Use of an in-house trypsin-based method to resolve the interference of daratumumab.

BACKGROUND: Daratumumab (DARA) is a monoclonal antibody for treatment of plasma cell myeloma targeting CD38, a surface molecule expressed on plasma cells and red blood cells (RBCs). This complicates blood bank testing, requiring dithiothreitol (DTT) to remove DARA interference. A simple in-house method of removing DARA interference without use of DTT, a potentially hazardous chemical, is desirable. We demonstrate a trypsin-based method to remove interference in antibody testing at a medical center (MC), with parallel testing at an immunohematology reference laboratory (IRL). STUDY DESIGN AND METHODS: Pre-DARA type and screen (T&S) samples were obtained from 61 patients for antibody testing and RBC phenotyping using untreated reagent RBCs. Subsequent post-DARA T&S testing was performed with untreated reagent RBCs to demonstrate interference and repeated after trypsin treatment. Positive trypsin-treated antibody screens were reflexed to antibody identification using trypsin-treated panel cells. Parallel testing was performed on the same post-DARA samples at IRL. RESULTS: DARA interference was detected in 61/61 (100%) samples by MC and IRL. After trypsin treatment, DARA interference was eliminated in 60/61 (98.4%) antibody screens by both institutions with an overall percent agreement of 96.7% (95% confidence interval [CI] 88.7%-99.6%). Identification of known alloantibodies was confirmed in 3/3 patients with 100% concordant results between MC and IRL. There were no false-negative results demonstrated by IRL's functionally CD38-negative controls. CONCLUSION: Our in-house trypsin-based method enables pretransfusion testing of patients receiving DARA in an accurate and cost-effective manner without missing clinically significant alloantibodies. This presents an additional testing option where DTT use is undesirable.

Transfusion reactions associated with COVID-19 convalescent plasma therapy for SARS-CoV-2.

BACKGROUND: Convalescent plasma (CP) for treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has shown preliminary signs of effectiveness in moderate to severely ill patients in reducing mortality. While studies have demonstrated a low risk of serious adverse events, the comprehensive incidence and nature of the spectrum of transfusion reactions to CP is unknown. We retrospectively examined 427 adult inpatient CP transfusions to determine incidence and types of reactions, as well as clinical parameters and risk factors associated with transfusion reactions. STUDY DESIGN AND METHODS: Retrospective analysis was performed for 427 transfusions to 215 adult patients with coronavirus 2019 (COVID-19) within the Mount Sinai Health System, through the US Food and Drug Administration emergency investigational new drug and the Mayo Clinic Expanded Access Protocol to Convalescent Plasma approval pathways. Transfusions were blindly evaluated by two reviewers and adjudicated by a third reviewer in discordant cases. Patient demographics and clinical and laboratory parameters were compared and analyzed. RESULTS: Fifty-five reactions from 427 transfusions were identified (12.9% incidence), and 13 were attributed to transfusion (3.1% incidence). Reactions were classified as underlying COVID-19 (76%), febrile nonhemolytic (10.9%), transfusion-associated circulatory overload (9.1%), and allergic (1.8%) and hypotensive (1.8%) reactions. Statistical analysis identified increased transfusion reaction risk for ABO blood group B or Sequential Organ Failure Assessment scores of 12 to 13, and decreased risk within the age group of 80 to 89 years. CONCLUSION: Our findings support the use of CP as a safe, therapeutic option from a transfusion reaction perspective, in the setting of COVID-19. Further studies are needed to confirm the clinical significance of ABO group B, age, and predisposing disease severity in the incidence of transfusion reaction events.

Convalescent plasma treatment of severe COVID-19: a propensity score-matched control study.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a new human disease with few effective treatments1. Convalescent plasma, donated by persons who have recovered from COVID-19, is the acellular component of blood that contains antibodies, including those that specifically recognize SARS-CoV-2. These antibodies, when transfused into patients infected with SARS-CoV-2, are thought to exert an antiviral effect, suppressing virus replication before patients have mounted their own humoral immune responses2,3. Virus-specific antibodies from recovered persons are often the first available therapy for an emerging infectious disease, a stopgap treatment while new antivirals and vaccines are being developed1,2. This retrospective, propensity score-matched case-control study assessed the effectiveness of convalescent plasma therapy in 39 patients with severe or life-threatening COVID-19 at The Mount Sinai Hospital in New York City. Oxygen requirements on day 14 after transfusion worsened in 17.9% of plasma recipients versus 28.2% of propensity score-matched controls who were hospitalized with COVID-19 (adjusted odds ratio (OR), 0.86; 95% confidence interval (CI), 0.75-0.98; chi-square test P value = 0.025). Survival also improved in plasma recipients (adjusted hazard ratio (HR), 0.34; 95% CI, 0.13-0.89; chi-square test P = 0.027). Convalescent plasma is potentially effective against COVID-19, but adequately powered, randomized controlled trials are needed.

How do we perform and bill for blood bank physician consultative services?

Transfusion medicine (TM) physicians provide medical services that benefit all patients such as providing 24-hour laboratory coverage, advising health care providers on test interpretation and selection, validating new methods, and supervising technical personnel. These services ensure delivery of accurate, reliable, and timely laboratory test results and blood products. TM physicians also provide consultations to individual patients by 1) interpreting and determining the clinical significance of test results (e.g., alloantibodies, direct antiglobulin tests), 2) recommending appropriate component therapy and approving deviations from laboratory policy, and 3) evaluating and recommending treatment of suspected transfusion reactions. The potential benefits of consultations are improved quality and cost of health care, enhanced provider education, and decreased inappropriate testing and product utilization. When physician services are delivered to individual patients, are appropriately requested, provide a diagnosis or recommendation, and are properly documented, TM physicians can receive professional reimbursement. While many TM physicians provide medical direction and oversight of apheresis procedures, billing in this area is sufficiently complex to be reviewed elsewhere. The objective of this article is for educational purposes to describe the 1) benefits of a consultative TM service, 2) development of reimbursement systems in the United States for professional component services and the current regulatory requirements, 3) current procedural terminology codes commonly used for TM physician services, and 4) examples of consultation documentation and daily workflow at tertiary care teaching hospitals. The information provided should help guide physicians to deliver and bill for these services.

G6PD Deficiency in an HIV Clinic Setting in the Dominican Republic.

Because human immunodeficiency virus (HIV)-infected patients receive prophylaxis with oxidative drugs, those with glucose-6-phosphate dehydrogenase (G6PD) deficiency may experience hemolysis. However, G6PD deficiency has not been studied in the Dominican Republic, where many individuals have African ancestry. Our objective was to determine the prevalence of G6PD deficiency in Dominican HIV-infected patients and to attempt to develop a cost-effective algorithm for identifying such individuals. To this end, histories, chart reviews, and G6PD testing were performed for 238 consecutive HIV-infected adult clinic patients. The overall prevalence of G6PD deficiency (8.8%) was similar in males (9.3%) and females (8.5%), and higher in Haitians (18%) than Dominicans (6.4%; P = 0.01). By logistic regression, three clinical variables predicted G6PD status: maternal country of birth (P = 0.01) and a history of hemolysis (P = 0.01) or severe anemia (P = 0.03). Using these criteria, an algorithm was developed, in which a patient subset was identified that would benefit most from G6PD screening, yielding a sensitivity of 94.7% and a specificity of 97.2%, increasing the pretest probability (8.8-15.1%), and halving the number of patients needing testing. This algorithm may provide a cost-effective strategy for improving care in resource-limited settings.

Acquired hemoglobin variants and exposure to glucose-6-phosphate dehydrogenase deficient red blood cell units during exchange transfusion for sickle cell disease in a patient requiring antigen-matched blood.

Red blood cell exchange (RBCEx) is frequently used in the management of patients with sickle cell disease (SCD) and acute chest syndrome or stroke, or to maintain target hemoglobin S (HbS) levels. In these settings, RBCEx is a category I or II recommendation according to guidelines on the use of therapeutic apheresis published by the American Society for Apheresis. Matching donor red blood cells (RBCs) to recipient phenotypes (e.g., C, E, K-antigen negative) can decrease the risk of alloimmunization in patients with multi-transfused SCD. However, this may select for donors with a higher prevalence of RBC disorders for which screening is not performed. This report describes a patient with SCD treated with RBCEx using five units negative for C, E, K, Fya, Fyb (prospectively matched), four of which were from donors with hemoglobin variants and/or glucose-6-phosphate dehydrogenase (G6PD) deficiency. Pre-RBCEx HbS quantification by high performance liquid chromatography (HPLC) demonstrated 49.3% HbS and 2.8% hemoglobin C, presumably from transfusion of a hemoglobin C-containing RBC unit during a previous RBCEx. Post-RBCEx HPLC showed the appearance of hemoglobin G-Philadelphia. Two units were G6PD-deficient. The patient did well, but the consequences of transfusing RBC units that are G6PD-deficient and contain hemoglobin variants are unknown. Additional studies are needed to investigate effects on storage, in-vivo RBC recovery and survival, and physiological effects following transfusion of these units. Post-RBCEx HPLC can monitor RBCEx efficiency and detect the presence of abnormal transfused units.

Quality monitoring of microbial contamination of cryopreserved parathyroid tissue.

Cryopreservation of parathyroid tissue (PT) provides patients undergoing parathyroidectomy with an option for delayed autologous heterotopic parathyroid transplantation. A standard protocol for quality monitoring of PT has not been established. This article describes a method for detecting the presence of bacterial contamination in PT tissue intended for autologous transplantation. PT was received in the tissue bank, processed under aseptic conditions, and placed into cryopreservation medium. Sterility testing was performed at 2 time points prior to cryopreservation. From January 2005 to October 2008, 47 PT samples were cryopreserved. The following bacteria were isolated from 11 PT specimens: Staphylococcus epidermidis, Staphylococcus capitis subspecies ureolyticus, Staphylococcus lugdunensis, Bacillus pumilus, and corynebacteria (diphtheroids). 23% of PTs were contaminated at the time of collection, predominantly with indigenous bacteria. Quality monitoring using this protocol is a useful tool to identify tissues contaminated with bacteria.

Comparative outcome analysis of ABO-incompatible and positive crossmatch renal transplantation: a single-center experience.

BACKGROUND: ABO-incompatible (ABOi) and positive crossmatch (XM) renal transplants pose special immunologic challenges. It is important to compare outcomes, study resource utilization, and attempt to risk stratify patients in these higher risk transplant settings. METHODS: We compared apheresis utilization and transplant outcomes in ABOi, XM, and combined ABOi-XM renal transplants. We also analyzed multiple parameters, including patient and laboratory variables, to identify predictors of transplant outcome. RESULTS: Incidences of early (< or =30 days posttransplant) antibody-mediated rejection (AMR) and acute cellular rejection (ACR) were similar among the three incompatible groups whereas they differed in allograft rejection for late (>30 days posttransplant) AMR and ACR. Notably, there were no episodes of late AMR among ABOi patients. Patients treated with more than four pretransplant plasmapheresis/intravenous immunoglobulin (PP/IVIg) had a greater likelihood of experiencing early AMR. The median number of posttransplant PP/IVIg treatments was greater than twofold higher in ABOi-XM and XM patients compared to ABOi patients. Patients who required more than five posttransplant PP/IVIg procedures and those with one or more prior renal transplants had higher incidences of late ACR. CONCLUSIONS: Our analysis aids in defining apheresis resource utilization and helps in risk stratification of incompatible renal transplantation. It also aids in predicting allograft rejection and provides an opportunity for preemptive monitoring and treatment.

Therapeutic plasmapheresis as a bridge to liver transplantation in fulminant Wilson disease.

Wilson disease is an autosomal recessive disorder of copper metabolism that leads to the accumulation of copper mainly in the liver, cornea, brain, and kidney. Rarely, Wilson disease can present as fulminant hepatic failure with direct antiglobulin test-negative hemolytic anemia and renal failure. In the absence of liver transplantation, this disease is uniformly fatal because medical therapy is ineffective. This report describes the successful use of plasmapheresis for a patient with fulminant Wilson disease as a bridge to transplantation. Five daily therapeutic plasmapheresis procedures using fresh frozen plasma as a replacement fluid were performed over 6 days. Serum copper, urinary copper excretion, and hemolysis were significantly reduced and renal function improved. The patient's clinical status improved and she remained clinically stable until a liver transplant was possible. Plasmapheresis can be a successful medical treatment in fulminant Wilson disease and should be considered as a therapeutic measure to stabilize a patient by decreasing serum copper, reducing hemolysis, and helping to prevent renal tubular injury from copper and copper complexes until liver transplantation is possible.

Glycosylation and cold platelet storage.

Platelets, unlike red blood cells and plasma, are stored at room temperature because platelets transfused after refrigeration at 4 degrees C are rapidly cleared from the circulation. Storage at room temperature promotes bacterial proliferation, however, and transfusion-transmitted bacteremia has become an increasing problem. Traditionally, the cold storage lesion has been attributed to a change in platelet shape from disc to sphere, but Hoffmeister et al. revisited this issue and have shown that the shape change induced by cold storage does not result in poor platelet survival. Instead, they showed that poor survival results from a virtually irreversible clustering of alpha subunits of glycoprotein Ib (GPIbalpha)) on the platelet surface. In a series of elegant papers, these researchers change the way we view platelet clearance. That is, they show that exposed, terminal, beta-linked N-acetylglucosamine (beta-GlcNAc) residues on clustered GPIbalpha are recognized by the lectin domain of type 3 complement receptors on liver macrophages, leading to rapid clearance by phagocytosis. They also demonstrate that phagocytosis of chilled platelets can be inhibited--and in vivo survival prolonged--by enzymatically galactosylating the terminal beta-GlcNAc residues on GPIbalpha. Disguising the exposed beta-GlcNAc residues on the N-glycans of the clustered GPIbalpha molecules by galactosylation is a promising approach to storing platelets at 4 degrees C without affecting platelet function. Cold storage would limit bacterial proliferation and extend the duration of platelet storage, reducing the incidence of transfusion-transmitted bacteremia and improving the availability of this scarce resource.

Progenitor cell expansion: an important source of hepatocyte regeneration in chronic hepatitis.

BACKGROUND/AIMS: Progenitor cell activation with subsequent maturation to hepatocytes and cells of the biliary lineage has been demonstrated in a variety of chronic liver diseases but the kinetics and magnitude of the progenitor cell response has not been adequately studied in detail in chronic hepatitis. We undertook this study to evaluate factors responsible for the progenitor cell/ductular response and further dissect the role of disease grade and stage as determinants of hepatocellular differentiation of bipotential progenitor cells in chronic hepatitis. METHODS: Cytokeratin 7 (and 19) stained biopsies from patients with chronic hepatitis C (n = 47), hepatitis B (n = 20), and autoimmune hepatitis (n = 20) were studied. Ploidy analysis and proliferation indices were evaluated in a subset of cases. RESULTS: Ductular reactions were present in the majority of cases (97%), appeared early in disease, and correlated with disease activity, while progenitor cell derived hepatocyes appeared later in disease and their extent correlated with disease stage. Proliferation indices of all cell types correlated with disease activity. CONCLUSIONS: Progenitor cell derived hepatocytes accrue in chronic hepatitis, possibly related to native hepatocellular dysfunction. However, the fate of these hepatocytes is unclear.

Renal oncocytomas with 11q13 rearrangements: cytogenetic, molecular, and immunohistochemical analysis of cyclin D1.

Two groups of renal oncocytomas have been cytogenetically defined by the loss of one or both of chromosomes Y and 1 or by structural rearrangement involving 11q12~q13. We report five renal oncocytomas with structural chromosomal rearrangements involving 11q13 with previously unreported partner chromosomes (namely, 1, 6, and 7). For two of the five cases, a t(6;11)(p21;q13) translocation was revealed; the others had t(1;11)(p13;q13), t(7;11)(q11.2;q13), and t(5;11)(q35; q13). Fluorescence in situ hybridization confirmed translocation of CCND1 at 11q13 to partner chromosomes 5, 6, and 7. Overexpression of cyclin D1, the protein product of CCND1, was detected in three of the five cases (60%) by means of immunohistochemical staining of formalin-fixed, paraffin-embedded tumor sections. In three cases for which fresh tissue was available, Southern blot analysis using the MDL-5 probe for the BCL1 breakpoint did not reveal rearrangement of BCL1. In addition, six consecutive renal oncocytomas diagnosed at our institution between 1999 and 2002 whose karyotypes did not show 11q13 translocations were all negative for cyclin D1 overexpression under immunohistochemical analysis. The findings of CCND1 rearrangement with FISH and correlation with cyclin D1 overexpression under immunohistochemical analysis suggest that cyclin D1 alterations play a role in the subset of renal oncocytomas with 11q translocations, although other genes may also be involved.

Evaluation of linearity in the clinical laboratory.

CONTEXT: Clinical laboratory assessment of test linearity is often limited to satisfying regulatory requirements rather than integrating this tool into the laboratory quality assurance program. Although an important part of quality control and method validation for clinical laboratories, linearity of clinical tests does not get the attention it deserves. OBJECTIVE: This article evaluates the concepts and importance of linearity evaluations for clinical tests. DESIGN: We describe the theory and procedural steps of each linearity evaluation. We then evaluate the statistical methods for each procedure. RESULTS: Visual assessment, although simple, is subjective. The lack-of-fit error and the 1986 NCCLS EP6-P G test are sensitive to imprecision and assume that the data are first order. Regression analysis, as developed as the polynomial method, is partly based on the experiences of the College of American Pathologists Instrumentation Resource Committee and has proved to be a robust statistical method. CONCLUSIONS: We provide general guidelines for handling non-linear results from a linearity evaluation. Handling linearity data in an objective manner will aid clinical laboratorians whose goal is to improve the quality of the tests they perform.

Cellular electrophysiologic properties of old canine atria provide a substrate for arrhythmogenesis.

OBJECTIVE: The incidence of atrial fibrillation increases with age. We hypothesized that aging-associated changes in the atrial action potential (AP) and conduction velocity provide a substrate for abnormal conduction and arrhythmogenesis. METHODS: We used microelectrode techniques to record AP from the endocardium of the right atrial wall of dogs aged 1-5 (adult) and >8 years (old). Conduction velocity was measured between two microelectrodes 3-10 mm apart. Histological study was carried out to assess fibrosis. RESULTS: Whereas resting potential, AP amplitude and V(max) did not differ with age, the plateau was more negative and AP duration was longer in old tissue. The L-type calcium current (I(Ca,L)) agonist Bay K8644 (10(-8)-10(-6) mol/l) elevated the plateau and shortened APD more in old than in adult, such that AP contour in old atria approached that of adult. In contrast, the I(Ca,L) blocker nisoldipine (10(-8)-10(-5) mol/l) depressed the plateau in adult and had no effect in old. There was no difference between the two groups in conduction velocity of normal beats, whereas for early premature impulses, reduced conduction velocity and a wider time window manifesting slow conduction were detected in old in comparison to adult tissue. A twofold increase in the amount of fibrous tissue was detected in old atria. CONCLUSIONS: Our data show significant differences in contour of AP in adult and old atria. The responses to Bay K8644 and nisoldipine suggest a decreased I(Ca,L) in old atrial tissue. The alterations in AP contour and increased fibrosis may be responsible for slower conduction of early premature beats in old atria. The age-related changes in conduction of premature beats are consistent with those observed in patients with paroxysmal atrial fibrillation and may contribute to the greater propensity to atrial fibrillation in the aged.

Interobserver Variability of Left Ventricular Measurements in a Population of Predominantly Obese Hypertensives Using Simultaneously Acquired and Displayed M-Mode and 2-D Cine Echocardiography.

Achieving low interobserver variability is a goal of echocardiographic determined left ventricular (LV) mass measurements. In a group of hypertensives, we evaluated interobserver variation using a method of simultaneously acquired two-dimensional (2-D) cine and M-mode images displayed in a split screen format. Sixty echocardiographic images from ongoing hypertension trials, including serial studies of the same patients, were obtained with an UltraMarktrade mark 6 Ultrasound System (Advanced Technology Laboratories, Inc., Bothell, WA, USA). Images were digitized online, coded with a random number, and pooled prior to the analysis to minimize observer bias. Studies were read by two independent observers in a blinded fashion and in a random order using a Color Vue II Nova MicroSonicstrade mark analyzer (Nova Microsonics, Mahwah, NJ, USA). The M-mode tracing and cine of three consecutive frames of 2-D parasternal short-axis views of the LV were simultaneously displayed in a split screen format. The 2-D cine was used as a reference image during M-mode measurements of LV dimensions. Measurements were obtained and the LV mass estimated according to the Penn convention. Interobserver variability for left ventricular internal dimension (LVID), interventricular septal thickness (IVS), posterior wall thickness (PWT), and left ventricular mass (LV mass) is low when either correlated (Pearson correlation coefficients of 0.94, 0.82, 0.75, and 0.93, respectively) or expressed as a percent of the mean (3.0%, 10.0%, 10.2%, and 8.9%, respectively). When read in a blinded fashion, interobserver variability (especially for LV mass) is small using digitized, simultaneously acquired and displayed cines of 2-D and M-mode echocardiograms. This is likely due to the ability to discriminate myocardial wall edges (endocardium) from other associated structures such as tricuspid and mitral apparatus. This method may prove useful in studies of LV mass.