Vox Sang. 35: 207-214 (1978)

Collection of Granulocytes for Transfusion The Effect of Collection Methods on Cell Enzyme Release

Neil Blumberg, Petrina Genco, Alfred Katz and Joseph Bove Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Conn., and Connecticut Red Cross Blood Center and University of Connecticut School of Medicine, Farmington, Conn.

Abstract. When granulocytes are collected by either discontinuous-flow centrifugation or filtration leukapheresis, lysozyme is released. More lysozyme is released during the filtration procedure than during the centrifugation procedure. A small amount of red cell lysis occurs during the centrifugation but not the filtration procedure. A rise in lactate dehydrogenase levels consonant with the amount of hemolysis is observed. These findings suggest that granulo- cytes collected by either filtration or discontinuous-flow centrifugation undergo degranulation but not lysis sufficient to cause cytoplasmic enzyme release during the donation.

Introduction

The collection of polymorphonuclear leu- kocytes from normal donors has become a widespread, albeit investigational practice. While there is both human and animal evi- dence that granulocyte transfusion is bene- ficial [l, 8, 13, 15, 25, 261 controversy ex- ists concerning optimal collection techniques. Yields are greater with filtration leuka- pheresis (FL), while cells collected by con- tinuous-flow centrifugation leukapheresis (CFCL) may provide greater posttransfu- sion increments [12], cause fewer recipient reactions and show fewer in vitro functional abnormalities [21, 291. Discontinuous-flow centrifugation leukapheresis (DFCL), a rel- atively newer technique, yields neutrophils

with similar properties to the CFCL cells, as far as has been determined [4].

Sciziffer et al. [24] have reported that lysozyme is released when granulocytes are exposed to nylon fibers. Some investigators claim that lysozyme release is a measure of cellular disintegration in vivo [lo]. How- ever, since lysozyme can be released by stimuli as disparate as calcium ion [ 5 ] , complement [6], phorbol myristate acetate [7] and conconavalin A [16], its release does not necessarily indicate cell lysis or membrane damage. To evaluate the role of cell damage as a cause of lysozyme release during FL or DFCL, we compared levels of lysozyme with those of the cytoplasmic enzyme lactate dehydrogenase (LDH) dur- ing the collection of leukocytes. We also

208 Blumberg/GencolKaWBove

measured hemoglobin to determine whether LDH release might be due to red cell lysis. LDIl release without increased plasma he- moglobin would suggest platelet or white cell lysis.

Metbods

Collection of Neutrophils by DFCL and by FL Collection was performed by previously de-

xribed methods [14, 181. DFCL was performed cmploying the Haemonetics 30 blood processor. ACD was employed as the anticoagulant and hydroxyethyl starch (6%) as a rouleaux-inducing agent [14]. 6-8 cycles of centrifugation using the 225-m1 disposable bowl were completed. Residual red cells were returned to the donor at the com- pletion of the collection so that the net loss of packed red cells was less than 20ml. FL was per- formed using a dual nylon wool filter harness and peristaltic roller pump, with anticoagulation by heparin [18]. Saline was used to wash residual red cells from the filters at the collection’s completion and return them to the donor. Granulocyte elution was performed with a solution of ACD, CPD plasma and saline, with gentle tapping of the filters [18]. All donors received either oral predni- sone (40 mg) the night immediately previous to donation or intravenous dexamethasone (6 mg) at the beginning of leukapheresis. None of the donors were studied more than once during the experi- ments to be described.

Collection of Samples Donors undergoing leukapheresis for patient

care needs gave informed consent to the collection of blood samples. All samples were collected in plastic syringes through three-way stopcocks and then emptied down the sides of tubes containing 3.8% sodium citrate. Specimens were centrifuged at 1,250 g for 10 min, the supernatant plasma re- moved and stored frozen at -20 OC. Samples were transported on dry ice from the collection site in Farmington, CT, to New Haven and immediately stored at -20 OC until assayed.

In 10 donors undergoing FL and in 10 under- going DFCL, pre- and postdonation samples were

collected from the withdrawal arm. In a second experiment involving an additional 10 FL and 13 DFCL donors, samples were obtained simultane- ously from the withdrawal and reinfusion lines. In the DFCL donors, reinfusion line samples were collected simultaneously with withdrawal line sam- ples after the plasma had been returned to the donor red cells, while reinfusion was in progress. Blood samples were obtained at least 1 h after the start of the donation (FL) or after at least two centrifugation cycles (DFCL) to ensure some de- gree of donor equilibration.

This latter experiment (withdrawal and rein- fusion line sampling) was repeated on an addi- tional 10 FL and 14 DFCL donors with identical procedures except that blood was collected in tubes without anticoagulant, allowed to clot at room temperature, centrifuged and the ‘serum’ separated. Since these samples contained either heparin (FL) or ACD (DFCL) they cannot cor- rectly be called serum, but since clotting did oc- cur within 0-2 h, we will use the term ‘serum’ to differentiate these samples from the plasma sam- ples previously described.

Lysozyme, Lactate Dchydrogenase and Hemoglobin The lysoplate method [22], which combines gel

diffusion and bacterial lysis was used to measure plasma lysozyme. Precautions were taken to ob- viate difficulties that others have experienced with this method [9]. These included assaying samples on plates made from one lot of agar at the same time, and running controls to assure day-to-day comparability. Human lysozyme standard was the gift of Dr. Eliott F. Osserman. The plasma lyso- zyme concentrations of 30 healthy persons were all less than 14 pglml. Plasma LDH was measured by a kinetic ’ method utilizing the oxidation of NADH [ l l ] adapted to the Perkin-Elmer KA-150. The LDH levels of 30 healthy persons were all less than 600 spectrophotometric Ulml. Plasma hemoglobin was measured using the oxidation of o-tolidine [2]. Employing a two-syringe technique healthy people have levels less than 5 mg/dl. Nei- ther sodium citrate (3.8%), ACD nor hydroxyethyl starch interfere with the above assays.

‘Serum’ LDH and hemoglobin were measured by the above methods and have identical ranges to plasma levels in healthy individuals. To mea-

Cell Enzyme Release during Granulocyte Collection 209

sure ‘serum’ lysozyme we used the turbidometric method of Litwack [20]. Utilizing a standard of egg-white lysozyme, healthy persons have levels less than 14 pg/ml.

In the DFCL procedure one part of ACD- hydroxyethyl starch is mixed with eight parts of donor blood during each centrifugation cycle. A correction for this dilution was made for all data from the DFCL reinfusion line samples. The aver- age donor hematocrit during DFCL was 40%. The plasmacrit was therefore 60%. Thus, the fraction of diluted blood that is plasma is approximately 0.6 (819) = 0.533. The fraction that is ACD-hy- droxyethyl starch is 119 = 0.111. The plasma space for lysozyme, LDH and hemoglobin has been expanded by approximately

0.533 + 0.1 11

0.533 = 1.2

in the reinfusion line samples. Analysis of variance (utilizing unweighted

means when there were unequal numbers of do- nors in the groups compared) and the Newman- Keuls test (when individual comparisons of means was appropriate and desired) were used to eval- uate data [28]..

Results

Pre- and Postpheresis Data from Donors Data and results of the statistical analysis

for the first study are shown in table I. Neu- trophil yields averaged 1.35 ? 0.32 x 1O1O (SEM) by FL and 1.23 k 0.18 x 10’0 (SEM) by DFCL. Hemodilution, as documented by fall in hematocrit, occurred with both col- lection methods and was significantly greater with DFCL. The mean fall in hematocrit was 3.4% with FL and 6.7% with DFCL. Plasma lysozyme fell in the DFCL but not the FL donors. Plasma LDH and white count did not change in either group of do- nors. Plasma hemoglobin was increased post- donation in the DFCL donors.

Withdrawal and Reinfusion Line Data from Donors Results of the study of simultaneous sam-

ples from the withdrawal and reinfusion lines are shown in table 11. Plasma lysozyme was significantly higher in the reinfusion line than in the withdrawal line for both leukapheresis techniques. Despite the greater change in plasma lysozyme with FL than with DFCL, there was no significant dif- ference between the two methods in the de- gree of withdrawal to reinfusion line change they produce, at least in this group of do- nors. Plasma LDH was decreased in the re- infusion line from the level in the withdraw- al line by the DFCL but not the FL method, but this difference was not significant. Plas- ma hemoglobin was increased in the rein- fusion line from the level in the withdrawal line by the DFCL but not the FL method. Individual plasma hemoglobin values in the withdrawal and reinfusion line samples are given in table 111. Levels are low in all cases but one when filtration was used but were much higher during centrifugation leuka- pheresis. In four donors extremely high val- ues in both the reinfusion and withdrawal lines were observed. It was determined ret- rospectively that these samples had been drawn by needle puncture of the withdrawal and reinfusion lines and then injected into evacuated tubes through the needle.

In order to confirm our findings of ly- sozyme, LDH and hemoglobin levels in the withdrawal and reinfusion lines of FL and DFCL donors we measured ‘serum’ levels in an additional 10 FL and 14 DFCL do- nors. These results are shown in table IV. Individually, all 10 FL and 12 of 14 DFCL donors showed increases in lysozyme level in the reinfusion line compared with that in the withdrawal line. In this group of donors,

Blumberg/Genco/Katz/Bove 210

Table I. Pre- and postpheresis data from donors undergoing filtration (FL) or centrifugation leukapheresis (CL); all data are f 1 SEM

Filtration leukapheresis Centrifugation leukapheresis Significance (n= 10) (n = 10) of differences

pre- post- signifi- pre- post- signifi- FL and CL' donation donation cance donation donation cance

between

Hematocri t 42.6f1.0 39.2f0.7 p<O.001 43.1f1.0 36.4f1.4 p<O.OOl p<O.OO5

White count 7,100f900 6,500f700 NS 6,400f700 6,000f700 NS NS

Plasma lysozyme, pg/ml 8.44Z0.6 8.7f0.5 NS 9.9f0.7 7.7f0.8 p<0.05 p<0.05

Plasma lactate dehydrogenase U/ml 4904~30 480520 NS 460&30 530f45 NS NS

Plasma hemoglobin mg/dl l . l f0 .4 3.1f0.5 NS 2.1 f0.5 19.7f5.1 p<O.OOl p<O.O1

1

change they produce. That is, the significance of the difference between the two methods in the degree of pre- to postinfusion

Table 11. Withdrawal and reinfusion line data from donors undergoing filtration (FL) or centrifugation leukapheresis (CL); all data are f 1 SEM

Filtration leukapheresis Centrifugation leukapheresis Significance (n = 10) (n=13) of differences

withdrawal reinfusion signifi- withdrawal reinfusion signifi- FL and CL' line line cance line line cance

between

Plasma lysozyme, pg/ml 8.8f0.9 11.64Zl.O p<O.OI 8.6f0.4 9.8f0.6 p<O.OI NS

Plasma lactate dehydrogenase U/ml 554f60 603f58 NS 574f58 465f45 NS NS

Plasma hemoglobin mddl 3.8f1.3 6.3f3.2 NS 20f5.1 110145 p<O.OOl p<O.OS

That is, the significance of the difference between the two methods in the degree of withdrawal to reinfusion line change they produce.

Cell Enzyme Release during Granulocyte Collection 21 1

Table III. Plasma hemoglobin levels in the with- drawal and reinfusion line of individual donors

~~~~~ ~

Centrifugation Filtration leu kapheresis leukapheresis

withdrawal reinfusion withdrawal reinfusion line line line line

2.5 3.1 3.9 5.2 8.6 12 12 21 21 25 29 46 64

24 1.3 11 30 21 39 39 8.2 I26 40 232 202 597

0.7 1 .1 1.2 1.6 2.0 2.0 3.0 3.6 8.3 14

1 . 1 1 . 1 3.0 2.9 2.6 3.3 1.4 2.5 5.1 40

the increase in lysozyme was of significantly greater magnitude in the FL donors than the DFCL donors. Individually, 13 of the 14 DFCL donors evidenced some red cell lysis, while the FL donors did not. There was significant elevation of reinfusion line LDH levels over those in the withdrawal line in the DFCL but not the FL donors.

Discussion

Neutrophils release granule contents af- ter various stimuli, but LDH is liberated only when cell membrane damage occurs [27]. Our data support previous observa- tions that leukocytes collected by filtration undergo degranulation [ 191. However, they do not undergo sufficient membrane disrup-

Table IV. Withdrawal and reinfusion line data from donors undergoing filtration (FL) or centrifugation leukapheresis (CL); all data are f 1 SEM

Filtration leukapheresis Centrifugation leukapheresis Significance (n=10) (n = 14) of differences

withdrawal reinfusion signifi- withdrawal reinfusion signifi- FL and CL’ line line cance line line cance

between

‘Serum’ I y sozy me m/ml

~~

5.0f0.28 8.6izO.43 p<O.O1 5.1iz0.27 5.7f0.27 p<0.05 p<0.005

‘Serum’ LDH U/ml 337f12 303f14 NS 321-115 441f20 p<O.O1 p<0.005

‘Serum’ hemoglobin mg/dl 4 .0 f2 .8 6 .9 f2 .8 NS 12f1 .5 46iz5.0 p<O.Ol p<O.001

That is, the significance of the difference between the two methods in the degree of withdrawal to reinfusion line change they produce.

212 Blurnberg/Genco/Katz/Rove

tion to allow LDH release. Cells collected by discontinuous-flow centrifugation under- go a similar, but significantly smaller degree of degranulation without membrane rupture.

The LDH determination should detect the release of this enzyme after lysis of as few as 1-2% of the granulocytes in normal whole blood. The LDH content of granulo- cytes is about 900 spectrophotometric units per 2 x 10‘ cells [7]. Using a figure of ap- proximately 4 x 10s granulocytes per micro- liter, each milliliter of whole blood contains granulocytes with a total of 1,800 U of LDH activity. Thus, if 1% of the cells are lysed and their LDH released, an increase in LDH of about 20U/ml is expected; this is well within the reproducibility and precision of the LDH assay used.

The failure to observe an increase in lysozyme postdonation (and, in fact, a de- crease in plasma lysozyme occurs postdona- tion in the DFCL donors, perhaps partly due to hemodilution) despite its release dur- ing pheresis is unexplained. One possibility is that lysozyme (molecular weight 17,000 daltons) is rapidly cleared by the kidney. We did not measure urinary lysozyme in our donors.

Hemolysis occurs during collection of granulocytes by DFCL, as documented by the increased plasma hemoglobin postphe- resis (table I) and the increased levels in the reinfusion line (table 11-IV). In DFCL, with- drawal and reinfusion line samples were col- lected after at least two centrifugation cy- cles. We believe the elevated withdrawal line hemoglobin levels (table 11-IV) repre- sent the hemoglobin released and reinfused during each centrifugation pass. Samples collected before donation and centrifugation did not show hemoglobin increase (table I). The origin of the red cell lysis during DFCL

is unknown. One possibility is that the shear forces present as the blood passes through the rotating seal are responsible. Hemolysis has not been shown to accompany leuka- pheresis employing continuous-flow centrif- ugation, to the best of our knowledge.

The full in plasma LDH in the reinfusion line of DFCL donors (table 11) was sur- prising, in view of the evidence of red cell lysis. However, others have noted the dif- ficulties that can be encountered in mea- suring LDH in plasma as compared with serum [23]. Thus, in our next group of donors (tableIV) we measured LDH on samples that had been allowed to clot and form ‘serum’. In this group of 14 DFCL donors the LDH rose commensurate with the degree of red cell lysis, but no more (i.e., approximately 3 U/ml/mg of hemo- globin released) [3]. We thus believe the original observation of falling plasma LDH with hemolysis (table 11) to likely be arte- fact. Similarly, the 4 donors undergoing DFCL (and possibly 1 of the FL donors) with unusually high hemoglobin levels (ta- ble 111) probably represent errors in sample collection methodology rather than massive hemolysis during DFCL such as has been previously reported [17]. None of the DFCL donors in our second group (tableIV) had reinfusion line levels higher than 74 mg/dl.

Whether cells that have released lyso- zyme are damaged will have to be shown by future studies that correlate lysozyme release with in vivo or in vitro function and viability. Our data do not enable us to state whether some granulocytes release all their lysozyme or whether most or all granulo- cytes release a portion of their lysozyme during leukapheresis. Lysozyme release, however, may be a reasonable measure of temporarily decreased neutrophil function

Cell Enzyme Release during Granulocyte Collection 213

(secondary to depletion of granular con- tents). If so, it may be one convenient mea- sure of the effects of procedural variations on the quality of the cells collected.

Acknowledgements

We would like to thank Ms. Judy Houx, RN for obtaining samples, technical advice and super- vising the leukapheresis procedures, Ms. Joanne Gaudioso and Mr. Robert Bonvini for technical assistance, and Mr. Robert Milstein for performing the statistical analysis.

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Received: January 6, 1978 Accepted: January 9, 1978

Dr. Joseph R. Bove, Department of Laboratory Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510 (USA)