8/10/2019 1.5 Platelet Antibodies in Immune Thrombocytopenic Purpura and Onyalai

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SA

MEDICAL

JOURNAL 6 JUNE 98 855

Platelet

antibodies

thrombocytopenic

In Immune

purpura and onyalai

S BRINK P B. HESSELlNG S AMADHILA H.

S

VISSER

ummary

A prospective study was undertaken to assess

the

nature, incidence and natural history of platelet

nt i od ies

in

p t ients with immune

thrombocytopenic purpura ITP) and patients with

onyalai,

using

an

immunofluorescent

technique.

Twelve patients under 14 years old and

patients

14 - 75 years old with ITP, and 24 patients with

onyalai were studied.

Aiternate younger

patients

were treated

with

corticosteroids

Ten

of

the

12

children

with

ITP had

IgG

platelet antibodies

in their serum, which

disappeared as the platelet count recovered.

Stero id therapy d id

not

change the

course of

the

disease or the antibody response.

Of

the 24 pat ients with onyalai, 23 had IgG

antibodies and 18 had IgM antibodies, which were

stil l present

after

14 days and unrelated to the rise in

platelet count. Steroid therapy did

not

affect the

platelet

count

or the antibody titre.

The difference in immune response of ITP and

onyalai points to a difference in aetiology.

The

clinical

presence

of

IgM antibodies in onyalai fits

the hypothesis

that

a toxin possibly acting as a

hapten

is

responsible

for

this

form

of

thrombocytdpen ia.

S.

Mr

me 59, 8SS

1981 .

In the serological investigation

of

immune thrombocytopenic

purpura ITP

it

is important to demonstrate

the presence

of

antibodies against platelets.

In vicro

studies for this purpose have

i nc lu de d a gg lu ti na ti on , c ompl eme nt fix at io n, i nh ib it io n of

complement f ixation, a nt ig lo bu li n c on su mp ti on and

125

1_

la belled a nt ig lo bu li n m ea su re me nt s in s er um . D ix on et al I

using a quantitative antiglobulin consumption technique, were

able

to

demonstrate increased

IgG on the

platelet surface,

but

the

method

is t oo c ompl ex for rou ri ne d ia gn osti c use.

Serological techniques have long

been

i n use bu t have led to

considerable problems.

2

Immunofluorescence tests

on

platelets

have in the past been

hampered

by nonspecific fluorescence

caused by non-immunological adsorbance

of

immunoglobulins

to

the

platelet surface. Recently it has

been

shown that fixation

with paraformaldehyde

PF

A) will prevent this without altering

cell surface antigens.

3

PF A fixation permits the stabilization of

Department

of

Haematology and Paediatr ics , Tygerberg

Hospital, ParowvaUei, CP

S.

BRINK, M B CH.B.,

L.F.

PAT S.A. ,

DIPL.

DATAMETRIE

P.

B.

HESSELING, M B CH.B., M.MED. PAED.

H. S. VISSER, DIPL. CLIN.

PATH.

HAEM B ECON

Oshakati

Hospital,

Oshakati , SWA

S.

AMADHILA, M B CH.B.

re

received: September 198

a nt ig en s at a ny d esired moment during the process of capping

and it inhibits non-immune binding

of

i mmun og lo bu li n to

platelets, granulocytes and lymphocyte . An alternative possibi

lity

is that PFA

inacri\ ates

IgG Fc

receptors

on

such

cell,

bur

does nor h in de r spe ci fi c b in di ng

of

antibodies

J

We r epor t on t he use

of

this immunofluorescent technique in

the study

of

patients with ITP and onyalai. The s er a f rom the e

patients were studied using pooled platelets, granulocytes and

lymphocytes from normal blood donors.

F ab ,-rabbit

antihuman

-RH

IgG-antiglobulin reagent was

u p p l ~

by th e

Department

of Immunohaematology, Central Laboratory of

the Netherlands Red Cross Blood Transfusion Centre,

Amsterdam.

In

principle this reagent

is

prepared

as

antibodies

against human IgG in rabbits.

The

serum was absorbed with

p ur if ie d p ro te in unt il it was s pec if ic for human y -c ha in s in a

passive haemagglutination test.

It

was t he n u bmirre d t o p arti al

enzymatic digestion

4

s

and

the

resulting F ab)2 fragments were

finally conjugated to fluorescein isothiocyanate

FITZ . The

background fluorescence obtained with these globulin fragments

is

significantly lower than with the native antibody molecule and

is

independent of the presence or absence ofFr receptor-bearing

cells.

atients and methods

Patients

The

sera from 47 patients with ITP or on ya la i we re

i nv es ti ga te d f or

platelet, granulocyte and lymphocyte

a nt ib od ie s. Twe lv e p at ie nt s, 7 fema les

and

5 m ales

under 14

years old,

and I I

patients,

9

female

and

2 males between 14

and

75 vears old, had a clinical diagnosis

ofITP. Two

female patients

with

subacute

lupus

e ry th emat os us SLE

and

t hrombo cy to pe ni a w ere also stu di ed . The sera from the 24

patients with onyalai were taken

on

t he 1st and t he 14th day of

t he a cu te illness.

The

l as t- na me d s er a we re

supplied

by

Dr

Amadhila from Oshakati Hospital, SWA,

and

transported

as

air

freight

in

a fro ze n sta te .

The

diagnosis

of

ITP in the a du lt s was ba sed on a bl eed in

o

disorder in the presence

of

thrombocytopenia, absence

of

preceding infection or drug ingestion, an increased number

of

megakaryocytes in the bone marrow aspirate

and

trephine biopsy

speCImens,

and

negative screening tests for

other auto-immune

disorders.

Controls

Twenty-six patients with thrombocytopenia

and

hypoplastic

or

a pl asti c a na emia o r p an cy to pe ni a a ssoc ia te d w it h va ri ou s

conditions were s tu di ed in a sim ilar m an ne r to t he above 47

patients, while another 26 patients with acute

or

chronic

leukaemia, lymphoma or metastatic carcinoma,

41

patients with

min or b lo od t ra nsfu si on rea ct io ns and 10 normal volunteers

from the medical

and

medical technology

staff

were screened for

the presence

of

platelet,

neutrophil

and lymphocyte antibodies.

Methods

The

i nd irec t p la te le t i mmun ofl uo re sc en t t est a d escrib ed by

von

dem

Borne el al

6

was u ed.

The

sera were examined with

8/10/2019 1.5 Platelet Antibodies in Immune Thrombocytopenic Purpura and Onyalai

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856 SA MEDIESE TYDSKRIF 6

JUNIE

9

commercial FITZ anti-l -globulin ehring Institute) an d

sheep-antihuman

IgG- Ig 1- and IgA-FITZ-antiglobulin

preparations Wellcome Laboratories).

Th e

sera

o f t h e

patients

with IT P an d onyalai were studied with the F ab)2-RH IgG

antiglobulin reagent.

Th e

indirect immunofluorescent test for

the determination of granulocyre antibodies and

of

lymphocyte

antibodies; was carried ou t with FITZ-Ig-antihuman globulin

reagent.

Fo r

each bat ch

of

testS the Western Province Blood

Transfusion Centre kindly supplied us with bloodfrom 5 normal

donors of blood group O. From these specimens a platelet pellet

was separated from the pooled platelet-rich plasma

an d

washed

thr ee times in EDTA-PBS hosphate-buffered saline).7 Th e

lymphocytes were separated on a Ficoll-Hypaque gradient

according to th e method of Boyiim.

8

In this gradient the pellet

contains the granulocytes with some red blood cells.

Th e

contaminating red cells are lysed for 5 minutes at 4 C with 2 ml

0,9 NH

4

CI-PBS containing

1

EDTA.7

Th e platelet, lymphocyte and granulocyte pellets from the 5

donors were pooled separately, washed three times in ED TA

PB S an d fixed by resuspension in 1 PFA for 5minutesat room

temperature. After a fur ther

three

washings,

th e

PFA-fixed

platelet, lymphocyre and granulocyte suspensions were

incubated with th e complement-inactivated test sera for 1 hour

at room temperature. After incubation th e cell suspensions were

again washed three times and incubated with th e appropriate

fluorescein-Iabelled antiglobulin preparation at optimal dilution

for 30 minutes

at

room temperature. Th e cells were then washed

twice, resuspended an d examined as wet preparations under an

immunofluorescence microscope. Fo r a negative control test ,

pooled group 0 cell suspensions were pu t

up

against

complement-inactivated

AB

serum. Th e positive control test

was a platelet suspension from a person of blood groupA pu t

up

against serum con taining ant i-A. A Wild-Le itz SM-LUX

microscope with epi-illumination using the

standard

filter set in

the

blue

e.xcitation range with excitation filters BP

45 49

+

FT

510 a nd b ar ri er filter LP 520 was used. Th e percentage of

fluorescent cells was judged by th e scoring of at least 200 cells,

using alternately blue incident an d phase cont rast light.

Reactions were scored

as

positive only when more

than 20

of

cells were fluorescent and if therewas definite evidence of ring

fluorescence Fig. 1 .

Platelets were counted

on C oulter

Model S Plus apparatus at

the

Department

of Haematology, Tygerberg Hospital, and on a

Coulter Model S at Oshakati Hospital.

Fig. 1. Membrane f luorescence o f human

platelets.

Incubation

was

performed with F ab ,-RH IgG-ant ig lobulin reagent af ter f ixation with

PFA, demonstrating

the

presence of IgG platelet antibodies x 1250 .

sults

T patients

u n d e r 14

years

Platelet antibodies were found in

th e

sera of 10 ofthe

12

ITp

patients under 14 years old. Th e antibody reacted most strongly

with th e

F I T Z ~ n t i l g l o b u l i n

preparation an d the F ab)2-RH

IgG-antiglobulin reagent, indicating

that

it was an Ig G

antibody. In 2 patients there was evidence of weak Ig M

antibodies and 1 patient had IgA antibodies. Th e investigations

were repeated

at

intervals during th e course of treatment Table

I). Statistically there was a significant association with th e Ig

antibody levels, which tapered of f as the platelet

count

improved

P

8/10/2019 1.5 Platelet Antibodies in Immune Thrombocytopenic Purpura and Onyalai

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SA

MEDICAL JOURNAL

6

JUNE

1981 857

500

ale

. . . . .

Truted

400

ONYALAI Results of 1st and

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(24)

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161*

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