Changes in the polymorphonuclear leukocyte function of blood samples induced by storage time, temperature and agitation.

We have investigated changes in polymorphonuclear leukocyte (PMN) functions of blood samples caused by such typical elements of laboratory handling as storage time, temperature and agitation. The blood of five healthy subjects was stored upright in test tubes at 4, 22 and 37 degrees C over periods of 20 min, one, two, six and 24 h. Controlled agitation was performed on a shaker. The following PMN functional parameters were measured: the white blood cell count (WBC), migration, elastase (EL) release, reactive oxygen species (ROS) production and lipid peroxidation. Migration was determined in a whole-blood membrane filter assay; ROS production by latex-stimulated, luminol-enhanced chemiluminescence (CL) in a whole-blood assay; EL as EL alpha 1-antitrypsin complex; and lipid peroxidation by malondialdehyde (MDA) generation. The reactions after handling were compared with the values measured immediately after blood withdrawal which served as reference values of 'genuine' PMN reactivity. The outstanding result was the marked scatter between the individual reactions. Overall, the proportion of migrating PMNs in the blood total decreased, while CL, correlating positively with MDA, increased with the time of storage. EL increased considerably in some of the samples. Agitation raised CL and MDA. The effect of temperature was apparent only after 24 h at 37 degrees C. There was evidence that inhomogeneities in the blood samples were another interfering factor, since resuspension of sedimented blood after storage can be incomplete. In order to obtain reliable results from PMN functional tests, whole-blood assays and processing of blood samples within 20 min after blood withdrawal are recommended.

Influence of age on the release of reactive oxygen species by phagocytes as measured by a whole blood chemiluminescence assay.

Polymorphonuclear and mononuclear phagocytes play an important role in host defense, but may also cause tissue injury through excessive inflammation. Reactive oxygen species (ROS) are not only directly ore indirectly involved in a wide variety of clinical disorders, such as atherosclerosis, reperfusion injury, pulmonary toxicity and cancer, but they are also important in the aging process. This process is associated with increasing susceptibility to infection. In this study we investigated the influence of age and sex on phagocyte activation by means of a whole blood chemiluminescence (CL) assay. Circulating phagocyte activity was measured in 55 healthy volunteers (24 females, 31 males) aged from 6 to 92 years. Using an automated luminescence system, phagocytes were stimulated by polystyrene beads and Luminol-enhanced CL was determined in terms of peak height and peak time in freshly withdrawn, peripheral venous whole blood. An extremely significant positive correlation (p < 0.0001) between the maximum of light emission after stimulation and increasing age was found. This finding is true for the total population of blood phagocytes as well as for a single cell. In contrast the time of the appearance of the maximum of light emission showed an extremely significant inverse correlation (p < 0.0003) with increasing age. The influence of sex on the CL-parameters showed no significant difference between women and men. It is concluded that the increased susceptibility of circulating phagocytes to oxidative burst in elderly subjects may be the consequence of several biological events. Senescent cells express more and also have new antigens on their surfaces that trigger an autoimmune response. Cellular senescence appears earlier in old organisms. Therefore phagocytes in aging individuals may be increasingly involved in their scavenger tasks that grow with the catabolic bias in cell turnover. Moreover, atherosclerotic alterations in the intima and endothelial lesions are physiologic concomitants of age and may lead to a stimulation of circulating phagocytes.

The influence of trauma on changes in neutrophil granulocyte function assessed by an analysis of granulocyte migration.

We examined the effects of trauma on polymorphonuclear leucocyte (PMN) migratory parameters and PMN elastase release, with the aim of tracing an acute inflammatory reaction from its very beginning to the phase of recovery. Fifteen patients who underwent monotrauma surgery, followed by uneventful healing, served as inflammation model. PMN activation was studied by measuring their readiness to migrate (TMI) and their penetration potency (DC) in a whole blood membrane filter device, in which a chemoattractant depot (FMLP) was integrated. Control chambers lacking FMLP provided parameters of the spontaneous migration. In healthy controls (n = 64), the numbers of invading PMNs decreased continuously from the outermost layer towards the interior of the filter device. FMLP did not influence the mobilization rate of PMNs immigrant from the blood into the filter, but those cells that did migrate penetrated deeper (P < 0.05). After trauma, the spontaneous and FMLP-stimulated DC was increased (P < 0.05). Trauma also tended to inhibit PMN migratory activity episodically; depression of the unspecific immune function (low TMI values) was found on the 3rd (P < 0.0001) and 12th (P < 0.01) postsurgical days. There was no correlation between the migratory parameters and the inflammation parameter, PMN elastase release. Preliminary results indicate that analyses of PMN migratory parameters by a whole blood membrane filter assay could provide a valuable adjunct in monitoring trauma-associated immunologic changes.

Assay of phagocyte activation by means of malondialdehyde and luminol-enhanced chemiluminescence during uneventful wound healing following trauma surgery.

The purpose of the study was the assessment of the acute inflammatory response in patients (N = 12) with comparable trauma severity and uneventful wound healing courses in the postsurgical period as a contribution to the search for objectifiable criteria in the monitoring of wound healing. Whole blood chemiluminescence (CL) on the one hand and the lipid peroxidation product malondialdehyde (MDA) on the other hand as tools for the detection of the respiratory burst activity of phagocytes were used as inflammation markers and were compared with the established marker PMN elastase. Blood samples were withdrawn daily from the day of surgery to the 14th postsurgical day. CL-parameters and PMN elastase increased postoperatively reflecting surgical trauma, while MDA remained within the normal range during the whole time of observation. A decrease of CL-activity in the postsurgical period correlated with decreasing PMN elastase levels (r = 0.52, P<0.0001) as well as with the tapering of local inflammation signs concerning the wound situs. MDA values neither correlated with PMN elastase nor with any CL-parameters. The results indicate that the measurement of the phagocytic activation by CL, used for the first time in traumatology to monitor wound healing, represents a promising marker for the assessment of the actual inflammatory status.