Journal of Clinical Apheresis 10:198-202 (1995)

Efficacy of Pentastarch in Granulocyte Collection by Centrifugal Leukapheresis Jong-Hoon Lee, Herb Cullis, Susan F. Leitman, and Harvey G. Klein

Department of Transfusion Medicine, National Institutes of Health, Bethesda, Maryland (J.-H. L., S. F. L., H. G. K.), and Biotech Division, Baxter Healthcare Corporation, Deerfield, Illinois (H. C.)

The efficacy of 6% hydroxyethyl starch (hetdstarch, HS) in enhancing granulocyte harvest by centrifugal leukapher- esis has been described by a simple equation which predicts the granulocyte collection efficiency (GCE) based on an intrinsic donor variable, the erythrocyte sedimentation rate (ESR): GCE (%) = 1.3 ESR (mm/hr) + 45. Ten percent low molecular weight hydroxyethyl starch (pentastdrch, PS) has been reported to be as effective as HS with potentially fewer adverse donor reactions (ADR). The derivation of an analogous equation for PS under conditions previously reported for HS may quantify PS efficacy and allow comparison to HS. We prospectively measured the in vitro and the in vivo effects of PS on the donor ESR in 53 granulocyte collections from 44 donors using the model CS-3000 Plus blood cell separator (CS). We then correlated the findings with the GCE of each procedure and derived an equation which expresses GCE in terms of baseline donor ESR. The in vitro addition of PS increased the donor ESR 2.4-fold, but its administration to a donor during a collection procedure did not appreciably change the ESR. Higher baseline donor ESR was more likely to result in more efficient cell collections: GCE (%) = 0.8 ESR (mmihr) + 20; (r = 0.37). For granulocyte harvests using the CS and PS as the sedirnenting agent 1) baseline donor ESR affects granulocyte harvests, but the poor correlation does not allow an accurate prediction of GCE and cell yield from the baseline donor ESR; 2) in comparison with HS (results from a previous study), PS may be less effective in vitro and not effective in vivo in elevating ESR, and may be less effective in enhancing granulocyte harvest; and 3) the parameters (slope, y-intercept, correlation coefficient) which define the linear relationship between baseline donor ESR and GCE may serve collectively as a quantitative measure of the effectiveness of different hydroxyethyl starch agents in enhancing granulocyte harvests. These parameters may be helpful in rapidly assessing the clinical efficacy of new, potentially useful hydroxyethyl starch agents prior to initiating a randomized, controlled clinical trial. 0 1995 Wiley-Liss, Inc.

Key words: erythrocyte sedimentation rate, granulocyte collection efficiency, hydroxyethyl starch

INTRODUCTION

Over the past decade, 6% hydroxyethyl starch (hetastarch, HS) has been preferred as the red cell sedi- menting agent for use during granulocyte collection by centrifugal leukapheresis. HS effectively enhances gran- ulocyte yield and has few donor side effects when admin- istered with conventional dose and frequency [ I ,2]. Sig- nificant blood levels of HS persist for weeks, however, and trace amounts can be detected for years following its administration [3-51. Delayed HS clearance and conse- quent long-term donor safety concerns resulted in the search for alternative agents.

Ten percent pentastarch (PS), a low molecular weight HS analog, has been reported to be as effective as HS for granulocyte collection and is rapidly cleared from the circulation [6,7]. Initial studies could not detect PS in the blood within a few days of its administration [ 11, and the safety profile has been inferred to compare favorably with the low risk of HS [8]. These initial studies have encouraged most donor centers to substitute PS for HS in collecting granulocytes by centrifugal leukapheresis.

We recently reported that the efficiency of granulocyte collection by centrifugal leukapheresis, using the tradi- 0 1995 Wiley-Liss, Inc.

tional HS as the sedimenting agent, may be summarized by a simple equation which predicts the granulocyte col- lection efficiency (GCE) based on an intrinsic donor vari- able, the erythrocyte sedimentation rate (ESR): GCE (%) = 1.3 ESR (mdhr) + 45 [9]. In the present study, we applied the previous granulocyte collection conditions to a protocol that substituted PS for HS. We derived an analogous equation for PS in order to quantify PS effi- cacy and compare PS with HS.

MATERIALS AND METHODS

We prospectively studied 53 consecutive granulocyte collections with PS as the sedimenting agent from 44 volunteer blood donors using the model CS-3000 Plus blood cell separator (CS, Fenwal Laboratories, Deer- field, IL). All donors were premedicated with 8 mg of oral dexamethasone 12 hours before each procedure, and received 500 ml of PS (McGraw, Inc., Irvine, CA) dur-

Received for publication January 7, 1995; accepted August 27, 1995.

Address reprint requests to Jong-Hoon Lee, M.D., Building 10, Room 1C-71 I , National Institutes of Health, Bethesda, MD 20892.

Granulocyte Collection Efficiency 199

Granulocyte Collection Efficiency

The GCE of each procedure was calculated by divid- ing the granulocyte yield by the total number of granulo- cytes processed. The yield was calculated as the product of component cell count and component volume. The number of granulocytes processed (GP) was calculated as follows: GP = (WBC)(%G)(L), where WBC represents the donor peripheral white blood count (per liter), %G the granulocyte fraction, and L the total volume of processed whole blood (liters). We used the average of the two absolute neutrophil counts, obtained immediately before and following each procedure. The accuracy of averaging the two cell counts has been previously verified [9]. All cell counts were performed with the Coulter Counter@ Model S-PLUS V (Coulter Electronics, Inc., Hialeah, FL).

TABLE I. Biochemical and Pharmacokinetic Characteristics of 10% Pentastarch

Weight average molecular weight (MW)

Hydroxyethyl starch concentration (g/dl) 10.0

Time to reach 50% peak blood level (hours) 24-hour plasma distribution (% dose)

24-hour extravascular distribution (% dose)

264,000 63,000 Number average molecular weight (MN)

Hydroxyethyl groups per glucose residue 0.45 2.5 7

70 33

24-hour urinary excretion f% dose)

Overall survival in blood 96 hours

TABLE 11. Apheresis Parameters for Granulocyte Collection on the Model CS-3000 Plus Blood Cell SeDarator

Whole blood flow rate 50 mlimin

Blood volume processed 6.5-7.0 L Centrifuge RPM 1,000

Interface detector setting 33 Program 2 Chamber type Granulo

ing each procedure. Thirty milliliters of 46.7% trisodium citrate added to 500 ml of PS were infused in a fixed ratio (1:13) over approximately 7 liters of processed whole blood. Informed consent for the apheresis granulocyte donation included obtaining research blood samples. The biochemical and pharmacokinetic characteristics of PS are summarized in Table I, and the specific collection parameters are detailed in Table 11.

Erythrocyte Sedimentation Rate The ESR was measured with the automated Diesse

Vesmatic-20 analyzer [ 101 following verification of its accuracy and precision within 4 mm/hr against the stan- dard Westergren method. For each procedure, up to 3 duplicate 1 .O-ml peripheral blood samples were collected and prepared as follows: 1) pre-procedure baseline sam- ple, collected at the establishment of intravenous access (all 53 procedures), 2) 100 pl of PS added in vitro to pre-procedure sample (25 procedures), and 3) post-pro- cedure sample obtained at completion of PS administra- tion to the donor immediately prior to discontinuing the intravenous access (approximately 140 minutes after ini- tiating the procedure; 27 procedures, not necessarily from the same donors studied for the in vitro effect). The amount of PS added in vitro (100 p1 to 1 .O ml of blood, 1: 10 volume ratio) was selected to reflect the in vivo ratio (1 : 13 administration ratio of PS:blood, arbitrarily adjusted to account for uneliminated, recirculated PS). All sam- ples from a given procedure were stored temporarily at 4"C, equilibrated at room temperature for 20 minutes, and ESR was measured simultaneously within 6 hours of collection. The starch-modified ESR was plotted against baseline donor ESR.

Predicting the Granulocyte Collection Efficiency

For nine donors undergoing repeat procedures, we examined the baseline ESR and GCE to determine if these values remained constant for each donor. For all 53 procedures, we plotted GCE against baseline ESR and derived a simple empiric linear relationship between the two quantities within the ESR range between 0 and 20 mmihr. The derived relationship was then compared with the previously reported equation for HS determined under conditions identical with the present study.

Statistical Methods The significance of the results on ESR was analyzed

using the sign test, and the adequacy of the sample size was inferred from the resulting degree of significance. For all plots, we used linear regression analysis (sum of least squares method) to generate trend lines.

RESULTS Erythrocyte Sedimentation Rate

The two plots of the starch-modified ESR as a function of the baseline ESR are shown in Figure 1. The baseline ESR values were scattered widely within the range be- tween 0 and 20 m d h r . Adding PS in vitro resulted in a 2.4-fold increase in the ESR (P < .OOl). The administra- tion of PS (500 rnl) directly to a donor, however, did not appreciably change the ESR when measured immediately after each procedure (upon completion of pentastarch infusion).

Predicting the Granulocyte Collection Efficiency

Table I11 demonstrates that both the ESR and the GCE are consistent and reproducible for a given donor. De- spite a wide distribution in GCE among the 44 donors ranging from 1% to 61% (Fig. 2), the GCE consistently remained within 3% for a given donor in five of nine

200 Lee et al.

ESR with Pentastarch (mdhour ) Collection Efficiency (%)

f IN VITRO *IN VlVO I------ 50

40

10

A

4 2 0

1 0

n I I 0 2 4 6 8 10 12 14 16 1 8 20

Baseline Donor ESR (mm/hour)

Fig. 1 . The effect of pentastarch (PS) on the donor erythrocyte sedi- mentation rate (ESR). The in vitro addition of PS resulted in a 2.4-fold increase in the ESR (upper line, N = 25), but the in vivo administra- tion of PS in proportionately equivalent amounts during a collection procedure did not appreciably change the ESR, even when measured immediately after the procedure at peak PS blood level (lower line, N = 27).

(55%) repeat donors, and within 12% in all nine donors. The ESR likewise varied widely among donors but re- mained constant for a given donor within 5 m d h r in six of seven (86%) assessable repeat donors, and within 8 m d h r in all seven assessable repeat donors.

The plot of GCE as a function of donor baseline ESR (Fig. 2) shows that widely scattered baseline ESR values are correlated with GCE. The poor correlation (r = 0.37, standard deviation = 14%), however, resulted in the der- ivation of a linear relationship which does not consis- tently allow an accurate prediction of GCE (%) based on the donor baseline ESR (mdhr ) , within the ESR range between 0 and 20 m d h r : GCE = 0.87 ESR + 20. The 35 & 15% GCE which generated a mean yield of 1.5 ? 0.8 X 10" granulocytes compared unfavorably with analogous collections using HS as the sedimenting agent, where 58 ? 9.4% mean GCE generated an aver- age yield of 2.6 & 0.8 X 10" granulocytes per proce- dure [9].

7(

6C

SC

40

30

20

10

0

A

A A

A A A A

0 2 4 6 8 10 12 14 16 18 20 22 24

Baseline Donor ESR (mm/hour)

Fig. 2. The dependence of the granulocyte collection efficiency on the donor baseline erythrocyte sedimentation rate (ESR). Within the ESR range between 0 and 25 mm/hr, higher ESR values were more likely to result in higher GCE. The linear relationship GCE = 0.87 ESR + 20 (r = 0.37) only roughly predicts the GCE based on the ESR.

DISCUSSION

The granulocyte yield depends on both the donor's level of circulating granulocytes and the efficiency with which the cells are extracted from the circulation during an apheresis collection procedure. Granulocyte extrac- tion efficiency in turn depends on the relative sedimenta- tion rates of granulocytes and red cells, and the granulo- cyte sedimentation rate relative to the upward plasma flow resulting from downward cell displacement. The similarity in red cell and granulocyte sedimentation rates under centrifugation [ 1 1,121 has necessitated the use of macromolecules which selectively enhance red cell sedi- mentation [ 13,141. Red cells, with highly negatively charged outer cell surface, are more susceptible to polar macromolecules than are neutral granulocytes. By pro- moting red cell rouleaux formation which increases the effective cell mass-to-cell surface area ratio, the macro- molecules selectively increase red cell sedimentation through both direct cell and indirect plasma effects [ 151. Although the precise molecular mechanism of red cell

Granulocyte Collection Efficiency 201

TABLE 111. A Comparison of Baseline Donor ESR (mm/hr) and GCE (%) for Repeat Collections From the Same Donor*

D Procedure 1 Procedure 2

GCE

44 41 23 23 44 30 25 26 8

ESR

8 9 4

17 30 9 3 9

18

GCE

42 38 17 25 52 42 37 23 10

ESR

11 5 2

15 25

8

10

-

-

Time

28 21 14 14 14

236 20 1 165 143

Mean GCE

43.0 t 1.0 39.5 t 1.5 20.0 2 3.0 24.0 t 1.0 48.0 t 4.0 36.0 5 6.0 31.0 * 6.0 24.5 t 1.5 9.0 t 1.0

Mean ESR

9.5 2 1.5 7.0 ? 2.0 3.0 t 1.0

16.0 t 1.0 27.5 2 2.5

5.5 * 2.5

14.0 t 4.0

-

-

*Repeated procedures on the same donor resulted in consistent GCE despite wide inter-donor variations (mean & 112 range). The reproducibility probably arises from intrinsic baseline donor ESR which remain relatively constant over time period up to 6 months. GCE = granulocyte collection efficiency (%); ESR = erythrocyte sedimentation rate (mm/hr); D = donor; Time = time interval between two donations (days).

rouleaux formation is unknown, kinetic studies using nu- clear magnetic resonance relaxation methods have shown that HS rapidly induces over 20 seconds the formation of aggregates containing four red cells on average at equilib- rium, which effectively doubles the mass to surface area ratio in comparison to a single red cell [ 161.

The macromolecules thus far used include hydroxy- ethyl starch preparations, dextrans, and modified fluid gelatins, and of these HS has been favored since it most effectively enhances the granulocyte yield with few ad- verse donor reactions [ l ] . Significant HS blood levels persist for weeks, however, and trace amounts may be detected for years following its administration [3-51. In- cidence estimates of clinically apparent HS-induced ad- verse reactions range from 0.09% to 0.7%, and the reac- tions consist almost entirely of mild dermatologic symptoms, fever, tachycardia, hypotension, respiratory disturbance, and nausea [ 17,181. With a dose less than 1,500 ml, HS induces only transient, mild, clinically insignificant laboratory alterations including prolonged partial thromboplastin and prothrombin times, decreased platelet counts and fibrinogen levels, von Willebrand- like syndrome, and hyperamylasemia [ 1,19,20]. Serious reactions including severe pruritus, disseminated intra- vascular coagulopathy, and shock, presumably as a result of HS administration, have been reported, however [21- 231. Although exceedingly rare, these reports along with a concern about long-term toxicity as a result of delayed HS clearance have prompted a search for a sedimenting agent with a more favorable elimination profile.

The initial studies on PS have suggested that it is as effective as HS [6,7] in centrifugal leukapheresis and has the advantage of more rapid elimination [8,20,24]. Both HS and PS are heterogeneous, highly branched, polysac- charide compounds closely resembling glycogen, and con- tain molecules with a range of molecular weights and degrees of hydroxyethyl substitution (Table I). The rela- tively rapid PS clearance is consistent with its lower degree of hydroxyethyl substitution and lower average

molecular weight. Its efficacy in enhancing granulocyte collection previously reported to be comparable to that of HS is surprising, however. The effect of hydroxyethyl starch preparations on red cell sedimentation and their rate of elimination from the peripheral circulation both depend on their common biochemical characteristics; the desirable effect on red cell sedimentation is not expected to be easily separated from the undesirable delayed elim- ination [ 1,15,16].

A recent earlier report established and quantified the dependence of GCE on baseline donor ESR in granulo- cyte collections by centrifugal leukapheresis, using HS as the sedimenting agent. By duplicating the study condi- tions and by substituting PS for HS, the present study utilizes the earlier report as a control in quantitatively comparing the efficacy of PS and HS. The linear relation- ships which summarize and quantify the efficacy of HS and PS are HS-GCE = 1.3 ESR + 45 (r = 0.65),9 and PS-GCE = 0.87 ESR + 20 (r = 0.37), where HS-GCE and PS-GCE represent GCE using HS or PS as the sedi- menting agent, respectively. Although the two agents were not directly compared, our results clearly show that PS procedures are less predictable (lower correlation co- efficient), less dependent on the donor ESR (lower coef- ficient for ESR), and significantly less efficient overall (lower y-intercept, 20 vs. 45) in comparison to HS proce- dures. PS is less effective in elevating ESR in vitro than is HS, the in vitro effect is also not maintained in vivo in comparison to HS [9], and its relative lack of potency may result in less effective granulocyte collection. The less predictable GCE using PS is consistent with un- changed donor ESR when measured immediately after a procedure, probably at peak PS blood concentration. In contrast, the equivalent in vivo and in vitro HS effects on donor ESR support the predictability of GCE when HS is used [9]. Donors apparently degrade, excrete, and dis- tribute PS sufficiently such that ESR does not appreciably change within the time frame of a single collection proce- dure. ESR measurements apparently cannot discriminate

202 Lee et al.

among the differences in donors’ ability to metabolize PS within approximately 2.5 hours, but do confirm donors’ uniform inability to inactivate HS within the same time period [9]. Routine PS use will more likely result in fewer granulocytes than the criteria established by the Ameri- can Association of Blood Banks Standards Committee, or 1 .O X 10” granaulocyteshnit in 75% of tested units [25].

CONCLUSIONS

The theoretical benefit from more rapid PS clearance should be balanced against the expected lower granulo- cyte yield. For granulocyte collection using the model CS-3000 Plus blood cell separator and PS as the sedi- menting agent, we conclude that 1) both baseline ESR and GCE for a given donor are consistent over multiple equivalent procedures; 2 ) in comparison with HS, PS increases ESR less effectively in vitro, and the in vitro increase in ESR is not maintained in vivo; 3 ) baseline donor ESR affects granulocyte harvests, but the poor correlation does not allow an accurate prediction of GCE and yield from the donor baseline ESR; 4) simple mathe- matical parameters (slope, y-intercept, correlation coeffi- cient) which define the linear relationship between base- line donor ESR and GCE suggest that PS may be significantly less effective in enhancing granulocyte har- vest than is HS; and 5 ) these parameters may serve as reliable indicators of the effectiveness of hydroxyethyl starch agents in general and may be helpful in rapidly assessing the clinical efficacy of new, potentially useful hydroxyethyl starch agents. The relative efficacy of PS and HS using other blood cell separators requires further study.

ACKNOWLEDGMENTS

The authors sincerely thank Sandra Bangham, Phyllis Byrne, Rolande Grammont, Jeanette Rothberg, Kathleen Swisher, and Margaret Weller for their skilled hemapher- esis operation and assistance in data collection, Gail Carter and Virginia Morgan for diligent donor recruitment, and Charles Carter for helpful technical insight.

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