J.G. Jones

Effects of preparative procedures and of cell activation on flow of white cells through micropore filters

Using newly developed filtration techniques to analyse the flow resistance of white blood cells (WBC), the effects of preparative procedures and of various treatments (including activation) have been tested. Flow rates of WBC were measured using both 5 and 8 μm pore filters. It was found that EDTA was the anticoagulant of choice over heparin and sodium citrate, and that calcium‐containing media should be avoided because they promoted cell‐cell aggregation. Exposure to density separation media did not significantly alter filtration rates, but storage of prepared WBC for even 1 h caused deterioration in flow. Storage of whole blood before preparation of the WBC had a much lesser effect. Storage appeared to be linked to cell activation, and use of the activating agents phorbol ester (PE, 4.5 × 106 M/l) and f‐methyl‐leucyl‐phenylalanine (FMPL, 10‐7 and 10‐9 M/l) also increased WBC resistance to pore transit. The effect of PE was greater, causing rapid pore blockage, but was nonspecific, while the lesser effect of FMLP was restricted to granulocytes and was concentration dependent. Cell volume was increased by these agents, but measurement of the filterability of hypotonically swollen granulocytes showed that volume changes alone only partly explained the activation effects. These results suggest that activation in vivo would have a significant rheological effect, detectable by filtration methods.

Total circulating red cells versus haematocrit as the primary descriptor of oxygen transport by the blood

Summary Peripheral haematocrit (PCV) is the traditional target and monitor in many transfusion regimens. Without negating the importance of PCV as a determinant of whole blood viscosity, the present article outlines two important reasons why the red cell volume (RCV) should replace PCV in the central target role during blood transfusion in intensive care and other emergency situations:

Methods and theory for analysis of flow of white cell subpopulations through micropore filters

Methods have been developed for analysing the resistance of WBC to flow, by measuring their transit rates through 5 and 8 μm pore filters at constant pressure. Unfractionated WBC and separated subpopulations have been compared. For either pore size, lymphocytes exhibited least resistance to flow, followed by granulocytes, with monocytes being most resistant. A theoretical model, which represents WBC suspensions as made up of three particle types (a relatively fast and a relatively slow population, plus a pore blocking population) adequately describes the data for flow rate versus volume filtered. For 5 μm pores, this theory indicates that a majority of WBC have transit times ±0.5 s. Unfractionated and mixed mononuclear samples contained a proportion of particles with transit times an order of magnitude longer, whereas, for granulocytes, no slow flowing population was evident. Removal of monocytes by plating out, reduced the proportion of the slow particles in the mononuclear preparation. Unique values for transit times could not be determined for 8 μm pores, but it could be concluded that the great majority of WBC made a very quick transit, with the flow becoming dominated by a small number of much more resistant cells. Simple flow parameters (initial relative flow rate and slow particle resistance) are described which characterize these two populations. Both 5 and 8 μm pore data indicated that few cells became permanently trapped within pores.

Evaluation of the contribution of red and white cells to the flow of suspensions of washed blood cells through 3 μm Nuclepore membranes

Summary. This study was designed to investigate a method of analysis which can quantitate the contribution of white cells to the flow of washed suspensions of blood cells. Such an analysis would obviate the need to remove white cells when studying the filterability of normal and abnormal red cells.

The flow of blood cell suspensions through 3 μm and 5μm Nuclepore membranes: a comparison of kinetic analysis with scanning electron microscopic examinations

Summary This study was designed to investigate the effect of red and white cells on the flow of dilute suspensions of blood cells through 3 μm and 5 μm Nuclepore membrane filters.

The Effects of Temperature on the Filtration of Diluted Blood Through 3 µm and 5 µm Filters

Abstract The effect of temperature on the flow of diluted blood [Hct = 0.21], through 5 μm Nuclepore filters, is described by the Arrhenius equation with an energy of activation of 27.7 kJ/mol. Plasma, diluted with PBS, is almost three times less sensitive to temperature, with an energy of activation of 9.8 kJ/mol, while red cells are of intermediate sensitivity, with an energy of activation of 14.7 kJ/mol. The most sensitive elements to changes in temperature are leukocytes, with energies of activation of 31 kJ/mol and 35 kJ/mol for fast-flowing leukocytes (granulocytes and lymphocytes) and slow-flowing leukocytes (monocytes) respectively. Hence, the major determinants of the decline in filterability of blood through micropore filters are the leukocytes. This effect is compounded when blood is kept for 10 minutes or more at 10° C due to activation of granulocytes, which leads to permanent pore blocking when the affected blood is filtered at room temperature. The combination of increased passage time of leukocytes through peripheral areas at abnormally low temperatures and subsequent activation might influence the flow of blood in non-affected tissues. The effect of temperature on the filterability of red blood cells through 3 μm filters is not described by the Arrhenius equation and the deviations are seen as a gradual change of slope rather than a sharp break between two straight lines. The data are consistent with a gradual shift in rate limiting step away from the entry event into pores, which dominates at low temperature but becomes progressively less important at elevated temperatures. The changing parameter is probably the volume of the red cell, which is less important when flow is measured through 5 μm pores.

Examination of a rheological profile for blood using micropore filters

Various techniques have been used to assess the flow properties of blood and blood cells in a range of clinical situations. Filtration through microfilters offers a single technique for measuring the flow properties of all cellular components of blood in one experiment but depends on an assumed ability to recognize cells that make up <10% of leucocytes. The remaining leucocytes, labelled fast leucocytes, were previously presumed to be lymphocytes and granulocytes. This study confirmed the identities of these fast leucocytes as those of lymphocytes and granulocytes in undiluted blood. The transit time for lymphocytes (1.2 s) and granulocytes (1.6 s) is close to that recorded for fast leucocytes (1.7 s). The resistance of each type of blood cell to flow through 5 μm filters was defined in this study as the product of the concentration of that cell in blood and its transit time through a pore in the filter. The total resistance to flow of healthy blood through the filter is 4.46 × 107 s/ml and is attributed to plasma (2.7%), red cells (25.9%), fast leucocytes (25.3%) and slow leucocytes (46.1%). In a cohort of 21 men with peripheral arterial disease the total resistance was increased to 7.82 × 107 s/ml and attributed to plasma (1.5%), red cells (14.5%), fast leucocytes (21.0%) and slow leucocytes (63.0%). This analysis therefore provides a single test for assessing the flow properties of all the cellular components of blood and plasma.