Flow-based sorting of neonatal lymphocyte populations for transcriptomics analysis.

RATIONALE: Emerging data suggest an important role for T lymphocytes in the pathogenesis of chronic lung disease in preterm infants. Comprehensive assessment of the lymphocyte transcriptome may identify biomarkers and mechanisms of disease. METHODS: Small volume peripheral blood samples were collected from premature infants enrolled with consent in the Prematurity and Respiratory Outcomes Program (PROP), at the time of discharge from the hospital. Blood samples were collected at two sites and shipped to a central laboratory for processing. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque gradient centrifugation and separated into individual lymphocyte cell types by fluorescence-activated cell sorting. Gating strategies were optimized to ensure reproducible recovery of highly purified lymphocyte populations over a multi-year recruitment period. RNA was isolated from sorted cells and characterized by high-throughput sequencing (RNASeq). RESULTS: Blood volumes averaged 2.5ml, and sufficient PBMCs were collected from 165 of the 246 samples obtained (67%) from the 277 recruited subjects to complete sorting and RNASeq analysis on the resulting sorted cells. The number of total lymphocytes per ml of blood in the neonatal subjects was approximately 4 million/ml. Total lymphocyte frequencies recovered following sort varied widely among subjects, as did the frequency of individual lymphocyte and NK cell sub-populations. RNA yield from sorted cells varied according to cell type, but RNA of sufficient quantity and quality was recovered to enable RNASeq. SUMMARY: Our results describe a validated procedure for the generation of genome-wide expression data from isolated lymphocyte sub-populations obtained from newborn blood.

Influenza vaccine antibody responses in lung transplant recipients.

CONTEXT: Lung transplant recipients are at high risk of morbidity and mortality from influenza infection because of altered lung physiology and immunosuppression. Annual influenza immunization is recommended, but the ability to mount an antibody response may be limited by immunosuppressant medications. OBJECTIVE: To compare the antibody response rate to influenza vaccine in lung transplant recipients to healthy controls. DESIGN: Open label study. SETTING: Lung transplant clinic and General Clinical Research Center at a university hospital. SUBJECTS: Sixty-eight single and bilateral lung transplant recipients and 35 healthy controls were enrolled in October and November 2002. METHODS: Each individual underwent blood sampling before receiving the 2002-2003 influenza vaccine and 4 weeks later. Influenza antibody concentrations were measured by hemagglutination inhibition assay. Vaccine response rates (antibody concentration >40 hemagglutination units and at least 4-fold increase in antibody concentration) were compared using chi2. The influence of specific immunosuppressants on vaccine response was compared. RESULTS: The influenza vaccine response rate for lung transplant recipients was 29/68 (43%) and 22/35 (63%) for the healthy individuals (P < .05; chi2). Among the recipients, mycophenolate mofetil was associated with poorer influenza vaccine antibody response (> 40 hemagglutination units) (62% vs 91%; P = .01), whereas sirolimus (91% vs 63%; P = .02) was associated with better influenza antibody response compared to those not taking mycophenolate mofetil or sirolimus, respectively. CONCLUSION: Lung transplant recipients had lower influenza vaccine response rates than healthy individuals. Influenza vaccine antibody response is influenced by concomitant administration of mycophenolate mofetil or sirolimus. Future studies should measure protection from influenza infection conferred by immunization and alternative vaccination strategies.

High-dose hepatitis B vaccine in patients waiting for lung transplantation.

STUDY OBJECTIVE: To increase the response rate to hepatitis B vaccine in patients awaiting lung transplantation. DESIGN: Historically controlled, open-label study. SETTING: Lung transplant clinic at a university hospital. SUBJECTS: Twenty-seven consecutive individuals with end-stage pulmonary disease who were enrolled to accrue 15 subjects who would complete the vaccine series before transplantation; and 27 lung transplant recipients who were immunized with the conventional dose before the study and served as historical controls. INTERVENTION: Intramuscular injection of high-dose hepatitis B vaccine 40 microgram at 0, 1, and 6 months. MEASUREMENTS AND MAIN RESULTS: Hepatitis B surface antibody (anti-HBs) concentrations were measured 1-2 months after completing the high-dose series. Individuals with undetectable anti-HBs received additional vaccine to a maximum of six doses. The response rate to the series was compared with that in the control group. Seventeen individuals in the high-dose group and 14 controls met the study criterion of complete vaccine series before transplantation. The former had a much higher response rate than the latter (9 [53%] vs 1 [7%], p<0.01). Four of six patients who received additional doses of vaccine seroconverted. Two of them underwent transplantation shortly after completing the three-dose series. CONCLUSION: The high-dose hepatitis B vaccine series produced a protective immune response in lung transplant recipients; however, the response was suboptimal, and alternative immunization strategies should be studied.

Cardiac arrest from tension pneumopericardium in a premature infant.

IMPLICATIONS: This case report describes a rare and potentially fatal anesthetic complication. It occurred during the care of a small premature infant as a result of improper use of medical equipment.