Journal of Clinical InvestigationVol. 46, No. 5, 1967

Relationship between Rho(D) Zygosity and Red CellRh0 (D) Antigen Content in Family Members *

S. P. MASOUREDIS,t MARYEDITH DuPUY, ANDMARGARETELLIOT(From the Cancer Research Institute and the Department of Medicine, University of

California Medical Center, San Francisco, Calif.)

Summary. The red cells of 63 members of 11 families were tested with125I-labeled anti-Rh0(D). Families with a history of hemolytic disease ofthe newborn due to fetomaternal Rh incompatibility were selected for study.In such families it was possible to determine the antibody binding to theRho(D) heterozygous red cells of the children and to compare within eachfamily this value with the antibody bound to the father's Rh0(D)-positivered cells and the mother's Rh0(D)-negative red cells. The fathers in all thefamilies studied could be assigned to two classes on the basis of the quantityof antibody bound to their red cells. One group bound about the same quan-tity of antibody to their cells as did their children, indicating that they wereheterozygous for the Rho(D) antigen. The other bound about twice as muchantibody to their cells as did their children, indicating that they were homozy-gous for the antigen. The Rh genotype of the father in all 11 families couldbe ascertained by using the children in each family as a reference point.The members of two families showed a poor correspondence between antibodybinding and zygosity. In one family an Rh heterozygous child (R1r) took up85% of the antibody bound to the father's homozygous cells (R'R'), and inthe other family an Rh heterozygous child (R1r) took up 20% more antibodythan did the cells of her father, which were of the same Rh phenotype (Rh,)and zygosity.

The quantity of antibody bound to the red cells of unrelated Rho(D) ho-mozygous individuals of the same Rh phenotype (Rh1) showed an almostsixfold variation. A consequence of this observation was that the cells ofRho(D) heterozygous children of high antibody uptake fathers took up moreantibody than did the cells of low antibody uptake Rho(D) homozygousfathers. The gene dosage effect for the Rho(D) antigen demonstrable withina family does not appear to apply when unrelated individuals are tested, eventhough they may be of the same Rh phenotype.

Introduction

Many serological and isotopic studies (1-5)have shown that a complex relationship exists be-tween the red cell Rho(D) antigen content andthe donor's genotype. Conventional serological

* Submitted for publication July 21, 1966; acceptedJanuary 3, 1967.

This study was supported by U. S. Public Health Serv-ice research grants HE-05071 from the National HeartInstitute and CA-04990 from the National Cancer In-stitute and in part by U. S. Public Health Service grant

techniques are unable to discriminate betweenRho(D) heterozygous and homozygous red cellsexcept for a few unusual red cell types such as the(D - -) cell (6, 7).

In a previous study (3) Rh, (D)-positive redcells obtained from a blood bank donor population

FR-00122 for consulting services, San Francisco MedicalCenter, University of California.

t Address requests for reprints to Dr. S. P. Masouredis,University of California San Francisco Medical Center,San Francisco, Calif. 94122.

681

MASOUREDIS, DUPUY, ANDELLIOT

were tested with 131I-labeled anti-Rh0 (D). Thefrequency distribution of the amount of anti-Rh0 (D) bound to the red cells derived from thispopulation was analyzed. Only those Rho(D)-positive red cell types that did not contain therh'(C) antigenic determinant segregated into twopopulations. There was a 1.6-fold difference be-tween the means of the two populations, suggestingthat the two red cell populations represented theRh0o(D) heterozygous and homozygous states.Red cells containing the rh'(C) antigenic deter-minant did not show the expected bimodal distri-bution.

The improvement in techniques that has oc-curred since the previous report and the identifi-cation of some of the factors (8, 9) that affect thereaction between the red cell and the anti-Rho(D)encouraged us to re-examine the relationship be-tween genotype and red cell Rh0o(D) antigencontent.

The present study differs from the previous re-port (3) in a number of important respects. Thered cells examined in this study were obtainedfrom the members of families in which there was ahistory of hemolytic disease of the newborn dueto fetomaternal Rho(D) incompatibility. In suchfamilies the Rho(D) heterozygous state was rep-resented by the Rho(D)-positive red cells of thechildren. Wechose these families to ascertain theheterozygous state and not to study the factorsinvolved in producing erythroblastosis.

Each sample of red cells was tested with dif-ferent concentrations of antibody so that the opti-mal proportion of cells to antibody was experi-mentally determined. Previously (3) the cellswere tested at only one antibody concentration,which, for some cells, may have been insufficientto establish equilibrium and, for other cells, mayhave been in sufficient antibody excess to produceinhibition of antibody binding (10). The anti-body bound to the red cells is presented in terms ofred cell numbers rather than, as previously, interms of red cell volume determined by centrifu-gation. Presentation of the results in this formavoids introducing into the data any differencesthat may be due to variations in the red cell vol-umes among the different donors. Finally, the125I-labeled anti-Rho(D) used in the presentstudy was derived from a gamma G fraction ob-tained by DEAEchromatography rather than from

a crude gammaG fraction contaminated with otherplasma proteins (11).

The Rh0 (D) red cell antigen content of 63members of 11 families was examined with 125I-la-beled antibody. The relationship between theRho(D) content of the children's cells and thatof the father as well as that among the childrenwas examined.

Methods

Family data. Eleven families were studied. The redcells of 41 children, 11 fathers, and 11 mothers weretested. Of the 63 red cells tested in these families, 49were Rho(D)-positive and 14 were Rho(D)-negative.All the 11 fathers were Rho(D) -positive, all themothers Rho(D)-negative, and, of the 41 children, 3were Rho(D)-negative. The probable Rh genotypes 1of the 49 (Rho (D)-positive red cells included: 29 R'r(CDe/cde), 6 R1R1 (CDe/CDe), 6 Ror (cDe/cde), 3R2r (cDE/cde), 2 R'r' (CDe/Cde), 1 R°R° (cDe/cDe),1 R1R' (CDe/cDE), and 1 R2R' (cDE/cDE). Theprobable Rh genotypes of the 11 fathers included abovewere 6 R'R1, 2 R'r, and 1 each of R°R°, R1R2, and R2R2.All the families were Caucasian except family Br, whichwas Negro.

Fourteen of the 41 children were treated by exchangetransfusion one or more times. In 6 of the 11 familiesthere were fetal or neonatal deaths due to Rh incom-patibility. The children in 2 families were only mildlyaffected and did not require exchange transfusions.

The family data are summarized in Table I. Theprobable Rh genotype of the members of these familieswas assigned on the basis of the family data (unequivo-cally in those families with Rho(D) -negative children).

The isotopic methods described previously (3, 13) wereused except for the procedures described below.

Fraction of the anti-Rho (D) serum. REZ serum wasobtained from a 34-year-old group 0, rr Caucasianhousewife who delivered an 0, R1r (CDe/cde) infantwith hemolytic disease in 1954. She had a Fallopian tubeligation and subsequently was reimmunized on four oc-casions between 1961 and 1964 by intravenous adminis-tration of 2 ml of whole blood from two different 0, R°(cDe) donors.

Three different globulin preparations obtained fromher serum were used for these studies. Globulin prepa-ration A-79 was obtained by ethodin (Rivanol) frac-tionation (14) and preparations A-87 and A-88 by DEAEchromatography using the technique of Levy and Sober(15). The globulin preparations were trace labeled with"I using iodine monochloride (16).

1The results are presented as probable Rh genotypes(in italics) rather than phenotypes. The genotype as-signment is based on the family data. As will be evidentfrom the results, the data obtained in this study do notfavor or support any of the three Rh nomenclatures incurrent use (6, 7, 12).

682

RHO(D) RED CELL ANTIGEN IN FAMILY STUDIES

TABLE I

Family data

Severity oferythro-

Family Probable blastosis Prepara-Family member Age Rh genotype fetalis* tion used

years

Mo Fa 25Mo 25

D-lt 6S-2t 4

Br$ Fa 32Mo 30

S-1 11D-2 8S-3 6S-4 5D-5 3D-6 2

Pe Fa 36Mo 35

D-1 12D-2 9S-3 7D-4 6S-5 4D-6 2

Sh Fa 38Mo 32

S-1 9S-2 8D-3 7D4 6S-5 2

Di Fa 34Mo 34

D-1 11D-2 7D-3 5S-4 4D-5 2

Go Fa 34Mo 29

S-1 7S-2 3D-3 1

Th Fa 41Mo 34

S-1 6D-2 5S-3 4S-4 2S-5 1

Is Fa 26Mo 24

S-1 7S-2 3D-3 2

Co Fa 30Mo 28

S-1 7S-2 6S-3 3S-411 0.1

Ma Fa 35Mo 34

D-1 15

Re Fa 32Mo 27

D-1 9D-2 9

0. R'R'0, rr0, Rlr0, Rlr

A, R°Ro0, rr0, ROr0, ROr0, R~rA, R~rA, ROrA, R~r

0, RIRIB, rrB, Rlr0, Rlr0, RlrB, Rlr0, RlrB, Rir

0. RIR'0, rr0, Rlr0, R'r0, Rir0, R'r0, Rlr

A, R'rA, rrA, R'rA, rrA, RlrA, RirA, R'r

0, RiRiA, rrA, R'rA, RirA, R'r

A, Rlr0, rrA, R'rA, RlrA, rrA, RirA, rr

0, RIR20, rr0, Rlr0, R2r0, R2r

0. RiRIA, rr0, Rlr0, Rlr0. Rir

Rho(D) +

A, R2R2A, rr0, R'r

0. RIRIA, r7rA, Rlr'A, Rlr'

NS-EX * 1

NNS-EXi 1S-EX *3S-EX -1

M

NNNS-EX -1

S-EX 1

S-EX * 1

NNM

M

S-EX * 1

NNM

S-EX * 1S-EX -1

NS-EX *3S-EX *2

NNNM

N

NNM

NNNS-EX *4

N

NM-EX* 1

* Severity of erythroblastosis fetalis indicated by the following code:N = normal; M = mild, no exchange; S = severe; EX * 1 = exchangedonce, and so forth.

. D daughter; S son.

I Negro family; all others Caucasian.i Acid eluates; all others were heat eluates.

I Not tested.

Radioautography of the immunodiffusion and immuno-electrophoresis patterns of ethodin globulin preparationA-79 showed that gamma A, gamma M, transferrin, andalbumin, in addition to gamma G, were present. All the'I in the DEAEglobulin preparations, A-87 and A-88,

was associated with the gamma G protein. The proper-

ties of the 'I-labeled anti-D-containing globulin frac-tions are shown in Table II.

Preparation of eluates containing .5I-labeled-Rho (D).The heat eluate derived from preparation A-79 was ob-tained by sensitizing a pool of lyophilized D-positive redcell stromata with the 'I-labeled globulin fraction at370 C for 90 minutes. The sensitized stromata were

washed four times and eluted by using the techniquesdescribed previously (3, 13). A total of 80 g of stro-mata derived from a pool consisting of 34 U of Rho(D)-positive red cells was used, and 0.31 mg globulin nitrogenper g of lyophilized stromata was used for sensitization.

The eluates obtained from the gamma G--15I prepara-

tions A-87 and A-88 were derived from sensitized intactred cells. A pool composed of 16 U of R' (CDe) and R'(cDE) red cells was used for A-87, and a 9-U pool was

used for A-88. The red cells were sensitized with thegamma G-S'I at 370 C for 90 minutes by using 2.78 mg

A-87 globulin nitrogen and 2.81 mg A-88 globulin ni-trogen per 200 ml of packed red cells. The sensitizedcells were washed and converted to stromata by freezingand thawing three times after the addition of 1 vol ofbuffered isotonic saline, pH 6.5, containing 2% bovineserum albumin (BSA). The stromata were recoveredby centrifugation in the Spinco model L at 73,000 maxi-mal g for 15 minutes and washed by centrifugation fourtimes with buffered isotonic saline, pH 6.5, containing0.1%o BSA.

The washed sensitized stromata derived from prepara-

tion A-87 were divided into two equal portions. One was

eluted with heat, 560 C for 20 minutes, using pH 5.8,0.15 M phosphate buffer containing 0.03% carrier hu-man gamma G. The other portion of sensitized stromataas well as the A-88 sensitized stromata was eluted withacid. The stromata were suspended in saline containing2% BSA, and the pH was gradually lowered at 230 C topH 3.2 using 0.2 N HC1. After adjustment of the pH, thesupernatant containing the eluted 'SI-labeled anti-Rho(D)was recovered by centrifugation and adjusted to pH 6.5with 0.2 N NaOH. The anti-Rho(D) was exposed to pH3.2 for 20 to 30 minutes by this technique.

All eluates were concentrated six- to eightfold withpolyethylene glycol (Carbowax), and the concentrated elu-ates were readjusted to pH 6.5 by dialysis against bufferedisotonic saline, pH 6.5, , = 0.16. The nitrogen content ofthe four anti-Rho(D)-containing eluates ranged from 0.571to 0.080 ,ug nitrogen per ml. The eluate nitrogen was cal-culated from the 'I content of the eluates and theI to nitrogen ratio of the anti-Rho(D)-containing

gamma G fraction. The nitrogen content of the gamma

G fraction was determined by the micro-Kjeldahl method(17). The anti-Rho(D) titers with the antiglobulinreaction ranged from 16 to 32. The original REZ se-

rum had an anti-rh' (C) titer of 1: 8, but the eluates were

683

MASOUREDIS, DUPUY, AND ELLIOT

TABLE II

Properties of "26I-labeled anti-Rho(D)-containing globulin fractions and eluates*

A-79 A-87 A-88

Globulin Heat eluate Globulin Heat eluate Acid eluate Globulin Acid eluate

Antiserum donor REZ REZ REZMg N iodinated 29.2 21.7 20.0Per cent usi1 bound 63.4 63.7 65.5Moles I per mole globulint 3.3 3.0 34Per cent 1261 TCA precipitablet 99.6 98.8 96.9 99.2 98.9 97.9 99.2Nitrogen, &g/ml 674 0.571 551 0.094 0.080 469 0.147,uc n25111g N§ 1.41 2.11 3.12Anti-Rho(D) titersll 256 32 256 16 32 512 32Per cent 1261 absorbed by

Rho(D)-positive cells 21.0 75.7 80.5 83.9Per cent 125I absorbed

by Rho(D)-negative cells 0.02 0.60 1.23 1.0

* Globulin fraction A-79 was obtained by ethodin fractionation (14), whereas globulin fractions A-87 and A-88 wereobtained by DEAEchromatography (15). Heat eluates were prepared by heating the sensitized stromata to 560 C for20 minutes at pH 5.8 and acid eluates by adjusting the pH to 3.2.

t Calculated with a molecular weight of 160,000 for the globulin fraction iodinated.t TCA = trichloroacetic acid.§ Activity at the time of iodination.11 Titers were determined with the indirect antiglobulin method using a commercial antiserum and group 0, R'R2 cells.

negative for anti-rh' (C) in both the antiglobulin reactionand isotopically as determined by reaction with r'r (Cde/cde) cells. These data as well as the per cent of "2I ab-sorbable by Rho(D)-positive and Rho(D)-negative cellsare shown in Table II. The differences in the propertiesof eluates derived from heat and acid elution of cells or

stromata sensitized with either ethodin- or DEAE-pre-pared gammaG are discussed elsewhere (18).

Reaction of red cells with l"I-labeled anti-Rho (D).Red cell suspensions, usually 10%, were prepared by us-

ing the microhematocrit technique (19). A 1% suspen-

sion by volume was used for the reaction with the anti-body. The number of red cells present in this dilutionwas determined by electronic counting with the Coultercounter (20, 21). In a typical study, 0.1 ml of a 1% redcell sample (about 107 cells) was incubated with differ-ent amounts of "2I-labeled anti-Rho(D) at 37° C for 60minutes. After reaction the sensitized cells were washedfour times with cold phosphate-buffered saline, pH 6.5,

==0.16. There was minimal dissociation of cell-bound"I during washing. Dissociation of 10 to 15%o of the

cell-bound '1I was possible only after incubation at 370C for 1 hour. The quantity of 'I bound to the red cellswas determined by well-type scintillation counting.Ionic strength was adjusted by diluting the mixture ofred cells and anti-Rho(D) at pH 6.5 with 0.29 Mglycine.

Determination of radioactivity. The '1I content of thedifferent samples was measured by gamma ray spec-trometry with a well-type scintillation detector (1i- X 2-inch thallium-activated NaI crystal) and an automaticsample changer (Packard). Counting of the 7.4 and 35.4key 'I photopeaks was carried out with a 55-kev windowcentered at 30 kev. With this mode of counting, 72% ofthe total 'I disintegrations were detected, and the back-ground counting rate was 50 to 60 cpm. All samplescontaining 1'5I were placed in thin-walled cellulose ni-

trate tubes (O X 2f inches, 43.4 mg per cm2) instead ofglass to reduce the absorption of the low energy photonsby the tube. The sample tube was then placed into an-other tube for automatic counting (Ai X 6 inches, 44.18mg per cm2). Unless indicated in the tables, all sampleshad counting rates greater than 10 times background.The coefficient of variation for replicate determinationswith the same red cell suspension was less than 1 %.There was a 5 to 10% variation when a fresh samplewas tested on different days.

Calculations. The evidence available (22-24) indi-cates that the reaction between red cells and anti-Rho(D)is reversible and conforms to the law of mass action.The data are presented both as cell-bound nitrogen atequilibrium and as values extrapolated with the Scatchardequation (25), as was done by Hughes-Jones and others(24-26). The equilibrium value represents the amountof cell-bound anti-Rho(D) under the conditions of reac-tion used for testing the cells; pH, ionic strength, time,temperature, proportion of cells to antibody, and soforth. The cell-bound nitrogen under the conditions ofreaction used in this study (equilibrium value) wasabout 80% of the extrapolated value. The assumptionsinvolved in converting radioactivity to nitrogen havebeen discussed previously (3, 11, 13).

We facilitated data processing by transcribing thedata from the automatic counter directly onto IBMcards by coupling an IBM 526 card punch summary tothe counter. The data on the IBM cards were processedby using an IBM 1620 or 1401 computer and a programdesigned for these studies.

Results

Cell-bound 125I-labeled anti-Rho (D) at equi-librium. The effect of progressively increasing the

684

RHO(D) RED CELL ANTIGEN IN FAMILY STUDIES

V

do

ac

0

zZ.

00I-Z

o.)

pg NITROGENADDED

FIG. 1. RELATIONSHIP BETWEENNITROGI

QUANTITY OF NITROGEN BOUNDTO RED CEL

MEMBERS. Preparation A-87, heat eluate.carried out at pH 6.5 and A = 0.16. Totalume was 1.0 ml with about 9 X 10' red cellwere so similar for the six children that or

are required to present the data. RBC= r

amount of added eluate nitrogen c

Rho(D) bound to a constant numberis shown for three different families i2, and 3. As the nitrogen added is incis a progressive increase in the nittuntil a plateau is reached. With theanti-Rho(D) used for these studies,quantity of cell-bound antibody occ

less than 5 %of the added nitrogen wa

indicating that only in antibody exces

2.5

do

z

0

I0cl,

z

Sa-I

0.02 0.04 0.06pg NITROGENADDED

FIG. 2. RELATIONSHIP BETWEENNITROG:QUANTITY OF NITROGEN BOUNDTO RED CEIMEMBERS. Preparation A-87, heat eluate.was carried out at pH 6.5 and u= 0.16.volume was 1.0 ml with about 9 x 10' re

for the father, mother, and only three of tiare shown.

GO. FAMILY- 2.0-

CrXA87, acid el.

wa. F *-eoX 1.0 S -2

0

Z 0.05 0.10jug. NITROGEN ADDED

FIG. 3. RELATIONSHIP BETWEENNITROGEN ADDED ANDQUANTITY OF NITROGEN BOUNDTO RED CELLS OF FAMILYMEMBERS. Preparation A-87, acid eluate. Reaction was

carried out at pH 6.5 and & = 0.16. Total reaction vol-ume was 1.1 ml with about 9 X 10" red cells.

EN ADDED AND.LS OF FAMILY mal uptake obtained. There was no marked inhi-

Reaction was bition of antibody uptake in antibody excess withI reaction vol- the degree of antibody excess used in this study.s. The values Two types of family red cell uptake curves areily two curvesed blood cells evident in the figures. In one pattern, shown by

homozygous fathers such as in the Br family in)n the anti- Figure 1 and the Go family in Figure 3, the anti-

of red cells Rho(D) bound to the father's red cells is aboutin Figures 1, twice that bound to his children's cells. In thereased, there other pattern, found with heterozygous fathersrogen bound as in the Di family in Figure 2, about the same

125I-labeled quantity of antibody is bound to the red cells ofthe maximal the father and his children. The relationship be-murred when tween the father and his children with respect tos cell-bound, antibody uptake appears to be maintained at all3s was maxi- concentrations of added eluate nitrogen (Figures

1 and 3).The data for all the red cells tested in this

study were graphically analyzed by using theScatchard equation, as in Figure 4, to obtain anestimate of the total antibody required to satu-

A87 HEAT ELUATE rate all of the antigenic sites. With the conditionsO FATHER ue* MOTHER used in this study, about 75 to 80% of the totalA SIB s* SIB 4 red cell sites appear to be occupied at equilibriuma SIB I (Table IV).

0.08 0.10 Reactizity of acid and heat anti-Rho (D) eluates.Seven families were studied with both acid and

ZEN ADDED AND heat eluates derived from preparation A-87. The,LS OF FAMILY antibody taken up from the acid eluate by the 22

Thel reaction Rho(D)-positive individuals in four families av-

d cells. Data eraged about 91% of the antibody taken up fromie five children the heat eluate. This small difference between

the heat and acid eluates was independent of the

685

1.5

0.51

MASOUREDIS, DUPUY, AND ELLIOT

0.10o

"I4-

GO. FAMILY

A-87, acid el.

F

S-IS-2S-30.05[

0--

0--

v-v

0-0

\

a I \t0.05 0.10 0.15

MOLES ANTIBODY BOUND X 10 12

FIG. 4. RELATIONSHIP BETWEENMOLESOF CELL-BOUND

ANTI-Rho(D) (r) TO THE RATIO OF CELL-BOUND TO FREE

ANTI-Rho(D) (r/A). The data shown in Figure 3 were

used to obtain this plot. A represents free anti-Rho (D).

Rh genotype, so that the relationship between thefather and his children with respect to antibodyuptake was maintained irrespective of the type ofeluate used.

Anti-Rho(D) uptake as a function of red cellnumbers and volume. The cell-bound anti-Rho(D) was expressed both on the basis of redcell numbers and red cell volume. The ratio be-tween red cell numbers and volume varied from0.82 to 1.22 and indicates that the two methodsof expressing the results are not equivalent. Ifthe Rh0o(D) antigen content is a function of indi-vidual red cells and independent of the red cellsurface area, then the best estimate of the red cellRh0o(D) content will be obtained by using red cellnumbers. If the antigen content is a function ofsurface area, then there would be an inverse re-

lationship between antigen content and mean cor-

puscular volume. The data, however, cannot beused to test this interpretation because there isvolume heterogeneity due to red cell age. A clear-cut answer to this problem must await measure-

ments of antibody uptake of individual cells.

Effect of ionic strength on the red cell binding of125I-labeled anti-Rho (D). The data in Table IIIshow that when the ionic strength of the reactionmedium was reduced from 0.26 to 0.12, there wasabout a 2.5-fold increase in the equilibrium valueof cell-bound anti-Rh, (D). The increased bind-ing at low ionic strength was proportional to thebinding at high ionic strength, so that the relation-ship between the father's red cells and those of hischildren was maintained at either ionic strength(see last two columns, Table III).

Results obtained with different 121I-labeled anti-Rho (D)-containing eluates. Members of the Brfamily were tested on different dates with twoheat eluates derived from two different globulinpreparations, A-79 and A-87. The two prepara-tions differed in many major respects (see TableII). Globulin fraction A-79 was a crude gammaG obtained by ethodin fractionation contaminatedwith albumin, transferrin, and other proteins.Globulin fraction A-87 was a homogeneous gammaG preparation obtained by DEAE chromatog-raphy. The A-79 eluate was obtained after sensi-tization of lyophilized stromata, whereas the A-87eluate was obtained after sensitization of fresh in-tact red cells. In spite of these differences in thepreparation of the two eluates, the relationshipbetween the father and his children with respectto quantity of cell-bound anti-Rho ( D) was al-

TABLE III

Effect of ionic strength on the binding of '25I-labeled anti-Rho(D) to the red cells of family members*

N bound to Fraction of1010 RBCt father

Family Probable Rhmember Age genotype ;4=0.26 ps=0.12 IA=0.26 i =0.12

years JugMo familyFa 25 0, R'R' 2.58 6.27 1.00 1.00Mo 25 0. rr 0.23 0.33 0.99 0.05

D-1 6 0, R'r 1.35 3.35 0.52 0.53S-2 4 0, Rlr 1.50 3.48 0.53 0.56

Br family:Fa 32 A, RORO 2.34 4.90 1.00 1.00Mo 30 0, rr 0.22 0.09

S-3 6 0, ROr 1.27 2.89 0.54 0.59S-4 5 A, R~r 1.25 2.93 0.54 0.60D-5 3 A, R~r 0.94 2.33 0.40 0.48

* Preparation A-79. Values represent red cell uptake of 125I as theeluate nitrogen added was progressively increased. About 5 X107 redcells was used for the reaction, and the total reaction volume was 1.45ml (Mo family) and 1.65 ml (Br family) at p =0.26. Ionic strengthwas adjusted by the addition of 0.29 M glycine at pH 6.5, and totalreaction volume at low ionic strength (0.12) was 2.45 ml (Mo family)and 2.65 ml (Br family).

t RBC=red blood cells.$ Negro family.

686

RHO(D) RED CELL ANTIGEN IN FAMILY STUDIES

TABLE IV

Quantity of '25I-labeled anti-D bound to the red cells of family members

Fraction of fatherN bound Ratio of equi- Molecules per

Family Probable Rh at equi- librium N to cell from N value at Extrapolatedmember genotype librium extrapolated N* extrapolated N equilibrium N value

pg N/1010RBC

Mo familytFaMo

D-1S-2

Br family:FaMo

S-1D-2S-3S-4D-5D-6

Pe family§FaMo

D-1D-2S-3D-4S-5D-6

Sh family:FaMo

S-1S-2D-3D-4S-5

Di familytFaMo

D-1D-2D-3S-4D-5

Go Family§FaMo

S-1S-2D-3

0, RIRi0, rr0, Rlr0, Rlr

A, RORO0, rr0, R~r0, Rr0, RKrA, R~rA, R~rA, RKr

0, RlR'B, rrB, Rlr0, Rlr0, RKrB, Rlr0, RlrB, Rlr

0, RlRl0, rr0, Rlr0, Rlr0, Rlr0, Rlr0, Rlr

A, RlrA, rrA, RlrA, rrA, R'rA, RlrA, Rlr

0, R1R1A, rrA, RlrA, RlrA, Rlr

2.580.231.351.50

3.470.071.741.742.032.091.711.98

2.000.041.151.651.311.611.751.64

3.300.061.862.012.502.122.38

1.970.102.460.232.061.972.26

2.200.051.391.091.20

0.60

0.580.61

0.78

0.790.810.820.810.800.81

0.75

0.780.780.730.780.780.74

0.75

0.760.760.770.800.75

0.78

0.85

0.800.790.80

0.75

0.710.680.68

10,500

6,0705,160

10,500

5,1805,0505,7506,0205,0405,760

6,220

3,5004,9604,2204,8505,3004,410

10,380

5,1705,4806,7605,6906,900

5,970

6,780

6,0505,8606,630

7,500

4,6003,8504,300

1.000.090.550.58

1.000.020.540.540.580.600.490.57

1.000.020.570.830.660.810.870.82

1.000.020.560.610.750.640.72

1.000.101.250.121.051.001.15

1.000.020.630.500.56

1.00

0.580.49

1.00

0.490.480.550.570.480.55

1.00

0.570.800.680.780.850.71

1.00

0.500.530.650.550.67

1.00

1.14

1.010.981.11

1.00

0.610.510.57

* Ratio of the maximal nitrogen bound to the red cells over the value derived with the Scatchard equation (25).t Preparation A-79, heat eluate obtained from sensitized stromata, total volume 1.45 ml, , = 0.26, about 4.5 X 107

RBCper reaction tube, and maximal nitrogen added = 0.80,ug.$ Preparation A-87, heat eluate obtained from sensitized red cells, total volume 1.0 ml, pL = 0.16, about 9 X 106 RBC

per reaction tube, and maximal nitrogen added = 0.084 pg.§ Preparation A-87, acid eluate obtained from sensitized red cells, total volume 1.2 ml, ,u = 0.16, about 9 X 106

RBCper reaction tube, and maximal nitrogen added = 0.077 p&g.11 Preparation A-88, acid eluate obtained from sensitized red cells, total volume 2.1 ml, ,u = 0.16, about 9 X 106 RBC

per reaction tube, and maximal nitrogen added = 0.218 ,g.

687

MASOUREDIS,DUPUY, ANDELLIOT

TABLE IV-( Continued)

Fraction of fatherN bound Ratio of equi- Molecules per

Family Probable Rh at equi- librium to cell from N value at Extrapolatedmember genotype librium extrapolated N* extrapolated N equilibrium N value

ug N/101ORBC

Th family§Fa A, R'r 1.76 0.70 5,150 1.00 1.00Mo 0, rr 0.03 0.02

S-1 A, R'r 1.61 0.75 4,920 0.92 0.96D-2 A, R'r 1.90 0.84 5,310 1.08 1.03S-3 A, rr 0.04 0.02S-4 A, R'r 1.67 0.84 4,700 0.95 0.91S-5 A, rr 0.03 0.02

Is family:Fa 0, R'R2 3.80 0.71 12,700 1.00 1.00Mo 0, rr 0.09 0.02

S-1 0, R'r 2.04 0.79 6,090 0.54 0.48S-2 0, R2r 2.55 0.78 7,700 0.67 0.61D-3 0, R2r 2.97 0.81 8,220 0.78 0.65

Co family IIFa 0, RIRI 2.43 0.78 7,340 1.00 1.00Mo A, rr 0.12 0.05

S-1 0, R'r 1.94 0.78 5,910 0.80 0.81S-2 0, R'r 1.75 0.76 5,560 0.72 0.76S-3 0, R'r 1.72 0.76 5,370 0.71 0.73

Ma familyFa A, R2R2 4.30 0.77 13,030 1.00 1.00Mo A, rr 0.17 0.04

D-1 0, R2r 2.71 0.80 8,000 0.63 0.61

Re familyllFa 0, R'R' 11.94 0.77 36,700 1.00 1.00Mo A, r'r 0.12 0.01

D-1 A, R'r' 7.95 0.75 24,820 0.67 0.68D-2 A, R'r' 7.99 0.76 24,750 0.67 0.67

most identical for the two eluates. The nitrogenvalues obtained with preparation A-79 were from55 to 68%o of those obtained with preparation A-87.This difference in large part was due to differencesin ionic strength. Tests with A-79 were carriedout at , = 0.26, whereas the tests with A-87 were

carried out at ,u = 0.16.Quantity of 125I-labeled anti-Rho (D) bound to

the red cells of family members. The data for all11 families are summarized in Table IV. TheTable shows the cell-bound nitrogen at equi-librium, the ratio of this value to the extrapolatednitrogen value, and the number of molecules percell calculated by using the extrapolated value.The last two columns present the cell-bound ni-trogen of family members as a ratio of the father's,using both equilibrium and extrapolated nitrogenvalues.

The 14 D-negative red cells took up from 1 to

10% as much of the 125I-labeled anti-Rh0(D) aswas bound to the fathers' cells at equilibrium,whereas the Rho(D)-positive cells of the childrenin these families took up from 49 to 125% of theanti-Rh0 (D) bound to the fathers' cells.

The cell-bound nitrogen at equilibrium for prep-arations A-87 and A-88 was 71 to 88% of thenitrogen value obtained by extrapolation, and60% for A-79. The nitrogen ratios among thefather and his children obtained by using the equi-librium value were almost identical to those foundwith the extrapolated value.

To facilitate comparisons within and between fa-milies, the data are presented by probable Rhgenotype in Table V. The results are shown -asaverage number of Rh0 (D) antigen sites per redcell, assuming that one antibody molecule binds toane antigen site. The zygosity of all the fatherscan be assigned unequivocally when the red cells of

688

RHo(D) RED CELL ANTIGEN IN FAMILY STUDIES

TABLE V

Number of Rho(D) antigen sites among thered cells of family members*

Number of sites X 10 (extrapolated values)

R'r R1R1 Rlr' R r RORO R'r RIRRRerR

6.07 10.50- 5.92 9.95b 8.00 13.035.16 5.25

4.725.40 5.87 11.00e 5.91

3.50 6.22d 4.76 7.234.96 4.554.224.855.304.41

5.17 10.38'5.486.765.696.90

5.71s6.195.555.986.84

4.60 7.50h3.854.30

5.154.925.314.70

5.91 7.34i5.565.37

36.70k 24.8224.75

Totals =28 6 2 6 1 1 1 3 1

*4 = Mo family, A-79; b = Br family, A-87; e = Ma family, A-88;d = Pe family, A-87-e = Is family, A-87; f = Sh family, A-87; * = Difamily, A-87 h =Go family, A-87; i = Th family, A-87; i = Cofamily, A-88; 7k = Re family, A-88. All acid eluates except Mo family.Father's value is underlined.

the father are tested simultanously with those ofhis children. Of the 11 fathers, 2 were heterozy-gous R1r, and 9 were homozygous (6 R1R1, 1R0R0, 1 R'R2, and 1 R2R2). No heterozygouschildren bound more than 85 %of the anti-Rho(D)taken up by the 9 homozygous fathers. The 31children of the homozygous fathers took up anaverage of 62% of the antibody bound to theirfathers' red cells.

The two heterozygous fathers were readily iden-tified because the red cells of their 7 children tookup, on the average, 103%o of the anti-Rho(D)bound to the fathers' cells (range 91 to 120%).The assignment of the heterozygous state was con-firmed independently of the isotopic data by the

presence of Rho(D)-negative children in thesetwo families.

There was at least a twofold variation in thequantity of Rho(D) antigen sites on the red cellsof the homozygous R'R' fathers. The R1R'father in the Pe family had 6,220 sites per cell,whereas the R'R' father in the Sh family had10,380 Rho(D) sites. If the unusually high inuptake Re family is included, there is an almostsixfold range in red cell Rho(D) antigen sites on

the R'R' cell, from 6,200 to 36,700. It is unlikelythat the high values in the Re family are due toexperimental error because each person was testedat four different concentrations of antibody and allmembers of the family showed the same highuptake.

Discussion

It is generally recognized that the serology andgenetics of the Rho(D) red cell antigen are ex-

ceedingly complex and poorly understood (6, 7).There is reason to believe that the Rho(D) anti-gen includes a spectrum of antigenic determinants.The antisera in use, therefore, are probably poly-valent and of different specificities, depending on

the Rh0 (D) antigens involved in the immuniza-tion of the donor. The difficulties associated withthe Rho(D) antigen in large measure are due tothe fact that the antigen consists of an unknownnumber of specificities and the antisera used forthe study of the antigen consist of unknown mix-tures of antibodies to different Rho(D) determi-nants. Hirschfeld (27) has discussed the problemsassociated with such an immunogenetic systemand has appropriately described it as a com-

plex-complex system, i.e., an antigen containingmultiple determinants and antisera with many

specificities. Unfortunately, our understandingof the Rho(D) antigen is handicapped further bythe paucity of information on the chemical natureof this antigen (28).

The present study has used iodine-labeled anti-Rho (D) to study the quantity of Rho (D) antigenon the red cells of family members. Before con-

sidering the significance of the results obtained itmay be useful to review some of the limitationsassociated with the use of this technique. An ob-vious one is that this method can only measure theantigenic determinants that are represented by an-

689

MASOUREDIS, DUPUY, ANDELLIOT

tibodies in the serum of our donor, REZ. In ad-dition, these antibodies may not be equally rep-

resented in the antibodies eluted. The resultsobtained by Rosenfield, Szymanski, and Kochwa(29), who showed that the first and second ab-sorption anti-Rh0 (D) eluates have different ca-

pacities to fix to the same cell, suggest that elu-tion may select different Rho (D) specificities.

The families included in the study were not se-

lected at random. They were identified and in-cluded in the study because there had been a his-tory of clinically significant hemolytic disease ofthe newborn due to Rh incompatibility. The de-gree of selection involved in using such a popula-tion is evident from the fact that only 2 to 5%o ofRh-incompatible pregnancies result in hemolyticdisease (30). Consequently, the families used forthis study may differ from the general popula-tion with respect to the Rh0o(D) antigen. Thefamilies also differed among themselves with re-

spect to another factor involved in maternal Rhsensitization. There is good evidence to indicatethat fetomaternal incompatibility in the ABOblood group system protects against primary ma-

ternal Rh sensitization (31, 32). Two families inthe present study had such fetomaternal incom-patibility in the ABO antigens. The type 0

mothers in the Br and Th families had type Achildren. Only one of the A children in the Thfamily had mild disease, since all the children were

A and incompatible with their 0 mother. The firstthree children in the Br family, however, were 0

and compatible with their 0 mother, so that themother was sensitized to the Rh0o(D) antigen.The ABO incompatibility of the last three A chil-dren did not protect the children in the Br familybecause the mother had been sensitized by previousABO-compatible pregnancies. There was no evi-dence that these two families with ABO incom-patibility were different from the other familieswith respect to the amount of red cell-bound anti-Rho (D).

The antigenic strength of the Rh0 (D) antigenof fetal cells has been implicated as an importantfactor in producing hemolytic disease (33). Ifclinical disease in the families studied was due toa greater antigenicity of the Rh0 (D) antigen, thenthe data obtained with the Rh0 (D) antigen foundin these families may not apply to the Rho(D)antigen found in the general population. An in-

sufficient number of families was available to de-termine if this may be the case.

Another factor to be considered is the disparityin age involved in comparing the red cells of thechildren with those of the fathers. The averageage of the 11 fathers was 33 years (range from25 to 41 ), whereas the average age of the 38Rho(D)-positive children was 5.8 years (rangefrom 1 to 15). Comparisons between these twogroups would not be valid if the Rho(D) red cellantigen is affected by the age of the donor. Grund-bacher (34) has shown that the A1 antigen in-creases in strength during the first 4 years. Noevidence has been produced in this study to showthat the Rho(D) antigen changes with the red celldonor's age.

There are three possible ways in which gammaG anti-Rho(D) may combine with antigenic de-terminants on the red cell. The calculation usedassumes that the antibody binds to the red cell withonly one combining site, so that the number ofmolecules bound represents the number of anti-genic determinants. If both combining sites ofthe antibody are fixed to the red cell, the numberof antigenic sites shown in our data would repre-sent only half of the Rho(D) red cell sites. Thismode of binding is unlikely unless the Rho(D)antigenic sites are grouped together in clustersor patches to allow the relatively short antibodymolecule (240 A) to bridge two sites (35). Thethird possible mode of binding would be withboth univalent and bivalent binding on a randombasis. If this should be the case, then there wouldbe no simple correspondence between the amountof anti-Rho (D) bound to the red cell and the num-ber of antigen sites. Our data do not support thisinterpretation, since within each family the re-sults were consistent with those to be expectedfrom genetic considerations.

Another assumption implicit in expressing theresults as the average number of molecules boundper red cell is that the circulating red cells allcontain the same quantity of antigen. Radioauto-graphic studies of the Rho(D) antigen contentof individual red cells indicate that under certainconditions the circulating red cells from a givendonor may not be uniform with respect to theirRho(D) antigen content (36). If the degree ofheterogeneity in different donors varied, the useof the average value for cell-bound anti-Rho(D)

690

RHo(D) RED CELL ANTIGEN IN FAMILY STUDIES

would be unreliable for comparing one donor toanother.

The estimates of the Rh0o(D) antigen content ofred cells made in the previous report (3) are inagreement with those in the present study. Thevalues ranged from 4,600 to 10,300 Rho(D) sitesper red cell, whereas in the present study the val-ues varied from 3,500 to 36,700. Only three of the49 Rh0 (D)-positive cells tested in this study hadmore than 11,000 sites per cell., This agreementbetween the two studies is fortuitous. The esti-mates in the earlier study (3) were subject totwo errors of equal magnitude but opposite direc-tion, which canceled each other and resulted inestimates that agree with the values found in thisstudy. In the previous study the red cell antigencontent was overestimated by using the iodine tonitrogen ratio of a globulin preparation (A-25)that was contaminated with albumin and otherproteins differing from gammaG in their tyrosinecontents (11). This overestimation was com-pensated for by the underestimation that resultedfrom the use of the equilibrium value rather thanthe quantity of antibody that would be requiredto occupy all of the antigenic sites.

The values found in the present study are inthe range reported by Rosenfield and Kochwa(29, 37), but are less than the estimates obtainedby Hughes-Jones and co-workers, who found24,000 sites per cell with 131I-labeled anti-Rh0 (D)(24) and by Rochna and Hughes-Jones, who foundfrom 10,000 to 33,000 sites per cell with 131I-labeledantigamma globulin (5). In view of the manyassumptions used in deriving these values, thevalues obtained in these different studies are inrelatively good agreement.

Evidence was obtained to show that by using125I-labeled anti-Rh0(D) under carefully con-trolled conditions, the dose relationship betweenthe father and his children with respect to red cellantigen content is reproducible under a variety ofconditions: ionic strength, different eluates, equi-librium vs. extrapolated values, and so forth.One of the striking findings was that it was pos-sible to assign unequivocally the Rho (D) geno-type of the father when the antibody uptake ofhis cells was compared with that of the heterozy-gous red cells of his children. It was impossible,however, to determine the zygosity of unrelatedred cell donors. There was a large overlap be-

tween the number of antigen sites on the Rh0 (D)heterozygous and homozygous red cells when un-related individuals were tested. The heterozy-gous RKr child (S-J) of a high in uptake R'R1father in the Sh family had more Rh0o(D) sites(6,900) than did the red cells of the low uptakehomozygous R'R' father in the Pe family (6,200).The wide variability in the strength of the Rh0 (D)antigen among unrelated individuals has been notedby others, most recently Greenwalt, Myhre, andSteane (1). The findings in this study differedfrom theirs in that they were unable to show anydifference between the fathers' homozygous R'R1red cells and the heterozygous R1r red cells oftheir children. These differences in results areundoubtedly related to differences in the techniquesused. These authors used agglutination of en-zyme-modified cells, whereas the present studymeasured the binding of antibody to unagglutinatedcells. The hemagglutination technique (38) un-doubtedly measures a different end point than theisotopic technique.

The marked variability of the Rh0o(D) contentwithin a given serologically defined Rh phenotype,Rh, (CDe), raises many questions. An obviousone concerns the manner in which the pheno-type was ascertained. Only the basic Rh pheno-type was determined with the fve common Rhantisera. It is conceivable that if tests for addi-tional Rh antigens were carried out, such as RhA,RhB, and so forth (39), these families would fallinto different serologically defined Rh phenotypes.An alternative related possibility is that the vari-ability of the same Rh phenotype within thesefamilies reflects a deficiency of antibody compo-nents in antiserum REZ. If our antiserum didnot possess antibodies to all the Rh0o(D) antigeniccomponents, the red cells with a high concentra-tion of these determinants would show a low up-take from this antiserum. The polyvalent natureof antiserum REZ is evident from the fact that itcan be fractionated by certain nonhuman primatered cells and by human type Du red cells (36).

The variability in strength of the Rh0o(D) anti-gen in different Rh phenotypes is well known(6, 7). There is weakening of the Rh0 (D) anti-gen when the rh'(C) antigen is present (1-4, 20).This effect is also found when an rh'(C)-positiveRh0 (D)-negative allele is paired with an Rh0 (D)-carrying allele (40, 41). This effect of the rh'(C)

691

MASOUREDIS, DUPUY, AND ELLIOT

antigen in the trans position was evident in two

families. In the Pe family, the six R'r childrenhad an average of 73% of the Rh0o(D) antigenfound on the father's R'R' cells, and in the Cofamily, the three R'r children had an average of77%o of the father's R1R1 Rh0(D) antigen. Inboth families, the antibody bound to the fathers'cells [double dose of Rh0 (D) antigen] was con-

siderably less than would be expected from theantibody bound to the cells of their children[single dose of Rh0(D) antigen]. This resultwould suggest that the presence of the rh'(C) an-

tigen in the trans position is associated with de-creased binding of antibody. Three families (Mo,Sh, Go), however, did not show this effect of therh' (C) in the trans position on the amount ofRh0 (D). The R1r children in these families tookup an average of 54 to 58% of the antibody boundto the R1R1 fathers' cells. The Re family alsoshowed no effect of the rh'(C) in the trans posi-tion. The two R'r children took up 68%o of theanti-Rh0 (D) bound to their father's R1R1 cells.It may be, however, that the father's two R1 chro-mosomes are dissimilar and only the high uptakechromosome was transmitted to the two daughters.This family was very unusual because the fatherand his two daughters took up almost three timesas much antibody as the highest value (R2R2)found in the other families. There is no obviousexplanation for this observation.

The results in these six families clearly showthat the presence of rh'(C) antigen on Rho (D)-positive cells is not consistently associated with a

reduction in the amount of Rh0 (D) antigen, as

measured with the isotopic technique. It is alsoevident that there is no consistent effect due tothe position of the rh'(C) gene. It would ap-

pear that factors other than the association ofthese two antigens are involved in reducing thequantity of red cell Rh0 (D).

Although the Rho(D) zygosity of the father was

evident from the isotopic data, a quantitative one

to one correspondence between antibody bindingand Rh0 (D) zygosity was not always obtained.One R1r child (S-5) in the Pe family took up

85% of the anti-Rh0 (D) taken up by the father'shomozygous R1R1 cells, whereas another R1rchild (D-1) took up only 56% of the anti-Rh0(D)bound to the father's R1R1 cells. In the Di family,an R'r child (D-1) took up 20%o more than her

R'r father. She should be genetically identical toher father, since she received his R' chromosome.She differs from her father only in the Rho(D)-negative chromosome, r, which she received fromher mother. These results suggest, if they arenot attributable to factors such as age differencesbetween children and father, that the r (cde)chromosome is not an amorph but may influencethe expression of the Rho(D) antigenic determi-nant. Boettcher (42) has recently proposed ascheme for the genetic control of the Rh antigensin which the r (cde) gene is not considered anamorph with respect to the Rho(D) antigen.

The previous studies (2-5) that have attemptedto relate Rho(D) zygosity to the quantity of cell-bound labeled anti-Rho (D) have used unrelatedindividuals and have not had the advantage of thegenetic resolution that is possible when the redcells from members of selected families are tested.Rochna and Hughes-Jones (5) studied unrelatedindividuals and had no way of unequivocally as-certaining the Rho(D) zygosity of the individualsincluded in their different Rh phenotype classes.For example, the wide range of 12 to 20,000 sitesreported for their R~r subjects may be due to thepresence of a homozygous ROROsubject in theirsample rather than to variability of the Rho(D)antigen in this phenotype. Another importantdifference between their study and the presentreport concerns the manner in which the cell-bound anti-Rho(D) was measured. Rochna andHughes-Jones (5) estimated the cell-bound un-labeled anti-Rho(D) from the uptake of 125I-la-beled antigamma G by the anti-Rh0 (D)-sensi-tized cells. This estimate may not be so reliableas the direct determination of the cell-bound anti-RhO(D) used in the present study because of theuncertainties involved in determining the combin-ing ratio of antigamma G with gamma G-anti-RhO(D).

Acknowledgments

We are grateful to Dr. Susie W. Fong, Departmentof Pediatrics, University of California, San Francisco,for making these families available to us for this studyand for the many hours she spent in obtaining the bloodsamples. We are also indebted to the families for theircooperation. The authors also acknowledge with grati-tude the numerous units of whole blood generously pro-vided by Dr. P. Sturgeon and the American NationalRed Cross, National Research Laboratories, Western

692

RHO(D) RED CELL ANTIGEN IN FAMILY STUDIES

Division, Los Angeles, Calif., and by the Irwin MemorialBlood Bank of the San Francisco Medical Society.

References1. Greenwalt, T. J., B. A. Myhre, and E. A. Steane.

Quantitative hemagglutination studies of the D(Rho) antigen in erythroblastosis fetalis. J. Im-munol. 1965, 94, 272.

2. Silber, R., M. B. Gibbs, E. F. Jahn, and J. H.Akeroyd. Quantitative hemagglutination studiesin the Rh blood group system. II. A study of theD (Rho) agglutinogen. Blood 1961, 17, 291.

3. Masouredis, S. P. Relationship between Rho(D)genotype and quantity of I' anti-Rho(D) boundto red cells. J. clin. Invest. 1960, 39, 1450.

4. Barnes, A. E., and R. S. Farr. The influence of raceand phenotype on the erythrocyte D antigen stud-ied by IP-labeled anti-D. Blood 1963, 21, 429.

5. Rochna, E., and N. C. Hughes-Jones. The use ofpurified I""-labelled anti-y globulin in the deter-mination of the number of D antigen sites on redcells of different phenotypes. Vox Sang. (Basel)1965, 10, 675.

6. Race, R. R., and R. Sanger. Blood Groups in Man,4th ed. Oxford, Blackwell, 1962.

7. Rosenfield, R. E., F. H. Allen, Jr., S. N. Swisher,and S. Kochwa. A review of Rh -serology andpresentation of a new terminology. Transfusion(Philad.) 1962, 2, 287.

8. Hughes-Jones, N. C., B. Gardner, and R. Telford.The effect of pH and ionic strength on the reac-tion between anti-D and erythrocytes. Immunol-ogy 1964, 7, 72.

9. Atchley, W. A., N. V. Bhagavan, and S. P. Masoure-dis. Influence of ionic strength on the reactionbetween anti-D and D positive red cells. J. Im-munol. 1964, 93, 701.

10. Masourqdis, S. P., M. E. Dupuy, and M. Elliot. In-hibition of specific binding of antibody to theRho(D) factor of red cells in antibody excess.Science 1964, 145, 1197.

11. Masouredis, S. P. Methodological problems associ-ated with the use of isotope labeled red cell iso-antibodies. Immunological Methods of BiologicalStandardization, Royaumont, France, October 26-28, 1965. In press.

12. Wiener, A. S. The blood groups. Three funda-mental problems-serology, genetics and nomencla-ture. Blood 1966, 27, 110. /

13. Masouredis, S. P. Reaction of I-131 trace labeledhuman anti-Rho(D) with red cells. J. clin. In-vest. 1959, 38, 279.

14. H6rejgii, J., and R. Smetana. The isolation of gammaglobulin from blood-serum by rivanol. Acta med.scand. 1956, 155, 65.

15. Levy, H. B., and H. A. Sober. A simple chromato-graphic method for preparation of gamma globu-lin. Proc. Soc. exp. Biol. (N. Y.) 1960, 103, 250.

16. Helnikamp, R. W., R. L. Goodland, W. F. Bale,J. L. Spar, and L. E. Mutschler. High specificactivity iodination of y-globulin with iodine-131monochloride. Cancer Res. 1960, 20, 1495.

17. Kabat, E. A., and M. M. Mayer. Experimental Im-munochemistry, 2nd ed. Springfield, Ill., CharlesC Thomas, 1961.

18. Fong, S. W., and S. P. Masouredis. Physicochemi-cal and immunological properties of eluates con-taining I-125 anti-Rho(D). J. Immunol. In press.

19. McGovern, J. J., A. R. Jones, and A. G. Steinberg.The hematocrit of capillary blood. New Engl. J.Med. 1955, 253, 308.

20. Mattern, C. F. T., F. S. Brackett, and B. J. Olson.Determination of number and size of particles byelectrical gating: blood cells. J. appl. Physiol.1957, 10, 56.

21. Brecher, G., M. Schneiderman, and G. Z Williams.Evaluation of electronic red blood cell counter.Amer. J. clin. Path. 1956, 26, 1439.

22. Evans, R. S., W. K. Mebust, and M. T. Hickey.Studies on the dynamics of the reaction between Rhantibody and antigen. Blood 1960, 16, 1469.

23. Evans, R. S., E. Turner, and M. Bingham. Studiesof I' tagged Rh antibody of D specificity. VoxSang. (Basel) 1963, 8, 153.

24. Hughes-Jones, N. C., B. Gardner, and R. Telford.Studies on the reaction between the blood-groupantibody anti-D and erythrocytes. Biochem. J.1963, 88, 435.

25. Scatchard, G. The attractions of proteins for smallmolecules and ions. Ann. N. Y. Acad. Sci. 1949,51, 660.

26. Hughes-Jones, N. C., B. Gardner, and R. Telford.The kinetics of the reaction between the blood-group antibody anti-c and erythrocytes. Biochem.J. 1962, 85, 466.

27. Hirschfeld, J. Serologic codes: interpretation ofimmunogenetic systems. Science 1965, 148, 968.

28. Green, F. A. Studies on the Rh (D) antigen. VoxSang. (Basel) 1965, 10, 32.

29. Rosenfield, R. E., I. 0. Szymanski, and S. Kochwa.Immunochemical studies of the Rh system. III.Quantitative hemagglutination that is relativelyindependent of source of Rh antigens and anti-bodies. Cold Spr. Harb. Symp. quant. Biol. 1964,29, 427.

30. Allen, F. H., Jr. Attempts at prevention of intrau-terine death in erythroblastosis fetalis. New Engl.J. Med. 1963, 269, 1344.

31. Levine, P. The influence of the ABO system on Rhhemolytic disease. Hum. Biol. 1958, 30, 14.

32. Murray, S., E. G. Knox, and W. Walker. Rhesushaemolytic disease of the newborn and the ABOblood groups. Vox Sang. (Basel) 1965, 10, 6.

33. Murray, S., G. Knox, and W. Walker. Haemolyticdisease and the rhesus genotypes. Vox Sang.(Basel) 1965, 10, 257.

693

MASOUREDIS, DUPUY, ANDELLIOT

34. Grundbacher, F. J. Changes in the human A anti-gen of erythrocytes with the individual's age.Nature (Lond.) 1964, 204, 192.

35. Edelman, G. M., and J. A. Gally. A model for the7S antibody molecule. Proc. nat. Acad. Sci.(Wash.) 1964, 51, 846.

36. Masouredis, S. P. Unpublished observations.37. Rosenfield, R. E., and S. Kochwa. Immunochemical

studies of the Rh system. II. Capacity of anti-genic sites of different phenotypes to bind andretain Rh antibodies. J. Immunol. 1964, 92, 693.

38. Solomon, J. M., M. B. Gibbs, and A. J. Bowdler.Methods in quantitative hemagglutination. VoxSang. (Basel) 1965, 10, 54.

39. Unger, L. J., and A. S. Wiener. Some observationson blood factors Rh', RhB and RhC of the Rh-Hrblood group system. Blood 1959, 14, 522.

40. Ceppellini, R., L. C. Dunn, and M. Turri. An inter-action between alleles at the Rh locus in manwhich weakens the reactivity of the Rho factor(DU). Proc. nat. Acad. Sci. (Wash.) 1955, 41,283.

41. Levine, P., M. Celano, S. Lange, and M. Stroup.The influence of gene interaction on dosage effectswith complete anti-D sera. Vox Sang. (Basel)1959, 4, 33.

42. Boettcher, B. The Rh 'deletion' phenotypes and theinformation they provide about the Rh genes.Vox Sang. (Basel) 1964, 9, 641.

NOTICE TO SUBSCRIBERS

Post Offices will not forward the JOURNALwhen you move.

Please notify THE JOURNALOF CLINICAL INVESTIGATION, TheRockefeller University Press, Box 261, New York, N. Y. 10021,when your address changes. Include your zip code number.

694