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State College of ﬁgricultura and [mum Science
in xmtml fulfillwmt of the “gunman

for the degrees of

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1951

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338515

Introauation

History of tho P11 Factor
material. and ffaﬁmdn
Result.

r‘ismalon

guaranty

Bibliograﬁzy

Eng.

1"”T2'?Ifi.“32‘l“2l

Tho 1-21: factor. oo called boosmso it was i’irot obeorvod in anti-
aonm prognrod in rabbits again“ tho blood of tho rhesuo monkey m
nggogga, dorivoo its immunological imyortanoo frmn.tho {not that it is
an iwiutinomn. Like the other major blood group factors, A and 3,
it is an antigen capablo of otimulnting antibody graduation whon intro-
duced parontornlly into on individual oboso collo do not contain tho
antigen. ﬁz.;roximtoly 15 per cent of the whito population of tho United
Statoa io Rb-nogntivo and no is, thonrotionll: at loaot. liablo to immun-
isation by tho Eh (water.

9onoitizotion of on Eh—noﬁativo inﬁividuol may occur in oovorul ways:
by puamgo of Ph-waitivo total orytiu'ooytoo through tho 1.:1woontn during
pregnancy. by trﬁnofuoion of ﬁhppo$1$1VI abolo blood. or by intramuscular
injection of whole blood. In onch onto the result to qualitativoly tho
sumo: otimulwtion.ot antibody production. ﬁne quantitative rooult.vorioo
with tho mmnt of antigen introduced and tho await: of tho individtml
to produce antibodies. Fortunately, only about 50 {or cent of tho Rh-
nogntivo persons aro ousoogtlblo to oonoitizotion by tho Eh.ontigen ovon
by rerouted intravenous injectiono (Loving 19%9). ?von though an Eh-
nugntivo person has a high Rh antibody titer, transfusions with Rh-nega-
tivo tholo blood are successful. Eowovor. introduction of more antigen-
contnining ﬁh-yoaitlvo blood result: in febrile ranctlon of Vunying lov-
ority. inns, with propor laboratory zrooautiono, thoxe nood to no diffi-

culty in giving transfusions to the Eh sensitized yntient.

The other and more viziely Lublicizod, osgeot of the Pi) imbim con.
come the effect of the mtarml antibodies on the Fix-gositive fetus 13
23:;2_(Jnvert 19h?, Levine, Eatzin and ﬁurnlnm.l3h9. narrow lGMT, ﬂuvia-
oohn 19h8, ﬁcCurdy 1950). Foot Rhnneantivo Iomon urn unduly worried about
the pose! ility of delivering a child effected with hemolytio diseaoo.

“f the 13 per cent of all matinee in which an Rh lncnmgntibility‘oxitta.
only one out of overy 35 or no iragnunciee results in on £h-;ositivo child
with homolytio discos. (fatter 1?k73.

?ho prognosis for on ﬁh-nagativo primigrnvida io‘uouully oxcollont.
Valeeo the has e.bietory of provioue trﬁnofuslon or abortion. prognonqy
and delivery should to oithout incident as far no the Hh factor io eon-
oernod. (Discombe 19%?) The reason for this nbeonoo of bomolytio dio-
oqee in tho firot child in not known. zottor (19¢?) foolo that tho moot
logical exylnnetion would be that either antibodioa are not rroduoed in
sufficiently high concentration to injure the fetus, or that “owmo con-
dition associated with delivery is responsible for the entrance of fetal
cello into tho'matornnl circulation. and immunization is not accomilished
until after the birth of the baby.” Baunlly a Immwn dollvoro at least
ten) mine-.1 children before she will have one affect-sci with iseiznolytio dio-
eoee. However. once immunization has progreosed to e dogroo sufficient
to muss hosmlytio diooo-ee in one child. all whooquent rah-positive chili-l-
ron will be effected. Thorn are no proved oxoeytiono to tilt rule. (Lo-
vino long). i

Although the eoturl number'of affected children in email. each ouch

Moe lo a tool tragedy for the family involved. If the child 19 born

alivo nod not too soverly affected, treatment by transfusion with 3h.
negative ibolo blood is usually ouccoooful ( loner 13%3). ﬁlth multiple
troanoncies however. tho Ehnpooitivo child is nfioctod tragreoeivol:
onrlior in intrauterine life, and, if not stillborn. may die shortly after
birth. it tho present time the obotstricinn can bo pro;nred to trout tho
child only after birth; he cannot :rovant tho condition. To irotoct the
fetus it sould be necessary to prevent antronoo of the antibodies from
the mother into tho fetal circulation. it is probably logosaible to al-
ter the placenta in such a I“! no to main it imxormoublo to the an anti-
bodies. 'E’ho magenta is regarded. no being on ingoruaooblo Irzolubrsmo, al-
though substances having a molecular weight of 300 to “00 are known to
pass through the placenta. ilasma irotoino. with their much greater molec-
ular weight. are not generally believed to traverse the placenta, although
it is known that tho Rh antibodies and several other antibodies are trunn-
{erred from uotbor to child through tho placenta (Aroy lQbI). ?ho univul-
ont Pb antibodies. thich have s probable molooulor weight of 155.050
(Wiener 1953b). are carried through the tiaouo by‘a mochnniom not yet
known. The yrovontion of antibody'tormotion would then argon: to be the
solution to the problouo

Vnriouo investigators have ottam;ton. by chemical and biochemical
moons. to neutralise the Hh-nntiboay i§,!;19, Knrihor (1347) reported
three cases in which be ouccosofully nood ethylene dieulfon to to lower
tho anti-Eh titoro of women who bod iroviously dolivorod erythroblaototio
children. Fbile under bis-core, they dallvered Eb-iositivo infants. two

of which sore normal and one thot recovered undor treatment. is quostione

the value of the ethylene diouli'omte became the amounts winini stored
”11’1"”. dilution in trille distilled water) were so ermll. He Bust-16833
tirmt the trigle distilled mter my two been recglonsiblo for the drop
in antibody titer.

Althom‘h it no known that the reticulo-endotholiol system could
resmnd to e. camber of truth-garlic otirisali at the mine tine, Manor and
form (17%) mmeatod that the coininietrztion of to strongly antigenic
mnterinl each as tylizoid-pzmtyﬁzoiri or tertuosie vaccine might ewlreso
t‘::e worker Rh antigen. Subcutaneous injectione of the vaccines eere
given but they were ineffectual in lowering the lib antibody titer. finger
(19.149) has among-ted to srevent eensitization of ﬁll-negative women by
"z“:‘iener'e vaccine thornpy. Ee found, in a ”rim of nine sensitised In-
men, that vaccine thorny was of no avail in lowering antibody titer.
However. of 25 non-eonsitized 3ri:::i;.nrne an: 100 non-sensitized mlti-_
frame treated by vaccine thorny, only one iuultigem delivered an affected
child and that one recovered under treatment. Von Loghom (1938) re,orted
that it we ”meaible to stimulate, even to novel-:9 Lrorluction of 77.11
antibodies by heteroomcific mitt-7on3 after e. shorter or loxt-zor course
eith specific Iii: antigens." Incidence of sensitization after stimula-
tion by the vaccinne r33 21 gar cont rather than the enacted 5nd} ree-
cent in You Laghem'e series. In i'ngor'e oyinion, the results of hie
study and those of Van Loghem'e are not ocmgumble einco Von Lo; $831..
mtiente were “irimd” with Fl: anti-gen before “ministration of the vec-
cine. The 0110711119an were e1 co undertaken to move different hygotheaoe.

Tlmt the Rb factor is [recent in the otrcrna of the normal red cells

had been amnatmud by Belicia and Menu (1914-4) and Calvin, Evans.
Bahrendt (anti Calvin (19.35). This was accomi'iished by 11311101311116 :12.
cells and concentrating iha strom by «hiring-ration. Tiny were able

to show agglutination inhibition titers for the Rb fractnr Iith the con-
centr'gted “mm which were eight to thirty-i150 times as great as they
met-'lutination imi‘bitian titers of the original cello. Calvin «at Ill.
(HMS) called this inhibitory m-xtwrinl “tannin." forever, olinin, in
amnion to the Fin substance, also containad tho A and 3 group syccii’io
subatﬂnces. In an 9?.th to concentrate further tho anti-111:. activity,
tm other extrxct of olinin was prepared. ‘i‘kzis process increased the anti-
31:12 titer to as high as 131.98. mm lowered the anti-A and anti-B titan.
”one showed th't: tha (’1')! 91min (1m;.‘hiliud‘) was hmt stab]... but that
the activity no lost on heating for ﬁve minute: at 55°C in tho :maonce
of minim”. Cadvin, at 9.1. (1956') rent-ted this mwrial to ‘ba a 11;!)-
rrotain. Us balioven mm the Rh factor may be It relatively litii;10 mol-
ecule attached. to he strum of the rev! can in such a. my that it is
emu: amtmyad by fractionation mathoda.

A different apzmnch to 1:11. problem is. reforted by Carter (FEW,
1939?. She obtained by alcohol-ather extraction of thxocitivn human
red bland call“, t 1111:! which sh: believed to be the ”9’1: tax-ion. $210
found m material to be non-antigenic ig- g‘Lv—t; but when conned with a
protein (can: albumin) ii elicit: graduation of specific antibodie; in

guinea 119:0. It is activo again“ anti-Eh serum in dilutions of 132000

 

’ The term ”anti-£13” refers to tit. aativity of the unmet-rd

material amimt the “h rantibady 5.31. vita.

(h

or'highor as measurea by the cnmglaiant fixation teat. Then injected
intmznusoulrarly during ”gammy {Lemaitre}? and Critter 1245. Carter 19°49).
it refused the anti-Eh titer of the mothera an! resulted in the delivery
of children who taro actual or who récovareé under treatment. Thasa fina-
iuga saro subatwntintod by Coldsnith {13%ﬁ, $50 n,b) and Frncklani (likﬁi.
ﬁn anwlysie o! the haytan by Erica. ¥e*é, ﬁwrﬂoa and Chen (1345) dhawad
it to be a fatty acid which on (Emma-Winn yieldad two tubstém-zel, active
in dilutitma of 125030 and l:i')"*.0¥30 rasgoutively as data-mined by the
cam;19msnt fixation test. Erica sug;eated that these fructione N#y bu
ra~ynnwibla for two of the Eh subgrnuya.

Other attemgrta to re;.ac"t Carter's '1an have not been succemzui.
Wong and Rustignn (1950) were unable to char any anti-ﬁh activity by ugh
glutinntion inhibition or oomglement fixation teats. Rosxowitt, Pundi-
liker, Calvin and. Evans (19????) and "E're-EBEFth (1’?1+9) were also nimble ts
yrnpérn active Iroducta. ”array. Lathe, Clark and Levine (1956) r030rted
that "nttamgts to confirm Carter‘s wnrk.hwva baan cam;lotaiy unsucceasful.“

This work is a razort of attornpts to Lrepnra tho Eh haztan using both
an adngtntion of Carter's original tachnicuo (19393 and Goldsmith‘a (1939b)

modificatian of Carter's tedhnique.

Althsmgh Lanziatoiner'o di waver} in 1353-0 of than four main blood.
zrmps hood mr‘mdly rod‘aced tmmfuaion reactions, it had not clizaimatod
than: (Maintainer 19313. Occasiumlly the rsoipiont of apgmrontly cox-a.
xoti'blo blood would have a rmction whoa thora- I-‘as no roasibility or on
error in ty‘ang. Even thaw-1h tho own or ‘i’i‘ reaction: was unknown, it
goon bosom eviient that there more certain features comma to all of
them. S‘irat trauma! ms, nxcegt in the 0:53. of rooontly delivered “3551631.
taro rarely involved. A reaction ms morn 111::on if a. tarpon Md recs-her!
multipio transfusion: Lrior to t"... one causing the reaction. Following
parturition a woe-inn might have a levers ro‘iction if tmnsfuoori oi th her
hogan”; blood oven thou-7h it was of the some tn'o. Introduction of
blood from another donor was usually rithout inci-icnt. Iamdatoinor. Lo-
rin» and Jones (1323) amonv'xtrnted tho ”Home of sits/5.1091 agglutiﬁino
following lame transfusions by using tho donor's cello and the recigiento
sorta, but thorn we no may of groéictin: whathor or not a. roci;.ient
“0,3,3 3.2‘OMJCQ the“ waglutininu.

mother, and "windy numb-ﬁtted, corriition in tho one known as
erthroblmtosio fotnlia. ‘Z‘Lio is :1 distrust! of tho fetus or the newborn.
child, then manta-ms of mm are due to a di star-bemoan of tic-o homomiotia
system. Erior to 1930 than van-ion:a clinical forms of hamiytio di goose,
knot-m as fatal deropa. ictema grwia. and anemia. of tin.» newborn. one
thought to have» «print. otiolmﬁea. 1"‘iom-md, Eilncicfon and Enty hotter

1’1“?) Lroaented the conceit that tmao more! all mifostutiom of a aim-51o

mbrgtancoa from tho father which. if lncldn; in the mthor. my atizhlato
ho: to produce isoantibndlas. '?ho {allowing year Levine, Purnham, Kntzin
and Vocal (1931) ahnwod that tie serum of mot are, whose chilﬁrwn had
bnwn stillborn. cantnined a Gabat“ac which re cteJ like the anti-1h auti-
bodiao. leseguont teats share! th"t tro affected c3113 and father were
almost oithnut etomztion “b-ynaitivo and the mother ﬁh—negwtivo. Thoao
circa-mt noes my 3110!: the [anther to ire-.2339 antibodies agacific for

tho Eh oungonnnt of tﬂe fetal cells. 919 aatibodiao «nigh Levine first
dascribod an brain: reagnneihlo for 2;e-.-:..:l;,rtic diaryme, and 3-53 31'- ho called
as lutinlna, were native 1n orlino eusgenaiona of red blood cells. rhu-
ovor. in mm mama of hematio (£198 33, w‘uidl 39mm]. to bo duo to Eh
incnmgwtibility. no antibodies could be do» untr tad. It seemed for a
time thwt this very ogosific agnﬁrona would have to be uodifled. Yianar
{1?%“?. ”too VlQQS) and Race an: Tnylar {13443, snrling inieiondently,
discovered tho clutinins or blocking antiboiiea. These are the antibodies
thvt actually trwvnrao tho Llwcenta and attack the fetal cells. Davidsotn
(1335} reviewed the various tasts for detocting these antibodies. Tho

two tygoo of antibodies are com;urod in ?ablo I.

JLon_tho Eh factor vqa diwcnvorad it was thauptt to be a nimble Han-
éolian awninﬁnt controlled by ono gnir of genes, the gano for tho grotenco
of t"o factor hoina dnmln“nt to tho can for its nbsqnco. In the course
of toasting: 131000.33, it I'm obeorvvd that not all 66118 ram. 5213911 in the
cafe mvnnor to certain antiaore. rurt er invwotigntian revoqloé that there
more two aziiitinml typos of anti-’11 new, one tar-win" o‘ecificity for 70
gar cont “3% tho other for 30 $6? cent of tho 3 its vaulution. 'ith

\
‘!

thoao throa- antL-"h 59m it boo-man moeiblo to iris-nth}; ol<;.;:-.t I'm-tugs.

10

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08.3w Scamnw
:3 8a 98 8 3.: 39:59. an! ta 3.3 3 93 33o
:18 go .3338 339:! '38 1.50038 no .5333 3:63.!
P 322323 ridding». P ".331383 33%..
32:»..- opSoooBN .8335 no: 5382,.“
8833. 833m 353.: .8323 2:33....“
3:3... 3331 £325 .83.. 382: 233 .5 3 Mai 3.5

.38
0.300 :93 an... aging .5... g .3 agdgo MC n8 :8
hOﬂdL g

.38 no Cl: 23 15¢ 93' .38 No Oahu one
3398 a agngugolaom 3—3 3536 agaoI-Oaﬁoomm

ooauhl
003.3. :3» 3 0.33.. 3 239 :3». Bo.“ «3.33.3 haunt”

. nouoaucadhaoo P .300 3,998 33.333.5ch .3 .32.. 358

I! 83.33 as? .3. 308 533.33- B .38 £35.
338.35: 33333 3258.85 2323:»

33 8‘
83:3 9532 33588 5333

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ﬁlming can. rad-332:.

.55!— «in!

.3398: 3 30¢

9.31 5 3oz

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unmade 90 has: «95m
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“mu—dang oaﬁdbnnl and .544»? g En

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11

On this evidence. Wiener {1935) ;royoood that thorn nro oight olloloo
to the Rb gone. Ho based his theory on tho {not that when too of thee:
antlgans are [recent in tho blood of o Latent, they are almost alvao
inhoritod by tho child on a unit. Only very rwrely to one antlgon in.
horitod without the other.

This in in oLLooition to Fiohor'o throo linkod gono thoory of in-
herit'znco (Face, .‘Eoumnt and atllendor 1346, Allott and Nolan: 191*?)-
Fiahor bollavol that throo gait: of closely linkod gone: are roozonalblc
for tho oxgrosoion of tho Rh factor. If tho antigens IQTI controlled
by genes at separate loci on tho chromooOmo, thay could bo inherited as
xnrta instead 0! as o tholo (crossing-over). Foo ouch cases have been
royortod (lotto: 19373. This controvoray has given rioo to two oystaoo

of nomﬂnclaturo (?ablo II}.

Tzﬁlt 11’

cowmmmr or Ft. *1. 05‘ 95-22: (mt/coo) aromas

   

?iohor~?nco Wiener

W
odo th
Cdo rh'
odﬁ rh"
8:13 thy
c3. fﬁﬁ,
CV3 :rh).
c1£ Eta
.2172 lib,

 

" 35133-3: 1257115;

Lavina, Burnhnm, Retain and V0591 leUl). thilo investigating various
onti-?h corn. found one thut cg lutinutod all ﬁh-negotivo and some Eh.

goaitivo cello. Becauso of tto roctgroo l notoro to tho Rh geno, ho called

1?

it "Er”. TLoco antibodies roact with tho cello of those indiviﬁuals who
do not possooo tho two gonea dotormining tho yrosonoo of Rh' (Tablos I!
and 2115. ?ho anti-serum is designated ”Kr'”. Fouraat (1955) discovered
o sooond Fr antibody, "Hr"". ani although ?iohor (Waco 123%) predicted
tho discovery of an Bro antibody. this has not boon definitoly confirmod
“tribal-mun 19%. i’iionor 13138, 1950133. If those 3th anthem wore avail-
able. it would bo possibls to identify 27 types of blood with regard to
Wh factor. foo Hr ontigono oro also out bi. of stimulating antibody pro-
duction and they'hnvo boon shown to ho roogonoiblo for scrotal casos of

homolytic disoooo and for transfusion reactions (Fisk lQHS; Eionor 13%5).

13

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Swastﬁoug . , V. G. 33a
£3-35 .3433. $3.33. .3733 3.7.33 paﬁﬂﬁ .aowoazoaﬁééé «no.3 333%?8
wxuavacmmotné ouaocsavoum
.5 Room um r13: manmwuuocm r; nﬁmﬁ my ”my? muomorocuwtt mwnﬁw

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t 8““ cl, 5‘9‘ .I I‘ll}: 14...).01t Irlltﬁl' {lit-(l. I! Is}..tssvllu’1-|i

 

.3 .2. .43 ‘4. our. 1 Is; 0 . s I! HI about A a t 9-. a.
.‘nwtsrhvr‘ '.. H G {v M F ﬂ u. 1.. ”it? .lili‘ p” n twhu

OF: wwq.&

v ‘mtv-av‘. ‘ s. “v~. "--"’-1'.g-... a
34f... :. 21.. {a 24.. .... r. A

The blood used in tl.‘ gragxzmtlon of the tout mitten-1:11 \ma obtained
from tho ﬁlchigan Bogartnant a. Health through the courtuay‘cf 3r. ﬂ. 0.
Anderson. Cello trom.outdated bunk blool of all Lcnﬂstoinor blood grougo
were used after removal of as much plmama an gosaiblo. Tho only rozuiro-
manta were that the call: be intact ani th-gnoltivo. The anouthotlc
grade other (Squibb) usud 1n the yrapqrntion ot the first two lots of
motorinl who provided by tho ijobn Comrany of Kalamazoo through the cour-
tocy of Dr. ﬁn. ”airman

In all, (on: lots of material were grepnrod; the first too by Carter's
(131V?) method (Tr-b1. “'3. the second two by hldcmlth'o (1950b) m-ﬂiflort-
tion ’?ablo V) of Carter's method. A: can be soon from the Tablas, thorn
in very little differenco bctwoan the two grocoases. Goldsmith used lor—
ger volumes of alcohol and other, and stirred the aucxonoiona for a longer
ﬁlm than Carter did. We (fblilmuit?) chat-med that! tho nee of a high Igeud
electric stirrer to agitoto the other cuagonaion resulted in an incroaood
yield of tartan.

In both mt‘mdo. filtrate I'J’. which contains the active fraction. is
ovnporqtad to drynooo rather by vacuum cictlllatlon at touyorotureo below
“0°C, or undor a hood by rn;antod fillings of the name watched Iotrl dish.
In the latter grooms: a current of olr blown over the surfoco of the £11-
trate by an electric {on will hasten evmyorntlon. Carter (13“?) dissolved

tho yellow to white waxy rations in absolute alcohol and otorod it in

m a ‘3' If
‘1‘." _.‘...o

(«tr-31

—:» .....

V!:I"~

.
=====Ellllll=====L~ *7:

1,

%h Calls fl‘lit;r3

1’1!

.' -' ‘.I~ "' An.
C . ‘ 1
3 I ' 1-,“

l
"*7.

V w w V; .7 v—t‘th
w A‘— ._, v__

wr—v “—7 w..—

L&u¢ t% .5 0811: With 5”? ml. dis.

illoa voter.

gti:rirlw:. :4 11.9;3 Of 95

Add slowly with
gar cont

ethyl alcohol. and stir :or F0

 

minun.¢s. Allow to atxnd at 35 O
OVGX'nifﬁl‘ o
Seiimgnt I. Filtratc I.
Filtor on fuchnor fannex. fr 'i v' wanoéﬁx V

!

suopwnc in 4 lltoro of
50 yer cont othgl alcohol
and allow to stand at hOG
ovarni.;;?:t.

’odiment XI.

'T-"rmt no obovo. using
25 got cont ethyl alcohol.

ﬂﬂlLJéﬁz XIX.

:ry t Horzaxialy on tho funool.
endear! 1n uneothetic gr?-‘--:19
other uni sunko tan mioutos a
day for from two to fivo dag-o.

Sedimeab IV.

ﬁlocﬁrd

 

Filbrﬁb. II.

l—A ML

disocraﬁ

Filzrcto III.

__ _ m

iioccrd

Elltrato IV.
2 ”.AI!

15

"253.1:

XIT'ﬁ '3

00L?

V

1.; "'-

7"V3
a‘._-J

W
Rh Cello foWO cc)

ﬁ—vww

._—_'_

Lake with 300 cc of distilled I
enter. etlr and refrigerate at
L93 for 30 minutes. it tie end
of tile time. 361 h .500 cc of 9"
eth3l alcohol. stir for 30 minutes
and rofrigorate at ¥°C overnight.

 

 

 

 

 

Soclment I Plltrute I
Roonerond in §,500 no of 505 r dlloerd
ethyl alcohol. stir for 30

nlnntoo and roturn to the re-

frigerator ovornight.

Sediment l! Filtrate 1!
Trent as above. using 25h discard
ethyl 9.100hal.

Sediment III Filtrnte III
riizéét. rumored unoﬁcie- ﬂ» discard
carded. Sediment snapendod

in “.500 cc of ethyl ether.

U.S.l.. mixed 30 mdnuteo and

refrigerated overnight.

null? mlxing repeated for the

next four days.

Sodlnent I? flltrete I?
dieoard ' £33113

17

bottles. ”oldmadt‘n (1930b) conducted all drying: proceceec under ultra-
violet light to help prevent virus contamination. He also (.11 uolved me
crock of the ethereal reei'iue as ioesi’ole in ram 96 per cent alcohol,

oi scnrded the elcohol insoluble material and 54min evaporated to dryness
in e weighed I‘etri dish. This regimes 1m: redissolved in 96 per cent
alcohol to mice a concentration of 200 mg. per cubic centineter of alcohol.

«icon: for anti-Eh activity was by Carter's (191+?) modification of
the Kolmer (19345) comelement fixation test for the tern-diagnosis o!
sn‘hiiie. It is possible to use the egglutimtion inhibition teat (Carter
1149) but the 50 per cent alcohol need to infirm! the antigen dilutions may
inhibit ngalutination.

'T‘ne tangents need in the comuemwt fixation test were titrated ac-
cording to Holmer‘e {Ni-65) method. iiemolyein was purchmed from imam
and Tnhme, £30.. itiiadelpin. Iennsylmnia. The sheep red blood. oelle
ﬁnd the anti-Rh (anti-I?) timing: new xwmui’nctureri by the Ortizo inﬁrmi-
cwticnl Gonorntion, Imriten, New Jersey were obtained from the iii-:higun
"garment of health. 'l’ne ample-meat ma wagered in the laboratory M
follows: eight to ten guinm gin were starved for 12 hours. then bled
”seiticzﬂly from the heart. The blood um allowed to clot, tint-med and
centrifuged. The seam m3 removed, yluced in aterile 2 ml. soft glue
ﬂtztgillﬁﬁ and izzr'zedintely shell frozen in n dry ice-alcohol bath. The my.
3.11103 were then sealed and stored in the Gas-p freeze at 40°C. Comple-
ment preyarad by thin method contained 2 units in 1 ml. of a 1:60 dilu-
tion of the Mm.

L’mne and Eiuetigim (1330) had noted that "lowerim; the tern; erature of

18

\

the filtrate: from the successive alcoholic extractions to 009 or below
resulted in the zrecixitation of Varying amounts of lipid-carbohydrate-
nitrogenous material shich showed i3 activity, but no Rh activity." To
:revent this loss of ligid the firct two extroctions were carried out at
~5°C, all other cOnditione being the some as outlined by Carter (l9h9).
The first two lots of cells each yielded about ?00 mg. of lipid. This
agrees with Carter's (19“?) yield.

”nly reagent groan ethyl alcohol and anesthetic (Squibb) grade or
U.?.T. ethyl ether were'used in maxing the extrectionc. All ginsesnre
was rinsed with trifle distilled enter and sterilized in the autoclave
for one hour at 15 pounds pressure. $ri;le distilled eater ens used in
making the alcohol dilutions to grevent introduction of metallic substan-
ces which might inoctivote the antigen. All sterility tests were negative.

Goldamith'e (lﬁgﬂbl method was followed in detail for the grnyarotion
of the next two lots. The yields were not as large as those obtained by
the low tangeroture extraction. but since ioldsuith hoe not indicated
the size of his yield, there is no bani: for coaoarison.

The ethereal residues from filtrate IV (Table V) sore treated in the
following toys:

LG? I.

?he wet ether filtrate. which had been reduced in volume to about
1?) ml. and distributed in weigxod maul”, r-u lyoyhilized and the amines
roeeighed. The cornice were then cooled and stored at M90. For assay,

05 per cent alcohol was added to the lycpnilised som;le to mate e one
per cent solution. Thin was then diluted with saline to the concentra-

tions indicated in Table VII.

1‘3

L7? II.

"he ether filtrate from this lot was divi-zied into two zortions.

"ne portion use treated in the some runner :13 Lot 1. The resiéue from
this lot is deeimteﬂ lie in Table Vii. After the etZ-r‘er was evoynmted
from the eeconri portion, the residue (111), Table VI!) was redissolved
in 10 m1. of other. and. 30 ml. of 1.”.3‘... entone gal-Jet to zrreciiitute
the rhoa,holig..ide {Ix-ice, 39nd,. awoos: sand Eben 13343). After centrifup
gntion for one hour in the cold at ks,ﬂnﬁ rgm. the eugernrtent fluid
decanted, distributed into eei ices! amulet no! crayon-ted to dramas: in
or vacuum desicmtor.

Carter (1939) stat-5d that sorta active memorial could be recovered
from the first alcoholic filtrwte. The 7012mm of filtrate I (see Table
IE") lot 2, no therret'ore reluoed to about 2-?) .21.. five volume of ether
min-lied with stirring, an: he mixture refrigerated overnight. The ether-
insoluble resiiue was discarded and the ethere~l eolution processed in
the eras-e manner or Lot 1. ’E'lzie material wile desigmﬁted 116.

L333 111 ml I‘:’.

[These lots were both gremred by hiésndth‘e method (Table n exam}
that the other vet tenured by vacuwn distillation rather tiara being
evaporated from an cyan Ietri dish.

In orier to rend the comblownt fimti-m test wotainim; on omens
antigen, homolyein control tubes ('r‘o’ole "v1.5 were included With each new
of Lots III and 11’. Thus a standard we grovioled with Ions-an degrees of

hemlyeie for cougar-hon with the teat groper.

u: a. 0w v" t?!
. I ..,- ”143.4:

walla—m
#1121359!) (189000) 005 0.5 005 ”'5 0.5 0'5 0‘5 0’5
mm. (0.55“?) 0.75 (5.5 0.29 .. 1.95 1.0 0-15 0-5

(Triflﬁﬁiﬁﬁﬁ (Bilﬂlt! 0.25 0.5 0075 100 0.25 005 0.73 1.0
1n1.om.)

Femnlyatn (2 units) 0.5 0.5 9-5 0-5 0-5 0-5 0-5 065

aheep red cells 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
(2‘?)

v «fjuy ‘v 9‘"
J 1;: 3

‘31

"he r9¢u1ts of tha gogglement fixation testa {viled to iniicate any
anti-3h activity in the four lota cf Bﬁtﬂriﬁl 1ragnrsd, Oven though
C rter'a (1339) assay groceﬁure mus followed in detail with adequate con.
trols. Thq tsetz on tLo fir5t lot were unmatisfactory. in all tha tuhas
contﬁining tha test antigan, the cnu;1aaent Wu; fixed, regnrd183s of
whathnr or ﬂat the Rh antiboﬁv E13 present. The reagent controls were
eatiafnntory. It wna thought that gerhngs the 11;1d-Cnrbohyarmta~n1tro-
genaus mﬁtﬁrial described by How. (1&39}, and $reaent in this lot bécause
'of ﬁle 10w temger~tura irocesaing, Was anticou,lementary. If this u¢ra
the cage, it ”ﬂuid axylnin the unsatisfactory test. 3ayar, Ouler. rier
and Feidelberger (l?%63 ahoaed the anticomilamantnr: nativity of an
antigsn to be due to the binding on the ﬁg lens by citrLte nr ;yrophos-
yhaio. It ﬁns fov this reason 33:: Lot 11 was divided ané one pmrt (iib)
freed of yhoapholigida by acetone gracigit'tion. 5%13 acetone ;recigi-
tatinn Inn 3130 used by Erica, ﬁend. Bardos and Chen (lth) to concentrate
tLa nativity of the bapten.

The results of tho first assay of Lot lIb (Table VII) were very
encouraging. There anemed to ho s;ecific anti-Eh activity in the dilu»
tiqns below 133000 as detanmined by the cou;lament fixation teat. the
ncrmnl human aerum‘uaed as a central also bound can,1«ment in the ;reaenue
of antigon in the 1:1530 and lzlﬁﬁﬁ éiiutions. The serum was nonewhmt

'.31eoua and:may have haen slightly nation 3leaaniury. ”base zosiciv-

TABLE VII

COMPLEMENT FIXATION TEST
Hapten Assay Antigen Controls

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Normal Serum Controls
Eubo No. 1 2 3 1+ 5 6 7
Red Blood Cell Homolytic System Normal serum Antiserum

Antigen Dilution 1:100 1:500 121000 1:1500 1:2000 1:2500 1:3000 1:100 1:500 1:1000 1:1500 1:2000 1:2500 1:3000 Control Control 1:100 1: 500 1:1000 451.500 1:2000 132500 4133000 001113me > Control .
Qtigon 0.5 ml. 0.5 0.5 0.5 0.5 0.5 OJ 0.5 0.5 0.5 0.5 0.5 0.5 2,; «- -- 0.5 0.5 0,; 0.5 0.5 _ 0L5 0.5 -- ---
Antibod 1: 2) 0.2 ml. 0.2 0.2 0.2 0.2 0.2 0.2 - - -. .- -- -— -. -- ~— -- -- -- -- -- -- - -- 0.2
Normal Serum (1:32) -- - -- -- -- - —- - .— -- - -. —- — .— --- 0.2 0.2 0.2 0.2 . 0.2 0.2 0.2 92.2 ~---.
we -- --- -- -- -- -- --- 0.2 0.2 0.2 0.2 1.1a 9.5
21°C for 15 min.
Complement

(2 units) 1.0 ml. 1.0 1.0 1.0 1.0 1.0 1.9 1.0 1.0 1.0 1.0
ABC for 18 hours
3:10 for 10 min.
ﬁgmolysin (2 units) 0.5 ml. 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Red Blood Cells
__ (2%) 0.5 ml. 0.5 0.5 0.5 0.5 0.5 DJ 0.5 O.§ 0.5 0.5
Incubate at. 37°C until controls are clear -
giants
3:92.; ++++ I-f-4—l- +444. ++++ ++++ ++++ +++¢ ++++ +—+++ ++++ ++++
ELI; +++.— ++++ +4— —- —. -— — ‘ ++++ +++4 —— ~—
Lot. Ila ++++ +++~+ ++ — - —- - Jr... .0... ~ -
Lot 111) ++++ ++++ 44-4-1 +++—L ++++ +++.— ++ ++++ ++++ . + 4.
Mb (repeat) +++4 +.+++ - _. .1 __ ﬂ ++++ HH- Q __
lat. 11c ++++ ++++ +++¢ +++4 +++ 4— +1—+ + ++++ ++++ ++++ H—H- ++++

of, III <>‘***~'\“~J“'*‘-A +1 +++ —- — — - omlﬁecl +4 +7” ,_
M Omiﬁ‘mk “ ~ - ~ - — owﬁr‘tnc! .. —. 1
KEY + + H— 2 Ho Homolysis
.. = Complete Hemolysis

 

 

 

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i
.
. l . ' p I Q
.. __..~. ——-. —-—- -- .
l
-_ ——— - --

 

 

 

 

 

 

 

 

.gggg
.8335
.8835
.8335
.83a5

nauhaonq owdhauuqoooupaa.

£ng om
033l$
gagmo
£23... mm
3.2:... 3

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results could not be confirmd when the away was repaatal. The ligid.
IIo rocovarad from the first ole-'.whnlio filtrate was a1 :0 without anti-
Ch activity, no was fraction Ila. The nntignn groporoi by Solégwith'o
method {Lots 11! nno IV) was also inoctive.

The to tho small womat of material amenable, no £11 £3.33; tests
"to node. However, undies of Lot. 11:; and Lot 11! { 3‘15th 1) wore sub.
Jootod to ultracantrimmtion to entertains another or not any substance;
of high. mleoular night were prevent. Since no such aubotnncoo could
ho dotactod. it is Lrobnble that the material oould ho non—antigenic
L7). 13:112- 30:}: omen" gave the same type of pattern, so only one miter-n

(Lot 111.1 1! included.

Disohaelsu

It is difficult to determine whether the n;.;.-orant inactivity of the
lirid was due to a flaw in tho techni qua of tho coxylaamnt fination test.
to nor-.9 inpirity in tho rode-ants: or to s fault in tho taohniquo of ex...
trction. Fmilton and Rrooklnnd {105.0} rogortod that of fifty lots of
mtnrinl Irv-gored in thoir inbomtory. only nine ohm-rod anti-21h activity
in t‘ne c011'~lemont fixation test. 01‘30 of Hamilton's, “nag-who" miles
war-o sent to C‘r'rt-ar for assay. She rwmrted activity in dilutions of
121000. On another oonasion, ﬁoldsmith rQLOPtOd activity in dilutions
of 1:066!) in 3 91:71:19 Hamilton brad ”curiously found to has negative. It
goons rouoihlo. thereforo, that one or more of tho extracts gregorod in
this inborntory warts active, but tho activity mo not dntactar‘}. in the
bnsio of Cortar'o agony, IMmilt-on and Brochland {19303 adminintorﬁi, to
Fremont and non: remnnt lpn'dtimd woman, tho mot-axial 11-03;:er if: their
laboratory and .0319 rro-gxzred by a comraercinl ﬂoor-Worst. In no was was
be able to reduce the antiut‘h titer.

It i. not likely that the menus: of the cnmlmont fixation test
worn.- rmd incorrectly. 'i‘o augments for the ogjnci ty induced by the on.
than, and. to yrovido additional WE-lmreant controls, controls were not
or '12 in ’Pnhle VI sans! no: at the some ttzze m tho 5352.3. 5‘00 dilution
of conglemqnt was such that there was cwmzieto hamoiysis in only the lost
tube. Tho aotxml toot was read by cot01=:.xrin;: with these control tubes.

Th. fault would therefore seem to ha in the method of granting the

antigen. Since it is knawn th t the Vb f'ctor is associatad with the
strwmm of tLo red cell (Balhin an: Tianar 1943; Saivin, Tva s, Eetrundt
and Calvin 19§63. any grocaaa that would denature protein might dwatroy
thn Rh antigsn. In the mathOAa of sxtrnctiqn'uaad. either the 35 gar
cent alcnhol or the taz.erature (-ﬁo ans #95) at which the extraction!
wars o:rried out could be cagabla of dearturing yrotain. Cohn, Strong,
at al. (133%) in their methods for frfctionnting aarwm and 1133mm iro-
teins, used temperatures between 0° and -1” s and cthanol concentrations
(vnium9.;er cent at 25°C) halos be. She teaieruturo of the first tto
extrwctions was within Cohn‘l suggested range, but tho alcohol concen-
tration was almost taicn as are t as tnnt which ha uses. Hoskwwitz,
{lﬂlldilll’iui' Calvin and Iﬁvsans (1.955)) luv-n m; eater}. that the hanglohin.
which is preciLitntsd during the initial alonhai }rocipitntion in cartor's
(124;) method, may exert a irotectiva action on the Rb antigan and gre-
vant its dcnxturation by the alcohol.

“his failure to demﬁnatr“ta anti-Rh activity is in ngreament with
tLo findin a or many investigators includina Unis: (1&50). #trnttnn and
ﬁanton {1555). and others.

Jalvin's lip03rotain, eiinin. Lreparod by tater «struction of Rh.
gositivo red blood cells {Tosknwitz, Eanﬁiiixar, Calvin, can EVana 1950),
has been shown by ag-iutinatian-inhibitisn tests to possess Rh, A and 3
factors. the Eh olinin was non-antigenic in rabbits, guinea pig: and
hum-ma. However, intrmiuscular injection of 1.0 to 1.5 gram of motive
clinin failed to effect a chmngo in the titer of circulating Eh-antibody

in human subjects (ﬁvans. Yosaocitz and Calvin 1950).

PU?!" ’ f»???

1. Four nttmtu wer- mde. two by each of two methods. to extract

from Fla-positive "d blood calls a substance whi ch would inhibit tin

“h H-ntibOdy m m.

2?. “She test actuarial: were assayed by the: Home: mmgiment fixation

tnchnic as and for that taro-dinggmcis of lygiﬁlio.
3. 3‘70 specific anti-27h activity was o'baorv-«i in any of the extracts.

h. I’ltmcontrifmm pnttama of tm of the extmcts new mac.
5. 'E‘caciblo rocsnna {or the lack of activity of the xrnzmtions are

discussed.

l.

m

1'.

6.

7.

'9

C3.

”I.

10.

ll.

1?.

Bléllﬂéﬁﬁxﬁy

9

tray. 1,.3. 131+? rQVFlpj-“33ﬁr7; Loft-3:1, 5:11 God. '3. Saunders Co...
;hiladolghin.

 

illott. 33.34., and Holman, 0.3.. 19‘“? .no rhesus factor. A szxmxxry
based on 200 cases. Lancet FHQ, 2?Q.?14.

Balkin. 11.3.. and ii’ionar. A. S. 11551.4 ii-mmmstmtion of t‘io iroicrties
of 11.33.1313, and 3h in tin ran-cell Itromtc. Iron. Soc. "anti.
a101, .na ”ed. 85, 21h.217.

”roclz'lsmd, 3. 19h? Irhphmtinn and use of F1: };=.=;}.ton. hm. J. of.
Mad. Tﬂdho LL 1(9013‘00

"9‘1qu 5"“. Km”. 5‘4?” 19'9-353'F-‘niih V. cm Galvin. '3. 1":3'45 El: anti-
gm "‘33 Hi :93. lroco 500. Lil-‘10 .101. Md $1.011. it“. 151;»g19.

int-79mm, L. I’M? 1Siltnmin C (fez-Jaime; an 9 303311310 factor in tho
wthogcni us of onsthroblustouis i'etzzlis. A. J. Chat. and
Synge. & 933.991.

ant-tor. 13.8. 193.7 I. grolinizrw)’ retort on n cnpotI-z-ncc that inlxibits
ranti-Ph new. m. J. Clin. nth. LL, W099.

flatter, 8.26. 949 '3: hwtwn: its “visitation. case-x3, an.) nature.
1. of hwunal. ﬂ, 7-7....15.

Cohn, 3.3., Stronat, 1...?” Hughes. '-’v".'... Jr” ”nlfcr. Ila-1.. Ashfcrd,
J.!7., Edolin, 3*... and “it-sylor. ELL. 19W) trowmtion cm 1mg»
articl of serum and plastic proteins. IV. A system for thn sop-
Prntion into fretiona of the imtcin and. 115.0,;rotein cosmonauts
of bioloaicnl tissues and ﬂuids. J. Am. Chem. Soc. 24 #59.
~75.

Burrow, R. E. and Chain, J. I‘M] intimgenesio of passive 130961331-
t‘mtion in w, nawmmo m. 4‘ o :3130 'jlildn LL 37-6%}.

'hvidonhn. I. 19% Inter-rotation of tho {Pb antibodies. Am. J. of
93113- ;a‘ho E." 620—6-59. '
r“irsmomi, L.K. 15335 i-hgzaiochevsuical nni imrmnoioygieal char-actor of
T-‘h antibodies. 2-year mad at the international. Esmwoiosy and

Ph Conferonce. Enllﬂa. ﬁexns.

15.

16.

17.

13.

13.

m.

:7.

wilconxbo. G. 19158 Liar important is trnncfuuion m a cause of
hemolytic chance of the newborn? Brit. :‘cd. J. 2;, 339—330.

Swim, 2.5., E-‘os‘mvitz, 2.3., and Swivin. H. 1950 Studies of the
anticano of human red cells. 11 ontological progertiaa of tho
”in factor in clinic. é'roc. c~oc. “qt-.1. 3101. cm! Yam. 11,
677-542. ~

Vick, 3. T. 1133 On the significance of Hr sensitization_in Rh
antibody dotersuinctiona. 31111. J. Clin. I-e-tth. L8, [10.

goldgngth‘ J.a. 1§h3 Txiericnces with Lh.hngton. Univ. of 'inn.
Zia-d. School Bull. 23;.

:‘mldamith, J.W. 153131391 Elmer-imam: with 32h hogtcn. m. J. Chat.
and, bynec. T}... 173-171-

=301damitlg J'.‘r+'. {433-01) Exgoriences Iitn m: hnxten. Rico. 5596.. J.
an. Bax-w.

"Tiroonwoit. THY. 19'4"? lralizainnry rag-art of micriancos with F‘h
”no; ton. J. Lab. and Olin. sec. ’34, lbﬁyldﬁﬁ.

Sinker...- n, at". 191.3 "Filo demostmtion anti char-carnation of the
anti-D mrfintinin and antigen predicted by Fisher and Enos.
Eiood. .1, bad.

ﬁzzmilton, 2.". 3. and Z’mckhmd, 3'. ‘53. 1950 “Tue results of trmtmnt
“thy m bitten. .513. of. What. {Lind ‘i‘ynﬁco QB“ ELFVIQO

Song, 6., and Rustimn, ‘9... 11360 Sturiiss with the 321100?) antigvvn.

J. 0: 1131113010 2"" 5403-513.

Javert, G. '1'. We Further studies on orythroblaatocil neonautorm
of obstetrical Ii:gni:‘ica-:nce. m. J. Chat. and 37sec. ILL 9:31.

Karin". DJ. 19347 ﬁn the r-rozhlmxis of hmiytic diam” of the
newborn. m. J. of (fast. smo; wince. 5%, 1.17.

Fan-.131“, J'..«". and Boomer. E. 14913-45 MW”=%H on;
“th Odo 3. APE-“1‘03 O Cmtury ho.

   

“’o‘n-iotoinor, !., Lavina, 3%, and Joann, 1LT... 19:“! On dove-lament
of isoan-‘lutinins folio-Mn»: tr-moiuaiono. iroc. Soc. can.
1:101. and. 3-395. EV”. $73—‘Jf‘i‘o

Maintainer, Ff. 1’131 Inéividmi difforonces in 111m blood. Science

a, imeg.

28.

Lnndstalner. K. and ﬁienor, A.S. 1937 On the yreaanco of M aéﬁlnn
tinogans in the blood of unnneya. J. of Immunol. 33, 1%-:5.

Landsteiner, K. and hianér, A.S. 1?}? Th 2 agglutinogan cf rLagug
monkeys. J. of Iamzmnol. ‘32, 15-91-177.

Lavina, E”. 9nd Stat-am, $.12. 1?}? ﬁn unwzml was of intratw’ .15;-
egglutinution. J. Am. 39d. ﬁasco. 1;}, 126-137.

Levine, I. and Entzin, 3.5. 1940 lsoim$un12ﬁtion in yragnhuqy and
the vat-1:107; of isms-planning observed. lroc. 5206. Ex. t1.
5101. an: 399d. 4“... "5-15-345-

chino, f.. Burntam. L., Katzin, :.L. and $0561. i. 1%51 The roll
:3! isniwrwmﬂmtton in the gazing/3:26:53 of erywroblastoam £9-
taliao A“. J. lab-3‘0 and {Q'nQOO ‘9’?" 915.9370

Lavina, I” Kata-31:1. 32.2., and Léurnlwa, L. 193-12 Isoizrmwuzntion 1n
zregnancy: It: pos.1blo bearing on the otialng; of erythro.
blastosla fatalls. J. am. 49d. Laeoc. 115; EEJ'9970

Loving, P. 19kg 1301m$un139t10n by ﬁne an factor. #03: érﬁd. Red.
§,h51-h88.

nought-y. J. and Car-tar, 3.8. 11,345 Tun-or". of a case of lrytézro‘ulru-
touts recall. trebtad with an hatten. Am. J. Obst. ani Gynac.
55, 1951-1052.

”330?. ¥.ﬁ., and ﬁuler. A.}., Piar, 0.}.. nnﬁ Felﬂﬁlbarsar. lvhé
Th0 activating; effect of ﬁg and. other c‘ltions on $2.. hamlg‘tio
function of comgwmt. J. 131x; :1. Fed. Eu. 535.598,

"'c“‘zz.rd:;-'. I*i’c.".’3. 1750 '1’": T—szarn; «pm-35:. Eillirm Y‘ainezwmn. 173.331.1321
Book. Ltd. London.

ﬁo'knwitm' ”'- Ymndilikar, ﬁ-o levin, a.. and :vann, R. 1650
Etudiqa on tha antiﬁans 0f hmnun red 03113; I “ey‘rntion
frnm-hummn erythrocytes of a wa‘e: soluble fr ctian owntwin.
1m: tics Eh, A. and 3 fzaabnra. J. of Its: 2.4301. 9:" $3.339.

"'Ounmt, AJZ. 1‘31“; A new Emma antibody. 23?“th 1"”, 5132—54993.

"1'? ca, (3173.. ”and, $35., Farina, 13.7., ram? (ken, C. 17334-53 15301361031
of a substance with Rh Macon activity. J. Am. Chem. 5-00. m.
3577-359’5- '

Page, 3.3. lﬁhh An ”1ncoa;let¢" antibody in human ssrum. ﬁaturc

1.5.3.. 771-

51.

31

”ace, 2.”.. and “wylor, G.E. 10%“ The incom;1ats antibody in 2h
immunization. Brit. Wad. J. 2, 75b.

*nco. R.W.. Vaurﬂnt, A.’.. and ﬂmliandnr, S. 1946 Eh antigen and
“ntibndiea in man. ﬁvtura gig, hlﬂ.hll.

‘truttsn, ?., ani ﬁenton, ?.V. 1°?“ *ha ﬁh hngtun. Lancet P“?, 390.

“33:91., LHT. 11hr! otU’EIQS on fraY‘ﬁHti-qn and cur-gti‘lﬁ tFGﬂtﬁi‘PTﬂt {-9,
Yb Iansitization. km. J. Chat. and agnec. ﬁ?, 1155-120”.

?nn Lagkom, J.J. 19kg Ereliiinqry nwta on thn influanca of hstérn-
s,ecific immunimatinn on tn; grﬁiuctinn of En unbihcﬁiaﬁ.
nr:‘. 1.9355. J. 5:. B‘s-37o

’iener. A.3. 19h“ A.new t9=t (bincgina toat‘ for :-‘h aqnsiti’wtion
9'

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‘iener, A.?. ¢h5 ?‘aary and navan lature of the Eh blonﬂ frctwra
.‘Tcienca “‘3 hyg.l.:-‘Q.

'iener. A.*., ané €onn. ?.?. 1&46 Ext 0 aneaia of conénntial ham.
ulytic 6143.53. II Illnwtrhtiva cxwa hiSﬁﬁfiﬁB nf Eh aanui-
tization. #4. J. Via. $111. 1;, 65-30.

'1639r. A.?.. 13%8 Intrﬁgrﬁur 1EC°hrntibilit¥ with resyect to tho
fr bloﬁd fwctors as a onuaw af minor’hamclytic transfusion
reactions. 3. Lﬁh. ¢né Wiin. 'sd. 3}; 950-997.

1030?. 1.53., Mid 3.3.119!“ Io}. 113...: SianQn-‘vt-"Ll 39100510110: dODOI'B
7°? Bxcﬁﬁnxe irnxtfuziqn in errthrnbluateais fatvlis.
Anaat.oaiolcgy 3; 2~0_A09. a

‘ienar. A.S. 19%? The “h factnr. Brit. Van. J. 3, 584.285.

“ianar, A.S. 1950a Irauent status of tag Rh-Kr blood tyges.
”rit “93. J. L, 813-114. ”

”i3uer, A.?. lnﬁﬁb 7% ﬁloaqpry. Lab. ‘ig.

 

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