0095-1137/81/061026-05$02.00/0
Freeze-Dried
Erythrocytes
for
an
Indirect
Hemagglutination
Test
for
Detection of
Cytomegalovirus
Antibodies
N.CABAU,' R. CRAINIC,2 C. DUROS,' G. DENOYEL,3 A. GASPAR,3 C. BRONNERT,2 A.
BOUÉ,'
ANDF.HORODNICEANU2*
InstitutNational de la Santéetde la RechercheMédicale, Unité 73, Château deLongchamp, Paris
75016';
Unité deVirologieMédicale, InstitutPasteur, Paris 75724 Cedex 152; Service des Virus, Institut Pasteur de
Lyon,Lyon 69365Cedex23;France
Received 9 December1980/Accepted18February1981
An indirecthemagglutinationtestwithlyophiized, fixed, tanned,and cytomeg-alovirus
(CMV)-sensitized sheep
erythrocytes
for thedetectionofCMVantibodiesisreported.To avoidnonspecifichemagglutination, cellswerefixed with glutar-aldehyde orFormalin directlyin whole blood. The
lyophilized,
CMV-sensitizederythrocytes
obtained by thistechnique
werestable upto9monthsat37°C
andretainedthe samereactivityasfresh, CMV-sensitizedcells. Indirect hemaggluti-nation performedwith
lyophilized,
sensitizedcellswashighlyefficient indetectingCMV-antibodies ascomparedwithcomplementfixation andenzyme
immunoas-say.
The
multiple
avantagesof the indirecthemag-glutination
technique (IHA)
fordetectionof hu-mancytomegalovirus (CMV)antibodies ascom-pared
with otherserological
methodshavebeenreviewed
recently (4). IHA,
however,
poses prob-lemsas aroutinetestwithregard tothe prepa-ration of the reagentsand,
especially,
thesensi-tizing
oferythrocytes (RBC)
with virus. Anothermajor drawback in the standardization ofIHA
is the instability of sensitized RBC.
Yeager
(6)
developed
animproved technique
for IHA
by using
humanORBC fixed inglutar-aldehyde
and frozen inliquid nitrogen.
Wetried to
freeze-dry
fixed, CMV-sensitized
sheep
RBC,
and noticed that the main obstacletotheuseof such RBC in IHAwas anonspecific
agglutination
occurring
afterlyophilization
of RBC.We have been able to demonstrate that the
origin
of thisnonspecific
agglutination
wasthe aldehyde fixation of centrifuged and washed RBC. Ifthe RBCwerefixed byglutaraldehydeorformalin in thewhole blood before centrifu-gation,they could then be sensitized and lyoph-ilized without introducing nonspecific aggluti-nation. Suchlyophilized, sensitized RBC proved stable forlong periods of storage, even at 37°C. This paper describes in detail the procedure for the
preparation
oflyophiized,
CMV-sensi-tizedRBC andgives results obtained with such RBC for quantitative detection of CMV anti-bodiesinhuman sera.MATERIALS AND METHODS
Selection of an RBC donor sheep. In IHA an
important variable is the quality of the RBC from
differentanimals. We thereforeselectedagooddonor
fromsix young sheep. The donor animalwaschosen
accordingto the results of acomparative CMV-IHA
testperformed with RBC of the sixsheep,preparedas described below.
Processing and fixation ofRBC. Whole blood
wasdrawn from the donorsheep intoabottle
contain-ingAlsever solution (AS) (20.5 g ofglucose,0.2 g of
NaCl,8goftrisodium-citrate, 0.55 g of citric acid, in
atotal of1liter of distilledwater).BloodandASwere
mixed inequalvolumes.The mixture ofbloodand AS wasbroughtimmediatelyto the laboratory, and the
fixationof RBC was carried out the same day.
We used a 70% solution of electron microscope
grade glutaraldehyde (Janning, Paris, France). For
long storage, glutaraldehyde was diluted in distilled water to 10% concentration and distributed in 1-ml vialsthat were sealed and kept at 4°C. A 1:150 or 1: 250 final dilution was made in phosphate-buffered
saline (PBS) (pH 7) just before use and mixed with
sheepblood in AS in equal volumes. The mixture was
incubatedovernight at 37°C with gentle stirring. The
nextday, fixed RBC were sedimented at 1,000 x g for 20minand washed five times in PBS.
ForFormalin fixation, the commercial 30% solution (Prolabo) was diluted to obtain a 4% solution in tri-sodiumcitrate (0.15 M). Equal parts of 4% formalin andsheep blood in AS were mixed and processed as above.
Preparation ofCMV antigen. Human embryo
lungfibroblast (HEL) cells strain 809 grown in Eagle minimumessential mediumsupplemented with 10% newborn calf serum were usedthroughout this study.
Confluentcultures of HEL in 75-cm2 plasticflasks
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wereinoculated with 0.5 ml of AD-169 human CMV
stocksuspension, and 15 ml of serum-free minimum
essentialmediumwasadded. Four days later, when an
extensive cytopathic effect had developed, infected
cells were trypsinized and then inoculated into four
other HEL cultures in 75-cm2 flasks, and 15 ml of
serum-free mediumwasadded.
After another 4 to 5days, when an advanced
cyto-pathic effect wasobserved, all cells were trypsinized
andpassagedontofour Rouxplastic bottles (150 cm2)
containing confluent monolayers of HEL, and 50 ml of
serumfree minimum essential mediumwasadded.
After4to5days the medium was discarded, and all
thecells from thefour Roux flasks were scraped and
suspended in 8 ml of PBS (pH 7). The infected cell suspension was then frozen and thawed three times
andcentrifuged at 1,000Xgfor 20 min. After
centrif-ugation, the supernatant fluid, considered as CMV
antigen,wasdistributed in 0.5-mlvolumes in vials and
stored inliquid nitrogen.
The appropriate dilution of the antigen used to
sensitize RBC was determined in an IHA test as
describedbelow. Theoptimaldilution varied between
1:15and 1:60.
Preparationoftanned RBC. A 3% suspension (in PBS, pH 7) of fresh unfixed, or glutaraldehyde- or
Formalin-fixed RBCwasmixed inequalvolumes with
atannic acid solution(in PBS) and allowedtostand
for15minat37°C.
Theconcentration of tannic acid solution varied (1:
40,000to1:160,000) and hadtobe determinedfor each
donor sheep and for each batch of viral antigen,as
describedpreviously (1).
Sensitization oftanned RBC with CMV
anti-gen. Forsensitization, tanned RBCwerecentrifuged
andsuspendedat aconcentration of 3% in0.15MPBS (pH 6.8).
Equal volumes of tanned RBC and CMV antigen
diluted in PBSweremixed and incubated for 15 min
at roomtemperature.
The CMV-sensitized RBC (CMV-RBC) were
washed once in PBS andresuspendedto6% insterile
PBScontaining0.2%crystallizedbovineserum
albu-min.Lactose(5%)wasalso addedtoCMV-RBC before
lyophilization.
Lyophilization procedure. Thefixed CMV-RBC
suspensionwasdistributed in 0.5-ml volumes into
5-mllyophilization vials,stirredgently, andfrozen
im-mediatelyat-70°C. Thevialswerethenlyophilized
inanUsifroid(Paris)apparatus.
Foruse, each vialcontaining RBCwasreconstituted
with 0.5 ml ofsterile distilled water, and 2.5 ml of
sterile PBScontaining0.2% bovineserumalbuminwas
added to obtainafinal 1% RBC solution.
IHAtest.Seratobe testedwerefirst adsorbed with
freshsheepRBCovernightat4°C.Thenextday,the
RBCwereremovedbycentrifugationat1,000xg for
20min.
IHAtests wereperformedinmicroplatesas
previ-ously described (1). Sera to be tested were
serially
diluted 1:10 to 1:5120 in sterilePBS containing0.2%
bovine albumin.
In all IHAreactions, the following controls were
included:anegativeserumandtwopositivesera,one
ofthe high titer (1:1,280 to 1:2,560) and one of medium
titer (1:320 to 1:640). The same positive and negative
control sera wereused throughout this study.
In parallel with CMV-RBC, each serum wasalso
tested with nonsensitized RBC. In some comparative
IHA tests, unflxed but washed, tanned CMV-RBC
were used without beinglyophilized.
CF.Complementfixation (CF) was done by using
the microtiter system and commercial
glycine-ex-tracted CMV, strain AD 169-infectedcell antigen.
EIA. Enzyme immunoassay (ETA) was performed
by the technique already published for the measure-ment of herpes antibodies (2) with some modification
(Denoyel et al., manuscript inpreparation).
RESULTS
Critical
steps inprocessing
freeze-driedCMV-RBC.
It is
first
important to screen sheep
to
find a suitable donor. To
avoid nonspecific
agglutination of fixed, lyophilized RBC, it was
essential
to treat the sheep
blood mixed with
Alsever solution directly with glutaraldehyde or
Formalin. When the RBC were
first separated
from the plasma by
centrifugation,washed,
fixed, and freeze-dried, nonspecific agglutination
in the
IHA test was regularly observed. Evengentle centrifugation of
theblood followed
byresuspension of the
RBC in the samesuperna-tant plasma
rendered the RBC unsuitable for
subsequent fixation
andlyophilization. These
experiments
wererepeated
several times, and
the results consistently showed that it was
crit-ical to
fixRBC
inthe whole
blood plus
antico-agulant solution. Such fixed RBC could then be
tanned,
sensitized with a viral antigen,
freeze-dried, and used
in IHAwithout agglutinating
nonspecifically.
When the
fixation
wasperformed with
glutar-aldehyde,
wefound
thatthe
chemical
purity
ofthe
reagent was acritical factor
inobtaining
specific agglutination. Only a pure concentrated
product kept
insealed vials
at4°C gave
satisfac-tory results in
ourhands. Even with
such
apure
product, it
wasnecessary
tocheck every batch
of
glutaraldehyde
toavoid
oneproducing
non-specific agglutination of fixed RBC.
The fixation of RBC
by Formalin under the
conditions described in
Materials and Methods
was
easily and constantly
accomplished by
using
a
common, commercial
product.
Another critical step in
preparing fixed RBC
for sensitization with a viral
antigen
was their treatment with tannic acid. Ourresults,
in thisrespect,
were inagreement
with thefindings
ofYeager (6)
concerning
the choice oftannic
acid concentration.Thus,
optimal
concentrations
oftannic
acid were found to be between1:40,000
and
1:160,000,
depending
on the RBC donorsheep
and thebatch of viralantigen.
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Freeze-drying of fixed RBCwascarriedoutin
PBS (pH 7) containing 0.2% bovine serum
al-buminand 5% lactose. The reconstitution of the
dry productindistilledwaterwasgreatly
facili-tatedby thepresence of lactose.
Comparison
of IHAperformed
with freeze-dried,
CMV-RBC with CF andELA.
Two differentgroupsof humansera weretested
in two independent laboratories both by IHA
performedwith
lyophilized,
CMV-RBC andbyCF.Agoodcorrelation in CMVantibody
detec-tionwasfound between these twotechniquesin
bothlaboratories,asrevealedbythecoefficients
of correlation(r= 0.8041 andr=0.8048).
We also compared CMV antibody detection byIHA, CF,and EIAinagroupof 31 randomly chosenhumansera.Ail three methods detected
the presence of CMV antibodies equally well.
However, a difference was seen in the titers
obtainedbythese methods.Byusing geometric
meantiterratios, EIAwasfound to be about2.4
timesmorereactive thanIHA,whichwasabout
4.9-foldmorereactivethan CF.
Reactivity of freeze-dried CMV-RBC in
IHA. The use of freeze-dried, virus-sensitized
RBCinIHA raised thequestionof thereactivity
of sucha reagent indetectingCMV antibodies.
Toanswerthisquestion,weperformed
compar-ativeIHA tests with (i) washed, tanned
CMV-RBC; (ii) glutaraldehyde-fixed, tanned,
CMV-sensitized, and
lyophilized
RBC; and (iii)for-malin-fixed, tanned, CMV-sensitized,and lyoph-ilized RBC.
In a series of10 comparative IHA tests, we
compared the antibody titers of two positive reference sera and negative control serum, by
using simultaneously i, ii, iii, and control
non-sensitized erythrocytes. The results are
pre-sented in Table 1. Itcan beseenthat the
geo-metric meantiters of IHA antibodies obtained
withiiand iiiwerecomparabletothoseobtained
with i. However,asmall statistically significant
difference was noticed between iii and i, the
formerproving somewhatlessreactive in these
tests.
These results were confirmed in another
ex-perimentinwhich thethreetypesofCMV-RBC
were tested simultaneously against 58 selected
humanserathatwerepositive in CF (Table 2).
We found a very good correlation when we
compared the geometric mean titer obtained
with iand iii (r=0.9503),and withiand ii (r=
0.9431). From the same experiment (Table 2),
wecanconclude that therewasnotasignificant
difference between the geometric mean titers
obtainedby using differenttypesofCMV-RBC. The CMVantibody titers detected inIHA with
freeze-dried, sensitized RBCwere not different
TABLE 1. Comparison oftwosensitization methods
onthe RBCbehavior in IHA reaction withreference
antibodies CMV-RBC
Lyophilizedafterfixation
Reference No.ofwih
antibodies tests
Freshh:
Glutaralde-hyde
Formalin
Positive 10 9.30a (0.00) 9.00 (0.48) 8.30b (0.82) serum 1
Positive 10 11.10 (0.43)10.30b(0.00)10.10b(0.63) serum2
Negative 10 0 0 0
serum
a
Geometric
mean hemagglutination titers(log2)
and standard deviations (in parentheses) for 10 tests with each category of RBC (fresh or freeze-driedafter fixation with formalin orglutaraldehyde)against each reference serum.
bStatistically significant different (ttest, P<0.01)
geometric mean titer as compared to thatobtained
withfresh RBC takenasstandard.
from thoseobtained with fresh CMV-RBC.
Thermostability of
lyophilized
CMV-RBC. Vials
oflyophilized CMV-RBC
from fivedifferent
batches of ii and one batch of iii RBC wereheld in an incubator at37°C.After
various times of storage, vials were withdrawn, reconsti-tuted, and run in an IHA test with onenegative and two positive reference serum(Table
3). A control offreeze-dried,
but nonsensitized RBC was also included in each experiment. All sixbatches
of freeze-driedCMV-RBC
werestable
up to 9 months of
maintenance
at 37°C. Non-specificagglutination
inthe presence of control negative sera orof nonsensitized RBC was not observed.Repeated
IHA assays performed at variousintervals
with the same batch of freeze-driedCMV-RBC kept
at37°C
showedlittle
variation in titer (1 to 2 dilutions), which was not more than theinherent
variation of the system.DISCUSSION
IHA has multiple advantages as a routine
procedure
in the diagnosis of CMV (4) and in thescreening of blood donors to decrease trans-fusion- andtransplantation-associated CMVin-fections
(6). Moreover, thepossibility
ofusing small bloodsamples collected on PKU(phenol-keto-urea)
cards (1) and a microprocedure (3) make IHAthemost suitable method for epide-miological studies.Because
long-termpreservation
of CMV-RBC isthe main limitation of IHA, we developed aon February 7, 2020 by guest
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ERYTHROCYTES FOR IHA TEST FOR CMV ANTIBODY 1029
TABLE
2. Capacity of CMV-sensitized, freeze-driedRBC todetect
thepresence
of CMVantibodiesindifferent
humanseraaStandarddevia-
~~~~~Correlation
coef-CMV-RBC GMTb(log2) Standarddevin
t(level ofsignificance) ficient(r)fori-ndividualsamples
Formalin-fixed 8.97 2.54
0.3809
(P< 0.70) 0.9503Fresh 9.16 2.82
0.8416 (P<0.40) 0.9431
Glutaraldehyde-fixed 8.73 2.70
a IHA tests wereperformed with each kind ofCMV-RBC with 58 human sera with
different
CMV antibodytiters asdetermined by CF. The level of significance (P) ofdifference between twogeometric means was
evaluatedbyStudent'sttest.
bGMT, Geometrie mean titer.
TABLE 3. Stability of CMV-sensitized, freeze-driedRBCduring storageat37°C
Reference Antibodytiters'atmonth: RBCa batch
serab
1 2 3 5 6 8 9
A Pos. 1 640 640 320 640 640
Pos. 2 1,280 2,560 640 2,560 2,560
Neg. 0 0 0 0 0
B Pos.1 640 320 320 320 640
Pos. 2 1,280 640 1,280 1,280 1,280
Neg. 0 O O O O
J Pos. 1 640 640 640
Pos. 2 2,560 1,280 2,560
Neg. 0 O O
K Pos.1 640 320 320 640 640
Pos. 2 1,280 640 1,280 1,280 2,560
Neg. 0 0 0 0 0
L Pos.1 640 320 640
Pos. 2 640 1,280 1,280
Neg. 0 0 0
Y Pos.1 320 160 320
Pos.2 1,280 640 640
Neg. 0 O O
a TheRBCweresensitized with CMVantigenafter
fixation
withglutaraldehyde,
except for batchY, whichwasfixed with Formalin.
bThe titers of
reference
serawhen tested inparallelIHAtestwithCMV-sensitized,fresh RBCwere:positiveserum1(Pos. 1), 640;positiveserum 2(Pos. 2), 1,280 to 2,560; andnegativeserum(Neg.),0(c20).
eReciprocalof theendpointdilutionasdetermined withsensitized,lyophilizedRBC heldat
37°C.
means of
freeze-drying
suchRBC. The
mainobstacle
inthe
preparation of stable
reagentsforIHA
wasnonspecific
hemagglutination resulting
from the RBC
preparation
procedure.
Itbecameclear that the causeof this
nonspecific
hemag-glutination
wasfixation of RBCby
glutaralde-hyde
orformalin afterRBC had beenseparated
from
plasma by
centrifugation.
When wholesheep
bloodcollected in ASwastreateddirectly
with
glutaraldehyde
orformalin,
RBC couldthen be
tanned,
virussensitized,
andfreeze-driedwithout
showing subsequent
nonspecific
hemag-glutination.
We have confirmed our previous results (1)
and Yeager's
findings
(6) concerning the needfor determining optimaltannic acid
concentra-tions, which varyaccording to both the donor sheepand thebatchofCMVantigen.
Theuseof Formalin for RBC fixation wasless
critical than glutaraldehyde treatment.
How-ever, theFormalin-fixed
CMV-sensitized,
freeze-driedRBCinthe IHA test were somewhatless reactive than the
glutaraldehyde-f;xed
reagentindetecting CMVantibodies.
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1030
CABAU ET AL.It is important to stress that
freeze-dried
CMV-RBC
werestable
upto9months
at370C.
During
this
time their
behavior inthe
IHA testdid
notchange.
We
found
aremarkable
correlation between
IHA,
CF, and EIA,
illustrating the
equal
relia-bility of all three
techniques
inthedetection
ofCMV antibodies.
However, the levels of
anti-body detected
wereclearly
higher
in EIA and IHA.Another
advantage of
IHAisfor
detection of
those
classes of antibodies
which seems to ap-pear first inCMV
infection;
IHAreactions
wereshown
tocorrelate well
with the presenceof
antibodies
toCMV
early
antigens detected by
immunofluorescence (5).
Yeager
(6)
simplified the
IHA test forCMV
by
using
human groupORBC fixed
inglutaral-dehyde and stored frozen
at-70°C.
With
ourmethod it
was notpossible
to usehuman RBC
since
the methodof fixation
thatwedeveloped
necessitated
glutaraldehyde
treatmentof RBC
directly
in thewhole blood.
Wecould therefore
not
exclude
thepossibility
that CMV antibodies
present
in human
serawould
bind toRBC
dur-ing fixation.
On
theother
hand,
wedid
notfind
that
glutaraldehyde-fixed cells reacted with the
plastic
ofmicrotiter
plates, enhancing
nonspe-cific
hemagglutination,
asreported by Yeager
(6).
At the present
time,
twolaboratories
collab-orating
inthisstudy routinely
usetheprocedure
described
above.They
havefound that
IHAperformed with freeze-dried
CMV-RBC is
re-markable for its simplicity and highreproduci-bility.
ACKNOWLEDGMENTS
We thank our colleague Susan Michelson for the helpful advice in preparing this manuscript. We are grateful to our colleagues J. M. Huraux (Hôpital Pitié-Salpétrière), S. Olivier (Hôpital Ambroise Paré), and F. Vezinet-Brun (Hôpital Claude Bernard) for testing some of our reagents in their laboratories.
This work was supported by grants from Institut National de la Recherche Médicale, contracts 005 ATP 36-76-68 and 011AT 50-77-82 to F. Horodniceanu and CRL 76-5-149-8 to A. Boué.
LITERATURE CITED
1. Cabau, N., C. Duros, N.Ravise, M. Coulon, and A. Boué. 1976. Titrage des anticorps anti-cytomégalovirus sur le sang recueilli sur buvard par la technique d'hémagglutination indirecte. Pathol. Biol. 8:575-579. 2. Denoyel, G. A., A. Gaspar, and C. Nouyrigat. 1980.
Enzyme immunoassay for measurement of antibodies toherpes simplex virus infection:comparison with com-plement fixation, immunofluorescent-antibody, and neutralization techniques. J. Clin. Microbiol. 11:114-119.
3. Fucillo,D. A., F. L. Moder, R. G. Traub, S. Hensen, and J. L. Sever. 1971. Micro-indirecthemagglutination testforcytomegalovirus. Appl. Microbiol. 21:104-107. 4. Horodniceanu, F., and S. Michelson. 1980. Assessment
ofhuman cytomegalovirus antibody detection tech-niques. Brief review. Arch. Virol. 64:287-301. 5. Michelson, S., N. Cabau, A. Boué, and F.
Horodni-ceanu.1979.Comparison of occurrence of antibodies to human cytomegalovirus as demonstrated by immuno-fluorescence and indirect hemagglutination techniques. J. Clin. Microbiol. 9:149-151.
6. Yeager,A.1979.Improved indirecthemagglutination test for cytomegalovirus using human O erythrocytes in lysine. J. Clin. Microbiol. 10:64-68.
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