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During the coronavirus disease 2019 (COVID-19) pandemic, mannequin models have been developed to mimic viral spread using fluorescent particles. These models use contraptions such as a spray gun or an exploding latex balloon to emanate a sudden acceleration of particles, simulating a “cough” reflex. No models have been developed to mimic passive aerosolization of viral particles during a cardiopulmonary arrest simulation. Our novel approach to aerosolization of simulated viral spread allows for a continuous flow of particles, which allows us to maintain components of high-fidelity team-based simulations. Our simulated model emanated GloGerm (Moab, UT) from the respiratory tract using a continuous nebulization chamber. Uniquely, the construction of our apparatus allowed for the ability to perform full, simulated cardiopulmonary resuscitation scenarios (such as chest compressions, bag-mask ventilation, and endotracheal intubation) on a high-fidelity mannequin while visualizing potential contamination spread at the conclusion of the simulation. Positive feedback from users included the ability to visualize particulate contamination after cardiopulmonary resuscitations in the context of personal protective equipment usage and roles in resuscitation (i.e. physician, respiratory therapist, nurse). Negative criticism towards the simulation included the lack of certain high-fidelity feedback markers of the mannequin (auscultating breath sounds and checking pulses) due to the construction of the particle aerosolization mechanism.

BACKGROUND: We sought to describe adverse events associated with unplanned extubation (UE) and to explore risk factors for serious adverse events post-UE among infants who experienced UE. METHODS: Data were prospectively collected on all infants who had a UE event at a single institution over a 4-y period. Demographic information and information on outcomes were obtained retrospectively. We described the frequency of post-UE adverse events: success or failure of extubation trial if offered, rate of re-intubation, post-UE changes in ventilator settings, and serious adverse events post-UE (eg, need for cardiopulmonary resuscitation, clinical sepsis, and death or tracheostomy prior to discharge). We used a multivariate logistic regression model to identify the risk factors associated with serious adverse events. RESULTS: There were 134 documented UE events. Agitation was the most common known cause. After UE, 49% of the subjects were given a trial of extubation, and 65% of the trials were successful at 48 h. Cardiopulmonary resuscitation (CPR) was performed in 13% of cases. In subjects requiring immediate re-intubation, mean airway pressure (Paw) and oxygen requirement increased in 33% and 55% of the subjects, respectively. Post-UE clinical sepsis occurred in 17% of subjects. Higher pre-UE Paw and difficult re-intubation were associated with a need for CPR. Subjects who received CPR had increased odds (3.7×) of developing clinical sepsis. CONCLUSION: UE can result in serious adverse events, including hemodynamic instability and possibly an increased risk for clinical sepsis. Difficult re-intubation was associated with a higher risk of needing CPR and, later, tracheostomy and death.

Aim The complement interfering (CI) effect has been shown to cause falsely decreased or negative result in the Luminex LabScreen assay in solid organ transplantation. Complement bound by anti-HLA antibodies blocks the access of reporting antibody to anti-HLA antibodies. Pretreatment of serum with EDTA can abolish the CI effect by chelating calcium cations required for complement complex association. Only a few studies demonstrated the CI effect in hematopoietic stem cell transplant (HSCT) setting despite the fact that the presence of donor specific antibody (DSA), especially complement binding DSA, is associated with inferior engraftment and overall survival in HSCT recipients. We validated the EDTA pretreatment of serum to improve the quality of HLA antibody testing in both PRA and SAB tests using the samples from HSCT patients. Methods 21 and 20 specimens were selected and underwent PRA or SAB test with and without EDTA pretreatment, respectively. Dilution studies (1:8) were performed on all samples to verify the CI effect or other prozone effect by an alternative method. 9 SAB samples with high PRA were tested additionally with C1q test. The positive effect was defined as MFI (EDTA or Dilution) – MFI (Neat) × 1.2 ⩾ 400. Results In both SAB and PRA tests, 99–100% of the samples/beads detected with CI effect using the dilution method were confirmed by the EDTA method. In contrast, only 50–60% CI positive samples by EDTA method remained positive using Dilution method. In SAB assay, many antibodies tested negative with dilution (MFI r 2 = 0.91 vs 0.85, respectively). A lower regression slope is seen with the Dilution method (0.663) compared to that with EDTA method (1.093) due to the dilutional effect. Moreover, no significant prozone effect was observed in the non-complement binding antibodies suggested by negative C1q test. Conclusions High prevalence of CI effect was seen in the HSCT setting. Most if not all antibodies demonstrating the prozone effect were complement fixing. Compared to dilution method, EDTA treatment provides a better resolution in identifying CI effect and a single dilution is not sufficient to reveal all the CI effects.

Introduction Accurate HLA typing for patients in need of allogeneic stem cell transplant is crucial for finding a matched donor & improving graft outcome by preventing graft-versus-host disease. We report here a case of mystery while HLA typing for a patient. Method We routinely perform Luminex SSO typing of HLA-class I & II loci for the initial sample first, followed by sequence-based typing (SBT) on the CT sample per SSO results for appropriate selection of SBT primers. The case Day 1, two peripheral blood (PB) (1 for initial, 1 for CT) collected 90 min apart by 2 phlebotomists were received. SSO typing was obtained successfully from 1st PB. Day 2, buccal swabs were received with an indication of circulating blasts in patient’s blood. SBT were performed on the buccal DNA per SSO results on 1st PB, typing was obtained for only those with locus primers but failed with group primers. Moreover, the SBT results didn’t match with those of the 1st PB. We SSO-typed the 2nd PB (CT sample), the typing matched with those of the 1st PB but not the buccal. Day 3, a PB sample of the patient’s son was received, typing results showed one haplotype match with the patient’s buccal results but not with any of the 2 PB results. A sample switch? The first two PB were re-extracted, typing results matched with those of previously obtained, suggesting that no sample switch had occurred in the lab. Is it possible a sample switch at collection sites? It seems very unlikely per the collection procedures. Where is the problem? Medical history was reviewed and showed that the patient had a history of low WBC counts and had multiple granulocyte infusions prior to HLA sample collection including one in less than 22 h to the 1st PB to HLA. Typing of the granulocyte donor were conducted and results were concordant with those of the 2 PB. Retyping of the 1st PB by SBT using appropriate group primers confirmed a low level of patient’s DNA that was masked by the much higher levels of granulocyte donor’s DNA. Another buccal sample from 3 weeks later showed concordance with the first buccal typing. Conclusion It is very important for HLA laboratory to know the transfusion history and previous transplant history in order to apply appropriate test procedures. It’d be imperative and good practice to recommend to testing buccal swab sample for every patient in need of allogeneic stem cell transplant.

Introduction Unambiguous, high resolution HLA typing plays a central role in hematopoietic stem cell transplantation. Current methodologies, primarily Sanger SBT, often fails to resolve all ambiguities creating the need for additional testing and longer turnaround times. Next generation sequencing (NGS) has the potential to address these shortcomings by providing full length, phase resolved sequences of HLA alleles with a single test. The Immucor Mia Fora NGS TM is an HLA typing kit and analysis software package that allows for typing of multiple samples per run in multiplex using the Illumina platform. In this study, we evaluated the NGS system against Sanger SBT methods. Methods NGS was performed on 11 HLA genes (HLA -A, B, C, DQA1/B1, DPA1/B1, and DRB1/3/4/5) for 69 subjects (1221 alleles) in alpha test, 69 (1223 alleles) in beta, and 35 (618 alleles) in further test. Automated liquid handling instruments were used for long range PCR and library preparation. Size selection was performed using the Pippin Prep followed by real-time PCR quantification on the ABI 7500. The final library was sequenced on the Illumina using the 300-cycle v2 kit. FASTQ files were analyzed with the Mia Fora software. NGS alleles to the 3rd fields were compared to the typing obtained by SBT, SSO, and SSP. Results A panel of 173 subjects carrying 3062 HLA class I and II alleles (total of 369 unique HLA alleles including novel alleles) was selected and typed by NGS. The overall concordance between NGS (automatic allele assigned by the software without editing) and Sanger SBT typing ranged from 98.5–99.7% in class I and 96.7–100.0% in class II. After manual review using the software’s tools for flagged alleles, the overall concordance will reach even higher. Conclusions The current NGS HLA typing product is able to produce robust and reliable allelic level HLA typing at all 11 HLA class I and II loci with no or very few ambiguities. Discordance was primarily observed in class II, most notably certain DRB1 ∗ 04 and DQB1 ∗ 03 alleles which often suffered from low read coverage in the presence of other alleles, and the ability to assign novel alleles or ambiguous results (e.g. DPB1) by the software. Improvements on test condition to prevent potential preferential amplification of certain HLA alleles would improve even more the accurate assignment of alleles at all HLA loci.

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Accurate HLA typing and matched donor selection is essential for graft outcome in patients receiving a stem cell transplant. Misidentification of new alleles can result in graft failure or acute graft vs. host disease. This study identifies eleven novel HLA alleles at class I and class II loci. Method Unusual or potentially novel HLA alleles were detected during sequence specific oligonucleotide (SSO) intermediate resolution typing. Characterization of novel alleles was performed by sequence based typing (SBT) using group specific and locus specific primers. Results Eleven novel HLA sequences were identified and characterized in 14 individuals, including HLA-A∗11:01:64 in a patient and a haploidentical sibling, C∗04:195 in a patient and a fully matched sibling, and DRB1∗03:110 in two haploidentical sibling donors. Nine of the eleven novel HLA alleles resulted in an amino acid substitution. The null allele C∗01:98 N resulted in a premature stop codon in exon 2. Six out of the eleven novel alleles have nucleotide/amino acid changes in exon 4 and the rest in exon 2. Table 1 shows the differences of nucleotides and amino acids between these novel alleles and their most homologous allele and the location of the changes. Discussion Identification and characterization of novel HLA alleles is important for the selection of appropriate stem cell transplant donors aimed at improving graft outcome and patient survival. Download : Download full-size image

Aim Identification of novel HLA alleles has been vital in population studies and in HLA matching for patients in need of stem cell transplantation in order to search for the best matched donor, thereby improving graft outcome. The goal of the study was to describe the characterization of two newly identified HLA alleles. Methods Unusual or potential new variants of HLA alleles were identified using PCR/probe based methods, and characterized by SBT using group/allele specific primers. Results Two novel HLA class I sequences have been identified and characterized in three individuals (Table 1) including one novel null allele of HLA-A (A∗33:73 N) in a patient & a haploidentical sibling, and one novel HLA-C allele (C∗03:218) in a donor. HLA-A∗33:73 N differs from A∗33:03:01 by one nucleotide (NT) “C” insertion between NT 627-628, resulting in a stop at coding 196. HLA-C∗03:218 has one nt change from C∗03:04:01:01 at NT 637, where G->T (codon 189.1 GTG->TTG), resulting in a coding change, 189 valine (V) is changed to leucine (L) (Table 2). Conclusions Identification of null HLA allele is important in order to accurately perform virtual crossmatch and to select the best donor for HLA sensitized patients in need of an organ transplant. The mismatch between the alleles HLA-C∗03:04:01:01 and C∗03:218 may be better tolerated because the latter has non-immunogenic change in exon 4.

Aim HLA-C∗04:09 N is characterized by a point deletion at nucleotide position 1095 in exon 7. This mutation results in a frame shift and causes the addition of 32 extra amino acids and the loss of cell surface expression (Balas A., et al. Tissue Antigens 2002;59:95–100). The goal of this study was to determine the frequency of the B∗44:03-C∗04:09 N bearing haplotype in leukemia patients treated at MDACC. Method Patients were typed for both HLA Class I and Class II using PCR-SSOP and PCR-SBT methodologies. The HLA-C∗04:09 N allele was resolved using PCR-SSP method. Results We identified and evaluated 300 consecutive patients with various forms of leukemia treated at our center carrying the HLA-B∗44:03-C∗04:01:01G typing with the inclusion of C∗04:09 N allele (2.2% of the total patient population). Of the 300 B∗44:03-C∗04:01:01G bearing patients, 21 were identified as HLA-C∗04:09 N (7%), which accounted for 0.15% in the entire patient population. Discussion Of the 21 patients bearing HLA-B∗44:03-C∗04:09 N alleles, 9 had haplotype assigned. Five of them (55.6%) carrying the haplotype of HLA-A∗23:01-C∗04:09 N-B∗44:03:01-DRB1∗07:01-DRB4∗01:01-DQB1∗02:02 and 2 others carrying the same haplotype but with different HLA-A alleles (Table 1). The study explored the frequency of HLA-C∗04:09 N in the presence of B∗44:03 in leukemia patients. The identification of this null allele would be important in HLA matching for patients in need of stem cell transplant. Misidentification of a patients null allele could impact graft outcome with a mismatched donor carrying the expressed allele or visa versa.

Aim Killer cell immunoglobulin-like receptor (KIR) genes are receptors expressed on the surface of natural killer cells. KIR genes recognize polymorphic epitopes of HLA class I-A, B & C, called KIR HLA ligands (HLA-L). After binding specific ligands the KIR may transmit inhibitory or activating signals. NKC sense & kill target cells missing MHC class I molecules. Studies showed that hematopoietic stem cells (HSC) transplant patients may benefit from NK alloreactivity of the donor or HLA-C alleles (C1/x) of the recipients. Previously we reported the diversification of KIR HLA-L in CBU from MD Anderson Cord Bank. The goal of this study was to further explore the frequency (Freq) of HLA-L in selected CBU from the bank and their transplant recipients aimed at providing information for finding the best donor and improving graft outcome. Methods 640 CBU selected from MD Anderson Cord Bank and 589 recipients were included. HLA typing of class I & II loci were obtained using PCR/probe based methods & sequence-based typing as needed. Freq of Bw4, C1, and C2 group of HLA-C alleles were examined. Results KIR3DL1 recognizes allotypes with HLA-Bw4 motif; KIR2DL1 recognizes HLA-C epitope with lysine at codon 80 (group 2-C2); while KIR2DL2 and KIR2DL3 recognize C alleles with asparagine at residue 80 (group 1-C1). Among the 640 CBU and 589 recipients, 70.0% and 72.0%, respectively, carry Bw4 bearing phenotypes. HLA-C1 bearing phenotypes (C1/x) were found to be predominated in both CBU (85.2%) and their transplant recipients(81.5%). Missing HLA-C1 was lower (C2/C2, 18.5%) than missing C2 (C1/C1, 33.1%) in recipients (Table 1). Conclusion Here we show that CBU can be classified based on their KIR repertoire (licensed versus unlicensed) and the presence of KIR-ligand mismatch with the recipient. Analysis of graft outcome in these HSC transplant recipients is underway. The ultimate goal of our study is to develop algorithms based on the KIR profile of CB units to select the units with maximum antileukemic activity to improve the outcome of CBU transplant.

Aim Since SBT technologies were applied in HLA typing, more and more new nucleotide mutations have been being identified and reported as new HLA alleles. However, some of the nucleotide mutations may not exist in the patient’s somatic cells, but (but or because) they are caused by tumor blast cells in the peripheral blood. If so, should the typing results be reported as new alleles? In this study, we present three cases in which nucleotide mutations were found in the whole blood from leukemia patients, but not in their buccal cells. Methods DNAs were isolated from peripheral whole blood (PB) specimens, and buccal specimens separately, as needed. Sequencing-based typing (SBT) was performed by using locus/group/allele specific primers to separate the heterozygous alleles. Results Initial SBT results with DNAs from PB showed case #1 carries HLA -B ∗ 14:02:01, 18:01:01 with 1 mutation in codon 203.1 (from TGC to YGC) and the patient with AML had 47% blasts in PB; Case #2 carries HLA-B ∗ 44:02:01 with 1 additional “A” in codon 171.3 (from TAC to TAM) and the patient with CML had 59% blasts in PB; and case #3 carries HLA-A ∗ 02:01:01 with 1 additional “T” in codon 96.1 (from CAG to YAG) and the patient with lymphoma had 71% abnormal lymphocytes with irregular nuclei. None of these patients had previous transplant history. Confirmatory SBT results with DNAs from buccal cells indicate that none of those nucleotide mutations above exist (Table 1). Conclusions The accurate HLA typings are crucial to the outcome of stem cell transplantation. Whenever an unusual HLA typing from peripheral blood of a cancer patient is identified, the result of blood differentials should be checked and the confirmatory testing with buccal specimen should be followed.

Aim Natural killer cells (NKC) express many different cell surface receptors including KIR. KIR genes recognize polymorphic HLA class I including HLA-A, B & C allotypes. The KIR genes after binding specific ligands may transmit inhibitory or activating signals. NKC sense & kill target cells lacking MHC class I molecules & release various cytokines on activation. UCB is an increasingly used source of HSC for pts without matched adult donor for Tx. The goal of the study was to attain frequencies (freq) of HLA-L in UCBU aimed at providing information (info) for finding UCB with NK benefit for HSC Tx pts. Methods Total of 21,002 UCBU from Cord Bank at MD Anderson was included. HLA typing of class I & II loci were obtained using PCR/probe based methods & sequence-based typing as needed. Freq of Bw4 alleles, C1 and C2 HLA-C alleles were obtained. Results KIR3DL1 recognizes allotypes with HLA-Bw4 serological motif; KIR2DL1 recognizes HLA-C allotypes with lysine at codon 80 (group 2 - C2); & KIR2DL2/KIR2DL3 recognize C alleles with asparagine at residue 80 (group 1 - C1). Among the 21,002 UCBU, 70.7% carry Bw4 alleles. UCB bearing C1/C2 heterozygous genotype (GT) accounted for 45.6%, C1/C1 homozygous GT for 39.6%, & C2/C2 GT for 14.7% only. Conclusions Incompatibility of KIR HLA-L between pt & donor has been shown to impact graft outcome (GO) in HSC Tx. NKC from the donor greatly contribute to eradication of leukemia blasts escaping the preparative regimen & to clearance of residual host dendritic cells & T lymphocytes thus preventing graft-versus-host disease & graft rejection. This study showed high freq of Bw4-bearing GT, increased freq of C1/C2, & C1/C1 but not C2/C2 GT in UCBU. These freq info could be useful for prediction of the possibility for HSC Tx pts, especially those with Bw6 or C2/C2 GT, to find suitable UCB donor(s) with beneficial NK reactivity, thereby improving GO. Further study of diversification of KIR HLA-ligands in pts would be warranted to assess the importance of these findings.

Aim Identification of novel HLA alleles has been invaluable contributions to the literature of HLA genes. New HLA sequences will be imperative in population study & in transplantation. The goal of the study was to describe the characterization of newly identified HLA alleles. Methods Unusual or potential new variants of HLA alleles were identified using PCR/probe based methods, and characterized by SBT using group/allele specific primers & then sequenced. Results Novel HLA class I sequences have been identified and characterized including three new HLA-A alleles in patients and one in a healthy individual, one HLA-B allele in a patient, and one HLA-C allele in a donor. The profiles of these novel alleles are shown in Table 1. Conclusions Two new HLA-A alleles, A∗02:397 & A∗11:129, have amino acid (AA) substitution from their potential progenitor alleles in exon 4, meaning these changes are not likely to affect peptide binding or T cell receptors. The other four alleles, A∗01:114, A∗32:46, B∗44:160, and C∗07:307, had AA substitutions in either exon 2 or exon 3. These changes could affect peptide binding and/or T cell receptors. Therefore, mismatch for these alleles could result in an increased risk for graft versus host disease or graft rejection in stem cell transplant, or possibly elicit anti-HLA antibodies to these alleles in patients with stem cell or solid organ transplantation to their donors, thereby impacting graft outcome. Further investigation of graft outcome of patients need ingof transplant bearing these new alleles would be warranted. [ Table 1 ]

Aim Characterization and identification of new HLA alleles have been important contributions to bone marrow and hematopoietic stem cell transplantation (BM/HSCT) for optimal donor selection which in turn will improve graft outcome. They are also invaluable in solid organ transplantation for selecting compatible donors according to organ transplant recipients’ antibody profiles. Methods Routine HLA typing by sequence specific oligonucleotide (SSO) probe based techniques and unusual or potential new variant of HLA alleles were typed by sequence based method in which alleles at a locus were separated by sequence specific primers and then sequenced. Results Four new HLA alleles have been identified and characterized in a donor and three BM/HSC transplant recipients. The profiles of these new alleles are shown in Table 1. Conclusions The B ∗ 40:206 and C ∗ 02:58 was identified in Hispanic male patients; DQB1 ∗ 03:40 in a Caucasian male donor; and the DQB1 ∗ 06:50 in a Black patient. Finding a matched donor for patients bearing these alleles would be difficult and challenging in BM/HSCT. Patient may make antibody to these alleles which in turn would impact graft outcome in solid organ transplantation. Therefore, the identification of these new alleles is important addition to the HLA allele list. Further investigation of the transplant outcome of these patients bearing these new alleles would be warranted [Fig. 1]. Download full-size image