RESUMEN
B cells fulfill an important role in the adaptive immunity. Upon activation and immunoglobulin (IG) class switching, these cells function in the humoral immunity compartment as plasma cells. For clinical applications, it can be important to quantify (switched) B cells accurately in a variety of body fluids and tissues of benign, inflammatory and malignant origin. For decades, flow cytometry and immunohistochemistry (IHC) have been the preferred methods for quantification. Although these methods are widely used, both depend on the accessibility of B cell epitopes and therefore require intact (fixed) cells. Whenever samples are low in quantity and/or quality, accurate quantification can be difficult. By shifting the focus from epitopes to DNA markers, quantification of B cells remains achievable. During differentiation and maturation, B cells are subjected to programmed genetic recombination processes like VDJ rearrangements and class switch recombination (CSR), which result in deletion of specific sequences of the IGH locus. These cell type-specific DNA "scars" (loss of sequences) in IG genes can be exploited as B cell markers in digital PCR (dPCR) based quantification methods. Here, we describe a novel, specific and sensitive digital PCR-based method to quantify mature and switched B cells in DNA specimens of benign and (copy number unstable) malignant origin. We compared this novel way of B cell quantitation with flow cytometric and immunohistochemical methods. Through cross-validation with flow cytometric sorted B cell subpopulations, we gained quantitative insights into allelic involvement in different recombination processes in the IGH locus. Our newly developed method is accurate and independent of the cellular context, offering new possibilities for quantification, even for (limited) small samples like liquid biopsies.
Asunto(s)
Linfocitos B , Cambio de Clase de Inmunoglobulina , ADN , Genes de las Cadenas Pesadas de las Inmunoglobulinas/genética , Cambio de Clase de Inmunoglobulina/genética , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Treatment delay is an important indicator of access to tuberculosis diagnosis and treatment. Analyses of patient delay (i.e. time interval between onset of symptoms and first consultation of a health care provider) and health care delay (i.e. time interval between first consultation and start of treatment) can inform policies to improve access. This study assesses the patient, health care provider and total delay in diagnosis and treatment of new smear-positive pulmonary tuberculosis patients, and the risk factors for long delay, in Vietnam. METHODS: A cross-sectional survey of new patients treated by the National Tuberculosis Control Programme was conducted in 70 randomly selected districts in Vietnam. All consecutively registered patients in one quarter of 2002 were interviewed using a pre-coded structured questionnaire. RESULTS: Median (range) delay was 4 weeks (1-48) for total, 3 (1-48) weeks for patient and 1 (0-25) week for health care delay. Patients with long total delay (> or = 12 weeks, 15%) accounted for 49% of the cumulative number of delay-weeks. Independent risk factors (p < 0.05) for long total delay were female sex, middle age, remote setting, residence in the northern or central area, and initial visit to the private sector. For long patient delay (> or = 6 weeks) this was female sex, belonging to an ethnic minority, and living at > 5 km distance from a health facility or in the northern area. For long health care delay (> or = 6 weeks) this was urban setting, residence in the central area and initial visit to a communal health post, TB hospital or the private sector. CONCLUSION: Analyses of patient and treatment delays can indicate target groups and areas for health education and strengthening of the referral system, in particular between the private sector and the NTP.