Superior detection rate of plasma cell FISH using FACS-FISH.

OBJECTIVES: Fluorescence in situ hybridization (FISH) for plasma cell neoplasms (PCNs) requires plasma cell (PC) identification or purification strategies to optimize results. We compared the efficacy of cytoplasmic immunoglobulin FISH (cIg-FISH) and fluorescence-activated cell sorting FISH (FACS-FISH) in a clinical laboratory setting. METHODS: The FISH analysis results of 14,855 samples from individuals with a suspected PCN subjected to cytogenetic evaluation between 2019 and 2022 with cIg-FISH (n = 6917) or FACS-FISH (n = 7938) testing were analyzed. RESULTS: Fluorescence-activated cell sorting-FISH increased the detection rate of abnormalities in comparison with cIg-FISH, with abnormal results documented in 54% vs 50% of cases, respectively (P < .001). It improved the detection of IGH::CCND1 (P < .001), IGH::MAF (P < .001), IGH::MAFB (P < .001), other IGH rearrangements (P < .001), and gains/amplifications of 1q (P < .001), whereas the detection rates of IGH::FGFR3 fusions (P = .3), loss of 17p (P = .3), and other abnormalities, including hyperdiploidy (P = .5), were similar. Insufficient PC yield for FISH analysis was decreased between cIg-FISH and FACS-FISH (22% and 3% respectively, P < .001). Flow cytometry allowed establishment of ploidy status in 91% of cases. In addition, FACS-FISH decreased analysis times, workload efforts, and operating costs. CONCLUSIONS: Fluorescence-activated cell sorting-FISH is an efficient PC purification strategy that affords significant improvement in diagnostic yield and decreases workflow requirements in comparison with cIg-FISH.

Establishing NK-Cell Receptor Restriction by Flow Cytometry and Detecting Potential NK-Cell Clones of Uncertain Significance.

Natural killer (NK) cells develop a complex inhibitory and/or activating NK-cell receptor system, including killer cell immunoglobulin-like receptors (KIRs or CD158) and CD94/NKG2 dimers, which are variably combined to generate the individual's NK-cell receptor repertoire. Establishing NK-cell receptor restriction by flow cytometric immunophenotyping is an important step in diagnosing NK-cell neoplasms, but reference interval (RI) data for interpreting these studies are lacking. Specimens from 145 donors and 63 patients with NK-cell neoplasms were used to identify discriminatory rules based on 95% and 99% nonparametric RIs for CD158a+, CD158b+, CD158e+, KIR-negative, and NKG2A+ NK-cell populations to establish NK-cell receptor restriction. These 99% upper RI limits (NKG2a >88% or CD158a >53% or CD158b >72% or CD158e >54% or KIR-negative >72%) provided optimal discrimination between NK-cell neoplasm cases and healthy donor controls with an accuracy of 100% compared with the clinicopathologic diagnosis. The selected rules were applied to 62 consecutive samples received in our flow cytometry laboratory that were reflexed to an NK-cell panel due to an expanded NK-cell percentage (exceeding 40% of total lymphocytes). Twenty-two (35%) of 62 samples were found to harbor a very small NK-cell population with restricted NK-cell receptor expression based on the rule combination, suggestive of NK-cell clonality. A thorough clinicopathologic evaluation for the 62 patients did not reveal diagnostic features of NK-cell neoplasms; therefore, these potential clonal populations of NK cells were designated as NK-cell clones of uncertain significance (NK-CUS). In this study, we established decision rules for NK-cell receptor restriction from the largest published cohorts of healthy donors and NK-cell neoplasms. The presence of small NK-cell populations with restricted NK-cell receptors does not appear to be an uncommon finding, and its significance requires further exploration.

Risk of Adverse Outcomes Associated With Cardiac Sarcoidosis Diagnostic Schemes.

BACKGROUND: Multiple cardiac sarcoidosis (CS) diagnostic schemes have been published. OBJECTIVES: This study aims to evaluate the association of different CS diagnostic schemes with adverse outcomes. The diagnostic schemes evaluated were 1993, 2006, and 2017 Japanese criteria and the 2014 Heart Rhythm Society criteria. METHODS: Data were collected from the Cardiac Sarcoidosis Consortium, an international registry of CS patients. Outcome events were any of the following: all-cause mortality, left ventricular assist device placement, heart transplantation, and appropriate implantable cardioverter-defibrillator therapy. Logistic regression analysis evaluated the association of outcomes with each CS diagnostic scheme. RESULTS: A total of 587 subjects met the following criteria: 1993 Japanese (n = 310, 52.8%), 2006 Japanese (n = 312, 53.2%), 2014 Heart Rhythm Society (n = 480, 81.8%), and 2017 Japanese (n = 112, 19.1%). Patients who met the 1993 criteria were more likely to experience an event than patients who did not (n = 109 of 310, 35.2% vs n = 59 of 277, 21.3%; OR: 2.00; 95% CI: 1.38-2.90; P < 0.001). Similarly, patients who met the 2006 criteria were more likely to have an event than patients who did not (n = 116 of 312, 37.2% vs n = 52 of 275, 18.9%; OR: 2.54; 95% CI: 1.74-3.71; P < 0.001). There was no statistically significant association between the occurrence of an event and whether a patient met the 2014 or the 2017 criteria (OR: 1.39; 95% CI: 0.85-2.27; P = 0.18 or OR: 1.51; 95% CI: 0.97-2.33; P = 0.067, respectively). CONCLUSIONS: CS patients who met the 1993 and the 2006 criteria had higher odds of adverse clinical outcomes. Future research is needed to prospectively evaluate existing diagnostic schemes and develop new risk models for this complex disease.

Anteroposterior pacer pad position is better than anterolateral for transcutaneous cardiac pacing.

INTRODUCTION: Transcutaneous cardiac pacing (TCP) is a lifesaving procedure for patients with certain types of unstable bradycardia. We aimed to assess the difference in the pacing thresholds between the anteroposterior (AP) and anterolateral (AL) pacer pad positions. The second aim was to characterize the severity of chest wall muscle contractions during TCP. METHODS: In this prospective crossover trial, we enrolled patients presenting to the electrophysiology laboratory for elective cardioversion. After successful cardioversion, sedated participants were sequentially paced in both positions. The study procedure concluded after successful capture or inability to achieve capture by 140 mA (the pacer's maximum output) in both positions. Pacing thresholds were compared between positions, using a student's paired t-test, assigning a value of 141 mA to any trials with non-capture. RESULTS: Forty-one patients were screened; 20 were enrolled in the study. Seven participants were excluded from the paired analysis (three were prevented from pacing in the second position at the anesthesiologist's discretion, and 4 did not capture in either position). The study population consisted of 14 men and 6 women with a median age of 65 years. The mean pacing threshold was 33 mA lower (P = 0.001, 95% CI 20-45) in the AP (93 mA) versus the AL (126 mA) position. The median contraction severity score was 3 in the AL position versus 4 in the AP position (P = 0.005). CONCLUSIONS: Placing pacer pads in the AP position requires less energy to capture. Major resuscitation guidelines may favor the AP position for TCP. CLINICALTRIALS: gov Identifier: NCT03898050 https://clinicaltrials.gov/ct2/show/NCT03898050.

Abnormal CD13/HLA-DR Expression Pattern on Myeloblasts Predicts Development of Myeloid Neoplasia in Patients With Clonal Cytopenia of Undetermined Significance.

OBJECTIVES: Patients with clonal cytopenia of undetermined significance (CCUS) are at increased risk of developing myeloid neoplasia (MN). We evaluated whether a simple flow cytometry immunophenotyping (FCIP) assay could differentiate the risk of development of MN in patients with CCUS. METHODS: Bone marrow aspirates were assessed by FCIP panel in a cohort of 80 patients identified as having CCUS based on next-generation sequencing or cytogenetics from March 2015 to May 2020, with available samples. Flow cytometric assay included CD13/HLA-DR expression pattern on CD34-positive myeloblasts; CD13/CD16 pattern on maturing granulocytic precursors; and aberrant expression of CD2, CD7, or CD56 on CD34-positive myeloblasts. Relevant demographic, comorbidity, and clinical and laboratory data, including the type and extent of genetic abnormalities, were extracted from the electronic health record. RESULTS: In total, 17 (21%) patients with CCUS developed MN over the follow-up period (median survival follow-up, 28 months [95% confidence interval, 19-31]). Flow cytometry immunophenotyping abnormalities, including the aberrant pattern of CD13/HLA-DR expression, as detected at the time of the diagnosis of CCUS, were significantly associated with risk of developing MN (hazard ratio, 2.97; P = .006). Additional FCIP parameters associated with the development of MN included abnormal expression of CD7 on myeloblasts and the presence vs absence of any FCIP abnormality. CONCLUSIONS: A simple FCIP approach that includes assessment of CD13/HLA-DR pattern on CD34-positive myeloblasts can be useful in identifying patients with CCUS at higher risk of developing MN.

CinE caRdiac magneTic resonAnce to predIct veNTricular arrhYthmia (CERTAINTY).

Better models to identify individuals at low risk of ventricular arrhythmia (VA) are needed for implantable cardioverter-defibrillator (ICD) candidates to mitigate the risk of ICD-related complications. We designed the CERTAINTY study (CinE caRdiac magneTic resonAnce to predIct veNTricular arrhYthmia) with deep learning for VA risk prediction from cine cardiac magnetic resonance (CMR). Using a training cohort of primary prevention ICD recipients (n = 350, 97 women, median age 59 years, 178 ischemic cardiomyopathy) who underwent CMR immediately prior to ICD implantation, we developed two neural networks: Cine Fingerprint Extractor and Risk Predictor. The former extracts cardiac structure and function features from cine CMR in a form of cine fingerprint in a fully unsupervised fashion, and the latter takes in the cine fingerprint and outputs disease outcomes as a cine risk score. Patients with VA (n = 96) had a significantly higher cine risk score than those without VA. Multivariate analysis showed that the cine risk score was significantly associated with VA after adjusting for clinical characteristics, cardiac structure and function including CMR-derived scar extent. These findings indicate that non-contrast, cine CMR inherently contains features to improve VA risk prediction in primary prevention ICD candidates. We solicit participation from multiple centers for external validation.

A Novel Use of Noninvasive Registered Electrocardiographic Imaging Map for Localization of VT and PVC.

A 33-year-old woman presented with sustained monomorphic ventricular tachycardia (VT). The 12-lead electrocardiogram, 3-dimensional (3D) picture of chest electrodes, and cardiac magnetic resonance were used to create a noninvasive 3D electrocardiographic imaging map to identify the most likely site of VT origin. This map was integrated with a 3D mapping system to aid in VT ablation. (Level of Difficulty: Advanced.).

MCIR1: A patient-derived mantle cell lymphoma line for discovering new treatments for ibrutinib resistance.

BACKGROUND: Despite the unprecedented success of ibrutinib in lymphoma therapy, the development of ibrutinib resistance due to acquired BTK or PLCγ2 mutations has become a new clinical problem. However, not all resistance is mediated by these mutations and these mechanisms are poorly understood due to a lack of study tools that truly recapitulate this clinical scenario. METHODS: We established a novel patient-derived ibrutinib-resistant mantle cell lymphoma (MCL) line named MCIR1. Using immunological, molecular, and cytogenetic approaches, we comprehensively characterized MCIR1 and further demonstrated its utility in the study of resistance mechanisms and treatments to overcome this resistance. RESULTS: We show that MCIR1 is a bona fide ibrutinib-resistant MCL cell line with normal BTK-/PLCγ2 but ibrutinib-resistant ERK1/2 and AKT1 signaling. RNA-Seq analysis revealed a robust non-canonical NF-kB signaling that drives the ibrutinib resistance. We also demonstrate the potential utility of a MCIR1-based cell and mouse model for the discovery of new treatments to overcome BTK inhibitor resistance. CONCLUSIONS: We have established the first patient-derived ibrutinib-resistant MCL cell line MCIR1 that lacks BTK or PLCγ2 mutations but exhibits a hyperactive non-canonical NF-kB pathway. We further demonstrate its utility in the discovery and validation of new drugs to overcome this resistance.

Metabolic heterogeneous zone assessed by 18 FDG-PET is predictive of postablation mortality in patients with ventricular tachycardia.

PURPOSE: We sought to study the predictive value of the metabolic heterogeneous zone (HZ) as determined by 18 Fluorodeoxyglucose (18 FDG) positron emission tomography (PET) viability studies in ventricular tachycardia (VT) patients. METHODS: PET studies utilizing 82 Rubidium (82 Rb) tracer for perfusion and 18 FDG tracer for viability were analyzed using PMOD (PMOD Technologies) and further analyzed using 684-segment plots. 18 FDG uptake was normalized to the area with maximal perfusion on the rest 82 Rb study. Metabolic scar, HZ, and healthy segments were defined with perfusion-normalized 18 FDG uptake between 0%-50%, 50%-70%, and >70%, respectively. RESULTS: Thirty-four VT patients (age, 63 ± 12 years) were evaluated with 18 FDG-PET viability study. Most (n = 31) patients underwent VT ablation. Patients were categorized to HZ < median versus HZ ≥ median based on a median HZ area size of 21.0 cm2 . HZ size was significantly larger in the deceased group than the alive group (35.2 cm2 vs. 18.1 cm2 , p = .01). Deaths were significantly higher in HZ ≥ 21 cm2 group than HZ < 21 cm2 group (58.8% vs. 11.8%, p = .005). Survival analysis showed significantly higher mortality in the HZ ≥ 21 cm2 group than the HZ < 21 cm2 group (HR = 4.1, 95% CI: 1.3-12.6, p = .016). In a multivariable analysis, HZ was found to be an independent predictor for all-cause mortality (HR = 1.07, 95% CI: 1.02-1.12, p = .01) CONCLUSIONS: Increased HZ size of myocardium was associated with increased mortality. Metabolic HZ quantification may be of value in risk stratification and management of ischemic and nonischemic patients with VT.

Image Integration Using Intracardiac Echography and Three-dimensional Reconstruction for Mapping and Ablation of Atrial and Ventricular Arrhythmias.

This article reviews the basis for image integration of intracardiac echocardiography (ICE) with three-dimensional electroanatomic mapping systems and preprocedural cardiac imaging modalities to enhance anatomic understanding and improve guidance for atrial and ventricular ablation procedures. It discusses the technical aspects of ICE-based integration and the clinical evidence for its use. In addition, it presents the current technical limitations and future directions for this technology. This article also includes figures and videos of clinical representative arrhythmia cases where the use of ICE is key to a safe and successful outcome.

CD2 and CD7 are sensitive flow cytometry screening markers for T-lineage acute leukemia(s): a study of 465 acute leukemia cases.

T-lymphoblastic leukemia/lymphoma (T-ALL/LBL) is a rare acute leukemia that expresses cytoplasmic CD3 (cCD3) and frequently lacks surface CD3. Given that routine flow cytometric testing for cCD3 may not be feasible and cCD3 interpretation may be difficult, we investigate if surface CD2 and/or CD7 expression on blasts can be used by flow cytometry to screen for T-lineage acute leukemia. We retrospectively reviewed flow cytometric data from 233 acute leukemias (36 T-ALL/LBL, 8 mixed-phenotype acute leukemia T/myeloid, 80 acute myeloid leukemia, 97 B-ALL/LBL, 8 mixed-phenotype acute leukemia B/myeloid, and 4 acute undifferentiated leukemia cases). Uniform expression (≥75% of blasts) of CD2 and/or CD7 was seen in all 44 cCD3-positive cases but in only 11% (20/189) of cCD3-negative acute leukemias, thus demonstrating 100% sensitivity and 89% specificity in the identification of cCD3-positive (T-lineage) acute leukemia. To avoid selection bias, we prospectively studied 232 consecutive acute leukemias for which cCD3, CD2, and CD7 were automatically performed in all cases. Similar to the retrospective study, uniform expression of CD2 and/or CD7 on blasts showed 100% sensitivity and 88% specificity in the screening for cCD3-positive (T-lineage) acute leukemia. Therefore, acute leukemias with uniform expression of CD2 and/or CD7 warrant further testing for cCD3 to evaluate for T-lineage acute leukemia. Blasts that lack both uniform CD2 and CD7 expression do not require additional cCD3 testing. We propose that CD2 and CD7 could be utilized in a limited antibody flow cytometry panel as a sensitive, robust, and cost-effective way to screen for T-lineage acute leukemia.

Arrhythmias in Cardiac Sarcoidosis Bench to Bedside: A Case-Based Review.

Cardiac sarcoidosis is a component of an often multiorgan granulomatous disease of still uncertain cause. It is being recognized with increasing frequency, mainly as the result of heightened awareness and new diagnostic tests, specifically cardiac magnetic resonance imaging and 18F-fluorodeoxyglucose positron emission tomography scans. The purpose of this case-based review is to highlight the potentially life-saving importance of making the early diagnosis of cardiac sarcoidosis using these new tools and to provide a framework for the optimal care of patients with this disease. We will review disease mechanisms as currently understood, associated arrhythmias including conduction abnormalities, and atrial and ventricular tachyarrhythmias, guideline-directed diagnostic criteria, screening of patients with extracardiac sarcoidosis, and the use of pacemakers and defibrillators in this setting. Treatment options, including those related to heart failure, and those which may help clarify disease mechanisms are included.

Perioperative Imaging to Guide Epicardial Mapping and Ablation.

Accessing the epicardial space without a sternotomy or a surgical pericardial window to treat ventricular arrhythmias in Chagas disease became a medical necessity in South America. Since the introduction of the dry percutaneous epicardial access approach, epicardial access has been standard procedure for management of ventricular arrhythmias in ischemic and nonischemic cardiomyopathies and atrioventricular accessory pathways after failed conventional endocardial ablation. Understanding the epicardial space and neighboring structures has become an important subject of teachings in electrophysiology. The evolution of complex ablation procedures to treat atrial and ventricular arrhythmias and device interventions to prevent cardioembolic stroke requires thorough understanding of pericardial anatomy.

Cytoplasmic Expression of CD3ε Heterodimers by Flow Cytometry Rapidly Distinguishes Between Mature T-Cell and Natural Killer-Cell Neoplasms.

OBJECTIVES: Distinguishing between T-cell and natural killer (NK)-cell neoplasms could be difficult given their overlapping immunophenotype. In this study, we investigated whether a flow cytometry assay with cytoplasmic staining for CD3 could be used for this purpose. METHODS: Flow cytometry immunophenotyping was performed on 19 surface CD3 (sCD3)-negative mature T-cell neoplasms, 10 sCD3-positive mature T-cell neoplasms, 13 mature NK-cell neoplasms, and 19 normal controls. In addition to routine antibody panels (CD2, sCD3, CD4, CD5, CD7, CD8, CD16, CD45, CD56, CD57, CD94, CD158a, CD158b, CD158e, NKG2A TCRγ/δ), cytoplasmic staining for a monoclonal CD3 antibody (clone SK7/Leu-4) was assessed in all cases. A molecular study for T-cell receptor (TCR) gene rearrangement and an immunohistochemical study for TCRß were performed. RESULTS: Our data showed all T-cell neoplasms were uniformly positive for cytoplasmic CD3 (cCD3) regardless of sCD3 expression, whereas 85% of NK-cell neoplasms completely lacked cCD3 expression. The 2 cases with classic NK-cell immunophenotype but partial cCD3 expression showed no molecular genetic features of T-cell lineage by TCR gene rearrangement studies. CONCLUSIONS: Uniform cCD3 positivity and homogeneous cCD3 negativity highly suggest T-cell and NK lineage, respectively. When partial cCD3 expression is encountered, additional confirmatory studies should be pursued for the most accurate lineage assignment.