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Blood, Vol 64, No 2 (August), 1984: pp 329-340 329

REVIEW

Advances in the Prenatal Diagnosis of Hematologic Diseases

By Blanche P. Alter for the WHO International Registry for Prenatal Monitoring of Hereditary Anemias

T HE MODERN ERA ofprenatal testing for hema-

tologic diseases began in 1974. Prior to that time,

the only test available consisted of determination of

fetal sex following amniocentesis in cases at risk for

sex-linked disorders, such as the hemophilias. Methods

for obtaining fetal blood in utero in ongoing pregnan-

cies were initiated in 1974, and these developments led

to the possibility of prenatal diagnosis of any blood

disorder that was expressed in utero and for which the

assay could be miniaturized. The first group of diseases

for which such testing was utilized was the hemoglo-binopathies. Recent advances in molecular biology

have now resulted in a return to the use of amniocente-

sis for many of these prenatal tests (see Orkin’ for a

recent review), although sampling of fetal blood

remains the method of choice for detection of hemato-

logic diseases in utero for which deoxyribonucleic acid

(DNA) probes are not yet available.

The World Health Organization (WHO) supports

an International Registry for Prenatal Monitoring of

Hereditary Anemias, which attempts to document all

cases examined in utero by fetal blood or DNA studies

for hematologic diseases. This Registry is maintained

on PROPHET, a computer resource sponsored by the

Division of Research Resources of the National Insti-

tutes of Health. Analyses of the Registry data are

complete for the period of June 1974 through Decem-

ber 1982 and will be referred to here.

Prenatal diagnosis of hemoglobinopathies was last

reviewed two years ago,2 and relevant references maybe found there, as well as in a detailed chapter.3 Theprevious review cited data from the Registry that

ended in March 1981 and included 1,856 cases that

had been studied by fetal blood analysis because of therisk of hemoglobinopathies. The current Registry

includes 4,1 33 cases studied for hemoglobinopathies,

or more than 2,000 additional cases, as well as more

than 300 cases investigated for other hematologic

disorders. A total of 4,47 1 prenatal studies were

reported through December 1982. The centers partici-

pating in the Registry are listed in Table 1 , and the

physicians of record at each center are listed in the

Acknowledgment section. All but the Scandinavian

centers began with examination of fetal blood in casesat risk for thalassemia. Several have now begun to

examine the DNA in such cases, which is obtainedcurrently by amniocentesis and, in the near future, will

be obtained from chorionic villi. Some centers have

also extended their studies of fetal blood to include

disorders other than hemoglobinopathies. This review

will summarize the major techniques and results

reported to the Registry.

FETAL BLOOD FOR HEMOGLOBINOPATHIES

Fetal Blood Sampling

Fetal blood can be obtained during the midtrimester

by several methods. Most of the procedures are done

with local anesthetic. The first technique involves

aspiration of blood from the placenta under ultrasound

guidance.4 A long 20-gauge spinal needle is introduced

transabdominally into the placenta, and several small

samples of blood are aspirated. The red cell size

distribution of the samples is analyzed immediately

with a Coulter Channelyzer (Coulter Electronics,

Hialeah, Fla) to determine the proportion of fetal cells,

because fetal erythrocytes have a mean corpuscular

volume (MCV) of 140 fi, while adult cells are less than

100 fl.5 As soon as an adequate sample is obtained, the

placentocentesis needle is removed. This method often

produces samples of mixed fetal-maternal blood (see

Fig 1), which can be enriched in fetal cells (see below),

and usually suffice for globin chain synthesis studies.

Samples with maternal and/or amniotic fluid contami-

nation are not optimal for determination of fetal blood

counts, coagulation factors, or many other examina-

tions.

A more successful technique for obtaining pure fetal

blood is fetoscopy.6 A fiberoptic endoscope (Dyonics

Needlescope, Dyonics mc, Andover, Mass) is inserted

transabdominally into the amniotic cavity, and a sam-

pie of fetal blood is taken under direct visualization by

passing a long 27-gauge needle through the sidearm of

the fetoscope cannula. Specimens were initially

obtained from fetal vessels on the chorionic plate,

which were punctured and bled into the amniotic fluid,

From the Polly Annenberg Levee Hematology Center, Depart-

ments ofMedicine and Pediatrics, Mount Sinai School of Medicine,

New York.

Supported in part by the World Health Organization, NIH grant

HL26132. and an Irma T. Hirsch! Career Scientist Award.

Submitted Nov 14, 1983; accepted March 16, 1984.

Address reprint requests to Dr Blanche Alter, Division of Hema-

tology, Mount Sinai School of Medicine, One Gustave L. Levy

Place, New York, NY 10029.

© 1 984 by Grune & Stratton, Inc.

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330 BLANCHE P. ALTER

Table 1 . Center S for Prenatal Diagnosis of Hemat ologic Diseases

Fetal Blood. Fetal DNA. Fetal Blood.Hemoglobin- Hemoglobin- Other

Country City opathies opathies Diseases

Australia

Canada

Melbourne

Queen Victoria

Royal Women’s

Sydney

Montreal

Toronto-Hamilton

+

+

+

+

+

+

+

Cyprus Nicosia +

Denmark Copenhagen +

England

France

London

University College

King’s College

Oxford

Paris

+

+

+

+

+

+

+

Germany Munich + +

Greece Athens +

Israel Jerusalem + +

Italy Ferrara

Milan

Rome

Sardinia

+

+

+

+

+ +

Sweden Malmo +

United States Baltimore

Boston

New Haven

NewYork

San Francisco

+

+

+

+

+

+

+

+

+

from which the blood was aspirated. Recent modifica-

tions have led to blood being drawn directly from the

umbilical vein at the placental insertion of the cord.7’8

These samples contain pure fetal blood, with neither

maternal blood nor amniotic fluid contamination, and

are suitable for all hematologic tests involving cells,

serum, or plasma. Figure 1 compares the proportion of

fetal cells obtained by several centers by fetoscopy andby placental aspiration. More centers use the former

method, and the weighted mean percentage of fetal

cells is 96%, compared with 56% by aspiration.

A recent modification has used ultrasound without

‘4

FFTAL

lee.

80.

6e.

4e.

20.

0.

0.

FETOSCOPY ASPIRATION

2.

Fig 1 . Percentage of fetal red blood cells in samples obtained

by fetoscopy (�) or aspiration ( � ). Each point represents themean value reported by each center. Closed symbols indicate less

than 40 cases and open symbols more than 40 cases.

fetoscopy to obtain pure fetal blood from the umbilical

vein and has succeeded in more than 50 cases.9 A

20-gauge long needle was used, which is larger than

the 27-gauge needle used through the fetoscope, and

the risks to the fetus of this modification are not yet

known. Another group has used transvaginal place-

ment of the fetoscope in cases with extensive anterior

placentas.’#{176} Other infrequently used approaches have

included ultrasound-guided placement of a needle into

the hepatic portion of the umbilical vein” and ultra-

sound-guided fetal cardiac puncture.’2 The safety and

necessity of some of these techniques are open to

question.

Method of Analysis

The proportion of fetal erythrocytes, initially esti-mated with the Coulter Channelyzer, is subsequently

confirmed with the acid elution slide test.’3 The fetal

blood (and a maternal sample as a control) is incu-

bated with 3H-leucine to radiolabel newly synthesized

globin chains, which are then analyzed by carboxyme-

thyl cellulose (CMC) chromatography. Details of

these techniques are provided elsewhere.’4”5 Mutant

f3-globins, such as 5, C, E, 0-Arab, and Lepore, are

detected because they separate chromatographically

from the normal f.�-chain.

The diagnosis of thalassemia is made when the




N0

0F�

CASFS

tame

000

t 00

to. -

0

00

0�

A’

0�

20 40 60 80 tOO 20

TOTAL MONTHS

PRENATAL DIAGNOSIS OF HEMATOLOGIC DISEASES 331

normal a- or j3-chain is absent or substantially reduced

in amount. Cividalli et aV6 suggested calculation of the

ratio of fl/7-globin synthesis. This value increases

slowly during the first and midtrimesters and is 0. 1 1 ±

0.05 (mean ± 2 SD) at 16 to 23 weeks in normal

fetuses)7 We found that the fl/”y-ratio in fetuses subse-

quently proven to have fl-thalassemia trait was 0.06 ±

0.04. The important aspect is the distinction of homo-zygotes from heterozygotes. In our experience, /3-

thalassemia major was diagnosed if the f3/’y-ratio was

below 0.025. Throughout the world, however, the

specific cutoff point ranges from 0.010 (where only

/3#{176}-thalassemia occurs) to 0.035 (where f3�-thalassemia

predominates). Each center has determined its own

value, depending on the “plusness” of the /3-thalasse-

mia in that region and the technical aspects of the

separation of /3- and �y-globin in its laboratory.

One problem that decreased with increasing obstet-nc experience is that of impure samples of placental

blood. Details of the many approaches that have been

used to enrich such samples for fetal red cells are

provided elsewhere)5 In the current WHO Registry,

many centers indicated that they now routinely obtain

pure fetal samples. Over the preceding years, I ,008

samples required enrichment, and 77% were done with

the �rskov procedure. This is a method for selective

hemolysis of adult red cells, which contain carbonic

anhydrase, and lyse in the presence of NH4C1 and

NH4HCO3. Fetal cells, with low levels of carbonic

anhydrase, do not lyse.’t In the past year, this was the

only method used when samples were impure.

Fetal Blood Studies for Hemoglobinopathies

Three thousand nine hundred fifty-nine cases werereported to the WHO Registry from June 1974

through December 1982, in which fetal blood was

examined because the fetus was at risk for a hemoglo-

binopathy. The most recent summary reported in the

literature from each center is included in references 19

to 3 1 . The number of cases tested increased signifi-cantly each year (Fig 2). Logarithmic transformation

of these data results in almost a straight line, with a

case number doubling time of approximately 15

months. By the end of 1982, 100 cases were being

studied each month worldwide. The disorders for

which the fetuses were examined are listed in Table 2.

Ninety-two percent were at risk for thalassemias, and

the rest were for sickle disorders. Of the thalassemias,

98% were /3 (j3#{176}and fl� are combined here, because the

specific genetic defect was often unknown), and 60% of

the sickle group were at risk for homozygous 55.

The results of the fetal blood studies for hemoglo-binopathies are shown in Table 3. Three hundred five

of the 3,959 cases required more than one attempt to

Fig 2. Log plot of cumulative number of cases reported to

each Registry. The first Registry ended in December 1 978. thesecond in March 1 980, the third in March 1 981 , the fourth inDecember 1981, and the fifth in December 1982. (-0-) Hemo-globinopathies by fetal blood. (---a---) other diseases by fetal

blood. ( . . . � . . . ) hemoglobinopathies by DNA.

obtain an adequate sample, but only 54 of the almost

2,000 cases studied in the past two years were in this

category, reflecting improved obstetric experience.

Three thousand eight hundred eighteen, or 96% of

cases, did result in an adequate sample eventually. Of

the adequate samples, 25% led to diagnosis of the

disease for which the fetus was at risk, which is the

proportion expected for autosomal recessive disorders.

Of the 961 fetuses found to be affected, only 1 6 (1.7%)

did not request an elective termination.

Errors were reported in 29 cases (0.8% of adequate

samples), of which 24 were false negatives. In these,

the fetal /3/’y-synthetic ratio exceeded the cutoff value

for the respective centers. Technical problems with the

/3- and 7-chain separations on the columns could be

implicated in only a few cases. Most of the errors

occurred in fetuses with f3�-thalassemia major, in

whom the fl/-y-ratio fell within the heterozygote range.

However, the error rates were slightly higher in centers

that had studied only small numbers of cases, perhaps

indicating some benefit from extensive laboratory

Table 2. Fetal Blood Testing for Hemoglobinopathies: Fetal Risks,

June 1 974 to December 1982

Disorder No. Percent

Thalassemia:

�3 3,548 97.6

#{244}�3 49 1.3

a 12 0.3

a+fl 6 0.2

E/fi-Thal 6 0.2

Lepore 15 0.4

Total 3,636 91.8 (of total)

Sickle:

55 193 59.8

S/fl-ThaI 126 39.0

SC 3 0.9

S/O-Arab 1 0.3

Total 323 8.2 (of total)

Total 3,959 100.0





TabI e 3. Fetal Blood Hemogl obinopathy Results, Ju ne 1974 t o December 1982

All All

Parameter

Thalassemias

No.

Sickles

%

Total

No. %No. %

Totalcases 3,636 91.8 323 8.2 3,959 100.0

Cases with > 1 attempt 291 8.0 14 4.3 305 7.7

No.ofadequatesamples 3,533 97.2 285 88.2 3,818 96.4

Affected:

Predicted 900 25.5 61 21.4 961 25.2

Delivered 13 1.4 3 4.9 16 1.7

Errors:

False negative 1 9 5 24

False positive 5 0 5

Total 24 0.7 5 1.8 29 0.8

Losses:

sl4d 161 4.4 11 3.4 172 4.3

>l4dinutero 42 1.2 1 0.3 43 1.1

Totalinutero 203 5.6 12 3.7 215 5.4

Termorinfant 27 0.7 2 0.6 29 0.7

Total overall 230 6.3 14 4.3 244 6.2

Maternal complications 62 1 .7 2 0.6 64 1.6

Prematures 169 6.6 13 5.1 182 6.5

Denominators vary throughout the table.

Corrected for terminations and losses.

experience in the technical aspects, as well as a clearercutoff point when large numbers of cases had been

examined.The overall fetal loss rate was 5.4%, representing

172 within the first 14 days following fetal blood

sampling, and 43 after the first 14 days. An additional

29 infants were lost at term or in the neonatal period,

for a total risk of 6.2%. The total loss rate following

fetoscopy was 5.4%, compared to 6.3% after placental

aspiration. More striking was the observation that the

loss rate in cases in which sampling was performed

only once was 4.8%, whereas it was 13.6% in the more

than 300 cases in which more than one attempt was

necessary to obtain an adequate sample. This differ-

ence may reflect anatomic difficulties in accessing the

fetal circulation, as well as the increased risk of

multiple invasive procedures. The loss rate was close to

5% in those centers with a large experience (more than

200 cases), and significantly higher in a few of the

centers in which smaller numbers of cases were stud-

ied. Thus, the presence of an experienced obstetrical

team is clearly important.

Maternal complications, usually leakage of am-

niotic fluid, occurred in I .6% of cases, mainly follow-

ing fetoscopy. This problem may arise because the

diameter of the fetoscope cannula, 2.7 mm, is substan-

tially larger than that of the 20-gauge needle used for

placental aspiration or for amniocentesis. There were

182 premature infants reported (which is an underesti-

mate, as not all babies were born at the time of the

Registry reports). These represent 4.6% of all cases

studied, and 6.5% of the cases that were neither lost

nor electively terminated. Forty-six babies were

reported to have problems at the time of birth.

Although the nature of these problems was not alwaysreported, at least three infants had healed superficial

needle marks.

In the series reported here, more than 90% of the

families benefited from the procedure by having an

adequate sample, no fetal loss, and correct informa-

tion. Prenatal testing for hemoglobinopathies has had

a significant impact in several regions with high gene

frequencies of /3-thalassemia. The birth rate of infants

with /3-thalassemia major declined to between 10% and

50% of the expected rate in Ferrara, Cyprus, Greece,

Sardinia, and among British Cypriots.32 Thus, fetal

blood sampling for prenatal diagnosis of thalassemia

has already made a major contribution to the public

health problems of those areas.

FETAL DNA FOR HEMOGLOBINOPATHIES

Fetal Tissue Sampling

Midtrimester amniocentesis is a well-established

technique, with a fetal risk of < 0.5% above the

spontaneous midtrimester loss rate.33 Twenty millili-

ters of amniotic fluid obtained at 16 to 20 weeks’

gestation contains sufficient fetal amniocytes to pro-

vide enough DNA for studies of globin genes. An

aliquot of the cells can also be cultured, both for

karyotyping and to provide a source of DNA if addi-

tional studies are required. The time when the amnio-





centesis is performed and the approximate two weeks

required for the analysis result in midtrimester termi-

nations of affected fetuses, as is the outcome when

fetal blood sampling is performed.

A new method is now being developed for obtaining

fetal tissue, which involves aspiration or biopsy of

chorionic villi at eight to 1 2 weeks’ gestation. This is an

outpatient procedure and is guided by real-time ultra-

sound. Sufficient DNA is obtained from the villi

(usually 10 to 20 �.ig) for examination of the globin

genes. This method has been reported in nine cases at

risk for hemoglobinopathies.3�36 In pooled early results

from centers just beginning to develop this technique,

the loss rate in 1 13 cases in which samples wereobtained for a variety of reasons was cited at 1 �

Although higher than that which occurs with amnio-

centesis or with fetal blood sampling, this rate may

decline with obstetric experience. A first-trimester test

has obvious psychologic and medical advantages.

Method of Analysis

The recombinant DNA technologies used to detect

hemoglobinopathies in utero were reviewed recently in

this journal, by Orkin.’ Globin gene deletion is the

cause of many cases of a-thalassemia, �f3-thalassemia,

rare /3#{176}-thalassemia (deletion of the 3’ end), and

Lepore. Linked restriction fragment length polymor-

phisms can be used to detect /35 and �C genes (HpaI),

/3#{176}-thalassemia in Sardinia (BamHI), and many other

fl-thalassemias.38 Extensive and time-consuming fam-

ily studies are often required to establish these link-

ages, however. A few DNA point mutations that cause

hemoglobinopathies are themselves restriction enzyme

cleavage sites, and thus can be diagnosed directly with

the appropriate enzymes. Examples of these are thediagnosis of flS with Dde!, MstII, or CvnI,36 all of

which cleave at the normal /36 codon, � with

EcoRI, and a few uncommon /3-thalassemia genes with

other enzymes. The most useful tool of molecular

biology will be the in vitro synthesized oligonucleotides

(19-mers), which are specific for each thalassemic

mutation. These probes identify either the mutant or

the normal /3-gene and have been used for prenatal

testing of cases at risk for the Sardinian /339 nonsense

mutation.39 Such methods can identify the most com-

mon Mediterranean fl�-thalassemia due to a mutation

in the first intervening sequence.”#{176} In families in which

the specific genetic defect is known, the oligonucleo-

tide method will be the most useful and accurate.

Fetal DNA Studies for Hemoglobinopathies

One hundred seventy-five cases were reported to the

WHO Registry to have had DNA studies prior to

December 1982, although additional cases had been

investigated.41�3 Table 4 summarizes the disorders for

which the 1 75 fetuses were at risk. Sixty-eight percent

were studied for sickle disorders and 32% for thalasse-

mias. The larger proportion for sickle risks reflects the

use of HpaI linkage studies during 1981 and the

introduction ofMstll in 1981 and 1982.

The results of the DNA studies reported to theRegistry are shown in Table 5. Most of the DNA

studies used material obtained directly from the uncul-

tured amniotic fluid cells. Twenty-six percent of the

fetuses were found to be affected, and there were no

errors reported. It should be noted that almost 40%

(17/45) of the affected fetuses were subsequently

delivered, in contrast to the < 2% (16/961) continuing

pregnancies among those where fetal blood sampling

was performed. This difference presumably reflects

the counseling given to the families, in which fetal

blood sampling is cited as having a > 5% fetal loss rate,

while amniocentesis has a < 3% loss rate. The differ-

ence may include differences in the attitudes of fami-

lies regarding termination of pregnancy for sickle cell

disease versus thalassemia. In the group studied by

fetal blood sampling, three of 61 sickle homozygotes

were carried to term, compared to 1 3 of 900 thalassem-

ics. In those analyzed by DNA methods, 1 5 of 37 sickle

and two of eight thalassemia homozygotes were dcliv-

ered. Thus, the proportion of homozygotes carried to

term following DNA analyses was in fact increased in

both disease categories. The loss rate in this series was

2.9%. The five cases shown, in fact, include a biased

sample. One of these cases required a fetoscopy to

provide a definitive diagnosis, and the fetus was lost

following that procedure. Two others had histories of

multiple miscarriages (one with an incompetent cer-

vix), and one of these plus the fourth had bloody

amniotic fluid at the time of the amniocentesis.

The latest methods in molecular biology can thus be

used on samples of fetal tissue obtained by amniocente-

sis or from chorionic villi and should replace fetal blood

Table 4. Fetal DNA Testing for Hemoglobinopathies: Fetal Risks,



Thalassemia:

fi 48 85.7

#{244}fl 2 3.6

a 6 10.7


Sickle:

SS 109 91.6

S/fl-ThaI 4 3.4

SC 5 4.2

5/0-Arab 1 0.8


Total 175 100.0




Table 5. Fetal DNA Hemoglobinopathy Results,


Table 6. Fetal Blood Testing for Other Diseases: Fetal Risks.


All

ThalassemiasAll

Sickles Total

Parameter No. % No. % No. %

Totalcases 56 32.0 119 68.0 175 100.0

Affected:

Predicted

Delivered

8 1 4.3

2 25.0

37 3 1 .0

15 40.5

45

17

25.7

37.8

Errors 0 0 0 0 0 0

Losses:

sl4d

>14d

Totalfetal

2

1

3 5.4

1

1

2 1.7

3

2

5 2.9

Prematures 0 0 0 0 0 0

sampling for prenatal detection of many of the hemo-

globinopathies. These methods can now be routinely

and reliably applied to cases at risk for sickle cell

anemia, Sardinian /339 nonsense mutation thalassemia,

gene deletion a- and #{244}$-thalassemias, and certain types

of /3#{176}-and /3�-thalassemia. The specific mutations in

the /3-thalassemias, or specific linked polymorphisms,

must be known for the successful application of the

recombinant technology. Thus, newly diagnosed thal-

assemic families may still require fetal blood globin

synthesis analyses until rapid methods for identifica-

tion of thalassemic defects are developed, such as

panels of oligonucleotides.

FETAL BLOOD FOR OTHER DISEASES

A large variety of hematologic and nonhematologic

disorders can now be detected in fetal blood. Diseases

other than hemoglobinopathies were first studied in

1979, and 338 cases were reported to the WHO

Registry through December 1 982. Of these cases, 243,

or 72%, were at risk for coagulopathies (Table 6),

mostly the hemophilias. The next largest group was at

risk for muscular dystrophies, followed by studies of

white cell disorders. For the sex-linked disorders, an

amniocentesis was performed first for fetal sex deter-

mination. Fetal blood sampling was then only done

from male fetuses.

For most of the disorders listed in Table 6, pure fetalblood was optimal, if not absolutely required. Assays of

clotting factor activity must be done with pure fetal

plasma, because amniotic fluid contains thromboplas-

tic activity. In fact, the need for pure fetal blood for the

coagulation diagnoses provided some of the impetus

for the development of techniques for sampling from

the umbilical vein. VIIICAS, VIIIRA5 and IXAg can be

measured by immunologic techniques in the presence

of amniotic fluid, provided the relative dilution of the

fetal plasma is known.



Coagulation:

Hemophilia A 208 85.6

Hemophilia B 19 7.8

von Willebrand 4 1.6

Amegakaryocytic 1 0.4

TAR 1 0.4

Wiskott-Aldrich 1 0.4

Alpha 1-antitrypsin 9 3.7

Total 243 7 1 .9 (of total)

Red cells:

HEMPAS 1 50.0

Rh 1 50.0


White cells:

CGD 3 6.8

lmmunodeficiency 3 6.8

Neutropenia 1 2.2

Rubella 2 4.5

Chromosomes 35 79.5


Other:

DMD

BMD

Total

Total

48 98.0

1 2.0

49 14.5 (of total)

338 100.0

TAR. thrombocytopenia with absent radii; HEMPAS, hereditary

erythroblastic multinuclearity with positive acidified serum test; CGD,

chronic granulomatous disease; DMD, Duchenne muscular dystrophy;

BMD, Becker muscular dystrophy.

Most of the hemophilia diagnoses were obtained in

three centers.’2’44�6 Two hundred eight cases were at

risk for hemophilia A (factor VIII deficiency), 19 for

hemophilia B (factor IX deficiency), and four for

homozygous von Willebrand’s disease (inherited as a

dominant). There was one error (Table 7), in which a

case with hemophilia was not detected in utero. There

were seven losses (3%), which indicates that the risk of

hemophilia does not increase the risk of fetal loss in

utero.

Two fetuses were studied for the possibility of

autosomal recessive thrombocytopenias by obtaining

platelet counts in fetal blood. One of these fetuses, at

risk for thrombocytopenia with absent radii, could

have been examined by ultrasound alone. This diag-

nosis has been reported in the past by means of fetal

x-ray studies.47’48 Another platelet disorder that has

been sought in utero recently is the Wiskott-Aldrich

syndrome.49 This diagnosis was excluded by the find-

ings of a normal fetal blood platelet count and normal

mean platelet volume in a male fetus whose affected

brother had thrombocytopenia and small platelets.

Red cell disorders other than hemoglobinopathies

can be detected in utero. Red cell grouping and typing





Table 7. Fetal Blood Other Disease a Results, June 1 9 74 to D ecember 1982

Muscular

Coagulation WBC + RBC Dystrophies Total

Parameter No. % No. % No. % No. %

Totalcases 243 71.9 46 13.6 49 14.5 338 100.0

Caseswith>1 attempt 5 2.1 0 0.0 8 16.3 13 3.8

Adequatesamples 236 97.1 46 100.0 45 91.8 327 96.7

Affected:

Predicted 80 33.9 13 28.3 5 11.1 98 30.0

Delivered 3 3.8 0 0.0 0 0.0 3 3.1

En’ors:negative 1 0.4 1 2.2 3 6.7 5 1.5

Losses:

�14d 4 1.6 2 4.3 3 6.1 9 2.7

>14d 3 1.2 1 2.2 2 4.1 6 1.8

Total 7 3.0 3 6.5 5 10.2 15 4.4

Prematures 10 6.3 1 3.3 2 5.1 13 5.7

WBC. white blood cells; RBC, red blood cells.5C�ected for terminations and losses.

can be performed adequately on fetal blood for diag-

nostic purposes,5#{176} for questions of paternity,5’ and for

treatment of hemolytic disease due to isoimmuniza-

tion.52 Intrauterine intravenous exchange transfusion

can be performed to treat the latter condition, using

fetoscopy and a needle placed in the umbilical vein.

Red cell enzyme deficiencies might be detected in

utero. Normal fetal red cell levels of several enzymes

were reported by Lestas et al53 in anticipation of

requests for diagnoses. In one case, the diagnosis of

galactosemia was ruled out by measurement of red cell

UDPgal.54 Fetal congenital anemias might be diag-

nosed, as fetal blood counts can be compared with

normal values for 20 weeks’ gestation (2.5 x 106

RBC/�sL, 37% hematocrit55). Defects in erythroid

stem cells could be detected, as Linch et al have shown

that normal fetuses have increased circulating cry-

throid progenitor cells (BFU-E, burst-forming units).56

Only hereditary erythroblastic multinuclearity with

positive acidified serum test (HEMPAS) and Rh

determinations were reported to the Registry.

Fetal white blood cells can be used to diagnose

several types of diseases. The first example involved

chronic granulomatous disease, usually an X-linked

disorder in which granulocytes and monocytes fail to

kill certain types of bacteria due to an inability to

generate superoxide radicals. The white cells of

affected fetuses were unable to reduce nitroblue tetra-

zolium on a slide test.57’58 Newburger and Latt noted

that samples of mixed fetal-maternal blood could be

used, because the male cells could be identified by

Y-chromatin fluorescence.59 Three such studies were

reported to the Registry. Since adequate numbers of

granulocytes are present by 20 weeks’ gestation, the

diagnosis of neutropenia could also be made, and one

such case was studied.

Another major category of white cell disorders

involves the immunodeficiency syndromes. Some

forms of severe combined immunodeficiency are asso-

ciated with adenosine deaminase (ADA) deficiency,and one affected fetus was detected by low levels of

fetal red cell ADA.�#{176}The risk of fetoscopy can usually

be avoided in these cases, because amniotic fluid cell

ADA is also reduced in affected cases.61’62 A more

general approach to the immunodeficient diagnoses

involves determination of the numbers and proportions

of fetal lymphocytes and T and B subsets, using

fluorescent antibodies and microscopic immunofluo-rescence assays or a fluorescence-activated cell sorter.

Four cases at risk for severe combined immunodefi-

ciency have been reported in the literature (three to the

Registry), and one was found to be affected.63�5

Fetal white cells obtained by fetoscopy can be

cultured, thus providing a source of chromosomal

analyses by 72 hours, much more rapidly than the two

weeks required to culture amniotic fluid cells.66 This is

particularly helpful when the amniotic culture fails or

the pregnancy is quite advanced when the test is

requested. This method was used to diagnose the

syndrome of mental retardation associated with fragile

x.67 Other chromosome defects would easily be deter-

mined from cultured fetal white cells, although in most

cases, amniotic cells should suffice (see below). Fetal

white cells were used for karyotyping in 35 cases

reported to the Registry, and eight were abnormal.

Fetal white cells were recently reported to be used to

exclude the diagnosis of Hurler’s disease by measure-

ment of leukocyte a-L-iduronidase.68 As with red cells,

many enzymes can be measured and diagnoses made

depending on the values obtained in relation to normal

fetal values.

Fetal serum or plasma is available if pure fetal blood

can be obtained. This material was used to measure

creatine phosphokinase levels in 49 fetuses at risk for





muscular dystrophies reported to the Registry (Tables

6 and 7)69.70 but false negative results were obtained in

three cases, and the assay is not considered to be

reliable.71’72 Current attempts to define restriction

enzyme polymorphisms that are closely linked to the

genes for the muscular dystrophies will be more valu-

able than fetal blood sampling.73’74

Fetal serum was used to diagnose Tay-Sachs disease

by measurement of hexosaminidase activities,75 but

this is a diagnosis that can be made with amniotic

fluid. Fetal plasma was reported to provide the diag-

nosis of alpha,-antitrypsin deficiency,76 and the investi-

gators indicated that this was necessary because most

of the alpha,-antitrypsin in amniotic fluid is derived

from the mother. Such studies were performed in nine

cases reported to the Registry.

The WHO Registry data, which extend through

December 1982, do not include all of the conditions

discussed above, because the use of fetal blood for

many other diagnoses is a rapidly growing area. The

summarized numbers of cases are small (Table 7). If

the cases at risk for muscular dystrophies are excluded,

the error rate is < I %, and the fetal loss rate is under

4%. This low loss rate may reflect the fact that most of

the nonhemoglobinopathy prenatal testing is per-

formed in only a few of the 1 1 centers that do any such

studies, and these centers have experienced obstetri-

cians.

OTHER HEMATOLOGIC DISEASES DIAGNOSED BY

AMNIOCENTESIS

The development of molecular probes for detection

of the hemoglobinopathies is advancing rapidly. Clon-

ing of the genes that code for the important coagula-

tion proteins and for red cell enzymes is also underway,

and has been accomplished for factor IX,7779 anti-

thrombin 111,80.81 and phosphoglycerate kinase.82’83

Prenatal diagnosis of deficiencies for these proteins has

not been performed with these probes and awaits

determination of whether the diseases are due to gene

deletion or whether mutant genes are linked to poly-

morphic restriction sites. The molecular approach to

prenatal diagnosis is under active investigation in

many laboratories, for both hematologic and nonhema-

tologic diseases.

Several hematologic diseases can currently be diag-

nosed by amniocentesis and nonrecombinant DNA

technology. One group in this category is the chromo-

some breakage syndromes, which have autosomal

recessive inheritance. The disorder with major hemato-

logic disease in this group is Fanconi’s anemia, in

which most patients develop aplastic anemia and/or

leukemia in childhood. All patients have increased

chromosome breakage, which is further increased fol-

lowing culture of cells with diepoxybutane (DEB), a

DNA crosslinker. Twenty-two pregnancies at risk for

Fanconi’s anemia have been monitored, and seven

affected fetuses were identified by the presence of

increased spontaneous and DEB breakage in cultured

amniotic fluid cells.84 Only four of the seven had

physical anomalies (two at birth and two of the five

terminated), and thus, ultrasound examination alone

would not have been diagnostic. One very recent case

had Fanconi’s anemia excluded in the first trimester by

the observation of no increase in breakage following

culture of chorionic villi alone or with DEB.85

Other chromosome breakage syndromes have an

increased incidence of neoplasia. Patients with ataxia

telangiectasia have immunodeficiencies and lymphoid

malignancies. One fetus was found to be affected,

based on finding a clastogenic factor in the amniotic

fluid that produced chromosome breaks in normal

lymphocytes, as well as increased breakage of the fetal

amniotic cells plus a translocation involving chromo-

some 14 in those cells.86 Patients with Bloom’s syn-

drome show growth retardation and develop leukemia

and other malignancies at an early age. Cultured cells

have increased breakage plus very high numbers of

sister chromatid exchanges (SCEs). Several pregnan-

cies at risk have been monitored by ultrasound and

amniotic cell SCE, but all were normal.87 Xerodermapigmentosum is characterized by skin sensitivity to

sunlight and ocular and cutaneous malignancies. Cells

from such patients fail to repair ultraviolet irradiation-

induced damage. Three pregnancies have beenreported to be monitored for xeroderma pigmentosum,

and two fetuses were found to be affected.88’89 All of

these conditions in which amniotic fluid cultures were

assayed might be detected in cultured chorionic villi.

Amniotic cells have been used to identify disorders

affecting cellular enzymes. One example is glucose

phosphate isomerase deficiency, which results in severe

congenital nonspherocytic hemolytic anemia. A fetus

at risk was diagnosed in utero by assay of the amniotic

cell enzyme level, and then was treated immediately

following birth by exchange transfusionsY#{176} Severe

methemoglobinemia due to cytochrome b5 reductase

deficiency was detected in amniotic fluid cells.9’ Both

of these enzyme deficiency disorders could presumably

have been diagnosed from studies of fetal blood or from

chorionic villi, if sufficient material were available.

Porphyria can also be diagnosed by analysis of

enzymes in amniotic cells. Sassa et al diagnosed auto-

somal dominant acute intermittent porphyria due to

deficiency in uroporphyrinogen I synthetase,92 and

Deybach et al excluded autosomal recessive congenital

erythropoietic porphyria by observing normal levels of

uroporphyrinogen III cosynthetase.93





Properties of white cells that are expressed in

amniotic cells can be used to diagnose nonhematologic

conditions as well. Pollack Ct al used HLA typing of

amniotic fibroblasts to determine paternity, assess

whether fetuses could be bone marrow transplant

donors or recipients, and diagnose HLA-linked dis-

eases, congenital adrenal hyperplasia due to 21-

hydroxylase deficiency, and complement C4 deficien-

cy.94 Forest et al also used HLA linkage, combined

with amniotic fluid hormone levels, to diagnose con-

genital adrenal hyperplasia in utero.95 HLA typing can

also be performed on white cells obtained from fetal

blood and perhaps on chorionic villi cells as well.

It should be stressed that the reliability of prenatal

testing for many of the hematologic and nonhemato-

logic disorders discussed here awaits the test of time.

The numbers of cases studied are often small. The

experience with muscular dystrophy (Table 7) has

shown that in utero tests may not be error-free. The

proper role of prenatal testing requires a reliable assay

that distinguishes affected from unaffected fetuses;

such assays are still being developed for some of the

conditions summarized in this review.

SUMMARY

Prenatal diagnosis of hematologic diseases can now

be performed with fetal blood, fetal amniotic fluid cell

DNA, and fetal chorionic villi DNA. Some hemoglo-

binopathies can be detected by all three methods, and

the choice will depend on the available obstetric and

laboratory techniques, as well as the time of presenta-

tion of the pregnancy. Hopefully, further development

of molecular probes and techniques will soon expand

these options to all of the globin disorders.

Detection of coagulation disorders in utero currently

requires samples of pure fetal blood. Gene cloning is

accomplished for some (factor IX and antithrombin

III) and is underway for others (factor VIII), and

further investigation is necessary to determine whether

deficiencies in these gene products are due to gene

deletion or to mutant genes linked to polymorphic

restriction enzyme sites of diagnostic use. Thus, molec-

ular biology may be applied to prenatal diagnosis of

the clotting problems, but this has not yet been accom-

plished.

Disorders affecting the number and/or function of

erythrocytes, leukocytes, and platelets can be diag-

nosed by analysis of fetal blood. Blood samples will

continue to be required until more is known about the

molecular biology of hematopoiesis.

Syndromes that can be diagnosed by chromosome

studies should be revealed in cultures of amniotic fluid

cells, fetal blood lymphocytes, and chorionic villi cells.

Cultured cells can be examined for karyotypes, Y-

chromatin, spontaneous or induced chromosome

breakage, DNA repair, SCEs, and translocations. The

techniques for culturing amniotic cells and fetal blood

white cells are established, and those for growing cells

from chorionic villi are improving rapidly. Direct prep-

arations of cells from villi only may suffice for some of

the above analyses.

The study of hematologic disease in utero has thus

come full circle, from the use of amniotic cells to

determine the sex in X-linked disorders, to fetal blood

sampling for the analysis of gene products, then back

to amniocentesis for DNA, and now earlier in gestation

to chorionic villi. All of this has occurred in less than

ten years, and it is anticipated that developments in the

next ten years will be equally dramatic. The future

should bring all prenatal testing into the first trimester,

use molecular probes, and provide for both early

diagnosis and early treatment of genetic hematologic

disease.

ACKNOWLEDGMENT

I am grateful to Dr Anver Kuliev for arranging for support from

the World Health Organization. Principal members of previous and

current International Registries are as follows, listed alphabetically

by country:

Australia: Melbourne, Queen Victoria Hospital, R. Matthews,

R. Sharma, W. Walters; Melbourne, Royal Women’s Hospital,

M. Cauchi, FR. Betheras; Sydney, V. Berdoukas. Canada: Mon-

treal, F. Congote, T. Perry; Toronto, R. Benzie; Hamilton,

S.C. Wong. Cyprus: Nicosia, M. Angastiniotis. Denmark: Copenha-

gen, i. Bang. England: London, King’s College Hospital, C. Rodeck,

R. Mibashan; University College Hospital, B. Modell, D. Fairweath-

er, R.H.T. Ward; Oxford, i. Old, Di. Weatherall. France: Paris,

M. Goossens. Germany: Munich, M. Jensen. Greece: Athens,

D. Loukopoulos, A. Antsaklis. Israel: Jerusalem, E. Rachmilewitz.

Italy: Ferrara, M. Lucci, C. Vullo; Milan, B. Brambatti, M. Ferrari;

Rome, A. Pachi, L. Tentori; Sardinia, A. Cao. Sweden: Malmo,

B. Gustavii, L. Holmberg, R. Ljung. United States: Baltimore,

H.H. Kazazian, Jr.; Boston, D.G. Nathan, S.H. Orkin; Hartford,

L. Hoyer; New Haven, Mi. Mahoney, iC. Hobbins, B.G. Forget;

New York, B.P. Alter, A. Bank; San Francisco, Y.W. Kan,

MS. Golbus.

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1984 64: 329-340

BP Alter Advances in the prenatal diagnosis of hematologic diseases

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