Alternative Methods for Screening

(1)

Alternative

Methods

for Screening

SUPPLEMENT

855 Michael

D. Garrick,

PhD

From the Departments of Biochemistry and Pediatrics, State University of New York, Buffalo

Alternative

methods

(Fig

1) are already

available

for screening newborns for hemoglobinopathies.

When

choosing

among

the

methods,

one

should

remember

three

aims:

(1) to identify

Hb

5; (2)

to

detect

when

Hb

A is absent,

suggestive

of an

S/S

homozygote

or a double

heterozygote

for

S/fl#{176}-thal-assemia or for

S/C

or S/another

fl-globin

variant;

and (3)

to

screen

for

other

potentially

pathologic

states. (Double heterozygosity for S/fl-thalassemia is also important to detect, for example.) Detection

of fl-thalassemia

major

by newborn

screening

would

be useful

but

is not

yet

reliable.

SPECIMENS

Blood

Both

liquid

(anticoagulated)

blood

and

dried

blood

spots

have

been

used.

Liquid

blood

is easier

to hemolyze,

but

dried

blood

spots

are

much

easier

to

mail

and

store.

Dried

blood

spots

are

already

used

in most

states

to screen

for other

disorders.

Age

In most

states,

a newborn

capillary

blood

sample

is obtained by heel puncture before the baby leaves

the

hospital

to

screen

for

phenylketonuria

(and

often

for

other

disorders

such

as hypothyroidism).

Cord

blood,

however,

can

be

obtained

in

larger

volume

and

is less

likely

to

be

contaminated

by

transfusion but more likely to be contaminated by

maternal

blood.

I prefer

the

newborn

specimen

because

of the

advantages

of coordinating

screening

programs both for thorough population coverage

and

for

economic

savings

at

several

levels.

In

a

coordinated

screening

program

for

multiple

disor-ders, hemoglobinopathy screening may help in

rec-ognizing specimens from infants in whom a

trans-fusion has taken place but the time the specimen

was taken for screening has not been noted. In

special cases, however, cord blood may be

prefera-ble,

eg,

when

accurate

appraisal

of

Hb

Bart’s

is

needed

or when

a research

program

is coordinated

with

screening.

LYSING

Hemolyzing

the

sample

with

buffer

containing

KCN still appears to be the best way to recover

hemoglobin

while

minimizing

artifacts

due

to

oxi-dation

of heme

iron

from

the

Fe2

state

to Fe3.

SCREENING

Screening

can

be done

by cellulose

acetate

elec-trophoresis,”2 by ion exchange chromatography,3

by isoelectric focusing,4’5 or by high performance

liquid chromatography.6 The four techniques can

be compared

as shown

in the

Table.

I recommend

cellulose

acetate

electrophoresis

mostly

because

of

the

larger

base

of experience;

however,

isoelectric

focusing

is promising.

Both

methods,

however,

are

labor

intensive

and

repetitive,

making

the

potential

for automation

another

criterion

for deciding

which

technique

to

use.

High

performance

liquid

chro-matography

is readily

automated,

although

other-wise

less

suitable.

CONFIRMATION

Because

other

hemoglobins

behave

like

Hb

S in

the initial screen with any of the previously cited

methods,

it is important

to confirm

the

presence

of

Hb

S. Confirmation

by

the

screening

laboratory

should not be confused, however, with diagnosis.

The

ability

of citrate

agar

electrophoresis’

to

distin-guish Hb S from similarly charged variants presses

inclusion of this method for confirmation in any

screening

program

for hemoglobinopathies.

Alternatives

to

citrate

agar

electrophoresis

po-tentially include the solubility test for Hb 57

and

the fragility test for Hb 58

but

neither

approach

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can

be argued

that

antibodies

will

be useful

only

as

an

adjunct

because

they

cannot

be

expected

to

detect

new

variant

hemoglobins,

and

it is difficult

to tell

when

Hb

A is absent

when

relying

just

on an

immunologic approach.

POLYMERASE CHAIN REACTION PCO3 tub#{149}amplification

r-ACACMCTGTGITCACTAGC.r

#{163}MOGTTGMGGTGGATGMG1TOGTG-3 i33LW30LVi10’3S

#{163}-33iLOO0iY3.LL0W0S (reprIsIntIng ttas. by Nnis)

856 TECHNIQUES

AND

EFFICACY

has been made functional for newborn dried blood

spots. High performance liquid chromatography is

another option, but there is a need to settle on

which high performance liquid chromatography

method should be used. Specific antibodies9 may

also be useful in confirmation.

THE FUTURE

New options that may prove more automatable,

less expensive, or more reliable in the future include

monoclonal antibodies and recombinant DNA

tech-nology. Polyclonal sera specific for Hb S and Hb A

have been reported,’#{176}” but the potentially

unlim-ited supply and the lot-to-lot reproducibility of

monoclonal antibodies are major advantages over

conventional sera. Anti-Hb S monoclonal

antibod-ies are clearly feasible, but specific anti-Hb

A-specific for the fl-6-Glu (where Hb S has a

Val)-is needed to make monoclonal sera even partly

suitable for screening. More monoclonal antibodies

specific for Hb C or Hb E are required before this

technology can be useful even for confirmation. It

ci2I

firm

agar

HPLC

eFectro-phoresis

specimens lysing screening

I

include KCN

I

blood age adapt

to

liquid dried cord newborn screening methods

CAEP IEC IEFHPLC

Fig 1

. Decision tree: Alternative methods for screening newborns for hemoglobinopathies. Abbreviations: CAEP; cellulose acetate electrophoresis; IEC, ion exchange chro-matography; IEF, isoelectric focusing; HPLC, high per-formance liquid chromatography. Modified from Garrick et al1’ and used with permission.

0’

m.ftaFann.

,,

4

riplicats w$Ih ICisnow fragn,., ofDNApoymirass +4 dTPs

I

4

repeat for 20 c ...t22o.ooox rspflcat#{232}on

hybddz#{149}b PS0 probss

j

CW1Cfl 19.4

cTGva iss

cncGYaTcT1 19c

0’4ythemaCthing probe bindsC

Fig 2. Amplification of DNA according to Saiki et al.”

High molecular weight, double-stranded genomic DNA is

mixed with excess of two single-stranded synthetic DNA

primers PCO3 and PCO4 as represented by DNA

Se-quences at top or lines below. DNA is melted and rena-tured so that primers bind to longer natural DNA; then

primers are used to initiate DNA synthesis off longer

templates. Newly made duplex DNA is again melted and

annealed to primer and doubling process repeated for 20

cycles. Amplified DNA is hybridized to labeled 19 mer

probes specific for fl-A in this region or for fl-S or /3-C.

TABLE.

Screening Methods*

Advantage Cellulose Acetate

Electrophoresis Ion Exchange Chromatog-raphy Isoelectric Focusing High Performance Liquid Chromatography Processing

Parallel + +

Serial -

-Resolution

High + +

Moderate ± ±

Separates by charge + + + +

Sensitivity to artifact

Insensitive + +

-Varying ?

Experience base

Large +

Moderate ± ± ±

* Symbols: +, definite; -, negative; ±, moderate; ?, unknown/varies. From Garrick” and

reproduced by permission.

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.

19A

AA

AS

Ss

SC

CC

AC

xx

.

S

.

19S

S

.

19C

SUPPLEMENT

857 Fig 3.

Detection of amplified DNA by dot blot analysis with specific hybridization probes. Human genomic DNA was amplified with approach shown schematically in Fig. 2, then hybridized to specific probes. Probe used is

mdi-cated on bottom and genotype on right. Reprinted from

Saiki et al3 and used with permission.

Recombinant

DNA

technology

is attractive

for

hemoglobinopathy

screening

in

the

long

term.

McCabe

et

al’2

have

recovered

restrictable,

high

molecular

weight

DNA

from

dried

blood

spots.

The

modest

quantities

of DNA

could

be

amplified

ac-cording

to Saiki

et al’3

(Fig

2) and

subjected

to an

oligonucleotide

probe

spot

test

as illustrated

in Fig

3. Automated DNA sequencing techniques’4 might

be an alternative

to spot

tests.

Reliable

methods

already

exist

to permit

newborn

screening for hemoglobinopathies. More

spectacu-lar

methods

may

be

developed,

but

there

is

no

technical

reason

to

wait

for

future

methods.

A

decision to proceed with screening should be based

on whether

there

will be sufficient

decrease

in

mor-bidity and mortality to justify the costs and risks.

ACKNOWLEDGMENTS

This paper is a reanalysis and updating of a previous

paper.” Scheme 1, excerpts from that paper and Fig 1,

which was modified from the original, are reproduced with permission. Fig 3 is reprinted by permission from Saiki et al.1’

REFERENCES

1. Garrick MD, Dembure P, Guthrie R: Sickle cell anemia and other hemoglobinopathies: Procedures and strategy for screening employing spots of blood on filter paper as speci-mens. N EngI J Med 1973;288:1265-1268

2. Schneider RG: Developments in laboratory diagnosis, in Abramson H, Bertles JF, Wethers DL (eds): Sickle Cell

Disease: Diagnosis, Management, Education, and Research.

St Louis, CV Mosby, 1973, pp 230-243

3. Powars D, Schroeder WA, White L: Rapid diagnosis of sickle cell disease at birth by microcolumn chromatography. Pe-diatrics 1975;55:630-635

4. Arad Y, Mayer TK, Aryan DA: Isoelectric focusing of

he-moglobins on thin-layer agarose. Am J Clin Pat/wi

1981;76:200-205

5. Ferrari M, Crema A, Canta-Rajnoldi A, et al: Antenatal

diagnosis of haemoglobinopathies by improved method of

isoelectric focusing of haemoglobins. Br J Haematol 1984;57:265-270

6. Wilson JB, Headlee ME, Huisman THJ: A new high-per-formance liquid chromatographic procedure for the separa-tion and quantitation of various hemoglobin variants in adults and newborn babies. J Lab Clin Med 1983;102:174-186

7. Nalbandian RM, Richols BM, Camp FR Jr, et al: Dithionite

tube test-A rapid, inexpensive technique for the detection of hemoglobin S and non-S sickling hemoglobin. Ciin Chem 1971;17:1028-1032

8. Asakura T, Ohnishi T, Friedman 5, et a!: Abnormal

precip-itation of oxyhemoglobin S by mechanical shaking. Proc

NatiAcad Sci USA 1974;71:1594-1598

9. Garver FA, Singh H, Moscoso H, et al: Identification of normal and variant hemoglobins after electrophoretic sep-aration and transfer to nitrocellulose membranes.

Hemoglo-bin1984;8:105-115

10. Noble RW, Reichlin M, Schreiber RD: Studies on antibodies directed toward single antigenic sites on globular proteins.

Biochemistry 1972;11:3326-3332

11. Tan-Wilson AL, Reichlin M, Noble RW: Isolation and char-acterization oflow and high affinity goat antibodies directed to single antigenic sites on human hemoglobin.

Immuno-chemistry 1976;12:921-927

12. McCabe ERB, Huang SZ, Seltzer WK, et al: DNA microex-traction from dried blood spots on filter paper blotters: Potential applications to newborn screening. Hum Genet 1987;75:213-216

13. Saiki RK, Bugawan TL, Horn GT, Mullis, KB, et al: Analy-sis of enzymatically amplified /3-globin and HLA-DQ DNA with allele-specific oligonucleotide probes. Nature

1986;324:163-166

14. Smith LM, Sanders JZ, Kaiser TM, et al: Fluorescence

de-tection in automated DNA sequence analysis. Nature

1986;321:674-679

15. Garrick MD: Technical options for screening newborns for hemoglobinopathies, in Therrell BL Jr (ed): Sixth

Interna-tioriai Neonatal Screening Symposium, November 16-19,

1986. Amsterdam, Elsevier, 1987, pp 417-423

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1989;83;855

Pediatrics

Michael D. Garrick