for platelet surface antibodies and then identify the
specific glycoproteins involved by using mono–
clonal antibody specific immobilization of plate–
let antigens (MAIPA). Such an approach may not
be fruitful since in a study of 65 chronic ITP pa–
tients comparing FCM analysis ofintact platelets
with a ELISA based examination of plateJet lysa–
tes (Stockelberg et al, 1996) both techniques iden–
tified approximately 45
o/o
of patients with ITP. In
addition, several patients positive by one assay
were negative by the other assay and the authors
concluded that both the assays have their merits
and demerits. In another study, conventional FCM
for surface immunoglobulins when comparison
toMAIPA had only a limited role in the diagnosis
of ITP (Joutsi and Kekomäki, 1997). In selected
cases it may however be useful to analyze plate–
lets by fluorescence resonance energy transfer
(FRET) on a flowcytometer as diseossed below.
2.3 Clinical background to HLA and anti–
platelet specific antigen antibodies:
Management of thrombocytopenic patients who
are refractory to platelet transfusions can be a di–
agnostic problem. This may be due to alloimmu–
nization and/or the presence of platelet reactive
antibodies against human platelet antigens (HPA).
In addition, fever, sepsis, bleeding and splenome–
galy can be other confaunding factors. Multiple
studies show that the incidence of alloimmuniza–
tion is 50-90
o/o
after transfusions with non-leuko–
cyte depleted blood as opposed to 0-28
o/o
when
using leukodepleted platelets (Novotnyet al, 1995).
In the TRAP study 3-5
o/o
ofpatients formed allo–
antibodies and as much as 6-11
o/o
developed an–
tibodies against platelet glycoproteins (Slichter et
al, 1997). In another study using apheresis plate–
lets 23
o/o
ofpatients developed antibodies against
HLA antigens, 2
o/o
against HPA and importantiy
of the patients alloimmunized to HLA antigens 9
o/o
also had antibodies against HPA (Kick!er et al,
1990). HPA's such asPJA,Bak, PlE, Ko, Br, PJ!, Pen
may also be of importance in neonatal alloimmu–
ne thrombocytopenia purpura and posttransfusion
purpura. The identification of HLA and HPA an–
tibodies can have therapeutic implicationswith re–
ference to need for HLA matehed transfusions,
splenectomy etc.
94
2.4Key issues in HLA versus HPA antibodies:
The problem of distinguishing HLA from HPA
antibodies can be approached in multiple ways
depending on the clinical situation. The simplest
approach is to look for the presence ofplatelet sur–
face immunoglobulin, which has been shown to
correlate with the platelet, corrected count incre–
ment (Rosenfeld et al, 1985). This approach may
be adequate if ITP is not clinically suspected.An–
other approach is to do a platelet cross-matching
proeecture by FCM and to look for platelet reacti–
ve antibodies without distinguishing HLA from
HPA antibodies (Gates and MacPherson, 1994).
Such an FCM approach (Köhler et al, 1996) ear–
relates highly with the MAIPA technique as weil
as with lymphocytotoxicity based assays and off–
ers a high sensitivity and specificity. However, in
certain conditions such as neonatal alloimmune
thrombocytopenias it may be important to distin–
guish matemal HLA from HPA antibodies. The
easy and inelegant method is to first identify the
presenceof platelet surface immunoglobulin. This
is followed by treating the platelets with an acid–
chloroquinine reagent to strip the HLA antigens.
Any remaining reactivity is due to HPA antibo–
dies (Marshall et al, 1994). This method requires a
careful testing of the acid-stripping conditions. A
more intellectually challenging approach is to use
FRET flow cytometry.
3.1 Fluorescence resonance energy transfer
(FRET):
The theory and wide range ofclinical and research
applications for FRET have been reviewed (Szol–
losi et al, 1998). FRET involves the radiationless
transfer ofenergy from an excited donormolecule
to an acceptor molecule under favourable condi–
tions. In simplistic terms, once the donor fluoro–
chrorne is excited it rapidly achieves a higher vi–
brational state and then drops to the lowest vibra–
tionallevel of the excited state. The molecule can
then either decay radiatively by fluorescence or if
a suitable acceptor fluorochrome is available ne–
arby then it will not decay but rather transfer its
energy to the acceptor fluorochrome. The accep–
tor fluorochrome wi ll then achieve a higher vibra–
tional leve! and subsequently decay radiatively
(fluorescence). Twoconditionsmust bemet for this
to occur. Firstly, the fluorochromes must be such
Klinisk Kemi
i
Norden4. 1999
