CRYOBIOLOGY
1.6, 105-111
Recovery
(1979)
of Functional Capacity of Human Freeze-Preservation with Dimethyl
R. S. HILL,
C. MACKINDER,
Department
S. CHALLINOR,
of Haematology, Wallace Auckland, New
Methods have recently become available which permit low temperature preservation of human haemopoietic cells in liquid nitrogen at -196°C and reconstitution to show recovery of functional capacity (4, 7, 12). Recent studies have shown that neutrophils can be reconstituted to show significant functional capacity as judged by Candida and bacterial phagocytosis, microbacidal activity, nitroblue tetrazohum (NBT) reduction, (1, 7, 11) and recovery of granulation in as many as 60 to 70% of cryopreserved cells (9). Such positive recent evidence documenting some capacity to preserve certain aspects of neutrophil function provided impetus for our studies in this field. We have recently shown during short post-thaw periods that a significant proportion of hurnan peripheral blood granulocytes can be freeze-thawed and reconstituted to show recovery of functional capacity as judged by Candida albicans candidacidal activity and phagocytosis, NBT reduction (5, 6). In the present studies we have compared preservativefree heparin (PFH) and acid citrate dextrose (ACD) anticoagulants in the cell freezing system, slow and rapid dilution techniques for cell reconstitution, and examined specific factors potentially capReceived 12, 1978.
March
2,
1978;
accepted
Granulocytes Sulphoxide
AND
Laboratory, Zealand
after
B. POSTLEWAIGHT Auckland
Hospital,
able of influencing cell recovery and viability including complement and activants of NBT activity (zymosan and E. coli endotoxin). Some experimental factors influencing leucocyte Candida phagocytosis and candidacidal activity were also studied. MATERIALS
Leucocyte
AND
METHODS
Collection
Thirty milliliters of human peripheral blood from healthy volunteers were collected by venepuncture with 25 units of PFH (Commonwealth Serum Laboratories, Melbourne) per ml of blood in a 50 ml plastic syringe. For comparison, some samples were collected with 5.6 standard units of ACD (USBP) per ml of blood. One half volume of macrodex (Dextran 70, Pharmacia, Uppsala) was added to all blood samples, the syringe inverted six times and left to stand vertically for 45 min. An 18 gauge needle was attached, bent to 90” and the plasma buffy coat expressed into plastic centrifuge tubes. An equal volume of heparinized Hanks buffered salt solution (PFH-HBSS) was added and the cells centifuged at 150 g for 5 min. The cell button was resuspended in PFH-HBSS and a total leucocyte count performed. A blood smear was made of the neutrophil-rich cell suspension, stained with Romanowsky stain and a leucocyte
September
105 OOll-2240/79/020105-07$02.00/O All
Copyright 0 1979 rights of reproduction
hy Academic Press, Inc. in any form reserved.
106
HILL
differential count carried out. Greater than 90% neutrophil counts were obtained by this method.
Freeze Preservation
of Cells
The cell suspension was diluted with PFH-HBSS to give 1 x lo7 neutrophils/ mls and the preserving agent DMSO (Merk) added rapidly with mixing, to a concentration of ,lO%. Sterile glass ampoules containing 2 ml of final cell suspension were immediately sealed and rapidly cooled to 0°C by placing them on ice. They were then cooled at l”/min in an ethyl alcohol (96% dehydrated, Kempthorne Prosser) bath to -60°C before being transferred to the liquid phase of nitrogen (-196OC) as previously described (4). Cells were stored for 24 to 72 hr before testing.
Leucocyte
Reconstitution
The reconstituting solution was made up of 50% PFH-HBSS and 50% Dextran 70 with the pH adjusted to 7.4 with 4.4% sodium bicarbonate. Cells were thawed rapidly in a 37°C waterbath, diluted immediately (rapidly) with 10 times the volume (20 ml) ‘of reconstituting media, centrifuged at 1506 for 5 min and washed once with a further 10 volumes of reconstituting solution before final reconstitution. The DMSO content after washing was considered to be 0%. A total cell count and differential were performed. For comparison samples of thawed cells were diluted with 10 volumes of reconstituting solution by way of a slow titration method over 20 min. These cells were tested after dilution without washing (0.9% DMSO). During studies to test the effect of the cationic local anaesthetic agent lignocaine on leuco-agglutination and the yield of reconstituted neutrophils, the pH of the reconstituting solution was adjusted to 6.7, 7.0, and 7.4 and the lignocaine hydrochloride (Douglas Pharmaceuticals) added
ET AL.
to celI aliquots in 1, 5, 10, and 20 mmol concentrations.
Qualitative Test
Nitroblue
Tetraxolium
(NBT)
The method has been detailed previously (7). Briefly, fresh and frozen leucocytes were tested qualitatively for NBT reduction on glass and after zymosan stimulation. Zymosan was obtained from S. cerevisiue yeast (Sigma). In absolute terms, percentage retention of NBT activity by freeze-thawed cells was calculated by the formula XY/Z: (X = s NBT activity of freeze-thawed cells, Y = % neutrophil yield after freeze-thawing and Z = s NBT activity of fresh cells).
Quantitative
NBT Test
This method, modified after Segal and Peters (14) has been detailed previously (7). A leucocyte/NBT mixture was run through a nylon fiber column after stimulation with zymosan. The phagocytes adhered to the nylon fiber in the column, Formazan was extracted from the nylon fiber with diethylene dioxide (1,4-Dioxan, Analar) and the optical density of the resulting blue solution was compared with that of a known formazan standard. One further modification was made. An absorption curve for formazan with maximum absorption at wavelength of 575 nm (Hill, unpublished data), provided the standard for spectrophotometric readings in this series of experiments. Samples ‘of fresh and reconstituted leucocytes were tested quantitatively for NBT reduction and formazan synthesis after endotoxin stimulation. Endotoxin was prepared by inoculating 100 mIs of TC 199 (GIBCQ) with a laboratory strain of E. coli. After 48 hr incubation the culture was millipore filtered (filter size 0.22 pm) and the filtrate stored in 5 ml aliquots at -22°C. Incubation for 10 min with 0.15
FREEZE
PRESERVATION
OF
mls of endotoxin was found to give optimal stimulation to fresh neutrophils. Canadida Activity
Phagocytosis
HUMAN
TABLE Effect
of Anticoagulant Functional Capacity
and Candidacidal
The method of Lehre and Cline 1969 (10) was followed using a leucocyte to Candida albicans cell ratio of 1:l. Incubation of the text mixture was carried out at 37°C for 75 min. Samples were taken from the tubes at 10 min intervals, films made and stained by Romanowsky and Gram’s stain. The percentage of leucocytes which ingested Candida (percent phagocytosis) and. the percentage of added Candida ingested (percent Candida uptake) were estimated by counting 400 cells. In absolute terms phagocytis retention by reconstituted cells was calculated using the same derived formula as for NBT retention (see above) substituting ‘$NBT activity with percent phagocytic activity. Candidacidal activity was assessed after release of ingested yeasts following disruption of leucocytes with sodium deoxycholate (2.5%). Equal volumes of 0.01% methylene blue added to the cell suspension distinguished intact (dye excluded) from dead Candida (dye taken up) and the proportion of yeast killed (the candidacidal activity) was calculated. The absolute candidacidal retention was calculated in a similar manner to phagocytic and NBT retention (see above). RESULTS
The effect of anticoagulants, PFH and ACD on leucocyte yields and NBT activity of frozen reconstituted cells is shown in Table 1. Using the Wilcoxon’s signed rank test on the data included in this table, an ‘overall improvement was found with heparin (P < 0.01). Compared to ACD, PFH increased all 5 comparative parameters; total leucocyte and neutrophil yields, NBT on glass and stimulated NBT with zymosan, and Candida phagocytosis.
107
GRANULOCYTES 1 on Leucocyte Yield after Reconstitution IIeparin
Total leucocytes Neutrophils NBT (glass) NBT (aymosan) Candida phsgocytosis Results are expressed *SD (five studies).
675 63.2 13.7 20.2 25.9 as the mean
* 7.3 + 12.8 f 4.4 f 7.0 zt 14.6 percent
and
ACD 56.3 50.5 7.9 13.7 21.9
rt 10.5 f 4.1 f 2.0 f 3.2 f 12.1
of the fresh
value
When reconstitution methods were compared in separate studies, slow dilution by titration over 20 min showed no advantage over a rapid dilution (see Materials and Methods) as judged by total leucocyte and neutrophil recovery, NBT glass and zymosan stimulated, and Candida phagocptic capacity (Table 2). The re,sults confirm that small quantities of DMSO (0.9% ) remaining after reconstitution are not detrimental to leucocyte functional capacity as tested in these studies. The rate of gelling and leuco-agglutination over a 75 min post-thaw period was not significantly reduced either by the addition to the reconstitution media of lignocanine hydrochloride reported to inadherence to nylon hibit granulocytes fibers (15) or by reducing pH. Reconstituted neutrophils showed selectivity in their response to stimulation as judged by the quantitative NBT test (Table 3). Incubated with zymosan they retained 20.9% of NBT activity. Cells incubated with E. coli endotoxin on the other hand retained only 7.770 of NBT activity of fresh cells. This contrasted to the quantitative NBT tests performed with fresh cells where zymosan and endotoxin gave similar results of 5.5 nmol formazan/ lo6 cells. When decomplemented serum was used as a constituent in the NBT test with zymosan stimulation, the results of the quantitative tests expressed as percent recovery of fresh were 7.6% compared to 20.9% when complement was added (Table 3). Similarly after endotoxin stimulation the results were 2.2’$ in the ab-
HILL
108
ET
TABLE: Effect
of Reconstitution
Method
AL. 2
on Leucocyte
Functional
Capacity”
Reconstituted Rapid 0.9% DMSO
Total leucocyte yieldb Neutrophil yield* NBT (glass)c NRT (zymosan)c Candida phagocyto& Candidacidal activity”
66.0 61.9 21.9 31.7 27.6 22.3
zk 5.7 f 6.7 f 5.7 f 8.2 f 16.5 f 10.4
Results are a mean f SD of five studies. a Leucocvtes collected in Heparin and Sedimented b Calculated as percent of fresh cells. c Calculated as percent retent,ion of fresh activit,y.
Slow 0.9% DMSO
56.5 53.3 16.6 26.0 21.1 10.2
with
Rapid 0% DMSO
f 4.3 f 6.2 f 2.1 f 4.0 f 11.4 f 9.3
Dextran
69.2 65.2 21.2 29.8 22.8
f f f f f
10.9 12.5 5.7 7.7 12.0
Slow 0% DMSO
57.5 54.4 16.8 23.6 22.7
f f f f f
8.9 10.7 3.6 7.2 10.4
70.
sence of complement and 7.770 with com- with continuing incubation since a rapid plement present. The results with decom- fall-off of reconstituted cell count occurred plemented serum were the same as when with incubation (Figs. 1 and 2). After 60 the incubation mixture contained no serum min incubation only 40% of reconstituted neutrophils could be counted in the sus(unpublished data). 27.6% of reconstituted neutrophils col- pension. This rapid fall-off in cell numbers lected in PFH, separated by dextran sedi- can be .attributed to leuco-agglutination and gel formation with continuing incubamentation and recovered after freezing tion and reflects the fragility of frozenwith DMSO (Materials and Methods) actively phagocytosed Candida albicans thawed celIs in this particuIar in &TO sys(Table 2). Reconstituted neutrophils re- tem. These studies incubated at 37°C were repeated at room temperature with comtained 22.3% of fresh candidacidal activity (Table 2). The phagocytic capacities of parable results (Hill et al. 1977, unpubfresh and reconstituted neutrophils, and lished data). the Candida uptake by those cells were DISCUSSION examined and compared (Figs. 1 and 2). Within 10 min in the presence of compleConflicting evidence for post-recovery ment, fresh neutrophils had ingested viability of neutrophils has been reported greater than 95% of free (Fig. 1) Candida after freezing. Some workers have sugand approximately 60% of neutrophils ex- gested that human polymorph leucocytes amined showed ingested yeast (Fig. 2). In cannot withstand freezing in the presence contrast with thawed cells maximum Can- of dimethyl sulfoxide (DMSO) but are dida ingestion was not reached until after irreversibly damaged ( 13). Such irreversapproximately 60 min of incubation with ible damage is reported to include loss of 12% of Candida being ingested (Fig. 1). motility, formation of cytoplasmic vacuoles Phagocytosis by freeze-thawed cells pro- and nuclear derangement with subsequent ceeded slowly over a longer period of time. disintegration ( 13). At 70 min the percent of phagocytic cells Such claims that human polymorph leuhad risen to a maximum of 12% (Fig. 2) cocytes cannot withstand freezing in the i.e. 20% of the fresh value. Both the presence of DMSO but are irreversibly Candida uptake and phagocytic capacity damaged are difficult to sustain when it appeared to be dependent on the cell yield is considered that motility, pseudopod
FREEZE
PRESERVATION
OF
formation and engulfment are necessary prerequisites for Candida or bacterial phagocytosis and given that up to 27.6% of neutrophils after freezing retain their phagocytic and 22.3% their candidacidal activity (5, 6). Recent studies have shown that a percentage of peripheral blood neutrophils retain their bactericidal activity for prolonged periods of time, if not indefinitely after appropriate freezing techniques (1, 11). Furthermore the recovery of neutrophils after freezing with intact myeloperoxidase granulation (9) suggests that as many as 60 to 70% of neutrophils should be capable of microbacidal activity. In each of four tests of functional capacity utilized in our present studies, namely qualitative and quantitative NBT, Candida phagocytosis and candidacidal activity, results show consistency with up to 31.7% of fresh cells after freeze-thawing retaining functional capacity as judged by one or other of the above criteria. In contrast to fresh cells where phagocytosis is completed within 15 min, ingestion of yeast in reconstituted cells proceeds more slowly over a much longer period. The numbers of phagocytic cells therefore will be influenced by any reduction in total neutrophil numbers during the period of incubation. The in vitro conditions of our studies lead to a rapid fall-off in neutrophils over the hour of incubation required for in vitro Candida phagocytosis. TABLE NBT Reduction Reconstituted
3 by Fresh Neutrophils
and
Stimulation
FreshReconstitutedb Complement Complement
present absent
ZplWX3~~
Endotoxin
5.5 *
1.8
5.5
f
1.9 1.7
7.6 2.2
* 1.8 * 1.5
20.9 7.7
f f
~1Results are expressed as nmol of formaaen/lO~ (mean f SD of not less than five studies). JJResults are expressed as percentage of (mean f SD of not less than four studies).
1.6
phagocytes fresh
activity
HUMAN
GRANULOCYTES 100
109
r - - - - -- - r-w
T
Y
20 Minutes
40
60
80
at 37’ C
FIG. 1. Candida uptake by neutrophils using a ratio of 1 Candida albicans: 1 leucocyte at 37°C. 0 -O denotes reconstituted leucocytes -C SD (4 studies). - - - - denotes fresh normal leucocytes. Superimposed is A-A denoting percent leucocytes remaining in suspension.
The present findings of strongly positive NBT activity in frozen reconstituted cells provides further evidence of their functional recovery after freezing given appropriate conditions. Furthermore the comparable leucocyte capacity to reduce NBT and synthesize formazan and its complement dependency appears to be a function probably involving metabolic processes closely related to those required for intracellular-tidal activity. During the metabolic burst that accompanies phagocytosis, hydrogen peroxide is produced by the oxidation of NaDH, by the enzyme NaDHoxidase ( 16). Reduction of NBT appears to be linked to these reactions (1). The production of an NaDH-inhibitor by freezing, although postulated as a possible mechanism for the previously uniformly negative NBT reactions of reconstituted neutrophils (2), cannot be supported in the light of current results with the NBT test. The recovery of significant NBT activity indicates that freezing and thawing
HILL
110 100
80
20
20
40 Minutes
60
80
at37'C
FIG. 2. Percentage phagocytosis using a ratio of 1 Candida albicans: 1 leucocyte at 37°C. O-O Denotes reconstituted cells * SD (four studies). --_Denotes fresh normal cells. Superimposed is A---A denoting percent leucocytes remaining in suspension.
by our methods do not necessarily inhibit oxidative processes or leucocyte-related NaDH reactions required for the reduction of the tetrazolium dye and formation of formazan in many freeze-thawed cells. The complement dependence of the NBT reaction is shown when E. coli endotoxin and zymosan are used as neutrophil stimulants, Although both substances produce comparable results in quantitative NBT tests carried out with fresh cells, strikingly different resuhs were obtained with reconstituted cells indicating selectivity of response for agents initiating the metabolic reactions leading to the reduction of NBT dye and the formation of reduced formazan intracellulary. It may thus be important when designing tests of functional capacity for reconstituted granulocytes to recognize that such differences may be present which are not discernable in the fresh. The results of our studies indicate that freeze-thawed cells collected in calcium-
ET
AL.
binding anticoagulants such as ACD show less NBT activity than those collected with heparin. Furthermore, calcium-binding anticoagulants reduce post-thaw leucocyte and neutrophil yields. Such findings confirm previous similar studies (3). Heparin, a sulphated mucopolysaccharide with strong acid properties has been shown to stimulate in vitro NBT activity in viable neutrophils (3). This observation has also been reported foll’owing the addition of methylene blue (8). However the use of 25 units/ml of heparin compared with ACD has resulted in our hands in a consistent improvement, not only in the results of NBT activity but also in total leucocyte and neutrophil yields, Candida phagocytosis and kill (5, 6). In our hands the precise co,oling rate of l”/min does not appear to be a critical factor in determining recovery of functional capacity as judged by NBT testing ( Hill, Kennedy, and Mackinder, 1977unpublished data) 1 Nor has slow titration as compared with rapid dilution methods during reconstitution improved cell-yield or functional capacity as judged by in vitro tests. For this reason as well as for technical simplicity we have used rapid dilution as the routine method for reconstituting human leucocytes after freezing, and dextran in all reconstituent media. The addition of lignocaine has not further improved cell yield or in vitro function tests. SUMMARY
Huma peripheral blood leucocytes (neutrophil rich) were collected either with preservative-free heparin (PFH) or acid citrate dextrose ( ACD), frozen with dimethyl sulphoxide (DMSO) at a controlled rate, stored in liquid nitrogen at -196°C and reconstituted in a solution containing dextran polymer 70. A battery of tests including nitroblue reduction, Candida tetrazolium (NBT) phagocytic and candidacidal capacity was used to compare anticoagulants and re-
FREEZE
PRESERVATION
OF
constitution methods as they affect functional capacity of freeze-thawed neutrophils during a short post-thaw period. Heparin showed an overall advantage over acid citrate dextrose. A slow titration reconstituticn method did not improve cell yield or functional capacity compared with a rapid dilution method and was a more cumbersome technique. The presence of complement greatly improved the capacity of reconstituted cells to reduct NBT and synthesize formazan. Freeze-thawed cells showed a selective response to stimulation as judged by the quantitative NBT test, responding strongly to zymosan in comparison with E. coli endotoxin. LignoCaine hydrochloride added to the reconstituent medium in concentrations up to 20 mmol/l did not have additional prvtective effect: ‘on post-thawed leucocytes as assessed by agglutination and leucocyte yields when compared with reconstitutent solutions containing only dextran. Reducing pH did not significantly slow the rate of gelling and leuco-agglutination or improve cell yields. Using these findings to optimize conditions, reconstituted neutrophils retained 31.770 ‘of fresh NBT activity, 27.6% of fresh phagocytic, and 22.3% of fresh candidacidal capacity.
HUMAN
4.
5.
6.
7.
8.
9.
10.
11.
12.
ACKNOWLEDGMENTS This work i:s supported by a grant from the Medical Research Council of New Zealand. The authors wish to thank Mrs. A. Hanson for her secretarial help.
13.
14. REFERENCES B. 1. Bannatyne, R. M., and Umamaheswaran, Bactericidal function of cryopreserved neutrophils. Cryobiology 10, 338341 ( 1973). 2. Fawcett, C. P., Ciotti, M. M., and Kaplan, N. 0. Inhibition of dehydrogenase reactions formed from reduced by a substance Biochim. diphosphopyridine nucleotide. Biophys. Acta 54, 202-212 (1961). 3. Hellum, K. B., and Solbert, C. 0. Influence of anticoagulants on the nitroblue tetra-
15.
16.
GRANULOCYTES
111
zolium test. Sound. J. lnfec. Dis. 5, 67-70 ( 1973 ) . Ifill, R. S., Carrington, V., Park, Diana, Fox, J. G. C., and Goldman, J. M. A new controlled-rate cooling apparatus for freezing haemopoietic cells for storage at -196°C Cryobiology 10, 1-9 (1973) Hill, R. S., and Maxine Kennedy. Problems with the preservation of blood cells. N. 2. Med. J. 83, 126 ( 1976a). Hill, R. S., and Maxine Kennedy. Recovery of functional capacity of granulocytes preserved at low temperature. Aus. N. 2. J. Med. 6, 360 ( 1976b). Hill, R. S., Kennedy Maxine, Mackinder Christine. Nitroblue tetrazolium (NBT) activity in human leucocytes after freezing. Pathology 10, 69-75 ( 1978). Humbert, J. R., Gross, G. P., Vatter, A. E., and Hathaway, W. E. Nitroblue tetrazolium reduction by neutrophils: Biochemical and ultrastructural effects of methylene blue. J. Lab. Clin. Med. 8, 20-30 ( 1973). Klebanoff, S. J. Myleroperoxidase: Contribution to microbicidal activity of intact leukocytes. Science 169, 1095-1097 ( 1970). Lehrer, R. I., and Cline, M. J, Interaction of Candida albicans with human leukocytes and serum. J. Bact. 98, 9961004 ( 1969). Lionetti, F. J., Hunt, S. M., Gore, J. M., and Curby, W. A. Cryopreservation of human granulocytes. Cryobiology 12, 181-191 (1975). Lowenthal, R. M., Park, Diana, S., Goldman, J. M., Th’ng, K. H., Hill, R. S., and White, G. The cryopreservation of human bone marrow and buffy coat cells. Morphological and functional changes. Bdt. J. Haemat. 34, 105-117 ( 1976). Malinin, T. I. Injury of human polymo’rphonuclear granulocytes frozen in the presence of cryoprotective agents. Cryobiology ‘7, 123-130 (1972). Segal, A. W., and Peters, T. J. The nylon column dye test: A possible screening test of phagocyte function. CZin. Sci. Mol. Med. 49, 590-596 ( 1975). Schiffer, C. A., Young, Virginia B., Aisner, J., and Sanel, F. T. Reversal of granulocyte adherence to nylon fibres by cationic anaesthetics. (Abstr. Dl) Sec. Int. Symp. on leucocyte separation and transfusion (1976). Stossel, T. P. Phagocytosis. N. EngZ. J. Med. 290, 744-781 (1974).
1.6, 105-111
Recovery
(1979)
of Functional Capacity of Human Freeze-Preservation with Dimethyl
R. S. HILL,
C. MACKINDER,
Department
S. CHALLINOR,
of Haematology, Wallace Auckland, New
Methods have recently become available which permit low temperature preservation of human haemopoietic cells in liquid nitrogen at -196°C and reconstitution to show recovery of functional capacity (4, 7, 12). Recent studies have shown that neutrophils can be reconstituted to show significant functional capacity as judged by Candida and bacterial phagocytosis, microbacidal activity, nitroblue tetrazohum (NBT) reduction, (1, 7, 11) and recovery of granulation in as many as 60 to 70% of cryopreserved cells (9). Such positive recent evidence documenting some capacity to preserve certain aspects of neutrophil function provided impetus for our studies in this field. We have recently shown during short post-thaw periods that a significant proportion of hurnan peripheral blood granulocytes can be freeze-thawed and reconstituted to show recovery of functional capacity as judged by Candida albicans candidacidal activity and phagocytosis, NBT reduction (5, 6). In the present studies we have compared preservativefree heparin (PFH) and acid citrate dextrose (ACD) anticoagulants in the cell freezing system, slow and rapid dilution techniques for cell reconstitution, and examined specific factors potentially capReceived 12, 1978.
March
2,
1978;
accepted
Granulocytes Sulphoxide
AND
Laboratory, Zealand
after
B. POSTLEWAIGHT Auckland
Hospital,
able of influencing cell recovery and viability including complement and activants of NBT activity (zymosan and E. coli endotoxin). Some experimental factors influencing leucocyte Candida phagocytosis and candidacidal activity were also studied. MATERIALS
Leucocyte
AND
METHODS
Collection
Thirty milliliters of human peripheral blood from healthy volunteers were collected by venepuncture with 25 units of PFH (Commonwealth Serum Laboratories, Melbourne) per ml of blood in a 50 ml plastic syringe. For comparison, some samples were collected with 5.6 standard units of ACD (USBP) per ml of blood. One half volume of macrodex (Dextran 70, Pharmacia, Uppsala) was added to all blood samples, the syringe inverted six times and left to stand vertically for 45 min. An 18 gauge needle was attached, bent to 90” and the plasma buffy coat expressed into plastic centrifuge tubes. An equal volume of heparinized Hanks buffered salt solution (PFH-HBSS) was added and the cells centifuged at 150 g for 5 min. The cell button was resuspended in PFH-HBSS and a total leucocyte count performed. A blood smear was made of the neutrophil-rich cell suspension, stained with Romanowsky stain and a leucocyte
September
105 OOll-2240/79/020105-07$02.00/O All
Copyright 0 1979 rights of reproduction
hy Academic Press, Inc. in any form reserved.
106
HILL
differential count carried out. Greater than 90% neutrophil counts were obtained by this method.
Freeze Preservation
of Cells
The cell suspension was diluted with PFH-HBSS to give 1 x lo7 neutrophils/ mls and the preserving agent DMSO (Merk) added rapidly with mixing, to a concentration of ,lO%. Sterile glass ampoules containing 2 ml of final cell suspension were immediately sealed and rapidly cooled to 0°C by placing them on ice. They were then cooled at l”/min in an ethyl alcohol (96% dehydrated, Kempthorne Prosser) bath to -60°C before being transferred to the liquid phase of nitrogen (-196OC) as previously described (4). Cells were stored for 24 to 72 hr before testing.
Leucocyte
Reconstitution
The reconstituting solution was made up of 50% PFH-HBSS and 50% Dextran 70 with the pH adjusted to 7.4 with 4.4% sodium bicarbonate. Cells were thawed rapidly in a 37°C waterbath, diluted immediately (rapidly) with 10 times the volume (20 ml) ‘of reconstituting media, centrifuged at 1506 for 5 min and washed once with a further 10 volumes of reconstituting solution before final reconstitution. The DMSO content after washing was considered to be 0%. A total cell count and differential were performed. For comparison samples of thawed cells were diluted with 10 volumes of reconstituting solution by way of a slow titration method over 20 min. These cells were tested after dilution without washing (0.9% DMSO). During studies to test the effect of the cationic local anaesthetic agent lignocaine on leuco-agglutination and the yield of reconstituted neutrophils, the pH of the reconstituting solution was adjusted to 6.7, 7.0, and 7.4 and the lignocaine hydrochloride (Douglas Pharmaceuticals) added
ET AL.
to celI aliquots in 1, 5, 10, and 20 mmol concentrations.
Qualitative Test
Nitroblue
Tetraxolium
(NBT)
The method has been detailed previously (7). Briefly, fresh and frozen leucocytes were tested qualitatively for NBT reduction on glass and after zymosan stimulation. Zymosan was obtained from S. cerevisiue yeast (Sigma). In absolute terms, percentage retention of NBT activity by freeze-thawed cells was calculated by the formula XY/Z: (X = s NBT activity of freeze-thawed cells, Y = % neutrophil yield after freeze-thawing and Z = s NBT activity of fresh cells).
Quantitative
NBT Test
This method, modified after Segal and Peters (14) has been detailed previously (7). A leucocyte/NBT mixture was run through a nylon fiber column after stimulation with zymosan. The phagocytes adhered to the nylon fiber in the column, Formazan was extracted from the nylon fiber with diethylene dioxide (1,4-Dioxan, Analar) and the optical density of the resulting blue solution was compared with that of a known formazan standard. One further modification was made. An absorption curve for formazan with maximum absorption at wavelength of 575 nm (Hill, unpublished data), provided the standard for spectrophotometric readings in this series of experiments. Samples ‘of fresh and reconstituted leucocytes were tested quantitatively for NBT reduction and formazan synthesis after endotoxin stimulation. Endotoxin was prepared by inoculating 100 mIs of TC 199 (GIBCQ) with a laboratory strain of E. coli. After 48 hr incubation the culture was millipore filtered (filter size 0.22 pm) and the filtrate stored in 5 ml aliquots at -22°C. Incubation for 10 min with 0.15
FREEZE
PRESERVATION
OF
mls of endotoxin was found to give optimal stimulation to fresh neutrophils. Canadida Activity
Phagocytosis
HUMAN
TABLE Effect
of Anticoagulant Functional Capacity
and Candidacidal
The method of Lehre and Cline 1969 (10) was followed using a leucocyte to Candida albicans cell ratio of 1:l. Incubation of the text mixture was carried out at 37°C for 75 min. Samples were taken from the tubes at 10 min intervals, films made and stained by Romanowsky and Gram’s stain. The percentage of leucocytes which ingested Candida (percent phagocytosis) and. the percentage of added Candida ingested (percent Candida uptake) were estimated by counting 400 cells. In absolute terms phagocytis retention by reconstituted cells was calculated using the same derived formula as for NBT retention (see above) substituting ‘$NBT activity with percent phagocytic activity. Candidacidal activity was assessed after release of ingested yeasts following disruption of leucocytes with sodium deoxycholate (2.5%). Equal volumes of 0.01% methylene blue added to the cell suspension distinguished intact (dye excluded) from dead Candida (dye taken up) and the proportion of yeast killed (the candidacidal activity) was calculated. The absolute candidacidal retention was calculated in a similar manner to phagocytic and NBT retention (see above). RESULTS
The effect of anticoagulants, PFH and ACD on leucocyte yields and NBT activity of frozen reconstituted cells is shown in Table 1. Using the Wilcoxon’s signed rank test on the data included in this table, an ‘overall improvement was found with heparin (P < 0.01). Compared to ACD, PFH increased all 5 comparative parameters; total leucocyte and neutrophil yields, NBT on glass and stimulated NBT with zymosan, and Candida phagocytosis.
107
GRANULOCYTES 1 on Leucocyte Yield after Reconstitution IIeparin
Total leucocytes Neutrophils NBT (glass) NBT (aymosan) Candida phsgocytosis Results are expressed *SD (five studies).
675 63.2 13.7 20.2 25.9 as the mean
* 7.3 + 12.8 f 4.4 f 7.0 zt 14.6 percent
and
ACD 56.3 50.5 7.9 13.7 21.9
rt 10.5 f 4.1 f 2.0 f 3.2 f 12.1
of the fresh
value
When reconstitution methods were compared in separate studies, slow dilution by titration over 20 min showed no advantage over a rapid dilution (see Materials and Methods) as judged by total leucocyte and neutrophil recovery, NBT glass and zymosan stimulated, and Candida phagocptic capacity (Table 2). The re,sults confirm that small quantities of DMSO (0.9% ) remaining after reconstitution are not detrimental to leucocyte functional capacity as tested in these studies. The rate of gelling and leuco-agglutination over a 75 min post-thaw period was not significantly reduced either by the addition to the reconstitution media of lignocanine hydrochloride reported to inadherence to nylon hibit granulocytes fibers (15) or by reducing pH. Reconstituted neutrophils showed selectivity in their response to stimulation as judged by the quantitative NBT test (Table 3). Incubated with zymosan they retained 20.9% of NBT activity. Cells incubated with E. coli endotoxin on the other hand retained only 7.770 of NBT activity of fresh cells. This contrasted to the quantitative NBT tests performed with fresh cells where zymosan and endotoxin gave similar results of 5.5 nmol formazan/ lo6 cells. When decomplemented serum was used as a constituent in the NBT test with zymosan stimulation, the results of the quantitative tests expressed as percent recovery of fresh were 7.6% compared to 20.9% when complement was added (Table 3). Similarly after endotoxin stimulation the results were 2.2’$ in the ab-
HILL
108
ET
TABLE: Effect
of Reconstitution
Method
AL. 2
on Leucocyte
Functional
Capacity”
Reconstituted Rapid 0.9% DMSO
Total leucocyte yieldb Neutrophil yield* NBT (glass)c NRT (zymosan)c Candida phagocyto& Candidacidal activity”
66.0 61.9 21.9 31.7 27.6 22.3
zk 5.7 f 6.7 f 5.7 f 8.2 f 16.5 f 10.4
Results are a mean f SD of five studies. a Leucocvtes collected in Heparin and Sedimented b Calculated as percent of fresh cells. c Calculated as percent retent,ion of fresh activit,y.
Slow 0.9% DMSO
56.5 53.3 16.6 26.0 21.1 10.2
with
Rapid 0% DMSO
f 4.3 f 6.2 f 2.1 f 4.0 f 11.4 f 9.3
Dextran
69.2 65.2 21.2 29.8 22.8
f f f f f
10.9 12.5 5.7 7.7 12.0
Slow 0% DMSO
57.5 54.4 16.8 23.6 22.7
f f f f f
8.9 10.7 3.6 7.2 10.4
70.
sence of complement and 7.770 with com- with continuing incubation since a rapid plement present. The results with decom- fall-off of reconstituted cell count occurred plemented serum were the same as when with incubation (Figs. 1 and 2). After 60 the incubation mixture contained no serum min incubation only 40% of reconstituted neutrophils could be counted in the sus(unpublished data). 27.6% of reconstituted neutrophils col- pension. This rapid fall-off in cell numbers lected in PFH, separated by dextran sedi- can be .attributed to leuco-agglutination and gel formation with continuing incubamentation and recovered after freezing tion and reflects the fragility of frozenwith DMSO (Materials and Methods) actively phagocytosed Candida albicans thawed celIs in this particuIar in &TO sys(Table 2). Reconstituted neutrophils re- tem. These studies incubated at 37°C were repeated at room temperature with comtained 22.3% of fresh candidacidal activity (Table 2). The phagocytic capacities of parable results (Hill et al. 1977, unpubfresh and reconstituted neutrophils, and lished data). the Candida uptake by those cells were DISCUSSION examined and compared (Figs. 1 and 2). Within 10 min in the presence of compleConflicting evidence for post-recovery ment, fresh neutrophils had ingested viability of neutrophils has been reported greater than 95% of free (Fig. 1) Candida after freezing. Some workers have sugand approximately 60% of neutrophils ex- gested that human polymorph leucocytes amined showed ingested yeast (Fig. 2). In cannot withstand freezing in the presence contrast with thawed cells maximum Can- of dimethyl sulfoxide (DMSO) but are dida ingestion was not reached until after irreversibly damaged ( 13). Such irreversapproximately 60 min of incubation with ible damage is reported to include loss of 12% of Candida being ingested (Fig. 1). motility, formation of cytoplasmic vacuoles Phagocytosis by freeze-thawed cells pro- and nuclear derangement with subsequent ceeded slowly over a longer period of time. disintegration ( 13). At 70 min the percent of phagocytic cells Such claims that human polymorph leuhad risen to a maximum of 12% (Fig. 2) cocytes cannot withstand freezing in the i.e. 20% of the fresh value. Both the presence of DMSO but are irreversibly Candida uptake and phagocytic capacity damaged are difficult to sustain when it appeared to be dependent on the cell yield is considered that motility, pseudopod
FREEZE
PRESERVATION
OF
formation and engulfment are necessary prerequisites for Candida or bacterial phagocytosis and given that up to 27.6% of neutrophils after freezing retain their phagocytic and 22.3% their candidacidal activity (5, 6). Recent studies have shown that a percentage of peripheral blood neutrophils retain their bactericidal activity for prolonged periods of time, if not indefinitely after appropriate freezing techniques (1, 11). Furthermore the recovery of neutrophils after freezing with intact myeloperoxidase granulation (9) suggests that as many as 60 to 70% of neutrophils should be capable of microbacidal activity. In each of four tests of functional capacity utilized in our present studies, namely qualitative and quantitative NBT, Candida phagocytosis and candidacidal activity, results show consistency with up to 31.7% of fresh cells after freeze-thawing retaining functional capacity as judged by one or other of the above criteria. In contrast to fresh cells where phagocytosis is completed within 15 min, ingestion of yeast in reconstituted cells proceeds more slowly over a much longer period. The numbers of phagocytic cells therefore will be influenced by any reduction in total neutrophil numbers during the period of incubation. The in vitro conditions of our studies lead to a rapid fall-off in neutrophils over the hour of incubation required for in vitro Candida phagocytosis. TABLE NBT Reduction Reconstituted
3 by Fresh Neutrophils
and
Stimulation
FreshReconstitutedb Complement Complement
present absent
ZplWX3~~
Endotoxin
5.5 *
1.8
5.5
f
1.9 1.7
7.6 2.2
* 1.8 * 1.5
20.9 7.7
f f
~1Results are expressed as nmol of formaaen/lO~ (mean f SD of not less than five studies). JJResults are expressed as percentage of (mean f SD of not less than four studies).
1.6
phagocytes fresh
activity
HUMAN
GRANULOCYTES 100
109
r - - - - -- - r-w
T
Y
20 Minutes
40
60
80
at 37’ C
FIG. 1. Candida uptake by neutrophils using a ratio of 1 Candida albicans: 1 leucocyte at 37°C. 0 -O denotes reconstituted leucocytes -C SD (4 studies). - - - - denotes fresh normal leucocytes. Superimposed is A-A denoting percent leucocytes remaining in suspension.
The present findings of strongly positive NBT activity in frozen reconstituted cells provides further evidence of their functional recovery after freezing given appropriate conditions. Furthermore the comparable leucocyte capacity to reduce NBT and synthesize formazan and its complement dependency appears to be a function probably involving metabolic processes closely related to those required for intracellular-tidal activity. During the metabolic burst that accompanies phagocytosis, hydrogen peroxide is produced by the oxidation of NaDH, by the enzyme NaDHoxidase ( 16). Reduction of NBT appears to be linked to these reactions (1). The production of an NaDH-inhibitor by freezing, although postulated as a possible mechanism for the previously uniformly negative NBT reactions of reconstituted neutrophils (2), cannot be supported in the light of current results with the NBT test. The recovery of significant NBT activity indicates that freezing and thawing
HILL
110 100
80
20
20
40 Minutes
60
80
at37'C
FIG. 2. Percentage phagocytosis using a ratio of 1 Candida albicans: 1 leucocyte at 37°C. O-O Denotes reconstituted cells * SD (four studies). --_Denotes fresh normal cells. Superimposed is A---A denoting percent leucocytes remaining in suspension.
by our methods do not necessarily inhibit oxidative processes or leucocyte-related NaDH reactions required for the reduction of the tetrazolium dye and formation of formazan in many freeze-thawed cells. The complement dependence of the NBT reaction is shown when E. coli endotoxin and zymosan are used as neutrophil stimulants, Although both substances produce comparable results in quantitative NBT tests carried out with fresh cells, strikingly different resuhs were obtained with reconstituted cells indicating selectivity of response for agents initiating the metabolic reactions leading to the reduction of NBT dye and the formation of reduced formazan intracellulary. It may thus be important when designing tests of functional capacity for reconstituted granulocytes to recognize that such differences may be present which are not discernable in the fresh. The results of our studies indicate that freeze-thawed cells collected in calcium-
ET
AL.
binding anticoagulants such as ACD show less NBT activity than those collected with heparin. Furthermore, calcium-binding anticoagulants reduce post-thaw leucocyte and neutrophil yields. Such findings confirm previous similar studies (3). Heparin, a sulphated mucopolysaccharide with strong acid properties has been shown to stimulate in vitro NBT activity in viable neutrophils (3). This observation has also been reported foll’owing the addition of methylene blue (8). However the use of 25 units/ml of heparin compared with ACD has resulted in our hands in a consistent improvement, not only in the results of NBT activity but also in total leucocyte and neutrophil yields, Candida phagocytosis and kill (5, 6). In our hands the precise co,oling rate of l”/min does not appear to be a critical factor in determining recovery of functional capacity as judged by NBT testing ( Hill, Kennedy, and Mackinder, 1977unpublished data) 1 Nor has slow titration as compared with rapid dilution methods during reconstitution improved cell-yield or functional capacity as judged by in vitro tests. For this reason as well as for technical simplicity we have used rapid dilution as the routine method for reconstituting human leucocytes after freezing, and dextran in all reconstituent media. The addition of lignocaine has not further improved cell yield or in vitro function tests. SUMMARY
Huma peripheral blood leucocytes (neutrophil rich) were collected either with preservative-free heparin (PFH) or acid citrate dextrose ( ACD), frozen with dimethyl sulphoxide (DMSO) at a controlled rate, stored in liquid nitrogen at -196°C and reconstituted in a solution containing dextran polymer 70. A battery of tests including nitroblue reduction, Candida tetrazolium (NBT) phagocytic and candidacidal capacity was used to compare anticoagulants and re-
FREEZE
PRESERVATION
OF
constitution methods as they affect functional capacity of freeze-thawed neutrophils during a short post-thaw period. Heparin showed an overall advantage over acid citrate dextrose. A slow titration reconstituticn method did not improve cell yield or functional capacity compared with a rapid dilution method and was a more cumbersome technique. The presence of complement greatly improved the capacity of reconstituted cells to reduct NBT and synthesize formazan. Freeze-thawed cells showed a selective response to stimulation as judged by the quantitative NBT test, responding strongly to zymosan in comparison with E. coli endotoxin. LignoCaine hydrochloride added to the reconstituent medium in concentrations up to 20 mmol/l did not have additional prvtective effect: ‘on post-thawed leucocytes as assessed by agglutination and leucocyte yields when compared with reconstitutent solutions containing only dextran. Reducing pH did not significantly slow the rate of gelling and leuco-agglutination or improve cell yields. Using these findings to optimize conditions, reconstituted neutrophils retained 31.770 ‘of fresh NBT activity, 27.6% of fresh phagocytic, and 22.3% of fresh candidacidal capacity.
HUMAN
4.
5.
6.
7.
8.
9.
10.
11.
12.
ACKNOWLEDGMENTS This work i:s supported by a grant from the Medical Research Council of New Zealand. The authors wish to thank Mrs. A. Hanson for her secretarial help.
13.
14. REFERENCES B. 1. Bannatyne, R. M., and Umamaheswaran, Bactericidal function of cryopreserved neutrophils. Cryobiology 10, 338341 ( 1973). 2. Fawcett, C. P., Ciotti, M. M., and Kaplan, N. 0. Inhibition of dehydrogenase reactions formed from reduced by a substance Biochim. diphosphopyridine nucleotide. Biophys. Acta 54, 202-212 (1961). 3. Hellum, K. B., and Solbert, C. 0. Influence of anticoagulants on the nitroblue tetra-
15.
16.
GRANULOCYTES
111
zolium test. Sound. J. lnfec. Dis. 5, 67-70 ( 1973 ) . Ifill, R. S., Carrington, V., Park, Diana, Fox, J. G. C., and Goldman, J. M. A new controlled-rate cooling apparatus for freezing haemopoietic cells for storage at -196°C Cryobiology 10, 1-9 (1973) Hill, R. S., and Maxine Kennedy. Problems with the preservation of blood cells. N. 2. Med. J. 83, 126 ( 1976a). Hill, R. S., and Maxine Kennedy. Recovery of functional capacity of granulocytes preserved at low temperature. Aus. N. 2. J. Med. 6, 360 ( 1976b). Hill, R. S., Kennedy Maxine, Mackinder Christine. Nitroblue tetrazolium (NBT) activity in human leucocytes after freezing. Pathology 10, 69-75 ( 1978). Humbert, J. R., Gross, G. P., Vatter, A. E., and Hathaway, W. E. Nitroblue tetrazolium reduction by neutrophils: Biochemical and ultrastructural effects of methylene blue. J. Lab. Clin. Med. 8, 20-30 ( 1973). Klebanoff, S. J. Myleroperoxidase: Contribution to microbicidal activity of intact leukocytes. Science 169, 1095-1097 ( 1970). Lehrer, R. I., and Cline, M. J, Interaction of Candida albicans with human leukocytes and serum. J. Bact. 98, 9961004 ( 1969). Lionetti, F. J., Hunt, S. M., Gore, J. M., and Curby, W. A. Cryopreservation of human granulocytes. Cryobiology 12, 181-191 (1975). Lowenthal, R. M., Park, Diana, S., Goldman, J. M., Th’ng, K. H., Hill, R. S., and White, G. The cryopreservation of human bone marrow and buffy coat cells. Morphological and functional changes. Bdt. J. Haemat. 34, 105-117 ( 1976). Malinin, T. I. Injury of human polymo’rphonuclear granulocytes frozen in the presence of cryoprotective agents. Cryobiology ‘7, 123-130 (1972). Segal, A. W., and Peters, T. J. The nylon column dye test: A possible screening test of phagocyte function. CZin. Sci. Mol. Med. 49, 590-596 ( 1975). Schiffer, C. A., Young, Virginia B., Aisner, J., and Sanel, F. T. Reversal of granulocyte adherence to nylon fibres by cationic anaesthetics. (Abstr. Dl) Sec. Int. Symp. on leucocyte separation and transfusion (1976). Stossel, T. P. Phagocytosis. N. EngZ. J. Med. 290, 744-781 (1974).
