British journal of Haernatology, 1981, 49, 371-381

Failure to Augment Primary Rh Immunization Using a Small Dose of ‘Passive’ IgG Anti-Rh

MARCELA CONTRERAS AND P. L. MOLLISON

North London Blood Transjiusion Centre, Edgware, Middlesex, and Department of Haematology, St Mary’s Hospital Medical School, London

(Received 20 February 1981; accepted for publication 27 April 1981)

SUMMARY. The immune response to the i.v. injection of 1 ml of D-positive (ccDEE, presumably cDE/cDE) red cells was studied in 12 D-negative (ccddee) subjects who received a simultaneous i.m. injection o f5 pg anti-D (the test group) and in a further 12 D-negative subjects who were not given anti-D (the control group). In all cases the red cells were labelled with Y r . Further injections of 1 ml of red cells were given at 7 and 12 months to subjects who had not made serologically detectable anti-D. In the test group the rate of clearance of the first injection of red cells was very variable, 99% of the cells being cleared in a period ranging from 3 to 20 d. Within 6-10 weeks of the first injection four subjects had produced anti-D; four more subjects produced anti-D after the second injection of red cells. In the control group Cr red cell survival following the first injection was normal in six cases and curtailed in the remaining six. Of the latter, four produced anti-D within 4-10 weeks ofthe first injection and two produced anti-D only after the second or third injection of red cells. Amongst the subjects who produced anti-D after the first injection of red cells antibody levels were lower in the test group than in the control group, indicating that the injection of 5 pg of anti-D with 1 ml red cells had not augmented the immune response and might have partially suppressed it.

Methods of augmenting the immune response are ofpractical as well as of theoretical interest. If the proportion of responders could be increased, or if more potent antibody could be produced, fewer Rh-negative donors would be needed, and if the immune response could be accelerated, fewer injections would be needed and effort would be saved.

Suggestive evidence was published by Pollack et a1 (1968) that when 5 ml of D-positive (ccDEE, presumably cDE/cDE) blood were injected i.v. into D-negative volunteers simultaneously with 10 pg anti-D i.m. the proportion of responders was increased; there was also evidence, discussed below, that the immune response had been ‘widened’. The present experiments were undertaken to see if augmentation of primary Rh immunization could be confirmed using a similar ratio of antibody to antigen.

Correspondence: Professor P. L. Mollison, Department of Haematology, St Mary’s Hospital Medical School, Paddington, London W2 1 PG.

0007-1048/81 / I 100-0371$02.00 0 1981 Blackwell Scientific Publications

37 1

372 Marcela Contreras and P. L. Mollison

SUBJECTS, MATERIALS AND METHODS

Su6jects. Twenty-four D-negative (ccddee) volunteers were recruited from amongst the regular donors of the North London Blood Transfusion Centre. Meetings were held at which it was explained to the volunteers that there were three known hazards of taking part. First, that of developing hepatitis, although it was thought that, because of the very thorough testing of the donor (see below), this risk was very small. Second, that of making anti-Rh and, just possibly, other alloantibodies; the chance of making anti-Rh was high and this might create difficulties in finding suitable blood for them in emergency, should they require blood transfusion. Finally, that of receiving 5'Cr, although it was explained that the doses to be used had been approved by the Medical Research Council's Advisory Panel on the Use of Radioactive Isotopes and subsequently by a corrcsponding panel set up by the Department of Health and Social Security.

The allotment of donors to test and control groups was carried out as follows: donors were arranged in order of age (in case immune response might be influenced by age) and then for each successive pair a coin was spun to decide which of the pair should be in the test group and which in the control.

At the time of recruitment, blood samples were taken from all volunteers for full red cell grouping, HLA-typing and screening for atypical red cell alloantibodies.

Donor. A single donor, K.B., a woman born in 1911, was used throughout. She was selected for two reasons, first because she was group 0, ccDEE, K-, k + , Fy(a - b +) and Jk(a - b +); her other groups were M -, N +, S -, s +, P, -, Lu(a- b +), Le(a - b +); the second because during the 10 months before her blood was first used in the current experiments (on 13 February 1979) her serum had been tested on 20 occasions and on each occasion had not only been HBsAg-negative by radioimmunoassay, but also had had serum liver enzyme levels (SGOT, SGPT and alkaline phosphatase) within the normal range. For this latter reason it was thought to be very unlikely that she was a carrier of 'non-A, non-B' hepatitis virus.

By the time that blood was taken from her for the second injection to the volunteers (on 18 September 1979), negative results with the tests mentioned above had been obtained on a further nine occasions and by the time that she gave blood for a third injection of red cells to a few of the volunteers (on 19 February 1980) negative results had been recorded on a further four occasions.

On each occasion on which red cells from K.B. were used for injection into the volunteers blood was mixed with ACD-A in the usual proportions and kept at 4°C for 24-36 h. The red cells were washed at least once and a saline suspension was then prepared for injection. The PCV of the suspension was determined so that the amount of suspension corresponding to exactly 1 ml of red cells could be calculated.

Estimation of C r red cell survival. V r was added to packed red cells from ACD blood from donor K.B. in sufficient amounts to label each millilitre with approximately 40 pCi "Cr. After incubating the mixture at 37°C for 15 min the red cells were washed once and a suspension was then prepared in saline. The PCV of the suspension was determined so that the amount of suspension corresponding exactly to 1 ml red cells could be calculated. After injecting the equivalent of 1 ml packed red cells i.v. into one arm, a sample was taken between

Failure to Augment Primary Rh Immunization 373 5 and 10 min after injection from the other arm. Thereafter samples were taken at various intervals as described below in 'Plan of Experiment'. On each occasion on which blood samples were taken following an injection of "Cr-labelled red cells, samples of approximately 4 ml blood were taken into EDTA tubes in duplicate. 2 ml of blood was pipetted from each of the duplicate tubes into a vial containing saponin, for counting, and the PCV on the remainder of the sample was then determined in a Coulter Counter. All samples were counted together in batches at various intervals of time (to monitor clearance) and were recounted at the end of each Cr survival study. Cr results were expressed as counts per ml red cells. No corrections were made for Cr elution because following the first injection of red cells given to the test group the rate of red cell destruction was very rapid so that corrections for Cr elution would have made little difference and because, following all the other injections of red cells, the object of the measurements with 51Cr was simply to distinguish between normal and diminished survival.

Anti-Rh immunoglobulin. A routine batch of anti-D immunoglobulin, stated to contain 50 pg anti-D in 2 ml, prepared by the Plasma Fractionation Laboratory (Elstree) of the National Blood Transfusion Service and intended for the prophylaxis of Rh immunization following abortion, was used. As one of the objects of the work was to compare results with those previously published by Pollack et a l (1968) , a sample of the material was sent to Dr Pollack who assayed it as containing 26 pglml.

The method used in preparing a dilute solution of anti-D for intramuscular injection was as follows:

A sterile rubber-capped vial was weighed; approximately 1 6 ml of sterile saline was injected from a syringe into the vial which was then re-weighed to determine the volume of saline precisely. 2 ml of the anti-D immunoglobulin preparation was added from a syringe and the vial was weighed once more. The protein concentration of the immunoglobulin had been estimated to be 10% and the specific gravity was assumed to be 1.027. Using this figure, the exact volume of anti-D immunoglobulin added could be determined and thus the amount of anti-D in 1 ml of the final solution could be calculated. Taking the anti-D content of the original vial as 26 pg/ml the volume of diluted immunoglobulin solution containing 5 pg could be calculated and this was the amount injected into each volunteer in the test group.

Plan of Experiment Test group. O n day 0 subjects received 1 ml of 5'Cr-labelled Rh-positive red cells i.v. and

immediately afterwards were given an i.m. injection of 5 pg of anti-D into the deltoid muscle. In most cases, blood samples were taken into EDTA tubes (for counting) at the following times: 5 min, 1 d, 2 d, 3 d, 7 d, 14 d and thereafter weekly until Cr survival was less than 5%. In cases where subjects had cleared more than 95% ofcells within a period less than 2 weeks, no further samples for counting were taken. However, samples were taken into plain tubes (for serology) from all subjects at 2 weeks, 4 weeks, 6 weeks, 8 weeks and 10 weeks and examined for the presence of anti-Rh. Subjects who had not made anti-D at 10 weeks were re-tested at 20 weeks and 30 weeks. Subjects who had made anti-D by 10 weeks were given a booster injection of unlabelled red cells from donor K.B. at 18 weeks and re-tested 1 week later.

374

Thirty-one weeks after the first injection, a second injection of 1 ml of SICr-labelled red cells from donor K.B. was given to the eight subjects in whose plasma anti-D could not be detected. Following this injection samples were taken into EDTA tubes and into plain tubes at 1 week, 2 weeks, 4 weeks and into plain tubes only at 9 wecks and 19 weeks.

Twenty-one weeks after the second injection (i.e. 52 weeks after the first injection) a third injection of S'Cr-labelled red cells was given to the six subjects who, following the first or second injection, had either not made detectable anti-D (1, 9, 10, 12) or had made anti-D detectable only with enzyme-treated cells (4, 7). Following the third injection, samples were taken into EDTA tubes and plain tubes at 1 week, 2 weeks, 4 weeks and a final sample was taken into a plain tube at 14 weeks. An injection ofunlabelled red cells was given to subject 1 1 and a sample was taken 1 week later.

Controlgroup. O n day 0, subjects received 1 ml of "Cr-labelled red cells from donor K.B. Samples were taken into EDTA tubes and plain tubes immediately before injection and at 5 min, 1 week and 2 weeks and thereafter at the same intervals as in the test group. Similarly, subjects who had made anti-D by 10 weeks were given a booster injection of unlabelled red cells at 18 weeks and re-tested 1 week later.

At 31 weeks, as in the test group, a second injection of 1 ml of "Cr-labelled red cells was given to those subjects who had not made serologically-detectable anti-D and samples wcrc taken at the same intervals as were used for the test group.

At 21 weeks after the second injection of red cells two subjects (16 and 21) received a third injection of S'Cr-labelled red cells. In subject 16 the injection was given because, despitc shortened survival following the first and second injections, anti-D was detectable only with enzyme-treated red cells; in subject 21 a third injection was given because of difficulty in interpreting results following the second injection of red cells. Following these injections samples were taken at the same intervals as in the test group.

Serological tests. Serum samples were screened for the presence of ailoantibodies using thc following tests: a two-stage enzyme test using papain, at 37°C; indirect antiglobulin test, firstly, using red cells in saline, secondly, using red cells in LISS, and thirdly, using papain-treated red cells. In addition, samples were tested for the agglutination of red cells in saline at 4°C and at room temperature. The sera were tested against a panel of six cells covering all the main red cell antigens; K.B. cells were included in the panel whenever possible. Samples were also tested in the AutoAnalyzer against CCDee and ccDEE cells using the manifold described by Marsh et ul (1 968) and the bromelin-methyl-cellulose technique; quantitation was performed by the method ofJudd &Jenkins (1970). The reference standard was the British standard for the quantitation of anti-D (National Institute of Biological Standards). The direct antiglobulin test was performed on the EDTA samples on the day of sampling, with very few exceptions.

The serological tests carried out after the first injection of red cells were read 'blindly'. The samples were coded with random numbers by one investigator and tested by another. Samples taken after the second and third injections were not coded and not read blindly.

Every sample in which anti-D was found was treated with dithiothreitol (DTT) ,and subsequently tested manually and in the AutoAnalyzer.

M a r c e h Contrerus and P. L. Mollison

100

50

c z Y u K

P Y

1

0 .5

0.2

Failure to Augment Primary Rh Immunization 375

TEST CROUP

0 1 14 21 28

CONTROL GROUP

L -1 0-2 0 7 14 21 28 84

DAYS

FIG 1. 5'Cr red cell survival in 12 D-negative subjects (test group) injected on day 0 with 1 ml ccDEE red cells i.v. and 5 pg anti-D i.m., and in 12 D-negative subjects (control group) receiving 1 ml ccDEE red cells i.v. without anti-D. Results not corrected for elution of Cr. * Anti-D detected in plasma by day 70; for further details see Table 1.

TABLE

I. C

r su

rviv

al a

nd t

ime

of fi

rst a

ppea

ranc

e of

ant

i-D a

fter

succ

essi

ve i.

v. in

ject

ions

of

1 m

l of

Rh-

posi

tive

(ccD

EE

) red

cel

ls in

12

Rh-

nega

tive

subj

ects

(tes

t gro

up) w

ho r

ecei

ved

an i.

m. i

njec

tion

of 5

/.ig

anti-

D a

t the

sam

e tim

e as

the

firs

t inj

ectio

n of

red

cel

ls a

nd in

12

subj

ects

(con

trol

gro

up)

who

rec

eive

d on

ly r

ed c

ells

. For

furt

her

deta

ils s

ee t

ext.

CJ 4

OI

Cr s

urvi

val

(Yo)

Firs

t in

ject

ion

Seco

nd in

ject

ion

Thir

d in

ject

ion

Subj

ect

Resp

onde

r (R

) D

ay

Day

D

ay

Day

D

ay

Day

D

ay

Day

s D

ay

Day

D

ay

Day

D

ay

Day

D

ay

Day

D

ay

or n

on-

1 2

3 7

10

14

21

42-7

0 0

7 14

28

13

3 0

7 14

28

re

spon

der(

N)

Tes

t gr

oup

1 2 3 4 5 6 7 8 9 10

li

12

Nor

mal

mea

n C

ontr

ol g

roup

13

14

15

16

17

18

19

20

21

22

23

-.

34

71.5

22

.6

9-1

0 62

-7

13.1

...

0.

5 94

-4

73-7

49

.1

10.3

...

0.

3 *

...

78.2

...

17

.2

7-4

0.9

90-2

67

.5

46.1

7.

8 ...

0

* ..

....

83.

6 53

.2

...

16.5

0-

8 ..

....

33

.0

2.1

....

..

34.3

5.

7 1.

4 ...

10

2-

9 *

99.7

83

.3

65.4

25

.5

...

1.1

99-3

87

.4

71.6

26

.5

...

0.9

103.

2 91

.1

80.9

40

.3

...

6.3

0.3

Day

D

ay

Day

D

ay

Day

D

ay

Day

7

14

28

42

56

70

84

83-5

73

.5

53-5

39

.0

81.2

68

.3

5.9

O*

84-2

74

.0

0.8*

84

-9

75.5

55

.6

39.8

31

.4

22.4

13

.8

83-9

74

.5

0-9

81-9

75

.6

53.3

41

.4

31.4

...

16

.1

76.1

70

-0

11*9

* 0

80.9

69

.6

40.7

25

.5

17.5

9.

7 6.

7 78

.9

68.2

46

.1

31.8

24

.4

15.4

10

.1

81.1

74

.1

51.3

38

.2

25.9

16

.8

7.9

83-8

73

-0

1.3

83.9

76

-0

1.0

* N

.4

69.8

50

.5

39.2

28

-0

19-0

13.

1

75.9

67

.2

47

79.1

67-7

46.0

80.6

70

-8

54.0

*

82-9

72

.9

4.2

* 64

-9

3.5

39.3

14

.5

7.4

* <

3

81.2

67

.2

48-1

80

.0

64.8

48

.2

81.4

73

.1

52.1

79

.0

63.7

48

.3

79.2

13

*1*

4 81

.3

69.6

48

.5

83.0

...

54.

4

Day

D

ay

Day

D

ay

Day

D

ay

Day

D

ay

Day

0

7 14

28

13

3 0

7 14

28

85.8

74

.2

55.1

52

.8

16.1

2.

5 *

64.2

38

29

83

-3

73.7

56

.2

79.9

72

.1

53.4

82

.0

71.1

53

.8

69.2

56

.9

36.3

*

804

73.1

55.8

....

....

. 84.0 7

1.4

55.2

N

R

R

R

R

R

R

R

N

N

R

N

R

R

N

R

N

R

N

N

N

R

R

N

Failure to Augment Primary Rh lmmunization 377

RESULTS

Table I summarizes both the 5'Cr red cell survival results and the time at which anti-D was first detected.

Test Group As Fig 1 shows, the rate of clearance following the first injection was very variable

although in all subjects C r survival was less than 1% at 21 d. Four subjects (3, 5, 6 and 8) developed serologically-detectable anti-D 6-1 0 weeks after the first injection of red cells. The remaining eight subjects received a second injection of red cells; in five subjects (1,2,9,10 and 12) survival was within normal limits although subject 2 developed anti-D 19 weeks after the injection. Subjects 1, 9, 10 and 12 received a third injection of red cells 1 year after the first injection; in these four subjects survival was again normal and these subjects were classified as non-responders.

Among the three subjects (4,7 and 11) in whom red cell survival was diminished following the second injection of red cells, anti-D was present 2 weeks after the injection in one case (1 1)

TABLE 11. Earliest time of appearance of anti-D in test and control groups, maximum concentration of anti-D recorded following the injection after which it was first detected and development of Rh

specificities other than anti-r)

Anti-D first detected Quantitation .f anti-D

Afier which After which Specificity of injection of Weeks after injection .f Weeks after Concentration other Rh antibodies

Subject red cells? injection red cells? injection (pglrnl) detected

Test group 3 5 6 8 2 4 7

11

First First First First Second Second Second Second

10* 6 8* 8*

19* 19* 19* 2*

First First First First Second Thirdt Thirdt Thirdt

10 6

10 10 19 4 1 1

0.01 0.1 0.04 0.01 1 .o 3.0 0.1 3.0

ES ES - E E+G G E E + G

Control group 13 First 6 First 10 2.0 E 14 First 4 First 8 2.5 E 18 First 4 First 6 3.8 E 23 First 10* First 18 0.01 G

22 Second 19 Second 19 0.0 1 E + G 16 Second 19* Thirdt 14 0.0 1 E

* Initially detectable only with enzyme-treated red cells; in the other cases anti-D was also detectable

t Concentration too low for quantitation after second injection. $ Transient.

by the indirect antiglobulin test.

378 Marcela Contreras and P. L. Mollison

and 19 weeks after the injection in the other two (4,7). In these latter two cases, following a third injection of "Cr-labelled red cells, survival was short (Table I) and anti-D increased in strength (Table 11).

Control Group Fig 2 shows the survival results following the first injection: survival was clearly normal in

five subjects (15, 17, 20, 21 and 24) and was normal or slightly subnormal in one further subject (19). In all of these six subjects survival following a subsequent injection given 7 months later (12 months later in case 21) was also normal. It was concluded that all these six subjects were non-responders.

In the other six subjects, following the first injection, all the red cells werc eliminated within 4-6 weeks; in four subjects (13,14,18 and 23) anti-D could be detected between 4 and 10 weeks after the first injection of red cells. In the remaining two subjects (16 and 22) red cell survival was subnormal after the first and second injections of red cells but anti-D was not detectable until 19 weeks after the second injection. In case 16 the antibody reacted extremely weakly and a third injection ofred cells was given; 14 weeks after this last injection anti-D was detected by the indirect antiglobulin test and the concentration was now estimated to be 0.01 Pglml*

Antibody Characteristics in Test and Control Groups As Table I1 shows, following the first injection of red cells, in the four subjects in the test

group who made anti-D, antibody concentrations were all between 0.01 and 0.1 pg/rnl, whereas in the four subjects in the control group antibody concentration was 2 pg/ml or more in three cases. The difference in the number of cases with an antibody concentration of 1 pg or more is suggestive but not statistically significant (P=0.07) of a partial suppression of the immune response by passive antibody. Following treatment with DTT there was no significant reduction in titre or in the estimate of antibody concentration in any case, indicating that IgM anti-D was absent or present only in low concentrations.

As Table I1 shows, anti-E accompanied anti-D in the serum of six of the eight test subjects and five of the six control subjects. Anti-G was present without anti-E in one test subject and one control subject and both anti-E and anti-G were found in two test and one control subjects.

N o red cell alloantibodies outside thc Rh system were detected in any of the 24 subjccts. The direct antiglobulin test was negative on all samples. There was no obvious relationship between HLA group and responsiveness to Rh.

DISCUSSION

There is some uncertainty about the criteria to be used in deciding that immunization has been augmented. The criteria proposed by Pollack et a1 (1968) were: (1) an increased probability of responding to the stimulus; (2) the formation, in those subjects who responded, of anti-D of more than one Ig class; and (3) a 'wider' specificity ofantibody. Judged by these criteria, in the series of Pollack et al, augmentation of the immune response was observed when 5 ml of ccDEE blood was injected i.v. together with 10 p g anti-D given i.m. Of 11 subjects, eight

Failure to Augment Primary Rh Immunization 379

produced anti-D at 3 months whereas of the subjects who received the same volume of red cells without passively-injected anti-D only one out of six produced antibody. Furthermore, amongst subjects receiving 5 ml of blood with 10 pg of anti-D, all but one of the eight responders produced IgM as well as IgG anti-D, whereas of four subjects out of 13 who made anti-D after having been given 5 ml of blood with only 1 pg of anti-D all produced only IgG antibody. Again, amongst the group receiving 10 pg of antibody, four made alloantibodies within the Rh system other than anti-D, whereas in the group receiving 1 pg of antibody none made any antibody except anti-D.

In the series of Pollack et a1 (1968) there was no evidence that antibody was produced sooner or in higher concentration in the group in which immunization appeared to be augmented. Nevertheless, these two criteria might be regarded as giving the most clear-cut indication of augmentation of the response. In any case, judged by any of the five criteria mentioned so far, augmentation was not observed in the test group in the present series. On the contrary, the figures shown in Table I1 for anti-D concentration after the first injection of red cells suggest the possibility that there may have been some suppression of the immune response in the subjects who received 5 pg of anti-D. One subject (2) in the treated group provided evidence of a different kind suggesting suppression of primary immunization; that is, although he was ultimately shown to be a responder, Rh-positive red cells given 7 months after the first injection survived normally (see Tables I and 111). As has been stressed elsewhere (Mollison et al, 1970), in responders it is almost invariable to find that when a second injection of Rh-positive cells is given about 6 months after the first injection, evidence of accelerated destruction ofthe red cells can be observed from the time ofthe injection onwards, suggesting that in fact small quantities of anti-D have been made following the first injection. Accordingly, if normal survival is observed following a second injection of red cells in a subject who ultimately makes anti-D there is a presumption that primary immunization did not occur following the first injection. In the present case it is possibly relevant that the subject concerned (2) was one of those who showed the most rapid clearance of red cells following the first injection (given together with anti-D).

Since it has been shown that estimates of anti-D concentration made by AutoAnalyzer methods are appreciably affected by variations in equilibrium constants (Gunson et al, 1976), it should be pointed out that the figures given in Table I1 assume that equilibrium constants did not vary widely in the samples tested. Moreover, it should be mentioned that although the British Working Standard, which is defined in international units, was used, results have been expressed as pg anti-D assuming that the relationship between i.u. and pg anti-D) (5 i.u. = 1 pg anti-D) was the same for this standard as for the international reference preparation. Reasons for preferring to express results in terms of pg per ml rather than i.u. per ml have been discussed elsewhere (Mollison, 1979, p. 461). Although the absolute values for the concentrations of anti-D given in Table I1 are subject to the sources of error just discussed, the inference that there was a difference in anti-D concentration between test and control groups seems justified.

The number ofresponders in the test and control groups (eight out of 12 versus six out of 12) is clearly not significantly different. Since both test and control subjects formed predominantly IgG anti-D and showed the same tendency to make antibodies of specificities other than anti-D within the Rh system, it is clear that the response was no ‘wider’ in one

380 Marcela Contreras and P. L. Mollison

group than in the other, using these terms in the sense in which they were used by Pollack t?t a1 (1968).

The very variable rate of destruction observed in the 12 subjects given 5 p g of anti-D with 1 ml of ccDEE red cells is surprising, although doubtless this is partly accounted for by the variable rate of uptake of immunoglobulin following intramuscular injection (Smith et al, 1972; Morel1 et al, 1980). A very similar variability in the rate of destruction of Rh-positive red cells by passively-administered anti-D was noted by Bartsch (1972) following the injection of 10 ml of Rh-positive red cells i.v. given together with 260 pg anti-D i.m.

It might be expected that there would be a relationship between the rate of clearance of the Rh-positive red cells by anti-D and the time of appearance of actively-produced anti-D. This question is analysed in Table 111, which indicates that of the four subjects ofthe test group who produced anti-D after the first injection of red cells three had shown clearance of 99% of the cells within 8-13 d of injection, the remaining subjects having shown somewhat slower clearance. None of the three subjects who showed the most rapid clearance of cells produced anti-D within the following 6 months.

There has been one other study in which, in an attempt to augment primary R h immunization, 5 pg of anti-D was injected (i.m.) with 1 ml of ccDEE red cells (i.v.). In this study there was one difference which may have been important, namely that the red cells were coated with complement by exposure to serum at low-ionic-strength before being injected. Two out of two subjects formed anti-D within the following 7 weeks but the authors concluded that immunization had not been significantly augmented (Gunson e l al , 1971).

The rate of clearance of the red cells was not estimated in the study just referred to, nor was it estimated in the study by Pollack et a1 (1968). There may bc a relation between the rate of clearance of red cells by antibody and the augmentation of primary immunization. It would

TABLE 111. Test group: relation between rate of clearance of first injection of red cells by passive-administered antibody and sub-

sequent development of immunization

6 months 12 months 99%

( d ) subjects Anti-D survival Anti-D survival Responders clearance No. .f Cr Cr

3-5 1 0 Normal 2 0 Normal

8-13 3 + 2 0 Reduced

14-20 1 + 1 0 Reduced 2 0 Normal

+ 1 0 Normal 0

3 + 2

1 + 1 0 Normal 0

12 8

Failure to Augment Primary Rh Immunization 38 1

therefore be very interesting to discover the effect on red cell clearance and on the immune response of giving a smaller amount of anti-D in relation to red cells than that used in the present study, using the same batch of anti-D and the same red cell donor. A study of this kind is in progress.

ACKNOWLEDGMENTS

We should like to thank Dr Jane Caseley of the West End Donor Centre for help in recruiting the volunteers, the volunteers themselves for unstinting co-operation and Dr M. M. Fisher of the Oxford Regional Transfusion Centre for collecting blood on many occasions from the specially-accredited and most helpful donor, K.B.

REFERENCES

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