Cryopreservation of antibody-coupled red cells for use in immunoassays

Journal of Immunological Methods, 54 (1982) 331-341

331

Elsevier Biomedical Press

Cryopreservation of Antibody-Coupled Red Cells for Use in Immunoassays D.E. Pegg, M.P. Diaper, S.E.G. Scholey and R.R.A. Coombs MR C Medical Cryobiology Group, Department of Surgery, and Division of Immunology, Department of Pathology, University of Cambridge, Cambridge, U.K.

(Received 25 January 1982, accepted 8 April 1982)

Red cells, trypsin-treated to render them more agglutinable and coupled with antiglobulin reagents, may be preserved by droplet freezing in liquid nitrogen. A 2% cell suspension in 0.45% w/v sodium chloride, 5% w/v sucrose and 10% w/v dextran 40 was used. After thawing the frozen pellets in phosphate-buffered saline at 40°C, more than 80% cell recovery was obtained. Sheep and ox red cells preserved in this way were as satisfactory in antiglobulin and in reverse passive haemagglutination tests as unfrozen indicator red cells. Trypsin-treated human red cells coupled with anti-IgE could likewise be frozen and on reconstitution used to assay IgE in human serum. Reconstituted ox red cells were slightly less efficient in rosetting than cells which had not been frozen. Key words: cryopreservation - - antibody-coupled red cells - - antiglobulin tests

Introduction R e c e n t l y a series of i m m u n o a s s a y s has been d e v e l o p e d in which a c o u p l e d red cell is used to label the a n t i b o d y reagent, usually a n t i g l o b u l i n ( C o o m b s , 1981). T h e a n t i b o d y is c o u p l e d to the red cell b y c h r o m i c chloride. R e c e n t a p p l i c a t i o n s o f i m m u n o a s s a y with red cell l a b e l l e d a n t i b o d y reagents have i n c l u d e d simple reverse passive h a e m a g g l u t i n a t i o n ( R P H ) to m e a s u r e IgE levels in h u m a n sera (Scott et al., 1981a) or r e s p i r a t o r y syncytial virus in n a s o p h a r y n g e a l a s p i r a t e s ( C r a n a g e et al., 1981), a n d the M r s P A H assay (mixed reverse (solid-phase) reverse passive h a e m a d s o r p t i o n ) to m e a s u r e a n t i b o d i e s of different Ig classes to a l c o h o l - s o l u b l e gliadin in coeliac p a t i e n t s (Keiffer et al., 1981) or a n t i b o d i e s to collagen ( C o o m b s et al., 1982). The p r o c e d u r e has sufficient sensitivity to m e a s u r e I g E a n t i b o d i e s (Scott et al., 198 lb). R e d cells labelled with a n t i g l o b u l i n in the direct a n t i g l o b u l i n rosetting r e a c t i o n ( D A R R ) also p r o v i d e a very sensitive m e a s u r e of surface m e m b r a n e i m m u n o g l o b u l i n on B l y m p h o c y t e s ( H a e g e r t a n d C o o m b s , 1979). A p a r t i c u l a r a d v a n t a g e of such red cell labelled assays is their simplicity in c o n c e p t a n d execution which gives t h e m great p o t e n t i a l for use in less s o p h i s t i c a t e d l a b o r a t o r i e s . However, little d e v e l o p m e n t for r o u t i n e p r o c e d u r e s is likely to take 0022-1759/82/0000-0000/$02.75

© 1982 Elsevier Biomedical Press

332

place until a satisfactorily preserved indicator cell, with a long 'shelf life', can be produced. At present coupled red cells last only 2-3 weeks, after which fresh cells have to be coupled and restandardized (although Ling et al. (1979) find that coupled red cells may last for 2-3 months). A technique for long-term preservation is urgently needed. The most commonly used method for long-term red cell preservation relies upon freezing in the presence of permeating neutral solutes such as glycerol (Smith, 1950) or dimethyl sulphoxide (Lovelock and Bishop, 1959). However, this approach has several disadvantages for laboratory studies: the use of a penetrating cryoprotectant results in osmotic disturbance during reconstitution, which makes the recovery of frozen cells a protracted and tedious affair. This is discussed more fully by Pegg (1976). Moreover, if only a few cells are required at one time, the thawing of a whole ampoule of frozen cells may result in considerable wastage. For these reasons, droplet freezing has become popular in blood group research laboratories (Huntsman et al., 1960; Rowe and Allen, 1965). In this technique drops of red cell suspension in an appropriate solution are allowed to fall on to the surface of liquid nitrogen where they cool rapidly, freeze, and sink to the bottom of the container. Thawing is carried out by sprinkling the required number of frozen droplets into warm saline. In preliminary experiments it was found that trypsin-treated sheep red blood cells (TTSRBC) responded to freezing in a similar manner to immunoglobulin-coupled TTSRBC. Optimal freezing conditions were, therefore, developed for TTSRBC first. We then cryopreserved coupled trypsin-treated sheep, ox and human cells (coupled TTSRBC, coupled T T O R B C and coupled TTHRBC). After reconstitution, each cell type was tested for % recovery and immunological function in the antiglobulin, RPH and D A R R assays.

Material and Methods The cryopreservation experiments were designed to investigate the effect on recovery of droplet-frozen TTSRBC of the concentration of sodium chloride, sucrose, glucose and a polymeric protein substitute in the suspending medium. (Protein was excluded for immunological reasons.)

Freezing and thawing A 2% suspension of TTSRBC was allowed to drop on to the surface of liquid nitrogen in a 1½ litre Dewar flask from a 100 ml burette, a 20 ml syringe with a 19-gauge needle, or from a hand-held Pasteur pipette, according to the volume being handled. The height above the nitrogen surface was not critical within the range 10-50 cm and the rate was approximately 50 drops/min. The frozen droplets, each approximately 20/~1, were collected in a funnel placed in the liquid nitrogen Dewar, and directed into a suitable container, usually a 30 ml clear plastic screw-cap bottle (Sterilin Universal Container) (Fig. 1). Each container was closed with a perforated cap (the perforation is important to avoid the risk of explosion) and was transferred to a LR 185 liquid nitrogen refrigerator (Union Carbide) for storage at less than

333

0

I

0

/

-I -

!

// Fig. 1. Diagram showingthe arrangement of funnel and collectingvial in a Dewar flask of liquid nitrogen used for droplet freezing of indicator red cells.

--160°C in the gas phase above the liquid level. When required, the appropriate number of droplets, one or two at a time, was thawed by adding them to 5 ml of a warm thawing solution. The composition of the freezing solution and the thawing solution varied according to the requirements of each experiment as described in the results. All solutions were prepared in terms of percentage w / v (g/dl).

Recovery of cells The surviving cells in the thawing solution were deposited by centrifugation at 1100 X g and the supernatant drained off. The cells were then resuspended in 5 ml of PBS, and were ready for use in immunological tests. If, however, the percentage recovery was to be measured, they were recentrifuged, the supernatant was removed and the cell pellet was lysed by the addition of 5 ml of distilled water. The

334 haemoglobin content of the lysed pellet and the supernatants was measured as cyanmethaemoglobin and the percentage cell recovery calculated.

Preparation of IgG fractions to be used for CrCI~ coupling Many of the reagents used for coupling were kindly prepared and donated from other laboratories as follows: Sheep Z652 anti-human C4, gift of Dr. A.R. Bradwell, Immunodiagnostic Research Laboratory of the Department of Experimental Pathology, Birmingham, and is described by Coombs et al. (1980). Sheep Z193K anti-human IgE, gift of Dr. A.R. Bradwell and is described by Scott et al. (1981a). Rabbit anti-human Fab donated by Dr. A.J. Munro, Department of Pathology, University of Cambridge, Cambridge and is described by Coombs et al. (1977). Sheep T379/4 anti-human Fab, a gift from Dr. A. Feinstein, ARC, Babraham. Normal rabbit IgG was prepared by ammonium sulphate precipitation followed by DEAE chromatography. Normal sheep lgG was prepared by the rivanol method of Weir (1978). The specificity of all immunoglobulins was established by the RPH test. If necessary reagents were absorbed with trypsin-treated red cell to which they were to be coupled. Sera used in the antiglobulin and reverse passive haemagglutination (RPH) tests were as follows: Sheep M33/3 anti-rabbit F(ab), gift of the ARC, Babraham. Rabbit M2 anti-sheep IgG 2, gift of M. Hobart, MRC, Cambridge. Normal human sera were used as the source of human Ig in the RPH tests. Coupling of IgG to trypsin-treated (TT) red cells with CrCI 3 Red cells (sheep, ox and human) were taken into acid citrate dextrose and stored at 4°C before use. The trypsinisation and coupling of red cells has been previously described (Scott et al., 1981a). The red cells were normally trypsin-treated and coupled with antibody within 3 days of bleeding. Lymphocyte preparation Human peripheral blood and tonsil lymphocytes were isolated using Ficoll-Hypaque sedimentation and carbonyl iron treatment (Eremin et al., 1976) and then made up to 2 × 106 cells/ml. The final 4 washes were in PBS with 1% BSA, which was also the suspension buffer. Antiglobulin and reverse passive haemagglutination (RPH) tests Serum dilutions were prepared in PBS and 1 drop (25/~1) added to wells (U-form) of polystyrene microtitre plates (Sterilin). One drop (25 ~tl) of a 1% suspension of coupled TTRBC was added to each well, and the plates were mixed on a micro-mixer (Triyo-Bussan) for 3 sec. The haemagglutination settling pattern of the red cells was read after 90 rain at room temperature.

335

Direct antiglobulin rosetting (DARR test, described by Coombs et al. (1977)) Rosettes were prepared by mixing equal volumes (25 #1) of lymphocytes (2 x 106 cells/ml) and 1% ( v / v ) red cells in 5 m m x 50 m m plastic tubes and centrifuging at 350 X g for 2 rain at 4°C. Following incubation for ½ h at 4°C, the percentage of reacting lymphocytes (with 3 or more erythrocytes adhering) was counted in suspensions stained with toluidine blue and mounted on siliconed microscope slides. The percentage rosettes was calculated from the total cell count of 200 and expressed as the mean of 2 duplicate tests. The D A R R test used to detect C4 on lymphocytes was slightly modified and is fully described by Wilson et al. (1982).

Results

Recovery after freezing and thawing Table I shows the effect of solution composition on recovery of TTSRBC after droplet freezing. In each case the droplets were thawed into the same solution at 50°C and then centrifuged and resuspended in PBS. The addition of either hydroxyethyl starch (HES) (weight-average molecular weight 450 kdaltons) or sucrose, or both, to saline increased recovery, whereas the addition of glucose was detrimental. When the concentration of sodium chloride was fixed at 0.9% and of HES at 6%, the optimal sucrose concentration was 5% (Fig. 2A). When the concentration of sucrose was fixed at 5%, the optimum salt concentration was found to be 0.45% (Fig. 2B). There was no evidence of significant interaction between sodium chloride and sucrose concentration (Pegg et al., 1982). The inclusion of 6% w / v of HES or one of several dextrans (weight-average molecular weights 40, 70, and 110 kdaltons, dextran 40, 70 or 110) with 0.45% sodium chloride and 5% sucrose all gave recoveries of 71-75%. Since the cells had ultimately to be resuspended in PBS it would clearly be most convenient if they could be thawed directly into PBS. A comparison of several thawing solutions (Pegg et al., 1982) showed that when the cells were subsequently transferred to PBS, recovery was similar to that obtained by thawing directly into

TABLE I THE EFFECT OF SOLUTION COMPOSITION ON RECOVERY OF TTSRBC AFTER DROPLET FREEZING Solution (% w/v)

Recovery

0.9% NaCI 0.9% NaCI+6% HES 0.9% NaCI+ 155[ sucrose 0.9% NaCI+ 15% sucrose+5% glucose 0.9% NaCI+ 15% sucrose+6% HES

1.3±0.3 43.5±3.5 30.4~1.5 9.1±0.7 37.0±1.2

336

100

s

J------

A

jJJ

•

75

50O

25.

I

0

5

i0

15

,SucROSE CONCENTRATION

20

(% W/v)

100

B

75

.

s

f

~

"~""~""~"~ ~

50 u

25

,

0

,

!

0,45

0,90

!

1,35

SODIUM CHLORIDE CONCENTRATION

1,80 (% W/V)

Fig. 2. The effect of varying the concentration of sucrose and sodium chloride in the freezing solution, in graph A the concentration of sodium chloride and HES were fixed at 0.9 and 6 g/dl, respectively. In graph B the concentration of sucrose and HES were fixed at 5 and 6 g/dl, respectively. The broken line shows recovery immediately after thawing, and the solid line, recovery after resuspension in PBS.

Unfrozen Dextran (F) b Dextran (S) ~ Dextran (P) d HES e

Unfrozen Dextran (F) Dextran (S) Dextran (P) HES

Normal sheep lgG

Sheep anti-human Fab (100) 80 82 81 81

(100) 80.7 82 81 78

% Recovery

6 (6) 5.5 (5-6) 6 6 5.5 (5-6)

5 (5) 5 (4-6) 6 6 4.5 (4-5)

Titre in antiglobulin test Well no. mean (range)

a Freezing solution, 0.45% NaCI + 5~ sucrose + variable colloid. b Dextran (F), 10% dextran 40 (Fisons). c Dextran (S), 10% dextran 40 (Sigma). d Dextran (P), 10% dextran 40 (Pharmacia). e HES, 10% hydroxyethyl starch. f RPH, reverse passive haemagglutination. s DARR, direct antiglobulin rosetting reaction. h NHS, normal human serum. i Well no., refers to x~ell no. in an X4 series dilution starti'ng at 1/40 (well no. 1).

Variable colloid in freezing solution

TTSRBC coupled with:

(0) (0) (0) (0) (0)

9.3 (9-10) 9 (9) 9 9 9 (9)

0 0 0 0 0

RPH f antigen titre (against NHS) h Well no. i mean (range)

16 16

10 (weak)

19 (weak)

0

0 0

Sample 2

19 20

0 0 0 0 0

Sample 1

DARR g % rosettes

29 31 29 32

0 0

Sample 3

THE EFFECT OF D I F F E R E N T F R E E Z I N G SOLUTIONS ~ ON THE BEHAVIOUR OF RECONSTITUTED TRYPSIN-TREATED SHEEP RED CELLS (TTSRBC) AS MEASURED BY % RECOVERY, ANTIGLOBULIN TESTS AND IN RPH A N D DARR REACTIONS

TABLE I1

Unfrozen Reconstituted Unfrozen Reconstituted

(100) 88 (100) 83

(100) 88 (100) 75

(100) 82 (100) 89

% Recovery

5 5 5 5

5 5 6 6

6 6 5 5

0 0 5 5

0 0 8 8

0 0 8 8

Titre in antiglobulin Titre in RPH test test (against N H S b) Well no. d Well no.

a Freezing solution, 0.45% N a C I + 5 % sucrose+ 10% dextran 40 (Sigma). b NHS, normal h u m a n serum. c DARR, direct antiglobulin rosetting reaction. d Well no., refers to well no. in an X4 series dilution starting at 1/40 (well no. 1).

Sheep anti-human C4

Normal sheep IgG

Rabbit anti-human Fab

Normal rabbit IgG

Unfrozen Reconstituted Unfrozen Reconstituted

Unfrozen Reconstituted Unfrozen Reconstituted

Normal sheep IgG

Sheep anti-human Fab

State of indicator red cell

TTORBC coupled with:

Peripheral blood lymphocytes 0 0 7 5 (weak)

Tonsil lymphocytes 0 0 64 54

Peripheral blood lymphocytes 0 0 17 15 (weak)

DARR ~ % rosettes

THE BEHAVIOUR OF F R O Z E N a T R Y P S I N - T R E A T E D OX RED BLOOD CELLS (TTORBC) C O U P L E D W I T H T W O A N T I - H U M A N Fab R E A G E N T S A N D AN A N T I - H U M A N C4

TABLE III

339 PBS. There was a slight improvement in recovery with increase in the temperature of the thawing solution, but the effect was small and in the range of 4 0 - 6 0 ° C was constant at 84.8-85.8%. A temperature of 40°C was chosen. The effect of increasing the concentration of HES in the freezing solution was studied and there was a small but significant improvement with increasing HES concentration (approximately + 1% in recovery for each g / d l of HES added). When dextran 40 was substituted for HES, similar recovery rates were obtained with T-I'SRBC (84-86%), but with human and ox cells ( T T H R B C and TTORBC) HES gave significantly lower recoveries than dextran 40 (40% vs. 82% and 63% vs. 86%, respectively). The combination of variables finally selected for all 3 cell-types was freezing in 0.45% sodium chloride with 5% sucrose and 10% dextran 40, and thawing into PBS at 40°C. Once reconstituted, the cells remained stable and no further lysis was observed.

Immuno-assays With TTSRBC. Table II shows that with coupled TTSRBC both the % recovery and the titres in the antiglobulin and R P H assays remained optimal with either 10% l I E S or 10% dextran 40 from 3 different sources. The variation from one test to another that we observed when testing unfrozen cells in antiglobulin and R P H assays was ± 1 well and hence the variation seen with the frozen reconstituted cells was within the normal range. In the D A R R tests cells preserved with all 3 brands of dextran 40 showed the same sensitivity, but cells frozen in 10% HES were less sensitive forming definitely weaker rosettes. Dextran 40 was, therefore, the polymeric colloid of choice and for convenience we used Pharmacia dextran 40 as it is supplied in powdered form. With TTORBC. Tests were then made with cryopreserved antibody-coupled TTORBC. As with coupled TTSRBC the recovery usually exceeded 80%; an unexplained exception (Table III) was the sample of T T O R B C coupled with rabbit anti-human Fab which yielded only 75%. In both antiglobulin and R P H tests the frozen cells behaved identically with the unfrozen sample. In the D A R R tests on both peripheral blood and tonsil lymphocytes the frozen antibody-coupled T T O R B C were less sensitive than the unfrozen cells and gave weaker results. Using TTHRBC. Trypsin-treated human red cells coupled with specific anti-IgE (Scott et al., 1981a) were then cryopreserved in medium containing 10% dextran 40. Recovery in 2 batches was 88% and in R P H tests the antibody-coupled red cells were, if anything, slightly more sensitive than the unfrozen cells in measuring IgE in 2 sera of atopic persons. Expressed simply as titres the figures were 2560 and 320 (compared with 1280 and 160 respectively with unfrozen cells). Control red cells coupled with normal I g G and frozen were perfectly negative in the R P H tests. Discussion

The droplet freezing method described in this paper gave more than 80% cell recovery consistently. The failure of the standard technique for native human red

340 cells (Rowe and Allen, 1965) to give adequate recovery of red cells was shown to be due to the use of glucose. Experiments have shown that glucose permeates these cells quite rapidly (Pegg et al., 1982) and it is, therefore, probable that the deleterious effect is mediated by an osmotic mechanism. The probable mechanisms of cryoprotection operating in this technique are discussed elsewhere (Pegg et al., 1982). The method described here allows permanent storage of red cell coupled reagents, within a laboratory, with the potential advantages of economy of labour and consistency. The method requires no special controlled-rate cooling equipment and is very simple to carry out. Storage in liquid nitrogen is essential, however, which admittedly increases the cost over that of conventional refrigeration. Interlaboratory exchange of reagents is thus possible and, for this purpose, non-spillable liquid nitrogen containers are convenient. Alternatively, samples can be transported for short periods at - 7 9 ° C in solid carbon dioxide with a loss of about 10% in 8-360 h (data for TTSRBC) (Pegg et al., 1982). We consider it important to develop stable carrier cells with a long shelf life in order to increase the general applicability of immunoassay methods which use the red cell as the label for antibody (Coombs, 1981). These assay procedures have great sensitivity and adaptability, and yet require little sophistication in handling or equipment. Freshly prepared antibody-coupled trypsin-treated red cells have remarkable sensitivity in antigen detection, e.g., 100 p g / m l IgE (Scott et al., 1982). The present investigation shows that it is possible to cryopreserve such red cell reagents and this is a considerable advance. With frozen, reconstituted red cells, we have obtained high recovery rates with similar sensitivities to those of unfrozen cells in the assay procedures. Preliminary experiments with cryopreserved cells in the MrsPAH test are very promising and this method further widens their applicability in immunoassays. The only test system where the cryopreserved cells proved to be less sensitive was that of the coupled TTORBC in the DARR, and is being further investigated. We contrast our attempts to preserve antibody-coupled TT native red cells with another approach, which is to achieve a long-life shelf product by coupling antibody reagents to 'fixed' red cells. Ling et al. (1979) found that glutaraldehyde or pyruvic aldehyde 'fixed' red cells could no longer be coupled with chromic chloride, but some success was achieved with dimethyl suberimidate-treated (fixed) red cells. Such red cells could be coupled and gave sensitive RPH tests but they did not store well and were too sticky for rosetting tests. Investigations such as these are important, but experience suggests that it will not be easy to achieve a carrier 'fixed' or preserved red cell with the delicate and sensitive agglutinability of the native cell. Such a preserved coupled red cell can be achieved, as reported here, by cryopreservation.

341

References Coombs, R.R.A., 1981, Assays Utilizing Red Cells as Markers in Immunoassays for the 80's, ed. A. Voller (MTP Press, Lancaster). Coombs, R.R.A., A.B. Wilson, O. Eremin, B.W. Gurner, D.G. Haegert, Y.A. Lawson, S. Bright and A. Munro, 1977, J. Immunol. Methods 18, 45. Coombs, R.R.A., A.B. Wilson and P.J. Lachmann, 1980, Int. Arch. Allergy 61, 371. Coombs, R.R.A., B.W. Gurner, G. Oldham, M.J. Barnes and M. Keiffer, 1982, Int. Arch. Allergy 67, 377. Cranage, M.P., E.J. Stott, J. Nagington and R.R.A. Coombs, 1981, J. Med. Virol. 8, 153. Eremin, O., D. Plumb and R.R.A. Coombs, 1976, Int. Arch. Allergy 52, 277. Haegart, D.G. and R.R.A. Coombs, 1979, Lancet, November 17th, 1979. Huntsman, R.G., B.A.L. Hurn and H. Lehmann, 1960, Brit. Med. J. ii, 118. Keiffer, M., P.J. Frazier, N.W.R. Daniels, P.J. Ciclitira and R.R.A. Coombs, 1981, J. lmmunol. Methods 42, 129. Ling, N.R., G. Stephens, P. Bratt and H.S. Dhaliwal, 1979, Mo[. Immunol. 16, 637. Lovelock, J.E. and M.W.H. Bishop, 1959, Nature (London) 183, 1394. Pegg, D.E., 1976, J. Clin. Path, 29, 271. Pegg, D.E., M.P. Diaper, S.E.G. Scholey and R.R.A. Coombs, 1982, Cryobiology in press. Rowe, A.W. and R.H. Alien, Jr., 1965, Transfusion 5, 379. Scott, M.L., M.J. Thornley, R.R.A. Coombs and A.J. Bradwell, 1981a, Int. Arch. Allergy 64, 222. Scott, M.L., M.J. Thornley and R.R.A. Coombs, 1981b, Int. Arch. Allergy 64, 230. Scott, M.L., T.G. Merret, K. Ishizaka. M.J. Thornley and R.R.A. Coombs, 1982, Clin. Exp. Immunol. 48, 417. Smith, A.U., 1950, Lancet ii, 910. Weir, D.M., 1978, Handbook of Experimental Immunology, Vol. 1 (Blackwell Scientific Publications, Oxford). Wilson, A.B., S. Prichard-Thomas, B.W. Gurner, P.J. Lachmann and R.R.A. Coombs, 1982, Clin. Immunol. Immunopathol. 22, 118.