THROMBOSIS RESEARCH 66; 43-53,1992 0049-3848/92 $5.00 + .OO Printed in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
PREPARATION
OF
PLASMA
AND
Kari
E.
Haematological
(Received
FOR
THE
DETECTION
ANTIPHOSPHOLIPID
Sletnes,
Karl
Research Hospital,
19.9.1991; accepted
OF
Gravem
and
Laboratory, N-0407
LUPUS
ANTICOAGULANTS
ANTIBODIES
Oslo
Finn Ulleval
4,
Wisloff University
Norway
in revised form 24.1.1992
by Editor P. Kierulf)
ABSTRACT The on
effect of the results
different of 1)
coagulant
a
methods of plasma preparation clotting assay for lupus antiactivated partial (th e dilute
(LA) detection and of 2) an ELISA test thromboplastin time, dAPTT), detection, was for anticephalin antibody (aCEPHA) evaluated. It is well known that platelet disintegration resulting from freeze-thawing of plasma samples may release procoagulant phospholipid - "LA-bypassing" activity. Even with fresh plasma, the dAPTT of LA positive samples the presence of was sensitive to residual blood platelets. This effect was accentuated by freezing and thawing: with test plasma that had been prepared by a centrifugation force of 3 000 g or less for 15 min shortening demonstrated platelet
led
to
at of
4OC, the
freeze-thawing (IAPTT. This
with
filtered
caused phenomenon test
plasma,
a significant could not which
be was
free. Surprisingly, ultracentrifugation also a substantial shortening of the dAPTT compared
to filtered method of
plasma, and should not be recommended as a plasma preparation for LA detection. The ELISA test was less sensitive to residual platelets than the dAPTT. Thus, plasma prepared by a centrifugation force of at least 1 500 g may be stored at -2O'C before performance of the ELISA test. For the dAPTT, filtering of test and plasma control plasma after centrifugation is recommended for maximum sensitivity, regardless of whether they are to be examined in the fresh state or after freezing and thawing.
Key
words:
Lupus APTT,
anticoagulants, platelets.
antiphospholipid
43
antibodies,
ANTICEPHALIN
44
ANTIBODIES,
dAPTT
Vol. 66, No. 1
INTRODUCTION Lupus the IgG pholipids have an
anticoagulants
(LA)
are
circulating
immunoglobulins
of
or
IgM isotype directed against negatively charged phos(1). Patients with the presence of LA are thought to increased risk of arterial and venous thrombosis (Z), and recurrent abortions and pregnancy loss (3). Thus, the laboratory diagnosis of LA is important. The tissue thromboplastin inhibition (TTI) test (4), the activated partial thromboplastin time (APTT) (5), the kaolin clotting time (KCT) (6,7), the dilute Russell's viper venom time (dRVVT) (8) and the platelet neutralization posed as tests for the phospholipids released platelets may reduce tests caused by LA processing in order stressed
(11).
test plasma the KCT is fresh plasma phospholipid recovery
of
High
procedure (PNP) diagnosis of LA. activation upon
the (IO).
(9) have all been proIt has been shown that or disintegration of of in vitro coagulation
prolongation
The importance to avoid this masking speed centrifugation
and control performed (14) was
plasma before for LA detection recommended. It
procoagulant LA activity
by
material simple
of
appropriate sample effect was recently (ll,lZ), filtration of
testing, (11,13), has also
may be extraction
particularly if or the use of been shown that inactivated with with chloroform
(15). Antiphospholipid antibodies (aPL) are commonly detected by an enzyme-linked immunosorbent assay (ELISA) (1). We have previously shown that anticephalin antibodies (aCEPHA) measured by have a high concordance with LA as detected with an ELISA test, time (dAPTT) on the results
the dilute activated partial thromboplastin effect of sample processing varying method has not been elucidated. The aim of the present study was different methods of plasma preparation the
samples
on
the
dAPTT
and
ELISA
MATERIALS
lest
to
(16). of
The this
evaluate the effect and of freeze-thawing
of of
results.
AND
METHODS
plasma
Venous blood and 2 patients containing 1.0 Dickinson, dAPTT with
obtained from 6 with aCEPHA only, ml of 0.105 mol/l
Meylan a I:1
Cedex, mixture
patients with both LA and aCEPHA, was collected into 10 ml tubes buffered sodium citrate (Becton The LA were diagnosed by a France).
of
test
plasma
and
fresh
control
plasma
(5,14,16), followed by PNP correction (9). The aCEPHA were diagnosed by an ELISA method (16). Three of the patients had aCEPHA of both IgG and IgM isotypes, 3 had IgM aCEPHA, whereas reference limit for aCEPHA of IgG 2 had IgG aCEPHA. The upper and IgM isotype is 1 ELISA unit (16). Thus, for the statistical results were treated as and IgM antibody evaluation, the IgG The tubes from each of the patients belonging to one isotgpe. 2 500 g, were centrifuged tit 4 C for' 15 tiin at 20 OOOg, 3 000 g, 1 500 each
g, 1 patient
000 5
g. ml
or of
500. g, venous
respectively. blood
centrifuged
Moreover, at
2
500
from g
for
Vol. 66, No. 1
ANTICEPHALIN ANTIBODIES, dAPTT
was filtered through a 0.22 urn filter 15 min, Platelet counts GS) to remove platelets (13). all plasma samples with an Ortho, ELT-EOO/WS.
Control
45
(Millipore Millexwere performed on
plasma
Venous blood from 6 healthy LA negative volunteers was colsodium containing a 0.105 mol/l citrate lected into tubes (9 parts blood to 1 part anticoagulant), and centrisolution obtain platelet poor fuged twice at 2 500 g for 15 min at 4’C to The median platele$. count in the control plasma plasma (PPP)9 Fresh control plasma was (range 3-17 x IO /l). was 10 x 10 /l used in each experiment.
Phospholipid
source
Crude cephalin (a mixture phosphatidylcholine, inositol, sphingomyelin) from bovine med,Pharma, Oslo, Norway.
Coagulation
of brain
phosphatidylserine/phosphatidylphosphatidylethanolamine was a generous gift
from
and Nyco-
tests
The APTT was performed as previously described (16). Briefly, 100 ~1 kaolin 5 mg/ml in 0.15 mol/l NaCl and 100 ul cephalin at standard dilution (1:200) were incubated with 200 1.11 test plasma initoiated by adafter which clotting was for 6 min at 37’C, 37 C. For a 1 :I to dition of 200 1.11 25 mmol/l CaC12, prewarmed ~1 of fresh control mixture, 100 ~1 of test plasma and 100 The plasma samples were plasma were mixed prior to incubation. concentrations (dAPTT) (16). also tested with lower cephalin The coagulation tests were performed on fresh plasma samples as described above. Each exprepared by different methods, freezing and thawing of the periment was repeated after 3 times plasma samples.
ELISA
for antiphospholipid
antibodies
aCEPHA were analysed as previously described (16), in an ELISA test where the wells of the microtiter plates were coated with diluted cephalin (I:1 000). Fetal calf serum (Sera-lab., Sussex, Ltd., England) diluted to 10% in phosphate buffered saline (pH 7.4) (FCS-10%) was used as a blocking agent and as a diluent. Plasma samples from 158 healthy individuals comprised the reference group (16). The 97.5 percentiles for IgG and IgM aCEPHA were chosen as the upper reference limits and allocated a value of 1 ELISA unit. A standard curve was calculated for each plate by eight twofold dilutions of plasma from a patient with strong IgG aCEPHA activity (IgG plate), and from a patient with strong IgM aCEPHA activity (IgM plate), respectively. A “fourparameter logistic” model for curve fitting was employed in a computer program (17), which also transformed the absorbances to ELISA units. The ELISA test was performed on plasma samples prepared by different methods, as described above. Each experiment was
ANTICEPHALIN ANTIBODIES, dAPTT
46
repeated samples.
after
Statistical The statistical considered
3
freezing
times
and
Vol. 66, No. 1
of
thawing
the
plasma
methods
Wilcoxon signed rank test evaluation. A p-value statistical significant.
(paired data) was used of < 0.05 (two-tailed)
for was
RESULTS
the with
Table 1 shows the results fresh plasma samples aCEPHA and/or LA.
of the prepared
TABLE
platelet differently
1
The Platelet Counts in fresh 8 patients with Anticephalin and/or Lupus Anticoagulants
Plasma method
= force
0 3 16 29 54
g g g g g g
of
3;:
(standard
x
109/1
(range)
(O1) (I11) (1520) (1643) (44-147) (47-244) (105-760)
centrifugation.
After centrifuaatinn of filtering for l-5 min at 4 C. 0.22 urn filter (Millipore
APTT
counts
Median
Filtering 20 000 3 000 2 500 1 500 1 000 500
g
Plasma Samples from Antibodies (aCEPHA) (LA).
Platelet
preparation
counts performed on from 8 patients
cephalin
venous
blood
was performed Millex-GS).
concentration,
at
2
500
through
g
a
1:ZOO)
filtering after centrifugation caused With fresh test plasma, a prolongation of the APTT (table 2, left side). Thus, compared shorter clotting significantly test plasma, filtered to the times were observed when the plasma samples had been prepared by a centrifugation force of 1 500 g or less (p
PREPARATION
OF
PLASMA
AND
Kari
E.
Haematological
(Received
FOR
THE
DETECTION
ANTIPHOSPHOLIPID
Sletnes,
Karl
Research Hospital,
19.9.1991; accepted
OF
Gravem
and
Laboratory, N-0407
LUPUS
ANTICOAGULANTS
ANTIBODIES
Oslo
Finn Ulleval
4,
Wisloff University
Norway
in revised form 24.1.1992
by Editor P. Kierulf)
ABSTRACT The on
effect of the results
different of 1)
coagulant
a
methods of plasma preparation clotting assay for lupus antiactivated partial (th e dilute
(LA) detection and of 2) an ELISA test thromboplastin time, dAPTT), detection, was for anticephalin antibody (aCEPHA) evaluated. It is well known that platelet disintegration resulting from freeze-thawing of plasma samples may release procoagulant phospholipid - "LA-bypassing" activity. Even with fresh plasma, the dAPTT of LA positive samples the presence of was sensitive to residual blood platelets. This effect was accentuated by freezing and thawing: with test plasma that had been prepared by a centrifugation force of 3 000 g or less for 15 min shortening demonstrated platelet
led
to
at of
4OC, the
freeze-thawing (IAPTT. This
with
filtered
caused phenomenon test
plasma,
a significant could not which
be was
free. Surprisingly, ultracentrifugation also a substantial shortening of the dAPTT compared
to filtered method of
plasma, and should not be recommended as a plasma preparation for LA detection. The ELISA test was less sensitive to residual platelets than the dAPTT. Thus, plasma prepared by a centrifugation force of at least 1 500 g may be stored at -2O'C before performance of the ELISA test. For the dAPTT, filtering of test and plasma control plasma after centrifugation is recommended for maximum sensitivity, regardless of whether they are to be examined in the fresh state or after freezing and thawing.
Key
words:
Lupus APTT,
anticoagulants, platelets.
antiphospholipid
43
antibodies,
ANTICEPHALIN
44
ANTIBODIES,
dAPTT
Vol. 66, No. 1
INTRODUCTION Lupus the IgG pholipids have an
anticoagulants
(LA)
are
circulating
immunoglobulins
of
or
IgM isotype directed against negatively charged phos(1). Patients with the presence of LA are thought to increased risk of arterial and venous thrombosis (Z), and recurrent abortions and pregnancy loss (3). Thus, the laboratory diagnosis of LA is important. The tissue thromboplastin inhibition (TTI) test (4), the activated partial thromboplastin time (APTT) (5), the kaolin clotting time (KCT) (6,7), the dilute Russell's viper venom time (dRVVT) (8) and the platelet neutralization posed as tests for the phospholipids released platelets may reduce tests caused by LA processing in order stressed
(11).
test plasma the KCT is fresh plasma phospholipid recovery
of
High
procedure (PNP) diagnosis of LA. activation upon
the (IO).
(9) have all been proIt has been shown that or disintegration of of in vitro coagulation
prolongation
The importance to avoid this masking speed centrifugation
and control performed (14) was
plasma before for LA detection recommended. It
procoagulant LA activity
by
material simple
of
appropriate sample effect was recently (ll,lZ), filtration of
testing, (11,13), has also
may be extraction
particularly if or the use of been shown that inactivated with with chloroform
(15). Antiphospholipid antibodies (aPL) are commonly detected by an enzyme-linked immunosorbent assay (ELISA) (1). We have previously shown that anticephalin antibodies (aCEPHA) measured by have a high concordance with LA as detected with an ELISA test, time (dAPTT) on the results
the dilute activated partial thromboplastin effect of sample processing varying method has not been elucidated. The aim of the present study was different methods of plasma preparation the
samples
on
the
dAPTT
and
ELISA
MATERIALS
lest
to
(16). of
The this
evaluate the effect and of freeze-thawing
of of
results.
AND
METHODS
plasma
Venous blood and 2 patients containing 1.0 Dickinson, dAPTT with
obtained from 6 with aCEPHA only, ml of 0.105 mol/l
Meylan a I:1
Cedex, mixture
patients with both LA and aCEPHA, was collected into 10 ml tubes buffered sodium citrate (Becton The LA were diagnosed by a France).
of
test
plasma
and
fresh
control
plasma
(5,14,16), followed by PNP correction (9). The aCEPHA were diagnosed by an ELISA method (16). Three of the patients had aCEPHA of both IgG and IgM isotypes, 3 had IgM aCEPHA, whereas reference limit for aCEPHA of IgG 2 had IgG aCEPHA. The upper and IgM isotype is 1 ELISA unit (16). Thus, for the statistical results were treated as and IgM antibody evaluation, the IgG The tubes from each of the patients belonging to one isotgpe. 2 500 g, were centrifuged tit 4 C for' 15 tiin at 20 OOOg, 3 000 g, 1 500 each
g, 1 patient
000 5
g. ml
or of
500. g, venous
respectively. blood
centrifuged
Moreover, at
2
500
from g
for
Vol. 66, No. 1
ANTICEPHALIN ANTIBODIES, dAPTT
was filtered through a 0.22 urn filter 15 min, Platelet counts GS) to remove platelets (13). all plasma samples with an Ortho, ELT-EOO/WS.
Control
45
(Millipore Millexwere performed on
plasma
Venous blood from 6 healthy LA negative volunteers was colsodium containing a 0.105 mol/l citrate lected into tubes (9 parts blood to 1 part anticoagulant), and centrisolution obtain platelet poor fuged twice at 2 500 g for 15 min at 4’C to The median platele$. count in the control plasma plasma (PPP)9 Fresh control plasma was (range 3-17 x IO /l). was 10 x 10 /l used in each experiment.
Phospholipid
source
Crude cephalin (a mixture phosphatidylcholine, inositol, sphingomyelin) from bovine med,Pharma, Oslo, Norway.
Coagulation
of brain
phosphatidylserine/phosphatidylphosphatidylethanolamine was a generous gift
from
and Nyco-
tests
The APTT was performed as previously described (16). Briefly, 100 ~1 kaolin 5 mg/ml in 0.15 mol/l NaCl and 100 ul cephalin at standard dilution (1:200) were incubated with 200 1.11 test plasma initoiated by adafter which clotting was for 6 min at 37’C, 37 C. For a 1 :I to dition of 200 1.11 25 mmol/l CaC12, prewarmed ~1 of fresh control mixture, 100 ~1 of test plasma and 100 The plasma samples were plasma were mixed prior to incubation. concentrations (dAPTT) (16). also tested with lower cephalin The coagulation tests were performed on fresh plasma samples as described above. Each exprepared by different methods, freezing and thawing of the periment was repeated after 3 times plasma samples.
ELISA
for antiphospholipid
antibodies
aCEPHA were analysed as previously described (16), in an ELISA test where the wells of the microtiter plates were coated with diluted cephalin (I:1 000). Fetal calf serum (Sera-lab., Sussex, Ltd., England) diluted to 10% in phosphate buffered saline (pH 7.4) (FCS-10%) was used as a blocking agent and as a diluent. Plasma samples from 158 healthy individuals comprised the reference group (16). The 97.5 percentiles for IgG and IgM aCEPHA were chosen as the upper reference limits and allocated a value of 1 ELISA unit. A standard curve was calculated for each plate by eight twofold dilutions of plasma from a patient with strong IgG aCEPHA activity (IgG plate), and from a patient with strong IgM aCEPHA activity (IgM plate), respectively. A “fourparameter logistic” model for curve fitting was employed in a computer program (17), which also transformed the absorbances to ELISA units. The ELISA test was performed on plasma samples prepared by different methods, as described above. Each experiment was
ANTICEPHALIN ANTIBODIES, dAPTT
46
repeated samples.
after
Statistical The statistical considered
3
freezing
times
and
Vol. 66, No. 1
of
thawing
the
plasma
methods
Wilcoxon signed rank test evaluation. A p-value statistical significant.
(paired data) was used of < 0.05 (two-tailed)
for was
RESULTS
the with
Table 1 shows the results fresh plasma samples aCEPHA and/or LA.
of the prepared
TABLE
platelet differently
1
The Platelet Counts in fresh 8 patients with Anticephalin and/or Lupus Anticoagulants
Plasma method
= force
0 3 16 29 54
g g g g g g
of
3;:
(standard
x
109/1
(range)
(O1) (I11) (1520) (1643) (44-147) (47-244) (105-760)
centrifugation.
After centrifuaatinn of filtering for l-5 min at 4 C. 0.22 urn filter (Millipore
APTT
counts
Median
Filtering 20 000 3 000 2 500 1 500 1 000 500
g
Plasma Samples from Antibodies (aCEPHA) (LA).
Platelet
preparation
counts performed on from 8 patients
cephalin
venous
blood
was performed Millex-GS).
concentration,
at
2
500
through
g
a
1:ZOO)
filtering after centrifugation caused With fresh test plasma, a prolongation of the APTT (table 2, left side). Thus, compared shorter clotting significantly test plasma, filtered to the times were observed when the plasma samples had been prepared by a centrifugation force of 1 500 g or less (p
