Brief Reports

Harvesting of Granulocytes using a Hydroxyethyl Starch Solution L. N.

S U S S M A N , W.

COLLI, AND C. PICHETSHOTE

From the Berh Israel Medical Cenrer. New York City. New York

The need for sophisticated component therapy has resulted in improved techniques for obtaining concentrates of platelets and granulocytes. The use of single donors as a source for these products is advisable to avoid multiple

similar. Thus. differential centrifugation is " usually unrewarding. To accomplish a satisfactory separation of these cells, %m3d

represents a problem because of the difficulty in sep arating granulocytes from red blood cells by differential centrifugation or sedimentation since the soecific gravities are similar. Hydroxyethyl starch (H&) maies the separation more effective. A solution made of 250 ml of 6 per cent HFS, 250 ml of distilled water, and 15 g of sodium citrate in 30 mi distilled water provided a satisfactory anticoagulant solution for this purpose. The granulocytes collected averaged 49 per cent of the total available in the processed blood; the platelets averaged 82 per cent. A satisfactory yield could thus be obtained from a single donor, and this could be repeated several times in a month. The value of ABO, Rh, and H L - A compatibility between donor and recipient probably increases the viability and safety of this procedure. The Haemonetic No. 30 Cell Separator provided an easy and rapid method for this procedure.

the IBM-NIH cell separator is moderately successful, but the Procedure is long (four to five hours) and requires heparinization of the donor.4 Another technique (Fenwal)* takes three to four hours, involves filtration separation,e and also depends on heparinization of the donor and subsequent elution of the granulocytes from the filter, with a good yield of neutrophils. The Latham method" requiring anticoagulation with ACD solution and subsequent differential centrifugation results in a shorter procurement time, but the yield of neutrophils is poor unless one uses as a donor a patient with chronic myelogenous leukemia.15.19There are some objections to the use of such leukocytes. Various pretreatments of the normal donor, such as the administration of steroids, or etiocholanolene,*.'O or vigorous exercise result in only moderate increase in the yield of granulocytes. The use of multiple donors to increase the number of granulocytes harvested raises the question of multiple sensitizations due to HL-A incompatibility as well as increasing the risk of hepatitis. It was found that when HESt normally used as a plasma expander was added to the donor's blood extracorporeally, it produced a more efficient separation of white blood cells and especially increased the yield of granulocytes.'2.'4*'s

THENEED for a readily available source of granulocytes to support the patient with a drug- or disease-related neutropenia has become an important problem. Intensive chemotherapy in the treatment of leukemia and other malignancies frequently results in severe bone marrow depressions that are theoretically reversible. Supportive component therapy then becomes essential. The beneficial and frequently life-saving effects of both platelet and granulocyte transfusion have been well d o ~ u r n e n t e d . ~ , ~ . ~ . ~ ~ The difficulty of harvesting the required large amounts of granulocytes from the other cellular elements is complicated by the fact that the sedimentation of erythrocytes, lymphocytes, and granulocytes is very Received for publication December 10, 1974; accepted February 8, 1975. Supported in part by The Samuel & Rose Hurowitz Research Fund and Melvin Mitchell Fund.

*Fenwal Laboratories Set, Fenwdl Laboratories, Morton Grove, 111. t(Volex) McGaw Laboratories, Glendale. Calif.

46 1 Transfusion SCPI.-OCI.1975

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This led to reinvestigation of the Latham Blood Processor, where a single donor is used, as a means for granulocyte harvesting. Heustis’ reported excellent yields of leukocytes with 6 per cent HES using ACD and a small dose of heparin as the anticoagulant. This was soon modified, and the heparin was eliminated. The great advantage of a single donor source, one who could be repeatedly used since only a small amount of RBC were lost to the donor, is obvious. An HL-A compatible donor, free of the hepatitis virus, who could repeatedly provide sufficient granulocytes would enable the patient to be supported during the period of neutropenia, without the several risks attendant on the use of multiple donors. Some attention was paid to the effects of the HES which was contained in the recycled blood returned to the donor after the leukocytes and platelets were r e m 0 ~ e d . IA~ significant plasma expansion is produced by the 6 per cent HES. This is manifested by a relative fall in hemoglobin, hernatocrit, total protein, albumin and calcium, and lasts approximately 24 hours. Method The Heustis formula was consequently modified, and a technique following the original Tullis method” for harvesting of platelets using intermittent flow differential centrifugation was used. The equipment is the Haemonetic No. 30 cell processor.1 The anticoagulant mixture consisted of a mixture of 250 ml of 6 per cent HES, 250 ml of distilled water, and 15 g of sodium citrates in 30 ml of distilled water. This results in a 3.0 per cent sodium citrate anticoagulant solution. The anticoagulant and HES solution (now lowered to 3 per cent HES) was delivered to the phlebotomy line almost at its origin from the donor’s arm in the ratio of I ml of the mixture for each 10 ml of blood. Approximately six to eight cycles using the large Latham bowl of 375 ml or eight to ten cycles using the small bowl of 225 ml gave adequate yields of platelets and leukocytes.

1Haemonetics Corporation, Natick, Mass. §Sodium citrate dihydrate 17g: citric acid rnonohydrate 0.8 g: water q.s.: = 56.6% trisodium citrate.

S c ~ l . - O c l1915 .

The platelets were easily visible during the centrifugation as a narrow band between the plasma and the red blood cell layer and were directed into the first collecting bag. The next 25 ml of blood, which consisted of concentrated leukocytes and erythrocytes was directed into the second collection bag. The remaining blood and plasma were returned to the donor through the opposite arm while the next cycle was begun. The total procedure of either six or eight cycles took approximately two hours. The various parameters that were monitored both pre- and postdonation were CBC, platelet count, differential count, sedimentation rate, clotting time, prothrombin time, activated partial thromboplastin time, blood chemistries (calcium, inorganic phosphorus, glucose, urea nitrogen, uric acid, cholesterol, total protein, albumin, total bilirubin, alkaline phosphatase, LDH, SCOT, CO,, chloride, sodium, potassium) urinalysis, blood pressure, and pulse rate.

Results The yields were measured by platelet counts, leukocyte counts, and differential counts on the final collecting bags. This was then related to the volume of blood processed, calculated as approximately 500 ml for each cycle using the small bowl and 700 ml for each cycle using the large bowl (Table I). In order to compare the findings, the number of granulocytes per liter of processed blood and the number of granulocytes harvested was determined (Table 2). It soon became evident that this yield of granulocytes was dependent on the predonation granulocyte count of the donor as shown in Figure I .

Discussion

Ideally, the patient could receive the maximum number of granulocytes if the final mixture of RBC and WBC in the second collection bag was administered intact. This requires that the donor and the recipient be of the same ABO and Rh grouping and that a compatible crossmatching be present, since approximately 100 ml of packed RBC would be infused. To avoid gross transfusion of RBC, the supernatent obtained by permitting the second collecting bag to settle for one-half hour resulted in a preparation containing only a minimal contamination with RBC. However, at least one third of the leukocytes

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G R A N U L O C Y T E HARVEST I N G

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Table 1. Yield of Platelets and Granulocytes Obtained in 23 Procedures Platelet Yield x 1011

7.8 4.6 7.6 7.0 6.0 4.6 8.4 8.5 9.0 3 8.3 X

11.2 6.2 8.8 5.6 9.0 7.5 8.7

9.0 8.4 9.5 10.4 Average 7.69 (for 22)

Per L x 1011

Per Cent

2.2 1.3 2.2 2.0 1.5 1.4 3.0 2.1 2.4 1.5 2.1

100 63 100 100 40 70 100 80 100 70 100

X

X

2.8 1.5 2.2 1.8 2.2 1.9 2.0 2.25 2.0 2.4 2.6 2.06 (for 22)

WBC Yield x 109

Per L x 109

90 80 115 110 96 84 100 100 92 99 93

18 9.6 19 12.5 24.4 12 9.5 11.5 13 5.8 25 8.8 18 18.3 22 9.5 9.35 17.3 8.5 13 12.4 26.5 16

2.4 2.9 3.2 3.7 6.0 2.0 4.5 4.6 5.3 3.2 2.0 4.3 2.24 3.2 3.1 6.6 4

82

14.8

3.85

5 2.7

5.4 3.1 6.0 3.0

remained in the unused residue which could then be returned to the donor. Table 2 indicates the average yield in total platelets, leukocytes, and granulocytes collected; the yield per liter of processed blood; and most importantly, the quantities of platelets, leukocytes, and granulocytes calculated as a percentage of the total available in the volume of blood processed from the donor. Thus, about 82 per cent of the available platelets and 49 per cent of the available granulocytes were harvested. The clinical value and survival of these products has been the subject of other reports. The importance of ABO compatible platelets and granulocytes has been discounted by several recent papers.1.3.9-13*1e However, the presence of some erythrocytes in the final preparation make it mandatory to crossmatch the donor and recipient. The further value of HL-A compatible products is another controversial subject. Such matching would seem essential in any situa-

Per Cent

Poly Yield x 109

Per L

3.1 1.4 3.4 1.9 3. 1.7 .8 1.15 1.1 1.7 2.4 .9 2.44 3. 2.4

56 64 67 80 50

11. 4.8 11.6 6.7 11.7 6.85 3.2 4.6 4.42 2.3 13.6 3.5 9.35 11.2 10.2 2. 2.3 9.24 2.74 4.2 2.5 13.3 12.1

1.05 .6 3.3 3.0

42 50 58 63 70 61 22 35 37 54 50 64 64 63 50 25 25 51 33 45 25 77 55

62

7.1

1.88

49

50 50 70 78 77 55

40 60 65 62 65 50 70 60 90 60 50

58

.7 .6 2.3

.7

Special Comments

Per Cent

3 cycles only Platelets lost

tion where future transplant is contemplated . The effects of the procedure on the healthy, normal donor also warrants conide era ti on.".'^ The plasma expansion is obvious from Table 3. These findings returned to normal within 24 hours, and apparently resulted from the expected increase in plasma volume. No adverse effects were reported by the donors, many of whom were examined the day following their pheresis. In addition, several of the donors were used repeatedly (some twice weekly) with a complete return to normal findings before each phlebotomy. No changes in chemistry, coTable 2. Average o f Yield of Platelets, Leukocytes, and Granulocytes of 23 Donors

Total Yield Platelets Leukocytes Granulocytes

Yield/L

7.69X lo1] 2.06X 1011 14.8 X 1011 3.85 X 109 7.1 X 109 1.88 X 109

Per Cent Recovered

82 62 49

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Transfusion Scpl.-Oct. 1975

SUSSMAN, ET AL.

8.

7.

-

W

6.

E Y

L

: 5 .

a

c .-

*

5

u

FIG. I . Granulocyte yield expressed as number per liter and the granulocyte count of the donor.

4.

nl Y x

9

3.

E

.a

(3

2.

1.

0.

.s

2.

1.5

1.

Granulocyte Y i e l d ( X

25

3.

l o l l ) per L i t e r

agulation studies, sedimentation rate, blood pressure, or pulse were noted except those indicated in Table 3. The effective anticoagulation by 3.0 per cent sodium citrate was tested on several occasions by determining the predonation plasma fibrinogen of the donor and comparing this finding with the fibrinogen content of the blood being reinfused to the donor. Again, no significant differences were found beyond those caused by hemodilution. The use of distilled water as a diluent for

the sodium citrate was decided upon when the osmolarity of the sodium citrate-saline solution was compared with the sodium citrate-distilled water solution. In the first case, the osmolarity was 522 and the second was 300, whereas that of normal saline was 290. The Ph of the reinfused blood varied from 7.3 to 7.6, which was considered acceptable. Using this technique, a satisfactory yield of granulocytes could be obtained from a single, preferably ABO, Rh, HL-A compatible donor.

Table 3. Effect of Hernodilution Caused by HES in Donor Per Cent

Hemoglobin Hematocrit Total protein Albumin Calcium Fibrinogen

Pre

Post

15. g/100 ml 44.7% 7.34 g/100 ml 4.60 g/ 100 ml 9.75 g/lOO ml 285 mg/ 100 ml

13.15 38.90 5.83 3.72 8.57 240

Change

-

-

-

- 1.85 5.80 - 1.51 - 0.88 - 1.18 -45 -

Change -

-

-

-

-12 -13 -21 - 19 -12 -15

Volume

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GRANULOCYTE HARVESTING

Number 5

Acknowledgments The authors are grateful to Dr. R. Stenger, Director of Laboratories and his staff for their assistance in clinical pathology determinations, to the McGaw Laboratories for supplies of Volex, and to the Haemonetics Corporation for their contribution of disposable equipment.

References I.

Cohen, E., H. Feliciano, and 0. Glidewell: Platelet increments following transfusion of ABO group specific and nonspecific platelets. Transfusion

evaluation of normal donors undergoing leukapheresis on the continuous .flow centrifuge. Transfusion 13:394, 1973. 12. Mishler, J. M., D. C. Hadlock, I. E. Fortuny, R. W. Nicora, and J. J. McCullough: Increased efficiency of leukocyte collection by the addition of hydroxyethyl starch to the continuous flow centrifuge. Blood (in press), 1974. 13. Pfisterer, H., S. Thierfelder, W. Stich: ABO Rh blood groups and platelet transfusion. Blut l7:l, 1968. 14.

Roy, A. J., W. B. Simmons, A. Franklin, and I. Djerassi: Hydroxyethyl starch for separation of normal granulocytes. Fed. Proc. 29:424,

15.

Szymanski, I. O., and A. Kliman: Transfusions of platelets and white cells. N. Y. State J. Med.

8:310, 1968.

Freireich, E. J.: Effectiveness of platelet transfusion in leukemia and aplastic anemia. Transfusion 650,1966. 3. Goldfinger, D., and M. H. McGinnis: Rh-incompatible platelet transfusions-risks and consequences of sensitizing immuno-suppressed patients. N. Engl. J . Med. 284:942, 1971. 4. Craw, R . G., Jr., G. P. Herzig, R. J . Eisel, and S. Perry: Leukocyte and platelet collection from normal donors with the continuous flow blood cell separator. Transfusion 11:94, 197 I . 5. -, G. P. Herzig, S. Perry, and E. S. Henderson: Normal granulocyte transfusion therapy, treatment of septicemia due to gramnegative bacteria. N. Engl. J . Med. 287:367, 2.

1972.

Herzig, G. P., R. K. Root, and R. G. Craw, Jr.: Granulocyte collection by continuous-flow filtration leukapheresis. Blood 39554, 1972. 7. Heustis, D.: Personal communication. 8. Higby, D. J., E. S. Henderson, and J. F. Holland: Report presented at American Society of Clinical Oncology, Houston, Texas. March 28, 6.

465

1970.

73:1859, 1973. 16. Tejada. F.. W. B. Bias, G. W. Santos, and P. D.

Zieve: Immunologic response of patients with acute leukemia to platelet transfusions. Blood 42:405, 1973. 17. Tullis, J. L.. R. J. Tinch, P. Baudanza. J. G.

Gibson I I , S. DiForte, G. Conneely, and K. Marthy: Plateletpheresis in a disposable system. Transfusion 11~368,197 I . 18. Vallejos, C . S., K. B. McCredie, and E. J. Freireich: Improved leukocyte collections with hydroxyethyl starch (HES) from patients with chronic myelocytic leukemia. (CML). Clin. Res. 20503, 1972. 19.

Wheeler, T. G., K . B. McCredie, E. J. Freireich, and T. V. Daniels: Increased efficiency of leukocyte collection from platelets with chronic myelocytic leukemia. Transfusion 14:253, 1974.

1974. 9.

Lohrman. H. P., M. I. Bull, J. A. Decter, R. A. Yankee, and R. G. Craw, Jr.: Platelet transfusions from HL-A compatible unrelated donors to alloimmunized patients. Ann. Intern. Med. 80:9, 1974.

10.

II.

McCredie, K. B., and E. J. Freireich: The use of etiocholanalone to increase collection of granulocytes with the IBM blood cell separator. J. Clin. Invest. 49:63a, 1970. McCullough, J., and I. E. Fortuny: Laboratory

Leon N. Sussman, M.D., Director of the Ralph Gore Blood Bank, Beth Israel Medical Center, 10 Nathan D. Perlman Place, New York, New York 10003; Clinical Professor of Medicine, Mt. Sinai School of Medicine of the City University of New York. William Colli, R . N., Chief Phlebotomist, Beth Israel Medical Center. C. Pichetshote, M.D., Fellow in Hematology, Beth Israel Medical Center.

Harvesting of granulocytes using a hydroxyethyl starch solution.

Brief Reports Harvesting of Granulocytes using a Hydroxyethyl Starch Solution L. N. S U S S M A N , W. COLLI, AND C. PICHETSHOTE From the Berh Isr...
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