A Simple Spot Screening Test for Fast Detection of ...

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A Simple

Spot

Red By

Screening

Cell

JEAN-CLAUDE

Test

KAPLAN,

rapid

spot

ANNE-MARIE

screening

diaphorase tilted ture

blood freshly

reagents. filter

Spots

are

and

ONGENITAL deficiency a relatively rare diagnosis of this of the

for

NICOLAS,

mixstock

made

at intervals for

estimation

cause

they

of

can

be

method of Hegesh,4 a relatively fresh

although preparation

prompted

us

to

described

for

fast

capacity

performed

by

measuring

or

of

\Vhole

In

the

the

blood

NADH,

NAD are

washings

of

an

easy

It is based

on

From

the

Center,

itut

Inst

February

Supported

in

man, of

330

rate

of

.

cells;2

the The

reduction

of

The methemoglobin time-consuming

he-

red

The

nitrite-treated

for

rapid

ultraviolet

cells.

diagnosis

spot

red

cell

test

of

NADH-

prmciple

enzyme

already

deficiencies.#{176}’7

METHODS

containing

dye

illuminated

by

Moleculaire,

1970;

a hemolyzing is reduced long

agent, by

France,

NADII

NADH.

wavelength

Paris,

de of

lIE

M.D.: Paris,

from

Associate

Paris,

20, the

ultraviolet

and

and

During

the

the light,

City

of

DCIP. reaction is

trans-

Medical

Hope

France.

Southern

of

Hope

Professor

of

Medical

NicoLAs:

ALENA

Biochemistry, Research

HANZLICKOVA-LEROUX:

Medical

California,

1970.

N1H.

ANNE-MARIE

Mol#{233}culaire, Paris, City

of

March

07449

France.

Pathologic

Medicine,

University

accepted

Grant

Mol#{233}culaire, institut

Medicine,

23, by

KAPLAN,

Division

the

red hemolysates.

specific, requires as a substrate hemoglobin. These drawbacks

other

the

when

Mol#{233}culaire,

Pathologic

INSERM

a mixture

de Pathologic

part

JEAN-CLAUDE

Pathologic

in

Calif.

Submitted

de

to

fluoresces

Duarte,

heterozy-

methemodobin-ferrocyanide

the

AND

nitrited

itself

( NADH ) tedious and

method the

various

of NADH-diaphorase,

which

of

Test

is added

presence

the

reduced of Scott

MATERIALS

Principle

of

simpler and more of enzyme-free

of

six defi-

correctly identibut it is not

detection

in intact activity

detection

of

to NADH-diaphorase rediictase deficiency” ) is by lifelong cyanosis.’ The several methods : the explora-

enzyme

devise

deficiency.

the

All

diaphorase

due

( DCIP)3

repeated

diaphorase

for

the

the presence of and the method

is oxidized. NADH

gotes.

reduction

require

have

suitable

methemoglobin characterized established by

disorder may be

dichiorophenol-indophenol in test

NADH with

METHEMOGLOBINEMIA clinical disorder

HANZLICKOVA-LEROUX

ciency studied have been fled using this technique,

on

( “NADH-dependent

determination

complex4’5 reduction

as

patients

defluores-

methemoglobin

quantitative

ALENA

cence

to a reaction from stable

of

BEUTLER

NADHNi-

Detection

Deficiency

ERNEST

described.

examined

C latter

is

is added prepared

paper

tion

test

deficiency

Fast

NADH-Diaphorase

AND

A

for

France.

ERNEST

Center, Los

BLooc,

Angeles,

VOL.

Calif.,

de

Institut

Research

Assistant

BEUTLER,

Duarte,

Institut

Technician,

M.D.: Clinical

ChairProfessor

Calif.

36,

No.

3

(SEPTEMBER),

1970

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SCREENING

TEST

Fig.

1.-UV

without

blood

DCIP.

3.

FOR

formed blood

Normal

2. Nitrited

blood blood

NADH-DIAPHORASE

without plus

NAD,

which

is not

with

sodium

nitrite

effect

upon

normal

normal

nitrite

complete

plus

plus

reaction

1. Reaction

reaction

mixture

mixture

mixture

without

without

DCIP.

4.

mixture.

under

oxidizes

blood.

blood

reaction

fluorescent

331

DEFICIENCY

NADH-diaphorase:

into

the

such

conditions.

hemoglobin,

Pretreatment

thus

preventing

of

its

the

direct

re-

DCIP.

Procedure

Five added

gil. of to

sample

ml.

blood

ml.

a freshly

0.1

of

is allowed

whole of

and

NADII

(Sigma).

10

of 10

examined

test

of

a drop

is

wave

a long

30

NADH,

on

with

a

jd.

of

cent

saponin

mM

DCIP

and

0.27

is freshly

to

blood

spotted

Twenty

Whatman

No.

of

0.2

sodium

Stal)le

mM

sodium

0.5

mixture

1 filter

nitrited

from

vial

reaction

the

and mM

0.27

preweighed

the

the

prepared

containing

are

mixed,

1 per

buffer

mg./ml.)

the

solution

thoroughly

of

mixture

Tris-HCI

aqueous

being

minutes. ml.

0.19

This

NI

ultraviolet

After

0.C4

7.6.

0.06

mM DCIP (6.25 tube containing

minutes,

under

mM

pH

ml.

for

nitrite

sodium

blood.

containing

0.70

buffer, 1.0

19

The

at 37#{176}C. Every

tube

)

( 1.24% whole

temperature

test

containing

adding

NI

ACD

room

a

NI Tris-HCI by

EDTA

at to

mixture

solutions

0.18 or

stand

added

a 0.06

in

.tock

prepared

heparinized

to

are

a reaction

EDTA

are

for added).

sample

ducing

Test

CELL

spot-test (nitrite

nitrited

Normal

RED

mg.

dry

is incubated

paper.

Dry

spots

lamp. RESULTS

Normal

is

blood

omitted,

or

hemolyzed reaction

mLxture, of

the

fluorescence

red

the

blood

a delay cells

defluoresces

in

in

cells

1’4luorescence

are

is heated there

at

is no

the

the

suspended time

30

no

56#{176}Cfor time.

Plasma

mixture. same

in various is

than

blood,

defluorescence.

reaction

gives

less

of

defluorescence of

saline

in

absence

proportional

result

hours

1). being

The

omission does

suspension unwashed

of plasma dilution

When

is

before

alone

A

the

(Fig.

defluorescence

as

amounts to

2

minutes

added

of not

of

DCIP

observed.

If to

nitrite

results

produce

any

washed

nitrited

nitrited whole

or

saline,

factor.

The

the same

the in dered

blood.

increased propor-

If

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332

KAPLAN

Fig.

2.-UV

perimental

plus

spot

the

for see

blood.

3 and

normal

tionality

test

conditions,

is observed

usefulness

diaphorase

than

defluorescence

the

other

clearly

for

of the

by

abnormal

less was

It

defluorescence

red

cell

subjects

tively,

is

time

gave

high

normal

cent.

hematocrit

reduction

This

spot

be

was

than

10 per to be

done

from

not

do

in

the

significantly

as compared

to

In

cent

did

24

per

an

hour,

On give

procedure

without infants

2). not

this

spite of blood. The and

subjects,

(Fig.

subjects

young

to six

it

NADH-

these

in

normal

11 per

removing

appropriate

cell

increased

samples, few

increased

counts,

normal.

recommend

to 70 blood that

reticulocyte

not

an

red

number

to prolong to normal

by applying

heterozygous

we

state. applied

of

influences cell

by

in

Their

considerably

two

red

either

cells

verified

cent

(For exmixture

value of

contributing the PCV

packed

test

found

noteworthy was

can

the

Therefore

NADH-diaphorase with

per

samples

results.

results.

The

combined

AL.

blood.

methemoglobinemia.

detection of the carrier The technique has been

positive

the

and normal blood. 2 and 4. Reaction

deficient

hemolysates.

ultraviolet

was blood

plus

suspending

congenital time

hand,

35

or

activity

the

deficient alone.

effect are additive, both We recommend readjusting

time.

with

mixture

mixture

because

the excess of plasma amount of saline.

The

Reaction

diluted

time

and hemoglobin-quenching the defluorescence whenever it is lower

diaphorase:

1.

5. Reaction

with

defluorescence

homozygotes

NADH

text).

ET

any

false

tested,

the

their blood

lowered of two

cent,

respec-

results. DISCuSSION

Since readily test

the

whole

available a simple

procedure equipment

and

fast

method

can and for

be

carried

reagents, diagnosis

out we of

within consider

the

NADH-diaphorase

using

only

fluorescent deficiency.

spot

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TEST

SCREENING

The

FOR

stability

of

the average NADH-diaphorase prepared,

the

are

for

is only deficiency.

this

can

be

for

many

stable

simply

it it

can quickly should be

cyanotic

the

of

as

in

in

a

the

an for

well

as

particular

importance,

to make solution

few

the must

state

and

the occasion presents itself.

of the

can

for

since

diagnosis of be freshly

All

minutes.

frozen

NADH when deficiency

provide valuable

infants,

is of

called upon the nitrite

months

333

DEFICIENCY

test

rarely Only

accomplished

added to a preweighed vial examination for NADH-diaphorase

Since samples,

NADH-DIAPHORASE

reagents

laboratory

and

solutions

CELL

RED

with

be

performing

all or none answer on capillary screening methemoglobineniic,

adults

other

merely

an

blood even

or

methemoglobinemia.

REFERENCES 1. Jaff, globinemia

E. R., and Heller, P.: in man. In: Moore,

Brown, E. B. (Eds.) Progress ogy, Vol. IV. New York, Grune 1964,

p.

2. in

The

reduction

erythrocytes

of

of

a patient

methemoglobinemia,

subjects

glucose-6-phosphate

deficiency

and

21:561, 3. of

in

Hematol-

human 1960.

4.

Hegesh,

5.

Scott,

72:339, E.

with

globin

reductase.

congenital erythro-

individuals.

for determining (NADH-methein erythrocytes. J. Lab. 1968.

M.:

A

NI.:

The

relation

erythrocytes

to

J.

Clin.

6. Beutler, procedures

of diaphorase Invest.

Clin. E.:

for

of 39:

ficiency

and

ficiency.

Blood

7. screening

E.,

Calmanovici,

N.,

of

two

DPNH-methemo-

Chim.

Acta,

23:49,

1969.

Blood

inheritance

comparison

determining

A series

and

NIed.

-,

and test

68:137,

of new

pyruvate

glucose-6-phosphate

E.

methemoglobinemia. 1176,

Med. of

with

method reductase

reductase)

Clin. methods

1963. Scott,

New

methemoglobin

dehydrogenase

normal

Avron, M.: fern-hemoglobin moglobin

& Stratton,

48.

-:

cyte

MethemoC. V., and

kinase

screening deficiency,

dehydrogenase glutathione 28:553, Baluda, for

de-

1966.

NI. C.:

galactosemia.

1966.

de-

reductase A

simple

spot

J. Lab.

Clin.

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1970 36: 330-333

A Simple Spot Screening Test for Fast Detection of Red Cell NADH-Diaphorase Deficiency JEAN-CLAUDE KAPLAN, ANNE-MARIE NICOLAS, ALENA HANZLICKOVA-LEROUX and ERNEST BEUTLER

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