Factors Affecting the Efficiency of Filtration Leukapheresis

A. J. KATZ, J . Houx, AND E. E. MORSE

From the Connecticut Red Cross Blood Center and the Department of Laboratory Medicine, University of Connecticut School of Medicine, Farmington. Connecticut

Continuous flow filtration leukapheresis was performed on normal volunteer donors with and without intravenous administraiion of dexamethasone. Although granulocyte counts were increased in steroid treated donors and almost 20 per cent more cells were presented to the filters, the final yield of granulocytes was slightly decreased and the efficiency of collection during a two- to three-hour leuka- pheresis was statistically significantly decreased.

E X P E R I E N C E W I T H L E U K A P H E R E S I S by continuous flow centrifugation has indicated that yields of granulocytes can be increased by ad ministering co r t ico s t eroids to th e donor.'.9 The granulocytosis produced by the steroid is considered responsible for the effect.',2,'0 In light of these reports, steroid stimulation of donors undergoing filtration leukapheresis was employed to try to increase granulocyte yield. This report deals with the effects of administering dexameth- asone to donors just prior to filtration leuka- pheresis, and some observations on the fac- tors affecting the efficiency of the procedure.

Materials and Methods

Filtration leukapheresis was performed on 44 healthy volunteer donors (30 male, 14 female, ages 19 to 55 years), using a dual nylon wool filter harness (Leukopak, Fenwal, Morton Grove, Illinois), and a peristaltic roller pump (Leukophe- resis Pump, Fenwal, Morton Grove, Illinois). Minimal modification of the basic procedure5 in- cluded administration of an intravenous bolus of Sodium Heparin (Organon, Inc., W. Orange, N.J.), 2,500 u at the start of the procedure. Dur- ing the filtration procedure, approximately one unit of heparin per ml of blood was added by continuous flow during the filtration procedure. At

Received for publication March 9, 1976; accepted June 12, 1976.

the end of the procedure which generally lasted between two and three hours, saline was pumped through the filters to return the red blood cells to the donor. After the leukapheresis was completed, the granulocytes were eluted with 1,500 ml of a mixture of one part ACD-A solution, one part CPD plasma and four parts saline. The filters were vigorously tapped during the elution procedure. The flow was adjusted to 100 ml/minute . Effluent granulocytes were concentrated to a final volume of about 300 ml by centrifugation.

Donors received steroids on a random basis, but a few did not receive the medication because they had borderline elevation of blood pressure, a his- tory of previous reaction to dexamethasone (including bladder fullness), or were reluctant to receive it. Dexamethasone (Merck, Sharpe and Dohme, West Point, Pennsylvania), 6 mg was administered intravenously within 30 minutes of beginning the filtration procedure. Blood samples were obtained from the donor prior to leukaphe- resis and immediately after the procedure, and from the final leukocyte concentrate. White blood cell counts were performed with a Coulter Model B cell counter and the percentage of granulocytes was determined by examination of Wright stained smears.

Results The data characterizing filtration leukapheresis

with and without donor stimulation with dexa- methasone, are summarized in Table I . Donors who received steroids had significantly increased numbers of circulating granulocytes a t the end of the procedure, and significantly increased average number of circulating granulocytes during the procedure as estimated from starting and ending counts. The rate of blood flow, the time of the collection, the total volume processed, and the total number of granulocytes (grans) processed was comparable in the two groups. However, there was a statistically significant difference in efficiency of granulocyte collection between the

67 Transfusion Jan.-Feh. 1977

Volume 17 Number I

KATZ ET AL. Transfusion JanwFeb. 1977

Table 1. Summary of Results Obtained by Filtration Leukapheresis of Donors with and without Steroid Treatment

No Steroid Steroid n Mean ? 1 SD n Mean t 1 SD

Start WBC (/mm3) Start % grans Start gram (/mm3) End WBC (/mm3) End % grans End grans (lmrn3) Rate (ml/min) Time (Min) Total volume processed (liters) Av. grans [/mrn3) Total grans in (X 109) Yield (X 109) Efficiency (%) Yield per liter processed

16 16 16 14 14 13 16 16 16 16 16 16 16 16

5594 f 1478 62.8 f 9.4

3474 f 824 6186 f 1462 67.8 f 8.6

4217 i 1305 54.4 f 7.3

142.5 f 25.7 7.63 5 1.10

3777 2 908 28.7 f 8.2 20.0 f 7.5 70.6 t 22.7 2.67 f 1.02

28 28 28 28 27 27 28 28 28 28 28 28 28 28

6961 f 7161 64.3 f 12.2

4402 t 3909 7141 k 2554 79.9 t 6.4 5731 ? 2094 54.3 t 9.7

135.0 f 22.4 7.24 f 1.28

4724 t 1361 33.7 f 9.4 17.8 f 6.0 54.8 t 16.2 2.53 f 0.92

NS NS NS NS

p < ,001 p < 0.05

NS NS NS

p < .02 NS NS

p < .02 NS

NOTE: grans = granulocytes; n = number analyzed; NS = not significant.

groups. A lesser proportion of those granulocytes passing through the filter were recovered in the eluted material from the steroid treated donors. The final yield of granulocytes was slightly, but not significantly greater in the non-steroid group.

The relationship of each of the variables to granulocyte yield, yield per liter of blood pro- cessed, and to efficiency, is presented in Table 2. Based on the levels of significance of the correla- tion coefficient^,^ the starting white blood cell count (WBC) and the starting number of circulat- ing granulocytes appear to be the best predictors of yield and yield per volume processed for donors who did not receive steroids. The percentage of granulocytes, rate of flow, length of procedure, and total volume processed are much less useful predictors of yield, given the ranges of these vari- ables (e.g. mean total volume processed 7.63 zt 1.10 I) in this study. Yield and yield per volume processed were significantly, positively correlated with the final and average circulating granu- locytes, and with the total number of granulocytes processed. Efficiency was determined by dividing the final yield by the number of granulocytes passed through the filters and expressing the result as a per cent. Efficiency decreased signifi- cantly as total volume processed increased, but was not significantly correlated with the number of granulocytes processed.

For donors receiving dexamethasone at the start of the procedure, only the average granu- locyte count during the procedure, and the total number of granulocytes processed were signifi- cantly correlated with yield. Yield per volume processed, as with the nonsteroid donors, was

positively correlated with final and average num- bers of granulocytes. Yield per volume was nega- tively correlated with rate and total volume processed. As the total number of granulocytes processed increased, the efficiency of t h e procedure significantly decreased when steroids were used.

Discussion

The results of this study suggest that steroid administration causes an increase in the number of circulating granulocytes pass- ing into the filter. Rubins, MacPherson et a1..8 have shown a decrease in the granu- locyte concentration in the blood of donors undergoing filtration Ieukapheresis after 6 mg dexamethasone IV, during the first 30 minutes o f the procedure, but it then increases, sometimes dramatically, in the second 30 minutes of the run and remains elevated. The granulocyte level in circulating blood is increased for approximately 75 per cent of the total time of a two- to three-hour procedure and calculations of average granu- locyte level based on beginning and ending counts may slightly underestimate the number of granulocytes presented to the fil- ters in steroid treated donors. In contrast to results reported with continuous flow centrif-

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70 KATZ ET AL Transfusion Jan.-Feb 1977

ugation leukapheresis, the actual yield during continuous flow filtration is unchanged and efficiency of collection is decreased following steroid administration.

Recent reports suggest that steroids (dexamethasone, prednisone), al ter the granulocyte membrane" and its adherence to foreign (nylon) surfaces.6 Thus it seems likely that steroid treated donors have some proportion of granulocytes in their circula- tion that adhere poorly to nylon filters. Either these granulocytes did not adhere dur- ing the filtration procedure, or they were lost from the filters during the flushing procedure prior to removing the leukapheresis ap- paratus from the donor. The present study suggests about 20 per cent of the granu- locytes in the circulation of steroid treated donors may not remain within the nylon filter for elution. Approximately the same yield was obtained (17.8 x log steroid vs. 20.0 x log nonsteroid), although about 20 per cent more cells were presented to the filters, (33.7 x lo9 steroid vs. 28.7 x log non- steroid). This observation, together with pre- vious that indicate that steroid treatment slows the exit of granulocytes from the marginating pool and increases the circulating granulocyte pool partly on this basis, raise serious questions about the use- fulness of collecting steroid treated granulo- cytes.

It has been suggested that longer collec- tion periods could be used in increasing granulocyte yields.4 Other studies have shown increased damage to granulocytes after prolonged filtration procedures, sug- gesting that two to three hours is the op- timum time for a filtration leukapheresis. It seems reasonable to conclude that methods used in the acquisition of granulocytes should be modified to include: 1) Addition of more filters to improve collection of more granu- locytes at short time intervals and, 2) the use of medications to increase granulocyte counts which do not act by altering the granulocyte membrane.

References I . Boggs, D. R., J. W. Athens, G. E. Cartwright, and

M. M. Wintrobe: The effect of adrenal glu- cocorticosteroids upon the cellular composition of inflammatory exudates. Am. J . Pathol. 44:763, 1964.

2. ~, J . W. Athens, G . E. Cartwright, and M. M. Wintrobe: Leukokinetic studies: IX, ex- perimental evaluation of a model of granulo- poiesis. J . Clin. Invest. 44:643, 1965.

Dixon, W. J . and F. J. Massey, J r . : Introduction to Statistical Analysis. N.Y., McGraw-Hill, 1969,

Harris, M. B., I . Djerassi, E. Schwartz, and R. K . Root: Polymorphonuclear leukocytes prepared by continuous flow filtration leukapheresis: via- bility and function. Blood 44:707, 1974.

Herzig, G. P., R. K. Root, and R. G. Craw: Granu- locyte collection by filtration leukapheresis. Blood 39554, 1972.

MacGregor, R. R., P. J. Spagnuolo, and A. L. Lentnek: Inhibition of granulocyte adherence by ethanol, prednisone, and aspirin, measured with an assay system. N. Engl. J. Med. 291:642, 1974.

7. Mishler, J. M., D. J . Higby, W. Rhomberg, E. Cohen, R. W. Nicora, and J . F. Holland: Hy- droxyethyl starch and dexamethasone as an ad- junct to leukocyte separation with the IBM blood cell separator. Transfusion 14:352, 1974.

Rubins, J. M., J . L. MacPherson, J. Nusbacher, and T. Wiltbank: Granulocyte kinetics in donors undergoing filtration leukapheresis. Transfusion 1655, 1976.

Shoji, M., and W. R . Vogler: Effects of hydro- cortisone on the yield and bactericidal function of granulocytes collected by continuous-flow centrifugation. Blood 44:435, 1974.

Soderlund, I . , L. Engstedt, and S. Paleus: Induc- tion of leukocytosis by ,hydrocortisone and/or muscular exercise. In: Proceedings of Interna- tional Symposium on Leukocyte Separation and Transfusion. London, 1974.

Wright, D. G., J. C. KaulTmann, M. J. Chusid, G. P. Herzig, and J . I . Gallin: Functional abnor- malities of human neutrophils collected by continuous flow filtration leukopheresis. Blood 46:901, 1975.

3.

p. 202. 4.

5 .

6.

8.

9.

10.

11.

Katz, Alfred J . , M.D., Director, Connecticut Red Cross Blood Center and Associate Professor of Labora- tory Medicine, University of Connecticut School of Medicine, 209 Farmington Avenue, Farmington, Con- necticut 06032.

Houx, J., R.N., Assistant Director, Nursing, Con- necticut Red Cross Blood Center, 209 Farmington Avenue, Farmington, Connecticut 06032.

Morse, E. E., M.D., Professor of Labora tory Medicine, University of Connect icut School of Medicine, Farmington, Connecticut 06032.