Copyright &y& Elsevier]

Aim The accuracy of HLA antibody testing is very important for entering UNOS Unacceptable donor antigens, evaluating organ offers by virtual and prospective crossmatch, and evaluating donor specific antibodies (DSAs) pre and post transplant. It is known that the Single Antigen Bead (SAB) assay can have HLA antibody false positives reactions, due to the exposure of cryptic, non HLA antigens, during the manufacturing process. This study was to determine the SAB false positive rate in our testing and evaluate how two other HLA antibody assays, FlowPRA Screen and LSPRA (phenotype bead), can help to improve the accuracy, by confirming or ruling out false positive SAB antibodies. Methods A Jan-March 2017 sample set of pre and post transplant patients with SAB HLA antibodies and LSPRA and/or FlowPRA confirmatory testing, was obtained from our HLA database. The three assays were compared to determine SAB false positive rate and how to improve the accuracy of this assay. Results Of 20 patients with SAB HLA antibodies, CPRA 10–99%, which also had a confirmatory LSPRA test, 95% (19) had one or more SAB false positive HLA antibodies. The number of false positive patient SAB antibodies ranged from 1 to 14, with a median of 3, MFI 900–3000. In 60% (12) of the patients, all of the SAB identified antibodies were all false positive, ranging from 1 to 14, with a median of 3. In 40% (8) of the patients, the LSPRA assay was able to confirm SAB HLA antibodies ranging from 1 to 20, with a median of 4. Conclusions It is unacceptable that alone, a SAB assay will result in about 3 false positive antibodies in 95% of patients who have HLA antibodies. The SAB assay is a very important test, but unless other testing platforms, like Phenotype beads and FlowPRA screen, are used to identify the false positive antibodies, the SAB assay will lose lifesaving organ offers for our patients or trigger unnecessary DSA treatments post transplant. [ABSTRACT FROM AUTHOR]

Aim The LABScreen single antigen bead (SAB) assay is routinely used for HLA antibody detection and allows for testing of multiple patient samples. Despite this, standard SAB assay is time intensive, taking 1.5–2 hr to perform, which is not optimal for urgent testing. Utilizing a multicenter study we compared the rapid optimized SAB (ROB) protocol (Liwski et al. ASHI 2014), with a 30 min turn-around time (TAT), to standard SAB LABScreen method. Methods Ten HLA labs (BCM, DCI, Edmonton, Halifax, Georgetown, Winnipeg, METC, PSBC, SWI, UPMC) participated in the study. Five ASHI 2014 AC-1 survey sera (AC 460-464) were tested in each lab with standard SAB LABScreen (SSL) vs the ROB protocol. Class I LS1A04 (lot 8) and class II LS2A01 (lot 10) HLA kits were used. Mean fluorescence intensity (MFI) values were collected. Protocols were compared with respect to the mean MFI, specificities reaching consensus (⩾90% labs; 2000 MFI cutoff) and coefficient of variation (%CV). Results HLA antibody reactivity patterns were similar between the protocols with an excellent MFI correlation (r2>0.99) and signal to noise differential. Average MFI was slightly reduced with the ROB protocol (by 5.0%). The number of specificities reaching consensus (⩾90% labs; 2000 MFI cutoff) was similar between protocols (97 SSL vs 94 ROB). When the ROB protocol cutoff was lowered to 1500 MFI, the number of consensus specificities improved to 99, including all 97 SSL specificities (Fig. 1). Importantly, the average %CV of raw bead MFI was significantly improved with the ROB protocol (ROB: 21.2+/−3.4 vs SSL: 34.4+/−5.6; p< 0.001; Fig. 2). A slight increase in background was noted with the ROB protocol (AC 462 serum) in some labs. However, this had no impact on antibody assignment and improved with one additional wash step. Conclusions ROB protocol reduces SAB LABScreen assay time by 70% and improves precision without compromising sensitivity or specificity. Implementation of the ROB protocol will expedite antibody testing in urgent cases and improve TAT for routine testing. [ABSTRACT FROM AUTHOR]