Britishjoitrrral of Haiwarology, 1980, 44, 627-634.

Frozen Red Cells in Rhesus Immunization I. A. COOK, ANNE L. ROBB, R. MKCHELL, ELIZABETH A. MCLAREN, S. URBANIAK

AND ANNE E. ROBERTSON

Nortlz of Scotland Blood TransJirsion Service, Raigmore Hospital, Inverness, Clasgow and West of Scotland Regional Transfusion Centre, Carluke, Lanarksliire, and

Ed in burgh and South- East Scot land Reg iona 1 Tra ns f . sio n Centre, Ro ya 1 It$ rrnar y , Edin hi4 rg it , Scotland

(Received 5 April 1979; accepted for publication 25June 1979)

SUMMARY. The use offrozen washed cells in varying doses in primary Rh immuniza- tion is compared in two groups of men with the use of fresh washed cells in a third group. In the first two groups, using frozen cells, doses ranging from 0.5 to 20 ml of whole blood (Group I) are compared with a 200.0 ml dose of red cell concentrate (Group I I ) , while Group 111 served as a control using a 20 ml dose of fresh washed red cell suspension (9.0 ml concentrated red cell equivalent). The response rate was 93%) in Group I1 compared with only 43% in Group I, suggesting the desirability of using relatively large doses of Rh-positive red cells for primary Rh immunization. The use of frozen washed cells from a special panel for ‘booster’ injections is also recom- mended.

This report presents the results of primary immunization in two groups of Rhesus negative male volunteers using frozen red cells. A change to the use of frozen cells was made in 1972 in the hope of reducing the risk of transmitting viral hepatitis. The project detailed is divided into three parts (Groups I, I1 and HI), and is part of the long-term policy in Scotland to supply high titre anti-D plasma to the Protein Fractionation Centre at Edinburgh, for the production of anti-D Immunoglobulin, to continue the prophylactic scheme against haemolytic disease of the newborn, begun in 1968.

METHODS

The criteria for the selection of male volunteers were as detailed previously (Cook, 1971). In the present scheme, the first group (Group I) was composed of53 Rh negative male volunteers. In this series frozen red cells were used throughout and were obtained from the Glasgow and West of Scotland Blood Transfusion Centre. The initial injection was 0.5,5.0, 10 15 or 20 ml red cells. No further injections were given for 6 months unless anti-D developed, when further injections of 0.5 ml were given at fortnightly or monthly intervals. The same protocol was followed after 6 months to a total of six injections, and ifanti-D did not develop thereafter, the donor was regarded as a ‘non-responder’.

Scotland. Correspondence: Dr I . A. Cook, North of Scotland Blood Transfusion Service, Raigmore Hospital, Inverness,

0007-1048/80/0400-0627$02.00 0 1980 Blackwell Scientific Publications

627

628 I. A . Cook et a1

At each monthly visit, the serum of the donor was tested as follows: (1) against a comprehensive panel of red cells by saline, bromelin, ficin and indirect antiglobulin techniques to detect the development of immune alloantibodies and identify their specificity; (2) the Rh antibodies were titrated against the same Rh positive cells each time by saline, ficin and indirect antiglobulin techniques. After 1975, the indirect antiglobulin test was done using a low ionic strength (glycine) medium in both (1) and (2) (Low & Messeter, 1974). In 1975 the anti-D screening and quantitative estimates ofanti-D were changed to autoanalyser methods (Gunson et a l , 1972). These methods were also used to follow the secondary response of anti-D to the booster injections. The following anti-D standards were used: (1) British working standard (72/229); (2) international anti-D serum standard (64/16); and (3) two local standards 'Law 5 and 7'; anti-D was measured to 0.001 pg/ml.

The donations of frozen red cells from Glasgow were tested for the following antigens: ABO, C, D, E, Z, e, M, N, S, s, K, k, P, Lea, Leb, Fya, Fyb, Jka and Jkb. These red cells were specifically matched to avoid the development of antibodies to S, K, Fya and Jka antigens. The general aim in the selection of red cell donors was to try to achieve complete compatibility outside the Rh system, even in systems involving M, N, P, Lewis and Lutheran, though not always feasible.

From each Glasgow donation, thawed recovered saline suspensions were prepared and dispensed aseptically in sterile universal containers for despatch by air freight to Inverness the same day for injection within 12 h of recovery (Mitchell & Muir, 1972; Mitchell et a l , 1973; Jenkins & Blagdon, 1971). Donors were chosen whose red cells had been stored for 6 months or more in liquid nitrogen, who had been used for routine transfusions in the past without adverse effect in the recipient, who were HBsAg negative by radioimmunoassay (RIA) (Abbott) at the time of donation, and had not reported any illness in the succeeding prethaw intervals.

On recovery, each donation was checked for sterility using aerobic and anaerobic culture up to 7 d at 21"C, 35°C and 37°C. Electron microscopy and light microscopy of recovered red cells was normal and earlier studies had shown red cell survival, post-thaw saline stability and osmotic fragility and post-thaw electrophoretic mobility to be normal (Mitchell, 1976) using the same methods.

In Group 11, 28 male volunteers at the Edinburgh Transfusion Centre received a 200 nil donation of red cell concentrate, stored previously in liquid nitrogen for 6 months. The processing of these cells is as already described by Pepper (1976), and booster injections followed the same time schedule as in Group I above. Fuller details of this scheme are recorded elsewhere (Urbaniak, 1979).

In Group 111, the Inverness Centre recruited 25 male donors but used fresh washed cells, as in the original scheme from 1968 (Cook, 1971). All the men received 20 ml of freshly washed red cell suspension equivalent to 20 ml whole blood or 9.0 ml concentrated red cells, as the primary dosage. It was hoped thereby to achieve the previous average response of around 70% to compensate for the poor response to frozen cells obtained up to 1975. The Inverness red cell donors used in Group I11 were medically examined by a consultant physician initially, and thereafter yearly. Such donors were subsequently seen at monthly intervals by Blood Trans- fusion Medical Staff when blood was taken for the following tests: (1) HBsAg radioimmuno- assay (RIA); (2) Coulter S blood cell counts and film; and (3) SMA12, SGOT, SGPT and gamma GT, within 1 week before taking a sample for injection.

Frozen Red Cells in Rh Immunization 629

By mid-1976 a small panel 0fR2R2 (SDE/CDE) and R2r (SDE/Ede) donors had been formed in Inverness with suitable MNS, P, Kell, Lu’, Le’, Leb, Jka, Jkb, Fy‘, Fyb antigen. These ‘walking donors’ have been used for subsequent booster injections to all Inverness anti-D donors.

RESULTS

The response of the 53 men in Group I in relation to both the dosage and Rh genotype of the cells used for primary injection is shown in Table I. Since individual donations of frozen blood were not selected on the basis of the Rh phenotype, but rather to avoid immunization of antigens outside the Rh system, most donations were of the commoner phenotypes such as

TABLE I. Response to frozen red cells with reference to (1) probable Rh genotype and (2) dosage

Initial Probable Rh genotype dose No. of Per cent (mi) Rlr RlR, RlRz R2r RzR2 responders response

0.5 - - 013 113 - 1 in6 16.7 1.0 1/4 - 1/1 - - 2 i n 5 40.0 5.0 - - 313 213 - 5 in 6 83-3

10.0 2/8 2/3 3/3 0/1 - 7in 15 46.7 15.0 016 - 214 011 - 2in 1 1 18-2 20.0 4/7 1/1 1/1 - 0/1 6in 10 60.0

Responders 7 3 10 3 0 23in53 43.3 Total 25 4 15 8 1

CcDe or CcDEe. Previous experience with fresh cells, suggesting possibly that an increased dose, e.g. 20 ml, was superior to 5.0 ml or less was not confirmed.

Analysis of the responders with reference to the first appearance of anti-D showed that five men had anti-D by 60 d, five by 90 d, three by 120 d and a further five by 150 d. Five men did not respond until up to 3 months after the second injection, anti-D appearing in three men at 7 months, and a t 8 and 9 months respectively in the other two men. The average time for the first anti-D to appear in this group was 127 d.

In Group I1 (Edinburgh series) the results using a standard primary dose of 200 ml red cell concentrate, involving five Rh genotypes, are shown in Table 11. There was no correlation in the very high response with probable Rh genotype involved, in that RIR, was as effective as R2R2.

Of the 28 volunteers, 24 (85.7%) produced anti-D after the primary dose, and a further two individuals responded after the first ‘booster’ injection to give a final response rate of 93%. The mean time for the first anti-D to appear was also 120 d, identical to Group I, but ranging over 2-9 months.

In Group I11 (Inverness series, Table II), the response to fresh washed cells showed the usual average (68%), but a poorer response to RIRl or RlR2 cells, compared with RZr or R2R2 was noted. The mean time to finding anti-D was 135 d, ranging over 2-15 months.

630 I. A . Cook et a1

TABLE 11. Anti-D response in Group I1 and Group I11

Group 11 (Edinburgh) Croup 111 (Inverness)

Probable

cefls Rkgenotype of No. of Anti-D Per cent No. of Anti-D Per cent

recipients responders responders recipients responders responders

- - Rlr 1 1 100 Ror 2 2 100

RiRi 9 8 89 7 5 71.0 R1R2 2 2 100 12 6 50.0

- 3 3 100.0 R2r RzR2 14 13 93 3 3 100.0

-

- - -

- -

28 26 93 25 17 68.0

Group I1 (Edinburgh): (1) Initial dose($ =200 ml cells (frozen recovered): (2) Boosting dose(s) =0.5-1.0 ml cells. Group 111 (Inverness): (1 ) Initial dose($ =20 ml fresh cell suspension equivalent to 9.0 ml concentrated red cells; (2) Boosting dose(s)=0.5 ml cells.

The anti-D response (pglml) in one donor to two ‘booster’ injections of frozen washed cells in the spring and summer of 1976 respectively is compared with fresh washed cells from a double and single injection in 1978 in Table 111. No appreciable difference in response is detectable.

Table IV shows both the pre-injection and ‘peak’ anti-D levels attained in six othcr responders to such stimuli. In these six men, and in a further six, frozen cells were similar in their antigenic effect to fresh cells.

In Group 11, after booster injections, seven produced anti-D levels over 50 pglml, four over 100 pg/ml and the remainder exceeded 10 pg/ml. It is noteworthy that these responders could be classified some 8 months after the primary immunization into low (< 10 pglml), medium (<50 pg/ml) or high (> 50 pg/ml) anti-D producers. In all cases in Group I1 in which R2R2 red cells were injected and in which anti-D was formed, anti-E, though admittedly in low titre, was also often formed. When R2R2 red cells were injected anti-G was also frequently detectable.

In Group 111, three responders showed a very weak anti-D response, 16, 13 and 15 months after the primary injection. The secondary injections had numbered 4,4 and 6 respectively, and were given between 6 and 9 months after the primary injection. Serology showed a very weak reaction by indirect antiglobulin techniques, and only when low ionic strength saline was used, a negative reaction to ficin treated cells using manual techniques and an autoanalyser assay of 0.02-0.04 pg/ml. Two further similar injections produced no increase in anti-D in two men, and the other increased only to 0.1 pg/ml.

In Group I , one donor made anti-Fyb transiently, which involved finding an RZr or R2Rz donor without the antigens S, Kell, Fyb and Jk“ for future injections. Another donor, who was found after 10 months to have a naturally occurring anti-M (i.e. of IgM type), unlikely to have

Frozen Red Cells in R h Immunization

TABLE 111. Example of anti-D response to ‘Booster’ injections of (a) frozen and (b) fresh (washed) cells: donor No. 65

63 1

(a) Frozen cells (b) Fresh cells

2.ii. 1976 19.ii. 1976 4.iii. 1976

25.iv.1976 30.iv. 1976

31 .viii.1976 14.ix.1976 27.x.1976 2.xii. 1976

Rrr 29.0 - 48 .O - 34.5 - 31.0

16.viii. 1977 23.viii.1977

7.ix.1977 5.x.1977

27.x.1977 24.xi. 1977 11.v.1978

28.viii. 1978 21.ix.1978 19.x.1978 2.xi.1978 7.xii. 1978

0.25 0.25 -

28.0 42.0 48.0 42.0 34.0 29.0 23.0

60.0 28.0 28.0 23.0

Primary dose 10.0 ml RlR2 15.v.1974.

TABLE IV. Anti-D (‘Peak’) response to booster injections of (a) frozen and (b) fresh cells

(a) Frozen cells (b) Fresh cells

Anti-D (pg/ml) Time to

Anti-D (pg /ml ) Time to

Probable (a) Pre- (b) ‘Peak’ ‘Peak’ Probable (a) Pre- (b) ‘Peak’ ‘Peak’ Donor NO. Date genotype injection level ( d ) Date genotype injection level ( d )

53 13.ix.1976 R2R2 490.0 640.0 8 16.iii.1977 Rzr 500.0 555.0 28 48 19.ii.1976 Rlr 34.0 56.0 13 9.ii.1978 R2r 39.0 72.0 21

28.viii.1978 R2r* 63.0 112.0 38 73 13.ix.1976 RzR2 19.0 40.0 14 3.x.1978 R2R2* 16.0 24.0 24 84 l.xi.1976 R2R2 40.0 83.0 23 3.ii.1978 R2R2 63.0 100.0 16

113 l.iii.1976 RlR2* 29.0 59.0 28 28.viii.1978 R;R2* 23.0 50.0 15

Volume of injection=0.25 rnl unless otherwise stated. * 0.5 ml.

been due to the injections, was given homozygous Ns cells thereafter even though the development of a warm immune anti-M would have been improbable. No antibodies outwith the Rh systems were produced in Groups I1 and 111.

DISCUSSION

The change to frozen from fresh Rh positive cells in 1972 was motivated by a number of developments. Firstly, the advances in our knowledge of the hepatitis B virus, along with the

632 1. A. Cook et al

screening of all blood donations since 1969, made staff more aware of the potential risks involved. Secondly, an outbreak of hepatitis B virus infection in an Edinburgh renal unit involving the deaths of eight patients and three staff, highlighted the inherent risks (Depart- ment of Health and Social Security, 1972; Bone er al, 1971; Robson, 1972). Also counterim- munoelectrophoresis (CIEP) was known to be insufficiently sensitive in detecting some of the weaker forms of this antigen. Admittedly, the ‘off-chance’ of any red cell donor incubating hepatitis at the time of donation, and despite previous negative tests for HBsAg, is remote, but from such a chance event the repercussions of injecting a group of healthy volunteers could not only be clinical but legal.

The practical issues involved in using red cell donors to immunized volunteers, now necessitate a special panel of such individuals, who should be accredited regularly both medically and by a series of biochemical and other blood tests, including RIA for hepatitis B virus (Cleghorn, 1979). These tests need to be repeated at intervals of not more than 1 month. From such a panel, based centrally, many Transfusion Centres can be supplied with red cells selected to avoid immunization by other non-Rh antigens.

In the Inverness series (Table I) 23 of 53 Rh negative men (43%) had developed anti-D. In the first series (Cook, 1971) using fresh washed cells, 23 of 34 men (68%) had responded, so the change to the use of frozen red cells resulted in a reduction of about one-third in the numbers expected to respond. At first the poor response in Group I seemed to be related to the predominant use of Rlr cells: 25 of the 53 (47%) volunteers had received such cells and only seven had responded (28%). This might be expected since the number of D antigen sites on Rlr cells is only 9900-14 600 compared with 14 000-16 600 and 15 800-33 300 in Rzr and RzRz cells respectively (Rochna & Hughes-Jones, 1965). However, this assumption seems to be negated by the excellent response to frozen cells, even of Rlr phenotype in Group 11. From their results, the Rh phenotype seems immaterial provided sufficient cells are given initially, and the greater the primary dose the better the final anti-D response. It is possible that since the reactivity of the D antigen is reduced by heating (Gunson et al, 1971) that the opposite effect of freezing might produce a similar loss of antigenicity. Against this argument is the fact that such cells give normal serological reactions with Rh antisera and the high response rate in Group 11.

Lee et al(1977) were unsuccessful in the primary immunization of 10 male volunteers using reconstituted frozen Ro (cDe) cells. These men received three to six injections of 1 ml packed red cells over a period of 8-25 months. The cells used had been stored for 18 months and showed only a 12% survival by a 51Cr red cell survival test, compared with 75% survival for recently frozen blood. It is therefore probable that the Rh antigenicity was poor in oivo though no loss of activity of the D antigen was found serologically.

The overall response rate in our series (43%) was well below the average response of60/70% in the literature (Gunson et al, 1970,1971,1976a, b; Jacobowicz et al , 1972; Gibson, 1973). In these references the response rate varied from 10-79% following a single stimulus to 35-100% after further stimuli. However, in the recent series by Eklund (1978) involving 66 men, there was only an 18% response to the primary dose and 44% after further injections using ACD/CPD blood up to 21 d old.

Eighteen of the 23 responders in Group I, and 24 of 28 responders in Group 11, had produced anti-D by 150 d, while in the first series of Gunson et al(1970), using washed red cells, nine of 11 men made anti-D between 37 and 130 d and in a second series (Gunson et al , 1971), using

Frozen Red Cells in R h Immunization 633

modified cells (treated with complement), eight out of nine men responded between 15 and 145 d. A further larger series (Gunson et al , 1976a, b) showed a similar time long before the development of serologically detectable anti-D. Thus our volunteers showed no appreciable difference in the time taken to respond compared with other reports.

Gunson et al(1974) have already shown that a series of repeated stimuli with Rh positive cells a t 2-4 week intervals gives a much higher anti-D level. It is the Inverness experience that there is a gradual fall in anti-D level in over 40% of the men being plasmapheresed weekly. As the Inverness red cell panel involves only ‘walking donors’, a recurrent programme of ‘booster’ injections is impracticable and, a frozen red cell bank might well be useful to maintain a maximum yield of anti-D in these donors.

The accuracy of anti-D assays in both serum and plasma can only be maintained by the use of the anti-D standards mentioned under Methods. Following an international collaborative study on the preparation of anti-D Immunoglobulin (68/419) a value of 150 iu anti-D per ampoule was assigned to the international standard (Bangham et al, 1978). Subsequent investigations, however, have shown that this value was in error and a revised value of 300 iu anti-D per ampoule has been substituted (Gunson & Thomas, 1979). Since each ampoule of 68/419 contains 60 pg anti-D, 1 pg is equivalent to 5 iu and not 2.5 as previously reported.

The use of large volumes of red cells (500 ml) was shown by Pollack et a1 (1971) to give an 82% response, and Cook & Rush (1974) in Australia have confirmed a similar high response following massive transfusion of Rh positive blood in open heart surgery, where 19 out of 20 Rh negative subjects (95%) developed anti-D. A similar response to a 200 ml primary dose of red cell concentrate (93%) in Group I1 (Table 11) by the Edinburgh male volunteers shows that the antigenity of different genotypes is not so relevant when a massive primary dose is used. The main advantages of this method are that fewer male volunteers will need to be placed ‘at risk’ and the search for suitable cell donors does not need to be restricted to those with an Rz antigenic complex.

The development of anti-Fyb in one donor was unfortunate, but a perfect ‘match’ was not always feasible. Our experience to date suggests that a minimum panel of 12-15 R2Rz red cell donors is needed to obtain a selection of red cells to avoid producing non-Rh antibodies such as anti-Kell, anti-Fya, anti-Jka or anti-S (Mollison, 1972). As one example of this difficult matching problem, another Centre has recently had to supply cells which were negative for the antigens S, Fy’ and Jk’, yet still compatible for other non Rh systems.

Gibson (1973) suggested that the response to Rh immunization might be genetically determined. HLA typing at A and B loci in over 110 of the Inverness volunteers (S. Myers, personal communication, 1978) has not revealed any major predominant HLA type in responders or non-responders. It is possible that typing at the D locus might be more informative.

ACKNOWLEDGMENTS

We wish to thank Dr W. D. Murray for undertaking the routine medical examinations of the Inverness donors yearly; Mr G. Grigor, F.I.M.L.S. (Edinburgh) for technical assistance with freezing/thawing of the frozen red cells in the Edinburgh series. Lastly our grateful thanks to Mrs R. Burns for typing the manuscript.

634 I . A . Cook et a1

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