Highly Sensitized Candidates Remain at Risk for Microvascular Inflammation Even When Donor-specific Antibody Is Avoided: A Matched Cohort Study.

BACKGROUND: Microvascular inflammation (MVI) is a key feature of antibody-mediated rejection (AMR) among patients with HLA donor-specific antibody (DSA), but MVI at AMR thresholds (Banff glomerulitis [g] + peritubular capillaritis [ptc] score ≥ 2) without DSA has been increasingly recognized. We aimed to determine the incidence of MVI among highly sensitized kidney transplant recipients without DSA. METHODS: We performed a single-center, retrospective, matched cohort study comparing outcomes of kidney transplant recipients with cPRA ≥90% with preexisting DSA (n = 49), cPRA ≥90% without preexisting DSA (n = 47), and matched controls with cPRA = 0 without preexisting DSA (n = 49). Controls were matched by age, donor type, and transplant date. Indication and surveillance biopsies combined with annual de novo DSA screening were obtained. RESULTS: Kidney transplant recipients with a cPRA ≥90% and no evidence of preexisting or de novo DSA had a higher incidence of MVI (glomerulitis + peritubular capillaritis ≥ 2) than patients with cPRA = 0 [35% (17/49) versus 12% (6/49), P = 0.0003] over a median (interquartile range) follow-up of 5 (4-6) y posttransplant. Among this cPRA ≥90% group without DSA, MVI persisted in 54% of cases on follow-up biopsy (7/13), and 24% (4/13) of cases developed transplant glomerulopathy (Banff cg score > 0). CONCLUSIONS: Highly sensitized transplant recipients have a high incidence of persistent and progressive MVI, even without DSA. The mechanisms underlying these histologic features needs to be elucidated, but this information is important to consider when making decisions about transplantation among highly sensitized individuals.

Bedside Ultrasound-Guided Percutaneous Cholecystostomy in Critically Ill Patients-Outcomes in 51 Patients.

Purpose The aim of this study was to report technical and clinical success of bedside ultrasound-guided percutaneous cholecystostomy (PC) tube placement in intensive care unit (ICU). Materials and Methods This is a retrospective study of 51 patients (36 males:15 females, mean age: 67 years) who underwent ultrasound-guided PC from May 2015 to January 2020. The indication for cholecystostomy tube placement, comorbidities, imaging finding, technical success, clinical success, timing of surgery post-cholecystostomy tube placement, indwelling catheter time, complications, and follow-up were recorded. Results Indications for cholecystostomy tube placement were acute calculous cholecystitis ( n = 43; 84.3%), perforated cholecystitis ( n = 5; 9.8%), and emphysematous cholecystitis ( n = 3; 5.9%). Most of the patients had multiple comorbidities; these were diabetes mellitus, hypertension, cardiovascular disease, chronic renal disease, underlying malignancy, and multisystem disease with sepsis. All patients had undergone PC through transhepatic approach under ultrasound guidance in ICU. Technical success rate of the procedure was 100%. Clinical success rate was 92.1% (47/51) and among these 44/51 (86.2%) patients underwent definitive elective cholecystectomy, 3/51 (5.9%) patients had elective tube removal. Three of fifty-one (5.9%) patients did not improve; among these two underwent emergency surgery, while there was 1/51 (1.9%) mortality due to ongoing sepsis and multiorgan dysfunction. There were no procedure-related mortalities or procedure-related major complications. One patient had bile leak due to multiple attempts for cholecystostomy placement. Mean tube indwelling time was 13 days (range: 3-45 days). Conclusion Ultrasound-guided PC can be safely performed in ICU in critically ill patients unfit for surgery with high technical and clinical success rates. Early laparoscopic cholecystectomy should be preferred after stabilization of clinical condition following cholecystostomy.

A Review of Laboratory Practices Using the HLA-B27 Survey by the College of American Pathologists: How Important Is Allele-Level Typing?

CONTEXT.­: Ankylosing spondylitis (AS) is an autoimmune disorder with a strong genetic risk, especially with HLA-B27. Clinical testing for HLA-B27 has been used to help diagnose patients with signs and symptoms of AS. Testing methods used by clinical laboratories for HLA-B27 fall into the broad categories of serologic/antibody- or molecular-based methods and have evolved over time. The College of American Pathologists (CAP) offers a proficiency testing survey for HLA-B27. OBJECTIVE.­: To analyze HLA-B27 testing trends and their performance in the past decade, using the proficiency testing survey data submitted to CAP. DESIGN.­: We analyzed the HLA-B27 CAP proficiency testing data from 2010 to 2020 for the method used, participant concordance, and error rates. Results from case scenarios to understand evolving scientific data around HLA-B27 risk alleles were also analyzed. RESULTS.­: Antibody-based flow cytometry is the most common method, though it has decreased from 60% in 2010 to 52% in 2020, with a corresponding increase in molecular methods. Among the molecular methods, real-time polymerase chain reaction has increased from 2% to 15%. Flow cytometry had the highest error rate (5.33%), and sequence-specific oligonucleotide (0%) is the most accurate (0%). Results of case scenarios demonstrated that most participants understood that allele-level HLA-B27 typing results inform clinical interpretation, for example HLA-B*27:06 is not associated with AS. CONCLUSIONS.­: These data demonstrated the changing trends for HLA-B27 testing during the past decade. HLA-B27 allelic typing provides a better understanding of AS association. This is possible by testing for the second field with methods like next-generation sequencing.

A Primer on Gene Editing: What Does It Mean for Pathologists?

CONTEXT.­: Gene editing-based therapies are currently in development in the areas of oncology, inherited disease, and infectious disease. These potentially life-altering therapies are derived from decades of research in both academic and industry settings that developed technologies rooted in principles and products of nature. However, with such technologic developments come many important considerations, including adverse risks, high cost, and ethical questions. OBJECTIVE.­: To educate pathologists about gene editing technologies, inform them of potential indications and risks, outline regulatory and practical issues that could affect hospital-based practice and laboratory testing, and advocate that pathologists need to be present at discussions among industry and regulators pertaining to gene editing-based therapies. DESIGN.­: A Gene Editing Workgroup, facilitated by the College of American Pathologists Personalized Health Care Committee and consisting of pathologists of various backgrounds, was convened to develop an educational paper to serve as a stimulus to increase pathologist involvement and inquiry in gene editing therapeutic and diagnostic implementation. RESULTS.­: Through multiple discussions and literature review, the workgroup identified potential gaps in pathologists' knowledge of gene editing. Additional topics that could impact pathology and laboratory medicine were also identified and summarized in order to facilitate pathologists as stakeholders in gene editing therapy administration and monitoring and potential use in diagnostics. CONCLUSIONS.­: Gene editing therapy is a complex but potentially transformative area of medicine. This article serves as an introduction to pathologists to assist them in future discussions with colleagues and potentially identify and alter pathology practices that relate to gene editing.

A Primer on Gene Editing.

CONTEXT­: Gene editing-based therapies are currently in development in the areas of oncology, inherited disease, and infectious disease. These potentially life-altering therapies are derived from decades of research in both academic and industry settings that developed technologies rooted in principles and products of nature. However, with such technologic developments come many important considerations, including adverse risks, high cost, and ethical questions. OBJECTIVE­: To educate pathologists about gene editing technologies, inform them of potential indications and risks, outline regulatory and practical issues that could affect hospital-based practice and laboratory testing, and advocate that pathologists need to be present at discussions among industry and regulators pertaining to gene editing-based therapies. DESIGN­: A Gene Editing Workgroup, facilitated by the College of American Pathologists Personalized Health Care Committee and consisting of pathologists of various backgrounds, was convened to develop an educational paper to serve as a stimulus to increase pathologist involvement and inquiry in gene editing therapeutic and diagnostic implementation. RESULTS­: Through multiple discussions and literature review, the workgroup identified potential gaps in pathologists' knowledge of gene editing. Additional topics that could impact pathology and laboratory medicine were also identified and summarized in order to facilitate pathologists as stakeholders in gene editing therapy administration and monitoring and potential use in diagnostics. CONCLUSIONS­: Gene editing therapy is a complex but potentially transformative area of medicine. This article serves as an introduction to pathologists to assist them in future discussions with colleagues and potentially identify and alter pathology practices that relate to gene editing.

Clinical impact of KIR haplotypes in 10/10 HLA-matched unrelated donor-recipient pairs undergoing allogeneic hematopoietic stem cell transplantation.

To evaluate the impact of killer immunoglobulin-like receptor (KIR) genotyping in allogeneic hematopoietic stem cell transplantation for myeloid disorders at our institution, retrospective KIR genotyping was performed on 77 patients and their 10/10 matched unrelated donors. In a multivariate model including donor age, HLA-DPB1 permissiveness, and presence of donor KIR B/x, an association with overall survival was observed (p = .047). Within the model, increasing donor age increased risk (RR 1.03 [1.00-1.06]/year, p = .046), while donor KIR and HLA-DPB1 permissiveness were not associated with risk (RR 0.51 [0.26-1.03] and RR 0.68 [0.34-1.36]). Grouping recipients by conditioning regimen or limiting the analysis to recipients of peripheral blood stem cells, no association between donor KIR and survival or relapse was identified. No significant associations were observed between overall survival, relapse, grade III-IV acute, or chronic graft versus host disease and presence of KIR B (B/x), quantity of donor KIR B haplotype motifs, or centromeric KIR type (all p > .05).

Seventy-five years of service: an overview of the College of American Pathologists' proficiency testing program in histocompatibility and identity testing.

The Histocompatibility and Identity Testing Committee offers an overview of the College of American Pathologists' (CAP) Proficiency Testing (PT) program, commemorating its significant 75th anniversary in 2024. The CAP PT program has undergone significant growth and evolution over the years, ultimately achieving Centers for Medicare and Medicaid Services approval. In 1979, CAP's partnership with the American Association for Clinical Histocompatibility Testing marked a pivotal moment, leading to the creation of the first proficiency testing survey in 1980. This laid the foundation for various PT programs managed by the CAP Histocompatibility and Identity Testing Committee, including HLA antibody testing, HLA molecular typing, engraftment monitoring, parentage/relationship testing, HLA disease associations and drug risk, and HLA-B27 typing. Each program's distinctive considerations, grading methodologies, and future prospects are detailed here, highlighting the continual evolution of histocompatibility and identity testing PT to support emerging technologies and evolving laboratory practices in the field.

Human Leukocyte Antigen (HLA) Testing in Pharmacogenomics.

The genetic region on the short arm of chromosome 6 where the human leukocyte antigen (HLA) genes are located is the major histocompatibility complex. The genes in this region are highly polymorphic, and some loci have a high degree of homology with other genes and pseudogenes. Histocompatibility testing has traditionally been performed in the setting of transplantation and involves determining which specific alleles are present. Several HLA alleles have been associated with disease risk or increased risk of adverse drug reaction (ADR) when treated with certain medications. Testing for these applications differs from traditional histocompatibility in that the desired result is simply presence or absence of the allele of interest, rather than determining which allele is present. At present, the majority of HLA typing is done by molecular methods using commercially available kits. A subset of pharmacogenomics laboratories has developed their own methods, and in some cases, query single nucleotide variants associated with certain HLA alleles rather than directly testing for the allele. In this chapter, a brief introduction to the HLA system is provided, followed by an overview of a variety of testing technologies including those specifically used in pharmacogenomics, and the chapter concludes with details regarding specific HLA alleles associated with ADR.

Death With Function and Graft Failure After Kidney Transplantation: Risk Factors at Baseline Suggest New Approaches to Management.

BACKGROUND: Improving both patient and graft survival after kidney transplantation are major unmet needs. The goal of this study was to assess risk factors for specific causes of graft loss to determine to what extent patients who develop either death with a functioning graft (DWFG) or graft failure (GF) have similar baseline risk factors for graft loss. METHODS: We retrospectively studied all solitary renal transplants performed between January 1, 2006, and December 31, 2018, at 3 centers and determined the specific causes of DWFG and GF. We examined outcomes in different subgroups using competing risk estimates and cause-specific Cox models. RESULTS: Of the 5752 kidney transplants, graft loss occurred in 21.6% (1244) patients, including 12.0% (691) DWFG and 9.6% (553) GF. DWFG was most commonly due to malignancy (20.0%), infection (19.7%), cardiac disease (12.6%) with risk factors of older age and pretransplant dialysis, and diabetes as the cause of renal failure. For GF, alloimmunity (38.7%), glomerular diseases (18.6%), and tubular injury (13.9%) were the major causes. Competing risk incidence models identified diabetes and older recipients with higher rates of both DWFG and nonalloimmune GF. CONCLUSIONS: These data suggest that at baseline, 2 distinct populations can be identified who are at high risk for renal allograft loss: a younger, nondiabetic patient group who develops GF due to alloimmunity and an older, more commonly diabetic population who develops DWFG and GF due to a mixture of causes-many nonalloimmune. Individualized management is needed to improve long-term renal allograft survival in the latter group.

Outcomes of Allogeneic Hematopoietic Cell Transplantation in T Cell Prolymphocytic Leukemia: A Contemporary Analysis from the Center for International Blood and Marrow Transplant Research.

T cell prolymphocytic leukemia (T-PLL) is a rare, aggressive malignancy with limited treatment options and poor long-term survival. Previous studies of allogeneic hematopoietic cell transplantation (alloHCT) for T-PLL are limited by small numbers, and descriptions of patient and transplantation characteristics and outcomes after alloHCT are sparse. In this study, we evaluated outcomes of alloHCT in patients with T-PLL and attempted to identify predictors of post-transplantation relapse and survival. We conducted an analysis of data using the Center for International Blood and Marrow Transplant Research database on 266 patients with T-PLL who underwent alloHCT between 2008 and 2018. The 4-year rates of overall survival (OS), disease-free survival (DFS), relapse, and treatment-related mortality (TRM) were 30.0% (95% confidence interval [CI], 23.8% to 36.5%), 25.7% (95% CI, 20% to 32%), 41.9% (95% CI, 35.5% to 48.4%), and 32.4% (95% CI, 26.4% to 38.6%), respectively. In multivariable analyses, 3 variables were associated with inferior OS: receipt of a myeloablative conditioning (MAC) regimen (hazard ratio [HR], 2.18; P < .0001), age >60 years (HR, 1.61; P = .0053), and suboptimal performance status, defined by Karnofsky Performance Status (KPS) <90 (HR, 1.53; P = .0073). Receipt of an MAC regimen also was associated with increased TRM (HR, 3.31; P < .0001), an elevated cumulative incidence of grade II-IV acute graft-versus-host disease (HR, 2.94; P = .0011), and inferior DFS (HR, 1.86; P = .0004). Conditioning intensity was not associated with relapse; however, stable disease/progression was correlated with increased risk of relapse (HR, 2.13; P = .0072). Both in vivo T cell depletion (TCD) as part of conditioning and KPS <90 were associated with worse TRM and inferior DFS. Receipt of total body irradiation had no significant effect on OS, DFS, or TRM. Our data show that reduced-intensity conditioning without in vivo TCD (ie, without antithymocyte globulin or alemtuzumab) before alloHCT was associated with long-term DFS in patients with T-PLL who were age ≤60 years or who had a KPS >90 or chemosensitive disease.

Prozone rates in the solid-phase platelet crossmatch assay and correlation with class I HLA antibody levels.

BACKGROUND: Solid-phase platelet crossmatch (PXM) testing is used to help manage patients with platelet transfusion-refractoriness. Recently, we published the first report of false-negative PXM results from prozone effect that was mitigated using sample dilution. This study aimed to describe the prevalence of PXM prozone effect and the levels of class I HLA antibodies (HLA-Abs) associated with positive PXM results and with false-negative PXM results from prozone effect. STUDY DESIGN AND METHODS: A cross-sectional study of patients undergoing PXM testing from July 2019 through December 2020 was performed. All PXM tests were run simultaneously using undiluted and 1:4 diluted patient plasma. Prozone effect was defined as a negative PXM result using undiluted patient plasma but a positive PXM result using 1:4 diluted patient plasma. RESULTS: Among 59 patients, 830 individual ABO-compatible PXM results yielded an overall positivity rate of 25.8% (214/830) and a false-negative rate from prozone effect of 4.7% (10/214). Among the 28 patients with class I HLA-Ab testing and no other anti-platelet antibodies, maximum HLA-Ab mean fluorescence intensity (MFI) was significantly associated with a positive PXM result (p < .0001; AUC approx. 0.9) and categorized into negative (<3700), indeterminate (3700-10300), and positive (>10300) maximum HLA-Ab MFI zones. Maximum HLA-Ab MFI, however, was not associated with prozone effect (p = .17; AUC approx. 0.6). DISCUSSION: While there is a strong predictive association between class I HLA-Ab levels and positive PXM results, PXM prozone effect is a common occurrence not associated with class I HLA-Ab levels, so additional testing with diluted samples should be considered.

Heart-After-Liver Transplantation Attenuates Rejection of Cardiac Allografts in Sensitized Patients.

BACKGROUND: In patients undergoing heart transplantation, significant allosensitization limits access to organs, resulting in longer wait times and high waitlist mortality. Current desensitization strategies are limited in enabling successful transplantation. OBJECTIVES: The purpose of this study was to describe the cumulative experience of combined heart-liver transplantation using a novel heart-after-liver transplant (HALT) protocol resulting in profound immunologic protection. METHODS: Reported are the results of a clinical protocol that was instituted to transplant highly sensitized patients requiring combined heart and liver transplantation at a single institution. Patients were dual-organ listed with perceived elevated risk of rejection or markedly prolonged waitlist time due to high levels of allo-antibodies. Detailed immunological data and long-term patient and graft outcomes were obtained. RESULTS: A total of 7 patients (age 43 ± 7 years, 86% women) with high allosensitization (median calculated panel reactive antibody = 77%) underwent HALT. All had significant, unacceptable donor specific antibodies (DSA) (>4,000 mean fluorescence antibody). Prospective pre-operative flow cytometric T-cell crossmatch was positive in all, and B-cell crossmatch was positive in 5 of 7. After HALT, retrospective crossmatch (B- and T-cell) became negative in all. DSA fell dramatically; at last follow-up, all pre-formed or de novo DSA levels were insignificant at <2,000 mean fluorescence antibody. No patients experienced >1R rejection over a median follow-up of 48 months (interquartile range: 25 to 68 months). There was 1 death due to metastatic cancer and no significant graft dysfunction. CONCLUSIONS: A heart-after-liver transplantation protocol enables successful transplantation via near-elimination of DSA and is effective in preventing adverse immunological outcomes in highly sensitized patients listed for combined heart-liver transplantation.

Is the level of HLA eplet mismatch a risk factor for graft loss among kidney transplant recipients who have already formed de novo donor specific antibody?

Eplet mismatches are associated with de novo DSA (dnDSA) and antibody mediated rejection (ABMR) among the general kidney transplant population. However, it is unclear whether the level of eplet mismatch can be used for risk stratification among patients with dnDSA. We performed a retrospective observational study of kidney transplant recipients with dnDSA (n = 44) transplanted between 10/2007 and 5/2014 to evaluate eplet mismatch as a risk factor for ABMR and allograft loss among dnDSA patients. High resolution typing was inferred from by imputation based on ethnicity and NMDP haplotypes, and the eplet mismatch was calculated using the Epvix algorithm. Biopsies (N = 151) from 95.3%(42/44) of patients were reviewed. The mean (SD) eplet mismatch was 69.8(22.8). The ABMR incidence was 71.4% (30/42) and 5 year death censored allograft survival was 67.4% during the mean (SD) follow-up of 5.3 (3.1) years. ABMR and death-censored allograft survival were not correlated with eplet mismatch among dnDSA patients. However, medication adherence and dnDSA MFI < 3000 were associated with reduced ABMR incidence. Among patients with both of these favorable characteristics, only 35.7% (15/42) developed ABMR. In conclusion, the level of eplet mismatch does not correlate with ABMR or allograft loss among high risk kidney transplant patients with dnDSA.

Daratumumab interference in flow cytometric anti-granulocyte antibody testing can be overcome using non-human blocking antibodies.

BACKGROUND: Daratumumab (DARA), a human IgG1K monoclonal antibody targeting CD38, is used to treat refractory multiple myeloma patients. CD38 is expressed on many cell types (RBCs, granulocytes, lymphocytes, etc.), and thus, DARA can interfere with serological tests. Information regarding how DARA affects anti-granulocyte antibody (AGA) testing and optimal neutralization of DARA will help laboratories perform accurate testing. METHODS: Screening of AGA was performed by the granulocyte agglutination test (GAT) and the flow cytometric granulocyte immunofluorescence test (Flow-GIFT). Samples were tested from patients on DARA (n = 7), non-transfused blood donors (healthy controls, n = 7) and AGA reactive samples (positive controls, n = 5). Two neutralization experiments, CD38 removal with DTT and DARA epitope blockage with mouse anti-CD38, were evaluated. RESULTS: Positive reactivity of human IgG binding was observed in 5/7 DARA cases when tested by Flow-GIFT; however, all 7 cases had negative GAT agglutination results. Further studies by Flow-GIFT revealed DARA concentrations >0·63 µg/ml bound to granulocytes. DARA binding was negated by DTT though a reduced Flow-GIFT sensitivity was observed in positive control samples due to increased background detection of human IgG. Mouse anti-CD38 neutralized the detection of human IgG observed in DARA-treated patient serum without effecting controls. CONCLUSION: We established that DARA can interfere with AGA testing, leading to false positive Flow-GIFT results without causing GAT agglutination. DTT treatment increased background binding of secondary antibodies causing a decrease in Flow-GIFT sensitivity. In comparison, blockage of the DARA binding epitope using mouse anti-CD38 antibody was effective in neutralizing DARA interference while maintaining Flow-GIFT sensitivity.

Chronic Histologic Changes Are Present Regardless of HLA Mismatches: Evidence from HLA-Identical Living Donor Kidney Transplants.

BACKGROUND: At 5 and 10 y after kidney transplantation, chronic histologic changes such as arteriolar hyalinosis and mesangial expansion are common; however, determining cause is difficult. We compared surveillance biopsies in living donor kidney transplants (LDKTx) from HLA-matched siblings (termed HLA-identical [HLA-ID]) with HLA non-ID to investigate which histologic changes were likely due to alloimmune injury and which were due to nonalloimmune injury. METHODS: We performed a retrospective, cohort study comparing HLA-ID sibling LDKTx (n = 175) with HLA non-ID LDKTx (n = 175; matched for age, sex, and year of transplant ±2 y) performed at a single institution from March 1999 to November 2018. RESULTS: Baseline characteristics and maintenance immunosuppression were similar. Mortality rates were similar, but in the HLA-ID group, 10-y death-censored graft survival was higher (93.8% versus 80.9% HLA non-ID LDKTx; P < 0.001), rejection rates were lower (after 1 y 9.6% versus 27.1%; P < 0.001), and Banff inflammation scores including glomerulitis and peritubular capillaritis were lower on surveillance biopsies at 1, 5, and 10 y. In contrast, chronic Banff scores (interstitial fibrosis, arteriolar hyalinosis, mesangial expansion, etc) were similar in prevalence and severity on surveillance biopsies at 1, 5, and 10 y. CONCLUSIONS: HLA-ID LDKTx have less inflammation and less transplant glomerulopathy, but most chronic histologic changes were similar to less well-matched LDKTx. We conclude that these types of chronic changes are not associated with HLA mismatches and may be due to nonimmunologic causes (hypertension, obesity, etc), suggesting that new management approaches to prevent these lesions may be needed.