RESUMEN
BACKGROUND AND OBJECTIVES: Our requirements for leukocyte-depleted platelet concentrates (LD-PC) for an adult patient are: platelets >240x10(9), leukocytes <5x10(6), volume of 150-400 ml; and at the end of storage a pH between 6.8 and 7.4 and presence of the swirling effect. Our aim was to develop a standardized, semiautomated method for the production of LD-PC, by pooling of buffy coats (BC), and prestorage leukoreduction by filtration. MATERIALS AND METHODS: Whole blood was collected in Top and Bottom systems, and separated automatically with the Compomattrade mark G3 equipment into a red cell concentrate, a plasma and a BC. Subsequently, a pool of 5 BC was made, and 200 g plasma from one of the donors was added. Then, after soft spin centrifugation, the platelet rich plasma was leukocyte depleted by filtration using the Autostoptrade markBC filter, and stored in a 1,000 ml polyolefin platelet storage bag. RESULTS: BC (n = 60) had a volume of 51+/-2 ml (mean +/- SD) with a hematocrit of 0.44+/-0.03 l/l and contained 80+/-5% of the platelets and 74+/-12% of the leukocytes of the whole blood. Routinely prepared LD-PC (n = 15,037) contained a median of 341x10(9) platelets (range 49-599x10(9)), with only 104/15,037 (0.7%) containing fewer than 240x10(9) platelets; the median volume was 263 ml (range 134-373 ml). In 118/917 (13%) LD-PC leukocytes were observed in the Nageotte hemocytometer, but only twice exceeding 1x10(6) leukocytes per unit, and none exceeding 5x10(6) (median <0. 6x10(6); range <0.6-1.41x10(6)). Storage experiments of the LD-PC (n = 12) revealed adequate oxygenation and maintenance of pH and swirling effect up to 9 days. CONCLUSIONS: This method warrants with 99% confidence that LD-PC contain more than 240x10(9) platelets; with 97.5% confidence that 100% of the LD-PC contain <5x10(6) leukocytes, and with 95% confidence that more than 99% of the LD-PC contain fewer than 1x10(6) leukocytes; these LD-PC can be stored satisfactorily for up to 9 days.
Asunto(s)
Plaquetas/citología , Separación Celular/métodos , Leucocitos/citología , Automatización , Centrifugación , Filtración/instrumentación , Humanos , Leucaféresis , Staphylococcus/aislamiento & purificaciónRESUMEN
BACKGROUND: Six filters were tested for their ability to remove white cells from buffy coat-depleted red cell concentrates at various temperatures. STUDY DESIGN AND METHODS: Cellselect FR, BPF4, and Sepacell filters were tested at both room temperature (RT) and 4 degrees C. The Leucoflex filter was tested only at 4 degrees C, while the Cellselect Optima Plus and Imugard filters were tested only at RT. Donor-dependent differences were excluded by pooling and subsequently dividing 9 red cell concentrates; 12 sets of experiments were performed. RESULTS: With all filters, red cell concentrates containing <5 x 10(6) white cells per unit were obtained. The lowest numbers of residual white cells were achieved with the Leucoflex (at 4 degrees C, 0.15 +/- 0.11 x 10(6), the Sepacell (at 4 degrees C, 0.23 +/- 0.14 x 10(6), the Imugard (at RT, 0.24 +/- 0.14 x 10(6), and the BPF4 (at 4 degrees C, 0.25 +/- 0.24 x 10(6); differences not significant). With the Cellselect FR, filtration at 4 degrees C resulted in 0.86 +/- 0.37 x 10(6) white cells per unit, a level not significantly different from that obtained with the BPF4 and Sepacell filters at RT (1.16 +/- 0.43 x 10(6) and 0.80 +/- 0.36 x 10(6) white cells, respectively). Filtration at RT with the Cellselect FR and Cellselect Optima Plus resulted in red cell concentrates with 1.79 +/- 0.69 x 10(6) and 2.29 +/- 0.69 x 10(6) white cells, respectively (p<0.01). CONCLUSION: All filters conformed to the current standards for white cell reduction; the process was less efficient at RT than at 4 degrees C. For routine application, the composition of the red cell concentrate, the temperature, and logistic preferences should be taken into account in the final choice of filter; before implementation, the chosen filter must be validated under routine conditions.
Asunto(s)
Separación Celular/instrumentación , Transfusión de Eritrocitos/métodos , Filtración/instrumentación , Leucocitos , Recuento de Células Sanguíneas , Estudios de Evaluación como Asunto , Hematócrito , Hemoglobinas/análisis , Humanos , Temperatura , Factores de TiempoRESUMEN
BACKGROUND AND OBJECTIVES: Whole blood can be separated by hard spin centrifugation into layers of blood components according to their specific gravity. The aim was to develop a program for an automatic separator to subsequently express the various components into their respective satellite bags in top and bottom systems with the following requirements: a red cell concentrate with a low leukocyte and platelet contamination, a 'cell-free' plasma, and a buffy coat with a volume of about 50 ml with an acceptable loss of red cells. MATERIALS AND METHODS: The Compomat G4 possesses an independently moving upper and lower press, to automatically express plasma or red cells to satellite bags of top and bottom systems. The influence of the extension of the lower press was studied by pooling and dividing two units of whole blood, and separating these units after centrifugation (2,960 g, 10 min) either with a program where the lower press was completely extended (program C), or with a program that left approximately 1 mm between the door and the lower press (program D). RESULTS: The program (program D), where the lower press was not completely extended, yielded a buffy coat with a volume of 52+/-1 ml (mean +/- SD, n = 36), which contained >75% leukocytes and >90% platelets of the original whole blood unit, with a red cell loss in the buffy coat of 21+/-1 ml (10.8+/-0.8% of the original volume). The leukocyte content of the red cell concentrates was 775+/-379x10(6) per unit, whereas the plasma contained 3+/-3x10(6) leukocytes and 4+/-3x10(9) platelets per unit. The pooling experiment indicated that complete extension of the lower press (program C) resulted in a significantly higher leukocyte contamination of the red cell concentrate (788+/-431x10(6 ) vs. 658+/-419x10(6); n = 12; p = 0.03), while there was no difference in the yield of red cells or plasma. The buffy coat produced with program D contained significantly more leukocytes (2,242 +/-396x10(6) vs. 2,065+/-327x10(6), p = 0.005) and more platelets (96+/-14x10(9) vs. 92+/-17x10(9), p = 0.02) per unit than with program C, probably because buffy coat cells sticking to the container wall are not expressed to the red cell concentrate, and thus remain in the buffy coat bag. Therefore, program D met our specifications for blood products. CONCLUSIONS: The Compomat G4 warrants reproducible separation of whole blood in top and bottom bags into red cells, buffy coat and plasma meeting our specifications.
Asunto(s)
Eliminación de Componentes Sanguíneos/instrumentación , Separación Celular/instrumentación , Automatización , Sistema Libre de Células , Centrifugación , Hematócrito , Humanos , Recuento de Leucocitos , Reproducibilidad de los ResultadosRESUMEN
A disturbed hypothalamus-pituitary-adrenal gland axis and alterations at the immune system level have been observed in patients with chronic fatigue syndrome (CFS). Glucocorticoids are known to modulate T cell responses; therefore, purified CD4 T cells from CFS patients were studied to determine whether they have an altered sensitivity to dexamethasone (DEX). CD4 T cells from CFS patients produced less interferon-gamma than did cells from controls; by contrast, interleukin-4 production and cell proliferation were comparable. With CD4 T cells from CFS patients (compared with cells from controls), a 10- to 20-fold lower DEX concentration was needed to achieve 50% inhibition of interleukin-4 production and proliferation, indicating an increased sensitivity to DEX in CFS patients. Surprisingly, interferon-gamma production in patients and controls was equally sensitive to DEX. A differential sensitivity of cytokines or CD4 T cell subsets to glucocorticoids might explain an altered immunologic function in CFS patients.
Asunto(s)
Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Dexametasona/farmacología , Síndrome de Fatiga Crónica/inmunología , Glucocorticoides/farmacología , Interferón gamma/metabolismo , Adulto , Linfocitos T CD4-Positivos/citología , División Celular , Células Cultivadas , Humanos , Interleucina-4/metabolismo , Persona de Mediana Edad , Células TH1/inmunologíaRESUMEN
Peripheral blood mononuclear cells (PBMC) from 40 consecutive patients entering a screening program on cognitive impairment were studied in vitro with respect to their sensitivity to dexamethasone (DEX). Phytohemagglutinin-induced proliferation by PBMC from patients with senile dementia of the Alzheimer type (SDAT) was less sensitive to the inhibitory effect of DEX, compared to PBMC from patients with multi-infarct dementia (MID) and PBMC from patients with miscellaneous causes of cognitive impairment (MISC). An intermediate sensitivity was found with PBMC from patients with clinical signs of both MID and SDAT (= MIXED). These differences could not be explained by differences in the composition of the CD4(+) T cell population, interleukin (IL)-2 or IL-4 production, or quantitative differences in the expression of glucocorticoid receptors as measured by flowcytometry. However, the expression of bcl-2 was higher in PBMC from SDAT patients than in cells from MID patients or from MISC patients, whereas the MIXED group showed an intermediate expression; a high bcl-2 expression correlated with a low DEX-sensitivity. These findings suggest that characteristics of PBMC reflect related changes in the central nervous system and indicate that PBMC may be a useful and accessible tool to obtain more insight into the pathogenesis of Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/inmunología , Demencia por Múltiples Infartos/inmunología , Dexametasona/farmacología , Inmunosupresores/farmacología , Linfocitos T/efectos de los fármacos , Anciano , Anciano de 80 o más Años , Apoptosis/efectos de los fármacos , Células Cultivadas , Resistencia a Medicamentos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-2/metabolismo , Interleucina-4/metabolismo , Activación de Linfocitos/efectos de los fármacos , Masculino , Fitohemaglutininas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Receptores de Glucocorticoides/biosíntesis , Receptores de Glucocorticoides/genética , Linfocitos T/metabolismoRESUMEN
To investigate the effects of the altered composition of the helper T cell compartment in ageing on the humoral response to influenza vaccine, we investigated correlations between helper T cell subsets and anti-influenza antibody responses in 23 JUNIEUR healthy young and 41 SENIEUR healthy elderly subjects. Naive helper T cell numbers (CD4+ CD45RA+) were negatively correlated with antibody production to two of the four strains investigated in JUNIEURS only. By contrast, memory helper T cell numbers (CD4+CD45ROhi) were positively correlated with in vivo IgG antibody titres to three of the four vaccine strains. Age-related differences in the composition of the helper T cell compartment, however, did not explain the lower IgG antibody response that was observed to two of the four vaccine strains examined.
Asunto(s)
Envejecimiento/inmunología , Anticuerpos Antivirales/biosíntesis , Isotipos de Inmunoglobulinas/biosíntesis , Vacunas contra la Influenza/farmacología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Formación de Anticuerpos/efectos de los fármacos , Formación de Anticuerpos/inmunología , Humanos , Isotipos de Inmunoglobulinas/sangre , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Persona de Mediana Edad , Análisis de Regresión , Subgrupos de Linfocitos T/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/efectos de los fármacosRESUMEN
Human CD45RA+ ('naive') and CD45RO+ ('memory') CD4+ T cells were compared with respect to their sensitivity to dexamethasone (DEX). In three different activation pathways, i.e. (i) immobilized anti-CD3, (ii) immobilized anti-CD3 plus soluble anti-CD28 and (iii) soluble anti-CD2 plus soluble anti-CD28, naive CD4+ T cells appeared more sensitive to DEX than memory CD4+ T cells. In the anti-CD3 system this difference in sensitivity was apparent at a suboptimal DEX concentration. Addition of anti-CD28 rendered the cells largely insensitive to DEX, indicating that the CD28 pathway is less dependent of the DEX-sensitive transcription factor AP-1. However, the alternative pathway of T cell activation through CD2/CD28 triggering was highly sensitive to DEX when naive cells were studied; in the case of memory cells, at least a 10-fold higher DEX concentration was needed to achieve a comparable inhibition. The strong inhibitory effect of DEX on naive CD4+ T cells stimulated via the alternative pathway was completely abrogated by activation of protein kinase C (PKC) with phorbol myristate acetate. Our data suggest that at least two different mechanisms contribute to DEX resistance, i.e. CD28 triggering and PKC activation, which may occur more effectively in memory cells making them less sensitive to DEX.
Asunto(s)
Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/inmunología , Dexametasona/farmacología , Memoria Inmunológica/efectos de los fármacos , Antígenos CD2/efectos de los fármacos , Antígenos CD28/efectos de los fármacos , Antígenos CD4/efectos de los fármacos , Linfocitos T CD4-Positivos/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Humanos , Activación de Linfocitos/efectos de los fármacos , Proteína Quinasa C/biosíntesis , Proteína Quinasa C/efectos de los fármacosRESUMEN
Cortisol levels in patients with Alzheimer's disease (AD) are relatively unaffected by a challenge with dexamethasone (DEX) in vivo. The present study demonstrates that DEX is less inhibitory for phytohemagglutinin (PHA)-induced T cell proliferation in AD patients as compared to age-matched controls. Since no significant differences were found between AD patients and age-matched controls with regard to the fraction of CD45RA+ or CD45RO+ CD4+ T cells nor the ability of peripheral blood mononuclear cells to produce IL-2 or IL-4, it is unlikely that the difference in DEX sensitivity is due to a changed lymphokine profile or a changed composition of the CD4+ T cell population. Sensitivity to DEX was negatively correlated with the ability to produce IL-2 and IL-4 in the controls but not in AD patients. This suggests that IL-2 and IL-4 synthesis in AD patients is less sensitive to regulation by glucocorticoids.
Asunto(s)
Enfermedad de Alzheimer/sangre , Dexametasona/farmacología , Linfocitos/efectos de los fármacos , Anciano , Enfermedad de Alzheimer/genética , Femenino , Humanos , Activación de Linfocitos/efectos de los fármacos , Masculino , Fenotipo , Sensibilidad y Especificidad , Linfocitos T/inmunologíaRESUMEN
The suppressive effect of the glucocorticoid dexamethasone (DEX) on purified CD4+ T cells was found to depend on the activation pathway. In contrast to anti-CD3- or PHA-induced T cell proliferation, the alternative pathway of T cell activation, i.e., through anti-CD2 and anti-CD28, appeared largely resistant to DEX. By titrating anti-CD28 or the protein kinase C (PKC) activator PMA in the DEX-sensitive systems, it was demonstrated that inhibition by DEX could be abrogated by enhancing the CD28 signal or by stimulation of the PKC-dependent pathway. Supraoptimal concentrations of PMA were inhibitory for proliferation and this effect was partly prevented by DEX. These data suggest that the outcome of the effect of DEX on CD4+ T cells is dependent on the activation pathway, in particular the role and composition of the transcription factor AP-1.
Asunto(s)
Antígenos CD28/metabolismo , Dexametasona/farmacología , Tolerancia Inmunológica/efectos de los fármacos , Proteína Quinasa C/metabolismo , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/enzimología , Linfocitos T CD4-Positivos/inmunología , Resistencia a Medicamentos , Activación Enzimática , Humanos , Tolerancia Inmunológica/fisiología , Técnicas In Vitro , Interleucina-2/biosíntesis , Interleucina-4/biosíntesis , Activación de Linfocitos/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The influence of ageing on phenotype and function of CD4+ T cells was studied by comparing young (19-28 years of age) and aged (75-84 years of age) donors that were selected using the SENIEUR protocol to exclude underlying disease. An age-related increase was observed in the relative number of memory cells, not only on the basis of a decreased CD45RA and increased CD45RO expression, but also on the basis of a decrease in the fraction of CD27+CD4+ T cells. Our observation that the absolute number of CD45RO+CD4+ T cells was increased, while absolute numbers of CD27-CD4+ T cells remained unchanged in aged donors, indicates that the latter subset does not merely reflect the size of the CD45RO+CD4+ T cell pool. The increased fraction of memory cells in the aged was functionally reflected in an increased IL-4 production and T cell proliferation, when cells were activated with the combination of anti-CD2 and anti-CD28, whereas IL-2 production was comparable between both groups. No differences were observed with respect to proliferative T cell responses or IL-2 production using plate-bound anti-CD3 or phytohaemagglutinin (PHA). The observation that IL-4 production correlated with the fraction of memory cells in young donors but not in aged donors suggests different functional characteristics of this subset in aged donors.