The Diagnosis of Thalassemia Trait by Starch Block

Electrophoresis of the Hemoglobin

By PARK S. GERALD ANI) LOUIS K. DIAM0Nn

T I1AIASSEIIA
anemia associated
trait
with
(thalassemia
abnormally
minsor,
shaped
nsicrocythemia)
erythrocytes
is a hereditary
characteristically
of a reduced average cell volume.’ It is the conisensus of opinion that this syni-
drome is the heterozygous state for the getsetic defect which ins the homozygous
individual results in the severe ansemia knsown as thalassemia major.’ Despite the
generality this
of belief, it is recognized that ansensias otherwise indistinguishable
from thalassemia have beens enscountered with clinical course instermediate ins
severity between the extremes of the major ansd minor disease, ansd hensce of
doubtful classifications as to genetic status.2 This divergence from the simple
clinical dichotomy predicted by genetic theory has led to the postulations of
gensetic and environmental nsodifying factors and to speculations conscernsimsg the
existence of variants of the thalassemia gene.2 Such theorizing, however, has
been as fruitless as it has been frequemst, largely due to the lack of a satisfactory
criterion for the diagnosis of “thalassemia trait.” Ins the absensce of a suitable
diagtsostic test, a homogeneous group of case material has not beens definsable,
nor the significansce ansd cause of the clinsical variations assessable.
Recemstly, a major advance ins knsowledge
our of thalassensia was nssade by
Kunskel and Wallenius who observed a characteristic alteration ins the hemo-
globin electrophoretic patterni in thalassemia heterozygotes.3 With their technsic
of hemoglobins electrophoresis ins a layer of starch prepared from a settled suspen-
sion, they were able to show that a small fraction of the hemuoglohimi ins all
normal individuals can be separated from the major portions. Inn thalassemia
trait patients the amounst of this minsor fractions was nsoted to be signsificanst.ly
increased above the normal value.
The present report details the results obtained with this technsic, ins additions
to conventional hematologic measurements, in a study of a group of adults with
thalassemia trait who are also the parents of children with classical thalassemia
major. An increase in the minor hemoglobin fractions ansd a reduced nseans cell
(erythrocyt.e) volume were found to be constantly presenst ins this patienst group,
and are suggested as beinsg suitable nsinsimum diagnostic criteria for thalassensia
trait.

M ETHOI)S

The patients studied were the parents of children with thalassemia major whso.se records
were in the Hematology Clinsic of the Children’s Medical Center. The diagnosis of thalas-

From the
1)epartment of Pediatrics, Harvard Medical School and the Children’s Medical
Censter, Boston, Mass.
This work was supported in part by Grant no. H-2405 from the National Institutes of
Health.
Submitted Aug. 7, 1957; accepted for publication Sept. 15, 1957.
* This technic is to be (listingulished from the starch gel electrophoretic method of
Smithies.4

61
62 1)IAGNOSIS OF THALASSEMIA TItAIT

semia Issajor was based ispni evidence of a severe, chronic, hsensolytic anemia hsaving onset
in childhood (buit after birth), amid associated with abnsormal ervthrocytes of a characteristic
appearance,5 the presensce of nsuncleated erythrocytes in the PeriPheral circulations, hepato-
splensomegaly, and nsarkedly elevated levels of alkali
hemoglobin. resistaist
The possibility
of ams abnsornsal hsensoglobins synsdronse ins these childrems was elimiflate(l in most cases by
electrophoresis of thse hensoglobins. The frequency of transsftzsion in several cases rendered
stu(lV of ensdogenotss hensoglobins iIsap)licable. In all .suschi insstances the hemoglobin of
both parents was examinsed an(l the absensce of an abnormal hsemoglobin trait in them was
verified.
Total hensoglobins wa.s determined pisotonsetrically as oxyhsensoglobin, erythrocyte coumits
were (Ionic with consventionsal techsnics, and the hsensatocnit was estimated using either a
macro or a microhsensiatocrit nssethod.6 ( )unr nsornsal valuses for red cell indices correspond to
those givens liv Winstrobe (82-92 cii. microns).7 Alkali-resistaist hemoglobin was measusred
by tise nsethod of Sinsger et al.5; values above 2.0 len censt are conssidered abnormal. In many
instalsces the routinse hiensatologic examssinationss had beens performed at the time of the
insitial (liagnsOsis of thalassensia in the family, and the alkali denatmnratmon and electro-
phonetic stmn(hies were (Ionic OhS more recenitly obtained specimens.
Elcctrophoretic fractionation of tise hemoglobin was performed usimsg a modification of
the technic (leveloped by Kunmskel amsd Wallensius.3 Subseqmient to their pmsblication, cyan-
mnethennoglobins was fotnnsd to be qunite stal)le mnnsder these cons(hitions’ and has been used rou-
tinsely by uss ins thse presenst insvestigsstions. Their original methodology was modified by
omitting the coverinsg over the starch block; the block was instead enclosed in a humidity
chamssber. The evaporations occmnrning umsder these co,sditions l)rodmnced an evaporative flow
of busffer, cotsnster to the direction of electrophoretic migration. The ne.smilting countercur-
rent actions facilitated separation of the hemoglobin components. The electrophoretic pat-
terns associated witis several consmons hsemoglobinopathies as obtained with this technic
are illustrated ins figure 1. Althomngh not illustrated, alkali-resistant hensoglobin in amounnts
greater thans 5 to 10 per censt is visible ins the starch electropisoretic pattern as a trail behind
the major consponenit. Thse niormal adult hsemoglobims patterns (at pH 8.6) shows three cons-
ponensts: (a) (lie misajor component (also knowns as A,); (b) the fast minor component mi-
gratimsg to the anode just in advance of A, and producing the characteristic “pointed”
shape of the zonse elect nophoretic patterns; and (c) the slow minor component with mobility
comparable to hensoglobin E. This E-hike hemoglobin, designated the A2 component, is the
portions characteristically increased in thalassemia trait. After completion of electrophore-
sis, each hensoglobims patterns was divided midway between the A, and A2 components and
the separated portions dusted individually by washing on a sinstered glass fu,nnel. The con-
centration of hemoglobins in the eltmate was then estinsated spectrophotometnicaily and the
A2 fraction calculated a.s per cent of the total hemoglobin. All analyses were done in dupli-
cate. The difference betweeni paired analyses, expressed as per cent of their average value,
was independent of the A2 level. The average discrepancy was found to be 8.7 per cent (based
on 50 pairs), ansd by calculations, the standard deviation of the mean of paired analyses was
estimated as 3.8 per cent of thse mean value. The nsormal range was established with a group
of 20 healthy admnlts (laboratory personnel) who were studied dmnring the course of this
investigations. Their A2 fractions (the result of single ansalyses omsly) ranged from 1.7 per
cent to 3.1 per cent, with an average of 2.4 per cent.

RESULTS

The results of the electrophoretic and hematologic analyses in 23 unrelated

adults, all parelsts of childreis with thalassemia major, are given in table 1. Al-
though the majority are descendants of Italian immigrants, a few claimed an-
cestral origins from other counstries, including Central Europe, France, and
Greece.
One of the group (a putative father) had a nsormal hemssatologic picture and an
A2 level in the normal range. Subsequent blood grouping tests gave consclusive
 PARK S. GERALD AND LOUIS K. i)IAMONI) (13

Thai. trait

Hgb C trait

0 Normal

1-1gb S trait

CA2 S A1
Anode
FIG. 1.-The starch block elect rophoretic pat termss of cyansmethemssoglobins, iii veronsal
buffer (pH 8.6, ionic strength 0.05). Du,rations of runmi, 22 hirs.

proof that this mans was not the gensetic father of tise child with thalassesssia
(exclusion was based upons homozygosity for the c factor ins the putative father-
aisd absence of this factor its the child as well as developnsent of anti-c followinsg
repeated transfusionss). (See case 4, table 1.)
The A2 levels ins table 1 range frons 3.3 per cemst to ans extrense of 6.8 per censt.
If this range is due to a ratsdotn distributions, then conssiderable overlap with
normals can be expected, atsd the diagisostic value of the test is lessensed. Close
inspection of the distributions of the A2 value insdicates, however, that a discon-
tinuous rather thans a random distributiots is presenst sinsce there is a defllsite gap
64 l)IAGNOSIS OF THALASSEMtA Tlt.’tIT

TABLE 1 .-Hematologu Findings in the Parents of (‘hildrcn with Thalassemia Major

Alkali-re- A2 Content
Propositus Parent
(GmIiOO ml.)
MCHC (fl;) Retics () sistant Hgb ( total
(%) Hgb)

Case 1 Fathser 13.6 58 32 1.6 4.6

Mother 9.3 62 29 3.2 5.2
Case 2 Father 12.1 56 30 1.8 1.9 4.8
Mother 11.4 80 29 2.1 3.6 5.0
Case 3 Father 14.0 64 33 1.9 5.1
Mother 9.4 68 31 2.6 4.5
* * * * *
Case 4 Father
Mother 11.3 67 29 2.9 4.0 4.8
Case 5 Father 13.6 68 32 2.5 5.3
Mother 10.5 65 32 3.0 5.3
Case 6 Father 15.1 67 32 1.7 4.5
Mother 13.6 79 32 1.6 3.6
Case 7 Father 12.5 57 34 1.7 3.8
Mother 14.0 65 37 0.2 3.3
Case S Fatiser 12.8 80 30 0.9 3.0 4.7
MoUser 10.3 76 32 0.6 2.0 3.4
Case 9 Father 14.0 65 32 1.3 2.0 5.1
Mother 11.4 73 31 1.4 4.8
Case 10 Father 12.3 65 31 2.3 2.7 4.5
Mother 11.0 70 31 3.0 1.8 4.8
Case 11 Fat lien 12.8 72 30 2.4 5.3
Mother 12.5 68 32 3.6 6.8
Case 12 Father 14.3 70 34 4.9
Mother 11.8 67 31 4.4

* See text (Results).

Frequency Distribution

.
#{149}. .
I I #{149}I
II I I IIIIIIIIII I

I I I I I

3.0 4.0 5.0 6.0 7.0

Hgb A2 Content % total Hgb)

FIG. 2.-l)istnibution of valunes for Hgh -‘.2 contenst ins parensts of childreni sviths thalas-
sensia misajor. Thse points correspond to the data given in table 1.

from 3.8 penS tnst to 4.4 penS ceist (fig. 2). The sigtsificamsce of this gap was further
attested vhens serial ansalyses ins four insdividuals, represensting a wide ransge of A2
constenst, showed essetstially nso change over periods varyinig frosss 3 to 18 weeks.
Such di.sconstinsuity suggested tise existence of gemsetic factors, and, accordingly,
 PA1tK S. GERALI) AND LOUIS K. DIAMONI) 65

6 oo%

0
5 0
a)
-o
E
4 0 0
z
1)
3 I 0

a)
2 10

I 10

3o 4.0 5o 60

Hgb A2 Content (% total Hgb)

FIG. 3.-The intrafamilial corresponidence of Hgb A2 valunes ins affected mensbers of
thalassemia trait pedigrees. I Proposituss; parenst of child with thsalassensia Issajol. 0 Pro-
posituns; clinical thalassemia trait. 0 Thalassensia trait relative of propositus. (Each poinst
is the average of duplicate ansalyses.)

fanssily studies to assess the instrafaissilial correlations of the A2 fractions ins thalas-
semia trait nsembers were l)egun. rfo date, six thalassenssia trait pedigrees have
beens examinsed, including a total of 14 affected mssensbers. The A2 value ins tise
affected nsensbers was very simssilar withins insdividual pedigrees (fig. 3). The wide
distribution of A2 values inn table 1 thus appears to he the comssequemsce of insciud-
ing genetically different groups of thalassenuia trait. Withins each group, the
hensoglobiss fractionsation results fall witisins a nsarrow range. This justifies draw-
ing a sharp distinctions between nsornual inidividuals ansd the group of tisalassenisia
traits with slightly elevated A2 values.

DISCUSSIoN

Ani attenspt to deterlssine nssinsinsunn diagnostic criteria for “thalassenssia trait”

insplies prior knsowledge of the definitions of “tisalassensia trait”-ans obvious
coistradictions. As a practical solutions to the problenss, we chose to study tise
most honsogenseous forns of a thalassensia synsdronse with which p were ac-
quainsted-namely, tisalassemia nssajor. If onse assulsses that thalassensia major is
the result of homozygosity for the thalassemia gense, tisens tise parents of such
children should all be affected with thalassenssia trait. As represensted by the
group of parents imscluded ins table 1, the heterozygous state for the thalassenssia
6(1 l)IAGNOSIS OF ‘rHAL.ssEMmA TRAIT

genie is insvariably associated with a reduced meant cell volume ansd an increase
ins theA2 fractions. ilonuogenseity with respect to both of these criteria lemsds
en-edensce to our belief that these cases are similarly homogeneous ins etiology.
Although the data givens above are ams adequate basis for insitiatinsg the hypoth-
esis that ans elevated A2 level is a nsecessary criterioms, they are inadequate for the
evaluations of the sufficiency, i.e. the pathogniomoniic value, of the test. Coiscur-
renstly witis the presenit study, several humsdred blood specimemss fronss pediatric
amid adult patients with assorted hemsiatologic amsd other diseases were examined.
Ins ever’ insstance of ini(reased A2 constetst for which hensatologic data were avail-
able, tise presumssptive diagnosis of thalassenssia trait could be nsade. Josephson,
however, has reported finsdinsg ati inscreased A2 value ins pernsicious amsennia in
relapse.” Ansy possil)le consftmsions with this disease would, of course, be prevented
lr (letem’Iflilsationl of the nsseaii tell volunsse.
flse rather snssall difference in A2 values hetweeis sonsse thalassemnia trait
l)atiensts amid soisse nsornnals is of (‘onisideral)le insterest. The clinical comssequemsces
of the presemsce of tise thalassemia gelse cant scarcely be the direct result of the
altered proportions of the hensioglobini (-onnponlensts, unsless a threshold value
exists for the effect of this alterations. Chimsically, we have nsot beens able to classify
the heterozygotes listed in table 1 nsor their progeny,
, ins ansy way to corresponsd
with tise grouping accordinsg to A2 level. If such a threshold exists, it would have
to he \‘ery nsarrow insdeed for the “low-valued” heterozygotes to exert their full
climsi(al eflect The altered henssoglobin
. fractioniations patteris observed ins per-
ssicious anensia ins relapse’0 likesvise argues againsst a close relations between
climsical symptomuatology atsd the disturbed hensoglobin symsthesis. From this ve
(OlsClU(le that the effect of thalassenssia ilponi the synsthesis of nsormnal henssoglobins
(-onssponsemsts is a se(onsdary onse.
Although mnicrocytosis is gemierally conssidemed to he a tssansifestationi of thalas-
selssia trait,’ absensce of nssicrocytosis has heems nsoted occasionsally ins pedigrees of
ansenssias ulinsicallyresensshlinsgthalassenssia. With tise discovery of the combitsed
abmiornual henssoglobims syndronnes, omse explamsat ion for these exceptions became
available. This explansation, however, has nsot sufficed to accounst for all excep-
tionss. Ins a nunssber of published pedigrees, thalassemssia has beens diagnosed in the
absenice of issicrocytosis whens other properties consmonsly attributed to the
thsalassenssia genie, suds as inscreased osmotic resistansce, increased number oh
target cells, or imsteractions with ahnsormal hensoglobin trait, have been pres-
enst.”’5 Admuittedly, nsonspensetransce of nsicrocytosis due to thalassemia has
apparemstly beeni observed’6 ansd has beets stressed as a possible explansation for
suds anomalous pedigrees.2 Nonpensetramsce does nsot seem reasonsable, however,
wheni noise of the so-called thalassensia trait nuesssbers of a family exhibit micro-
cytosis. We ourselves have not l)eens al)le to find any instansces of nsonpensetranice,
despite a rather instenssive search. Conssequeistly, we feel that tssany of the normo-
cytic abssormalities labelled “thalassenuia trait” are unknowns hereditary erythro-
cytic defects awaitinig identifications.
rfhe very recenit article by Singer et al.,’7 ins which a patient with “thalassemia
nsinsinsa” l)ut without nsicrocytosis is described, is most provocative. Ins this
paper, they report on a Negro family ins which the father has Hgb C+S disease
and the nsother has “thalassensia nsinsinsa.” Among their progensy are onse case
 PAItK S. GERALD AND LOUIS K. t)IAMONI) (17

each of ‘11gb S-thalassensia” and “Hgb C-thalassenssia” disease. The diagnsosis

of thalassemia minima ins the mother was established by the finsding of “some
ovalocytosis ansd the presence of nunsserous target cells (26 per cent) ansd by the “

elevation A2 cotstenst
ins (9.2 per (P1st !) The absetsce
. of nsi(rocytosis ins the pres-
ence of ams itscreased A2 is at variansce with our flisdinsgs. Tise explanations nssay lie
in the following unsusual features of this patietst : (1) the highs frequenscy of target
cells (its a survey of 20 thalassemia traits,’8 only one was foinnsd to show a conu-
parable tsumber of target cells; the rensaininsg 19 had but a few per (emit) ; (2) tlse
very high level of A2 (compared to a nsaximunu of 6.8 per (enst ins our series) ; ansd
(3) the racial origin (nso Negro patiensts were inscluded ins our series) As has beens .

suggested by Zuelzer et al.,2 there is clinsical evidence to insdicate that “thalas-

semia” ins Caucasiamss nsay differ fronu that observed ins Negroes. If we are correct
ins cotscludinsg that the elevation ins A2 constenst is onsly seconsdarily related to the
basic disturbansce us thalassemia (as we have defined the disease), thens it is
quite consceivable that variansts of thalassennia may be associated with ans dc-
vated A2 Value, but be otherwise distinguishable frotu tlsalassenssia trait.
This emphasis on the presence of nuicrocytosis ins tlsalassemia trait Issust nsot
be construed as indicating that a reduced nseans cell volunse is a sufficient (riterioni
for the diagnosis. During the preseist study of thalassenssia, two varieties of
hereditary nsicrocytosis electrophoretically distinsguishable from thalassenisia
were encountered.’9 Since they serve to illustrate the value of starch block
electrophoresis, a brief accounst is appropriate. The first of these was discovered
ins the fansily of a patienst with Hgb H disease. Onse parenst of the patienst was
hematologically msormal and the other resenssbled thalassemia trait--ill (015-

fornsity with published reports of this disease.2#{176} rFhe henssoglobins fractionsations

patterns of the microcytic relatives of the patietst revealed that a nsormal level of
the A2 conspotsenst was presenst, as opposed to the inscreased value founsd its
thalassemia major pedigrees. On the basis of this finsdissg, the hereditary defect
has beemi classified as a noms-thalassemic hereditary microcytosis.
A second microcytic abnormality transsmitted as a donssinsant characteristic
occurred ins the family of a child with classic thalassenssia nsajor. The tssother
and four of her relatives possessed nsicrocyt#{149}ic erythrocytes, anid ots electro-
phoresis of their blood a nsew abnornsal hemoglobins presenst ins low conscenstrations
(10 to 12 per censt of the total pignsent) was discovered. Subsequently, the henuo-
globin of the propositus was also found to contaims a small ansount (5 per censt) of
the abnsormal hemoglobin in addition to the increased alkali-resistamst fractions
(80 per cent). Both of these hereditary nsicrocytoses will he described inn detail
ins the near future.
Now that means is available for nsakiisg a positive diagmiosis of thalassenssia
trait, use of the term should be limited to those cases which fulfill the minsinsuns
diagnostic criteria given above. Variants of thalassemia synsdronsses, in particular,
should be reinvestigated to determimse if they are not actually nonsthalassensic
diseases, two of which have already beens recognized.

SUMMARY

Starch block electrophoresis of the hemoglobins has beens performed for a group
of adults who are the parents of childrens with thalassensia major. The henso-
(38 I)IAGNO5IS OF THALASSEMIA TRAIT

globimi electrophoretic l)atterml was founsd to he (onsstantly abmsormal ins this group,
ins that the nssinsor conssponenst with E-like nnohility (designsated the A2 consponsenit)
constituted a greater than nortssal proportions of tlse total hemoglobims. Reduced
nueans (dll (ervthrocvte) volunue was likewise foumsd to be presemst ins all mensbers
of the l)atielst group.
If it is assimnssed tisat typical childhood thalassemia major represensts the
honisozygous state for the thalasseissia gemse, thens the patiesst group studied cons-
stitutes a populations of adults heterozygous for thalassemia. Sinsce Inicrocytosis
and am inscrease in the A2 (onstelst were conistatstly present in this group, they are
suggested as sinitable nssinsinsiunss diagnsostic (riteria for thalassemia trait.
The degree of elevations of the A2 fractions was nsoted to have a disconstinsuous
distributions. Prelinsinsary studies have denssonsstrated that the degree of elevations
appears to l)e i(lenstical ins affected nsenuhers of sinsgle pedigrees. The discoistimsuity
ins distributions observed is thus apparemitly umsder gensetic constrol.
The rather small differensce ins A2 contenst hetveen some normal amid sonse
thalassenssia trait adults inuplies that the alterations of hemoglohims synsthesis is a
seconsdarv l)henionsselsons, 51l5(C 511(15 “low-valued” thalassensia traits were Isot
different ins tiseir (linsi(ntl expressions. I)espite the smnallnsess of this difference,
ouatstitationi of the A2 fractions sufficed to distinsguish these “low-valued” thalasse-
155 UI heterozygotes fronu nsormssal inidividuals.

SUMMAItI() IN INTEItLIN(UA

Electrophorese de lsensli)gloh)imsa per nssedio del technsica a hioco de ansylo esseva

effectuate pro ums gruppo de adultos qiti es le parenstes de patieistesde thalassensia
nssajor. E55eVt trovate (isle le (onsfigurations electrophoretic del hemoglobinsa
esseva utsiformssemssemste atsornssal its iste gruppo, i.e. le componsente nuinsor cots
nssohilitate sinssile a E (desigmsate
comO conspontenste A2) consstitueva un proportion
plus que nsornssaldel henssoglohinsa total. Uns reducite volunsine cellular medie (de
erytlsrocytos) esseva etiamss colsstatate insonssnse menssbros del gruppo.
Si tsos supponse que typic tlsalassenssia nsajor de juveniles representa le stato
honssozygotic pro le gent de thalassensia, il seque que le gruppo de insdividuos hic
studiate nonstitue uns population (IC adultos heterozygotic pro thalassemia.
Proque nsycrocytosis e uns augnssemsto del constensto de A2 esseva uniformememste
presenite its iste gruppo, il es suggerite que ille duo factores represensta un con-
venubile mimsimo de criterios diagnostic pro le tracto de thalassemia.
1’sseva nsotate que Ic grado de elevatiols del fraction A2 habeva ums distribution
discont insue. Studios I)relinsinsari ha denssonsstrate que illo es apparenitemenste
idenstic inn afficite nssensshros del nssesnse arhore gemsealogic. Assi le discontinuitate
del distributions que ha essite observate pare esser genseticamente determinate.
Le satis mssicre differenstias del contensto de A2 in certe adultos normal comparate
(011 certe alteres con le tracto de thalassemia significa que le alteration del
synsthese de hensoglobinsa es uns phenomemso secunsdari, proque tal casos de tracto
de thalassensia a “valor basse” nsons differeva ims br expressions clinic.

REFERENCES

l)AcIE, J. V.: The Hensolytic Anaemias. Congenital and Acqunired. New York, Grune
& Stratton, 1953.
 PARK S. GERALD AND LOUIS K. DIAMOND 69

2 ZUELZER, W. W., NEEL, J. V., AND ROBINSON, A. R. : Abnormal hennoglobimss. In TOCAN-

TINS, L., Ed. : Progress in Hematology. New York, Grune & Stratton, 1956.
3 KUNKEL, H. G., AND WALLENIUS, G. : New hemoglobin in normal adult blood. Science
122: 288, 1955.
4 SMITHIES, 0. : Zone electrophoresis in starch gels: Group variations ins the serunns proteins
of normal hunman adults. Biochem. J. 61: 629, 1955.
5 BLACKFAN, K. D., DIAMOND, L. K., (lie Blood
AND LEISTER, C. M. : Atlas of
ins Childrens.
New York,
The Commonwealth Fund, 1944.
6 MCGOVERN, J. J., JONES, A. R., AND STEINBERG, A. G. : The hematocnit of capillary blood.
New Eng. J. Med. 253: 308, 1955.
7 WINTROBE, M. M. : Clinical Hematology. 4th Ed. Philadelphia, Lea and Febiger, 1956.
8 SINGER, K., CHERNOFF, A. I., AND SINGER, L. : Studies on abnormal hemoglobins. I.
Their demonstration in sickle cell anemia and other hematologic disorders by nseans
of alkali denaturation. Blood 6: 413, 1951.
9 KUNKEL, H. G. : Personal communication.
50 JOSEPHSoN, A. : Discusssion given at the Conference on Hemoglobins, sponssored by the
National Research Council, Washington, D.C., May 2-3, 1957.
“AKSoY, M. AND LEHMANN, H.: Sickle-cell-thalassemia disease ins sousths Turkey. Brit.
Med. J. 1: 734, 1957.
12 ERLANDSON, M., SMITH, C. H., AND SCHULMAN, I.: Thalassensia-lsemssoglobins C disease
in white siblings. Pediatrics 17: 740, 1956.
‘ HUMBLE, J. G., ANDERSON, I., WHITE, J. G., AND FREEMAN, T.: A family illusst rating
the double inheritance of the sickle cell trait and of Mediterranseans anaensia. J. Clin.
Path. 7:201, 1954.
‘ NA-NAKORN, S., AND MINNICH, V.: Studies on hemoglobin E. III. Homisozygous hsemoglo-
bin E and variants of thalassemia and hemoglobims E. A fansily stindy. Blood 12: 529,
1957.
16 ZUELZER, W. W., AND KAPLAN, E.: Thalassemia-hemoglobin C disease. A nsew synsdrome
presunmably due to the combination of the genes for thalnussemssia ansd hsemssoglobims C.
Blood 9: 1047, 1955.
16 KAPLAN, E., AND ZUELZER, W. W.: Erythrocyte survival studies ins chsildhood. II. Stundies
in Mediterranean anemia. J. Lab. & Clin. Med. 36: 517, 1950.
“ SINGER, K., JOSEPHSON, A. M., SINGER, L., HELLER, P., AND ZIMMERMAN, H. J.: Studies
on abnormal hemoglobinss. XI!!. Hemoglobin 5-thalassemia (lisease ansd hemnoglobims
C-thalassemia disease in Siblings. Blood 1: 593, 1957.
i8 KAPLAN, E., ZUELZER, W. W., AND NEEL, J. V.: Fuirther sttmdies on hensoglobins C. II.
The hematologic effects of hemoglobins C alone and ins combinsation withs sickle cell
hemoglobin. Blood 8: 735, 1953.
‘ GERALD, P. 5.: Starch elect rophoresis of hemoglobin: Findings in tisalassensia synsdnonses.
Presented at the Conference on Hemoglobin, sponsored by the Nationsal Research
Council, Washingtons, 1). C., May 2-3, 1957.
20 MOTVLSKY, A. G.: Gemsetic ansd haematological signsificance of hsaensoglobins 11. Nature
178: 1055, 1956.