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Mul i-Tissue Con ols and Mul iplex Immunocy ochemis y in Pulmona y Cy ology
© 2024 The Au ho (s). Published by S. Ka ge AG, Basel
Published e sion
Vuo isalo, An i; Haapaniemi, Teppo; Kholo á, I ana
Vuo isalo, A., Haapaniemi, T., & Kholo á, I. (2024). Mul i-Tissue Con ols and Mul iplex
Immunocy ochemis y in Pulmona y Cy ology. Ac a Cy ologica, 68, 481-493.
h ps://doi.o g/10.1159/000540367
2024
Ac a Cy ologica
Techniques
Ac a Cy ologica 2024;68:481–493
DOI: 10.1159/000540367
Recei ed: Ap il 11, 2024
Accep ed: July 12, 2024
Published online: July 30, 2024
Mul i-Tissue Con ols and Mul iplex
Immunocy ochemis y in Pulmona y
Cy ology
An i Vuo isalo
a, b
Teppo Haapaniemi
b, c
I ana Kholo á
a, b
a
Facul y o Medicine and Heal h Technology, Tampe e Uni e si y, Tampe e, Finland;
b
Depa men o
Pa hology, Fimlab Labo a o ies, Tampe e, Finland;
c
Depa men o Biological and En i onmen al Sciences,
Uni e si y o Jy äskylä, Jy äskylä, Finland
Keywo ds
Mul i- issue con ols ·Mul iplex immunocy ochemis y ·
Immunocy ochemis y ·Pulmona y cy ology
Abs ac
In oduc ion: The Wo ld Heal h O ganiza ion 2021 lung
cance classifica ion highligh s he cen al ole o immuno-
his ochemis y (IHC) in diagnos ic pa hology. Despi e adi-
ional IHC being essen ial, i s limi a ion o one ma ke pe
issue sec ion b ings challenges, pa icula ly when acing
cy ological limi edly sized samples. To o e come hese
challenges, mul iplex immunocy ochemis y (mICC) ech-
niques o e he simul aneous de ec ion o mul iple ma ke s
om a single sec ion. These ad ances complemen he highly
complex imaging echniques ha enable addi ional analyses
o cellula in e ac ions. Me hods: The p esen s udy ou lines a
comp ehensi e mICC me hodology o an au oma ed mul i-
plex immunope oxidase s aining me hod and mul iple issue
hyb id con ols o ICC/mICC. P o ocols a e p esen ed in de ail
and demons a e a ca e ul app oach o op imizing a ious
ma ke s o diagnos ic wo kup including immuno he apy.
Conclusion: Mul iplex IHC/ICC eme ges as a ans o ma i e
o ce in biomedical diagnos ics and esea ch. Beyond si-
mul aneous ma ke de ec ion, i un a els complexi ies wi hin
issues –un eiling co-localiza ion nuances, deciphe ing ex-
p ession pa e ns, and enhancing unde s anding o cellula
popula ions. As pe sonalized ea men s gain p ominence,
he s udy emphasizes he heigh ened impo ance o diag-
nos ic ools and sample adequacy. The p esen me hodo-
logical s udy, encapsula ing an au oma ed mul iplex im-
munope oxidase s aining me hod, symbolizes a s ide o-
wa ds p ecision in pulmona y ca cinoma diagnosis. Mul i-
issue con ols ep esen a key elemen in quali y assu ance in
pa hology labo a o ies. © 2024 The Au ho (s).
Published by S. Ka ge AG, Basel
In oduc ion
O e he pas decades, he field o immunohis o-
chemis y (IHC) has made significan p og ess. The
Wo ld Heal h O ganiza ion (WHO) 2021 classifica ion o
lung cance inco po a ed IHC in he classifica ion sys-
em [1, 2].
The adi ional IHC is a aluable diagnos ic ool in
su gical pa hology, and despi e i s limi a ions, such as he
labelling o only one ma ke pe issue sec ion, i emains an
essen ial echnique in a diagnos ic wo kup. Examina ion o
all equisi e ma ke s may no be possible i he samples a e
An i Vuo isalo and Teppo Haapaniemi sha e 1s au ho ship.
[email p o ec ed]
www.ka ge .com/acy
© 2024 The Au ho (s).
Published by S. Ka ge AG, Basel Co espondence o:
An i Vuo isalo, an i. uo isalo @
uni.fi
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NonComme cial 4.0 In e na ional License (CC BY-NC) (h p://www.
ka ge .com/Se ices/OpenAccessLicense). Usage and dis ibu ion o
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deple ed, especially wi h samples acqui ed h ough mini-
mally in asi e me hods like co e needle biopsies o fine
needle aspi a ions. This is a challenge, pa icula ly in he
e alua ion o undi e en ia ed umou s, sa comas, and
lymphomas, which o en demand an ex ensi e panel ex-
ceeding 12 IHC s ains o accu a e diagnosis [3, 4].
Howe e , in he e a o a ge ed he apy and immuno-
he apy, an ex ensi e panel o p edic i e ma ke s is also
equi ed in he diagnos ic wo kup o ca cinomas [5].
The in oduc ion o mul iplex immunohis ochemis y/
immunofluo escence (mIHC/IF) echnologies has add essed
his limi a ion and is becoming mo e popula , enabling he
simul aneous de ec ion o mul iple ma ke s in a single
sec ion. Addi ionally, ad ancemen s in highly mul iplexed
imaging echniques o e comp ehensi e analyses o cell
composi ion and in e ac ions, inc easing diagnos ic po en-
ial. The abili y o assess mul iple ma ke s in one sec ion is
especially beneficial o low cellula samples [6, 7].
The mIHC/IF enables he applica ion o se e al di-
agnos ic ma ke s in a single issue sec ion. Fu he mo e,
he mIHC/IF p o ides de ailed, quan i a i e da a on he
quan i y and spa ial dis ibu ion o a ious cells including
immune cells wi hin umou s, se ing as a powe ul
in es iga i e ool o unde s anding he immune con ex
o he umou [6].
Since fixa ion and o he p e-analy ical s eps can a ec
an igenici y and quali y o he s aining, using con ol
samples wi h di e en p ocessing me hods can lead o
un eliable esul s. Va iabili y in s aining pa e ns caused
by inco ec con ols can complica e esul in e p e a ion
in cy ological samples p ocessed di e en ly om su gical
pa hology specimens. When using an inapp op ia e
con ol ype o con ols ea ed di e en ly om he
sample, he likelihood o ob aining a alse-nega i e
s aining esul is heigh ened [8–10].
Acco ding o he College o Ame ican Pa hologis s 2024
guideline upda e “P inciples o Analy ic Valida ion o Im-
munohis ochemical Assays,”labo a o ies should use alida-
ion issues p ocessed wi h he same fixa i e and me hods as
clinical cases whene e possible. The fixa ion and p ocessing
me hods can a ec ce ain epi opes in a manne ha may al e
he eliabili y o he assay, making consis en p ocessing
c ucial. Howe e , ha es ing con ol issues in-house has
become inc easingly challenging, leading many labo a o ies o
ely on comme cial supplie s, which may no eplica e he
labo a o y’s specificfixa ion and p ocessing me hods.
The e o e, main aining he flexibili y o using in-house
con ols emains impo an . Addi ionally, labo a o ies
should e i y assay pe o mance using a leas one known
posi i e and one known nega i e con ol issue when in o-
ducing a new an ibody lo o an exis ing alida ed assay [11].
In su eys conduc ed by he Eu opean Fede a ion o
Cy ology Socie ies (EFCS) and he College o Ame ican
Pa hologis s (CAP), he impo ance o posi i e and
nega i e con ols in ensu ing accu a e s aining esul s was
examined. Wi hou p ope con ols, i is di ficul o
de e mine i he s aining obse ed in he pa ien ’s sample
is eal o alse due o an e o in he s aining p ocess.
Con ol samples wi h di e en fixa ion and p ocession
me hods compa ed o he pa ien ’s sample can lead o
w ong in e p e a ion and diagnos ic mis akes [8–10].
In he CAP su ey, mo e han hal (59.2%) o labo-
a o ies used con ol samples ha we e p ocessed di e -
en ly om he pa ien ’s cy ology specimens. Only 40.8% o
labo a o ies used con ol samples ha we e p ocessed in
he same way as he pa ien ’s samples. Valida ion o non-
FFPE ( o malin-fixed, pa a fin-embedded) fixa ion
me hods is expensi e and ime and labou consuming.
Many cy ology es s a e low olume, meaning hey a e
pe o med in equen ly, which makes alida ion e en less
cos e ec i e. The high cos and ime in es men equi ed
o alida ion c ea e a challenge o many labo a o ies [8,
9]. Ne e heless, acc edi a ion sys ems equi e hose
quali y assu ance s eps, and he la es ISO 15189 is isk
p e en ion o ien ed [12]. As ea men s become mo e
indi idualized and case specific, he impo ance o diag-
nos ics and he adequacy o samples becomes e en mo e
c ucial, pa icula ly in immuno he apy, which is la gely
based on accu a e pa ien p ofiling and bioma ke analysis.
This me hodological s udy desc ibes an au oma ed
mul iplex immunope oxidase s aining me hod using
common diagnos ic ma ke s o he di e en ial diagnosis
and immuno he apy p ofiling o pulmona y ca cinomas.
The ho se adish pe oxidase (HRP) mul ime was used o
de ec ion o i s s abili y and ep oducibili y. The dis inc
ea u e o he mul iplexes in his s udy is enhanced by he
ac ha hese we e made o b igh field mic oscopy wi h
ch omogenic isualiza ion, hus p ese ing mo phology
and acili a ing in e p e a ion; anslucen ch omogens ha e
been used o s aining, allowing be e isualiza ion o he
colocaliza ion by highligh ing i as a e ac i e colou ; he
au oma ed me hod acili a es mul iple p o ocol s eps and
does no equi e da k field mic oscopy and fluo och omes
o isualiza ion. In addi ion, cy ology-specific mul i- issue
con ols we e de eloped and applied in he p esen s udy.
Me hods
Pulmona y samples we e e hanol-fixed biopsies ob ained om
486 pa ien s who unde wen endob onchial ul asound b on-
choscopy (EBUS) be ween Janua y 2017 and Decembe 2018 and
482 Ac a Cy ologica 2024;68:481–493
DOI: 10.1159/000540367
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we e e ospec i ely selec ed om C5Lims –labo a o y in o -
ma ion sys em o he Fimlab Labo a o ies Pa hology Depa men ,
Tampe e, Finland [13]. Con ol samples we e collec ed om esh
no mal issues om diagnos ic specimens. The s udy was ap-
p o ed by he E hical Commi ee o Pi kanmaa Hospi al Dis ic
and was pe o med acco ding o he guidelines o he Decla a ion
o Helsinki. This consen p o ocol was e iewed and he need o
w i en and in o med consen was wai ed by he E hical Com-
mi ee o Pi kanmaa Hospi al Dis ic , decision e e ence numbe
R17174.
Mul i-Tissue Hyb id Con ols o Immunocy ochemis y
A mul i- issue con ol block consis ing o bo h FFPE and
e hanol-fixed pa a fin-embedded issue co es was in oduced o
immunocy ochemis y (ICC). Con ol issues om he same
o iginal specimen we e sepa a ed o wo di e en fixa ion p o-
cesses, e hanol fixa ion, and o malin fixa ion (Table 1). The
compa ison be ween FFPE and e hanol-fixed pa a fin-embedded
con ol blocks was no ully in line, as he hyb id block was in-
ended o con ain panc ea ic issue, which could no be u ilized in
e hanol-fixed o m. The block s ill allowed o con ol he no mal
unc ion o he s aining, conside ing insulinoma-associa ed p o-
ein 1 (INSM1) exp ession le els, and o e i y he eliabili y o he
s aining.
Du ing he g ossing s ep, esh issue was dissec ed in o
mul iple 2–3 mm pieces o ensu e simila i y o cy ological ma e ial.
Di e en esh issue ypes ( onsil, placen a, skin, and small in-
es ine) as no mal issue emnan s om he g ossing labo a o y
we e collec ed.
A 3 mm punche o e hanol-fixed issue and a 4 mm punche
o o malin-fixed issue we e used as in e nal quali y assu ance and
con ol pa ame e s. By his me hod, we we e able o p ac ically
dis inguish hem om each o he wi h he naked eye du ing
p ocessing and unde mic oscopy, hus educing misin e p e a ion.
Fo malin fixa ion, o 24 h–48 h, was pe o med wi h neu al
bu e ed 10% o malin a oom empe a u e. E hanol fixa ion was
pe o med wi h wo di e en ime poin s: 1-week fixa ion in 50%
e hanol a oom empe a u e and 2-week fixa ion, espec i ely.
Pos -fixa ion o 4–6 h in o malin was pe o med o e hanol-
fixed issues be o e issue p ocessing.
The di e en fixa ion imes a e due o he ac ha o malin
fixes quickly, bu e hanol fixes slowly. In ou p ac ice, we check he
mo phology o he cy ospins fi s , and based on he findings,
pa hologis s o de he cell blocks (CBs) and ICC la e , so he
e hanol fixa ion is longe , and he ime in e al mimics he ac ual
labo a o y flow. In addi ion, he p e-analy ical fixa ion pe iod may
be long as ou cen al labo a o y se es a la ge geog aphic a ea o
se e al p o inces, and he dis ances a e long, so samples can each
he labo a o y in a ew days a e collec ion.
Fo malin- and e hanol-fixed issues we e p ocessed using
Pa hos Del a hyb id p ocessing echnology (Miles one Medical,
Kalamazoo, MI, USA). E hanol-fixed ma e ial was p ocessed wi h
a sho biopsy p o ocol, mimicking he cy ological ma e ial p o-
cess. Fo malin-fixed issues we e p ocessed wi h he ou ine
p o ocol o his ological specimens.
Embedded blocks we e sec ioned and s ained wi h hae-
ma oxylin and eosin o ensu e he quali y and co ec o i-
en a ion o he issue. Co es we e punched using a Kai medical
disposable biopsy punche (Kai Indus ies Co., L d., Osaka,
Japan). E hanol-fixed issue co es we e punched om dono
blocks wi h a 3 mm punche and o malin-fixed ma e ial wi h a
4 mm punche , espec i ely. Tissue co es we e embedded wi h
a simila o ien a ion as in he dono block as shown in
Figu e 1.
Mul iplex Immunocy ochemis y S aining
The au oma ed mul iplex immunope oxidase s aining me h-
odology was designed u ilizing he common diagnos ic ma ke s o
he di e en ial diagnosis and immuno he apy p ofiling o pulmo-
na y ca cinomas, including adenoca cinoma, squamous cell ca ci-
noma, and small cell ca cinoma. Two ch omogenic iple s aining
me hods wi h fi e di e en ch omogens we e designed. The syn-
ap ophysin, ch omog anin A, and INSM1 combina ion o he
diagnosis o small cell ca cinoma and he p og ammed dea h-ligand
1 (PD-L1), p40, and hy oid ansc ip ion ac o 1 (TTF-1) com-
bina ion o he diagnosis and immuno he apy p ofiling o pul-
mona y non-small cell ca cinomas we e in oduced. All p ima y
an ibodies we e fi s op imized wi h a single s aining me hod be o e
combining mul iplex s aining. The immunohis ochemical mul iplex
s aining consis s o h ee sequen ial s aining me hods, espec i ely.
Table 1. Con ol block composi ion
and fixa ions NTissue agmen Diagnosis Fixa ion, du a ion
1 Small in es inal smoo h muscle No mal issue Fo malin, 24–48 h
2 In es inal epi helium No mal issue Fo malin, 24–48 h
3 Skin No mal issue Fo malin, 24–48 h
4 Panc eas No mal issue Fo malin, 24–48 h
5 Tonsil No mal issue Fo malin, 24–48 h
6 Placen a No mal issue Fo malin, 24–48 h
7 Skin No mal issue E hanol 50%, 2 weeks
8 Tonsil No mal issue E hanol 50%, 2 weeks
9 In es inal epi helium No mal issue E hanol 50%, 2 weeks
10 Tonsil No mal issue E hanol 50%, 1 week
11 Small in es inal smoo h muscle No mal issue E hanol 50%, 2 weeks
12 Placen a No mal issue E hanol 50%, 1 week
13 Small in es inal smoo h muscle No mal issue E hanol 50%, 1 week
14 In es inal epi helium No mal issue E hanol 50%, 1 week
Con ols and Mul iplex
Immunocy ochemis y
Ac a Cy ologica 2024;68:481–493
DOI: 10.1159/000540367 483
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The designed hyb id con ol sec ions and EBUS biopsies [13] we e
sec ioned a a hickness o 3 μm on he posi i ely cha ged TOMO
®
slides (Ma sunami Glass Ind., L d., Osaka, Japan). IHC was pe o med
using a Ven ana DISCOVERY ULTRA au oma ed slide s aine
(Ven ana Medical Sys ems, Inc., Tucson, AZ, USA). Depa a finiza ion
and ehyd a ion we e pe o med on he ins umen . Cell Condi-
ioning 1 (CC1) alkaline an igen e ie al solu ion (Ven ana Medical
Sys ems, Inc., Tucson, AZ, USA) was used as a p e ea men solu ion
o hea -induced epi ope e ie al (HIER). HIER was pe o med only
be o e he fi s p ima y an ibody incuba ion s ep. Endogenous pe -
oxidases we e quenched using he DISCOVERY inhibi o (Ven ana
Medical Sys ems, Inc., Tucson, AZ, USA) o 8 min. Be ween he
sequen ial s aining s eps, he deac i a ion o p e ious p ima y an i-
bodies and he de ec ion sys ems was pe o med using he an ibody
dena u a ion s ep and applying Cell Condi ioning 2 (CC2) acidic
an igen e ie al solu ion o 8 min a 99°C (Ven ana Medical Sys-
ems,Inc.,Tucson,AZ,USA).A e hefinal ch omogenic isuali-
za ion, he slides we e coun e s ained wi h haema oxylin II, dehy-
d a ed, and co e slipped (Fig. 2, 3). The de ailed p o ocols and e-
agen in o ma ion a e summa ized in Tables 2 and 3.
In he p esen s udy, we es ed he e ec o an ibody sequence in
mul iplex s aining. Vendo s do no selec he an ibodies based on
easibili y o e hanol fixa ion. I was he eason behind we ha e bo h
o malin- and e hanol-fixed ma e ial in a con ol panel. We ound
ha s aining is impai ed in all clones o e hanol-fixed issuema e ial
compa ed o o malin-fixed ma e ial (Fig. 4, 5). This has also been
shown in p e ious s udies ha e hanol fixa ion impai s an igenici y
[14–20]. All ma ke s we e op imized, and mul iplex ICC esul s
we e compa ed o o malin-fixed IHC and e hanol-fixed single ICC.
The compa ison was made wi h DAB as ch omogen, and based on
his implemen a ion es ing p o ocols, an ibodies, and i s op imized
p o ocols we e implemen ed in he mul iplex p o ocols. An igen
deple ion was pa icula ly obse ed in onsilla ollicula mac o-
phages. Reduced s aining was also obse ed in onsilla epi helial
c yp cells. The weakes s aining esul was ob ained specifically
using he SP142 clone, which is ypically used in an IC algo i hm
ha conside s he s aining o immune cells in ela ion o he sample
su ace a ea. The clea es s aining esul and mos in ense s aining
we e ob ained wi h s aining using he SP263 clone. In placen al
syncy io ophoblas s, s aining was seen a bo h he apical memb ane
and he basal memb ane.
In ou s udy, we ound ha Ven ana DISCOVERY’s pu ple
ch omogen and yellow ch omogen anslucency allowed he co-
localiza ion esul ing in o ange s ain colou . This echnique allows
mo e accu a e analysis and diagnosis o an igens localized in he
same cell and he same cell compa men . In he pas , mul iplex
ch omogenici y has been mo e obscu ing and has no allowed o
mo e accu a e diagnos ics.
I should be no ed ha he Ven ana DISCOVERY ULTRA is an
open immunos aining au oma ion machine, which allows o
p o ocol amplifica ion and op imiza ion o e hanol-fixed sample
ma e ial. Fo iple s aining, he clone SP263 was selec ed as he
PD-L1 an ibody due o i s bes s aining esul s. The p o ocol
wi hou amplifica ion allows sensi iza ion o he p o ocol and hus
compensa ion o an igen loss in he in ensi y o s aining.
PD-L1-S ained Con ols
The pe o mance o PD-L1 s aining wi h hyb id con ols was es ed
using pa hology labo a o y diagnos ic p o ocols (Table 4). Table 4
p esen s he final op imized PD-L1 p o ocols o ou di e en clones.
In summa y, all HIER s eps we e pe o med on boa d wi h an alkaline
an igen e ie al bu e a 99°C. All PD-L1 an ibodies we e op imized
wi h hyb id block sec ions wi h Ven ana BenchMa k ULTRA in-
s umen o mul iplex p o ocol in Ven ana DISCOVERY ULTRA.
Clone SP263 was selec ed acco ding o i s obus and sensi i e s aining
pa e n du ing he alida ion o mul iplex s aining. Finally, fine- uning
o an ibody incuba ion and de ec ion me hod was pe o med.
PD-L1 s aining wi h clones SP263 and SP142 we e Ven ana
PD-L1 IHC assays, while 22C3 and 28-8 we e labo a o y-
p epa ed p op ie a y es s. Ven ana’s p o ocols a e closed
and in line wi h he Pha maco he apy Recommenda ion. The
22C3 and 28-8 a e labo a o y- alida ed p o ocols alida ed on
he Ven ana BenchMa k ULTRA pla o m. Ty amide ampli-
fica ion was used o s aining wi h SP142, 22C3, and 28-8 as
clones.
Fig. 1. Haema oxylin and eosin-s ained slide sec ion om a
hyb id block con aining bo h e hanol- o malin-fixed con ol
issues (3-mm punches, uppe ow) and o malin-fixed con ol
issues (4-mm punches, lowe ow). aIn es inal smoo h muscle,
o malin fixed. bIn es inal epi helia, o malin fixed. cSkin,
o malin fixed. dPanc eas, o malin fixed. eTonsil, o malin
fixed. Placen a, o malin fixed. gSkin, 50% e hanol fixed.
hTonsil, 50% e hanol fixed. iIn es inal epi helia, 50% e hanol
fixed. jTonsil, 50% e hanol fixed. kIn es inal smoo h muscle,
50% e hanol fixed. lPlacen a, 50% e hanol fixed. mIn es inal
smoo h muscle, 50% e hanol fixed. nIn es inal epi helia, 50%
e hanol fixed.
484 Ac a Cy ologica 2024;68:481–493
DOI: 10.1159/000540367
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Toimp o es aining esul s, hean ibodysequencewasconside ed.
We fi s applied hose an igens ha need o be coun ed and quan ified
(as PD-L1) as issue is changing wi h he ollowing subsequen p o-
cessing sequences, mo phology is less isible, and an igenici y weakens.
In addi ion, ch omogen cha ac e is ics should be conside ed: fi s , we
use opaque ch omogens and a e anslucen ch omogens.
Discussion
Analysis o se e al immunohis ochemical ma ke s
wi hin a single issue sec ion is a complex ask ha equi es
mul iplex ch omogen o fluo escence de ec ion. The
Fig. 3. mICC in pulmona y small cell ca cinomas diagnos ic
wo kup. Th ee neu oendoc ine ma ke s (INSM1, synap o-
physin, ch omog anin) we e applied as neu oendoc ine
ma ke s sensi i i y and specifici y a y. Pulmona y small cell
ca cinomas a e TTF-1 posi i e. aCase o small cell ca cinoma in
an EBUS- a ge ed lymph node in a 76-yea -old emale. INSM1
nuclea posi i i y in b own, synap ophysin cy oplasma ic
g anula posi i i y in pu ple, and ch omog anin A cy o-
plasma ic g anula posi i i y in yellow. No e ha despi e all
ma ke s a e posi i e in his case, no all cells exp ess all h ee
ma ke s; so wi h less su ficien samples, only one o wo ma ke s
can be p esen . Mul iplex immunos aining o INSM1, syn-
ap ophysin, and ch omog anin A (×200 magnifica ion). bTTF-
1 nuclea posi i i y in ed in a small cell ca cinoma in an EBUS-
a ge ed lymph node shown in a. No e a squamous cell
me aplas ic agmen wi h p40 nuclea posi i i y in g een. PD-
L1 is nega i e. Mul iplex immunos aining o PD-L1, p40, and
TTF-1 (×200 magnifica ion). cCase o small cell ca cinoma in
an EBUS- a ge ed lymph node in a 68-yea -old male. INSM1
nuclea posi i i y in b own in pa o ca cinoma cells, syn-
ap ophysin cy oplasma ic g anula posi i i y in pu ple in al-
mos all cells. Ch omog anin A is nega i e. Exp ession o
neu oendoc ine ma ke s is a iable in a ious cases bu also
wi hin he issue o a single case as case a. showed exp ession o
all h ee ma ke s bu no di usely. In his case, only wo
ma ke s we e exp essed wi h only one o hem o be exp essed
di usely. Mul iplex immunos aining o INSM1, synap ophy-
sin, and ch omog anin A (×200 magnifica ion).
Fig. 2. mICC in pulmona y non-small cell ca cinomas diag-
nos ic wo kup and immuno he apy: p40 an ibody nuclea
posi i i y dis inguishes squamous cell ca cinoma om TTF-1
an ibody nuclea posi i i y cha ac e is ic o adenoca cinoma.
PD-L1isappliedasama ke o immuno he apy.aCase o
squamous cell ca cinoma in an EBUS- a ge ed lymph node in a
73-yea -old male. PD-L1 memb anous posi i i y in b own in
30% o ca cinoma cells. Nuclea g een posi i i y o p40 in mos
ca cinoma cells in a iably dispe se agmen s. Backg ound
lymphocy es a e nega i e o all h ee ma ke s. Mul iplex im-
munos aining o PD-L1, p40, and TTF-1 (×200 magnifica ion).
bPD-L1 nega i e squamous cell ca cinoma in an EBUS-
a ge ed lymph node in a 70-yea -old emale. PD-L1 is nega-
i e in ca cinoma cells. Nuclea g een posi i i y o p40 in mos
ca cinoma cells in a igh shee . Mul iplex immunos aining o
PD-L1, p40, and TTF-1 (×200 magnifica ion). cAdenoca ci-
noma in an EBUS- a ge ed lymph node in a 57-yea -old male.
PD-L1 memb anous posi i i y in b own in he majo i y o
ca cinoma cells (95%). Nuclea ed posi i i y o TTF-1 in mos
ca cinoma cells in ubula and abecula g oups. Backg ound
lymphocy es a e nega i e o all h ee ma ke s. Mul iplex im-
munos aining o PD-L1, p40, and TTF-1 (×200 magnifica ion).
Con ols and Mul iplex
Immunocy ochemis y
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g owing popula i y o bo h ch omogenic and fluo escen
mul iplex s aining is due o he de elopmen o eliable
mul iplex s aining echniques and sophis ica ed mul i-
spec al imaging. The benefi s o mul iplex s aining include
he abili y o p ese e aluable issue samples o e en he use
o limi ed specimens and he abili y o co-localize an igens
and imp o e he accu acy o in e p e a ion. This is espe-
cially impo an o he eme ging field o immuno he apy as
i allows he iden ifica ion and de ec ion o immune cells, as
well as he cha ac e iza ion o esponse bioma ke s [21–27].
The mIHC/IF app oaches ha e become significan ly mo e
powe ul, p o iding enhanced insigh s in o disease he e o-
genei yand heunde lyingsys emsbiologymechanisms.
These app oaches also con ibu e o he p ese a ion o
limi ed issue ma e ial [6]. Tumou he e ogenei y and he
limi ed ep esen a i eness o small biopsy o cy ology samples
Table 2. Mul iplex p o ocol o non-small cell ca cinoma and small cell ca cinoma
S ep Reagen Time, min Tempe a u e, °C
Mul iplex: PD-L1 + p40 + TTF-1
Depa a finiza ion DISCOVERY Wash 12 70
P e ea men , HIER in CC1 CC1 64 99
Inhibi o CM o quenching endogenous pe oxidases Inhibi o CM 8 37
P ima y an ibody 1: PD-L1 (SP263) P ima y an ibody 1 32 36
De ec ion: An i-Rabbi -HQ DISCOVERY HQ HRP 16 Ambien
De ec ion: An i-HQ-HRP DISCOVERY HQ HRP 16 Ambien
Ch omogen 1: DAB DISCOVERY Ch omoMap DAB 8 Ambien
Dena u a ion s ep o deac i a ion an ibodies and HRP CC2 8 99
P ima y an ibody 2: p40 (BC28) P ima y an ibody 2 48 36
De ec ion: Op iView Linke Op iView DAB IHC De ec ion 20 Ambien
De ec ion: Op iView HRP Mul ime Op iView DAB IHC De ec ion 20 Ambien
Ch omogen 2: G een HRP Ki DISCOVERY G een HRP Ki 64 Ambien
Dena u a ion s ep o deac i a ion an ibodies and HRP CC2 8 99
P ima y an ibody 3: TTF-1 (SP141) P ima y an ibody 3 48 36
De ec ion: Op iView Linke Op iView DAB IHC De ec ion 20 Ambien
De ec ion: Op iView HRP Mul ime Op iView DAB IHC De ec ion 20 Ambien
Ch omogen 3: RED HRP Ki DISCOVERY RED HRP Ki 64 Ambien
Coun e s aining wi h Haema oxylin II Haema oxylin II 8 Ambien
Bluing o haema oxylin Bluing eagen 4 Ambien
Mul iplex: INSM1 + synap ophysin + ch omog anin A
Depa a finiza ion DISCOVERY Wash 24 70
P e ea men , HIER in CC1 CC1 64 99
Inhibi o CM o quenching endogenous pe oxidases Inhibi o CM 8 37
P ima y an ibody 1: INSM1 (A-8), 1:50 P ima y an ibody 1 60 36
De ec ion: An i-Rabbi -HQ DISCOVERY HQ HRP 24 Ambien
De ec ion: An i-HQ-HRP DISCOVERY HQ HRP 24 Ambien
Ch omogen 1: DAB DISCOVERY Ch omoMap DAB 8 Ambien
Dena u a ion s ep o deac i a ion an ibodies and HRP CC2 8 99
P ima y an ibody 2: Synap ophysin (SP11), RTU P ima y an ibody 2 52 36
De ec ion: Op iView Linke Op iView DAB IHC De ec ion 20 Ambien
De ec ion: Op iView HRP Mul ime Op iView DAB IHC De ec ion 20 Ambien
Ch omogen 2: Pu ple HRP Ki DISCOVERY Pu ple HRP Ki 32 Ambien
Dena u a ion s ep o deac i a ion an ibodies and HRP CC2 8 99
P ima y an ibody 3: Ch omog anin A (LK2H10), RTU P ima y an ibody 3 60 36
De ec ion: Op iView Linke Op iView DAB IHC De ec ion 20 Ambien
De ec ion: Op iView HRP Mul ime Op iView DAB IHC De ec ion 20 Ambien
Ch omogen 3: Yellow HRP Ki DISCOVERY Yellow HRP Ki 96 Ambien
Coun e s aining wi h Haema oxylin II Haema oxylin II 8 Ambien
Bluing o haema oxylin Bluing eagen 4 Ambien
Dehyd a ion wi h ising e hanol se ies, xylene clea ing, and co e slipping.
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may lead o disc epancies be ween cy ological specimens and/
o co e biopsy and he final his ologic diagnosis om e-
sec ion specimens [28, 29]. The mIHC/IF holds significan
u ili y in a ious aspec s o diagnos ics and biomedical e-
sea ch. Fi s , i enables he simul aneous de ec ion o nu-
me ous ma ke s in a single issue sec ion, ex ac ing op imal
in o ma ion om limi ed o p ecious samples. Second, i
allows he explo a ion o ma ke s co-localiza ion and in e -
ac ion.Thi dly,s udiesbenefi om mIHC/IF by he iden-
ifica ion o exp ession pa e ns, p o iding insigh s in o u-
mou o ganiza ion and enhancing ou unde s anding o he
p esence o a ious popula ions wi hin he umou . Las ly, by
e ealing spa ial ela ionships among cells and issues and
unco e ing disease he e ogenei y, mIHC/IF con ibu es o
inc easing diagnos ic and p edic i e accu acy as well as ad-
ances biomedical unde s anding o he diseases [7, 26, 30].
Du a ion o he e hanol fixa ion a ies in he labo a o y
based on he decision o he p epa a ion o he CB. In-
c easing he du a ion o he e hanol fixa ion, he s aining
in ensi y g adually ain ed o some an ibody clones.
Howe e , we no iced he same e ec using he hyb id
block-con olling me hod o he op imiza ion o PD-L1
Table 3. Mul iplex eagen s o non-small cell ca cinoma and small cell ca cinoma
(a) Mul iplex: PD-L1 + p40 + TTF-1
P ima y an ibody Clone Dilu ion An ibody
incuba ion, min
Vendo
PD-L1 SP263 P edilu ed 32 Roche Ven ana
p40 BC28 P edilu ed 48 Roche Ven ana
TTF-1 SP141 P edilu ed 48 Roche Ven ana
De ec ions S eps Dilu ion Incuba ion(s), min Vendo
DISCOVERY HQ HRP hap en-linked mul ime
de ec ion
2 P edilu ed 16 + 16 Roche Ven ana
Op iView DAB IHC De ec ion Ki (using only
de ec ion pa )
a
2 P edilu ed 20 + 20 Roche Ven ana
Ch omogens Colou Dilu ion Incuba ion(s), min Vendo
DISCOVERY Ch omoMap DAB Ki (HRP) B own P edilu ed 8 Roche Ven ana
DISCOVERY G een HRP Ki G een P edilu ed 32 + 32 Roche Ven ana
DISCOVERY RED HRP Ki Red P edilu ed 32 + 32 Roche Ven ana
(b) Mul iplex: INSM1 + synap ophysin + ch omog anin A
P ima y an ibody Clone Dilu ion An ibody
incuba ion, min
Vendo
INSM1 A-8 1:50 60 San a C uz Bioscience
Synap ophysin SP11 P edilu ed 52 Roche Ven ana
Ch omog anin A LK2H10 P edilu ed 60 Roche Ven ana
De ec ions S eps Dilu ion Incuba ion(s), min Vendo
DISCOVERY HQ HRP hap en-linked mul ime
de ec ion
2 P edilu ed 24 + 24 Roche Ven ana
Op iView DAB IHC De ec ion Ki (using only
de ec ion pa )
a
2 P edilu ed 20 + 20 Roche Ven ana
Ch omogens Colou Dilu ion Incuba ion(s), min Vendo
DISCOVERY Ch omoMap DAB Ki (HRP) B own P edilu ed 8 Roche Ven ana
DISCOVERY Pu ple HRP Ki Pu ple P edilu ed 32 Roche Ven ana
DISCOVERY Yellow HRP Ki Yellow P edilu ed 48 + 48 Roche Ven ana
a
Op iView de ec ion was pe o med using Ven ana use fillable dispense s.
Con ols and Mul iplex
Immunocy ochemis y
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clones. Tissue co es fixed 1 week in 50% e hanol s ained
mo e in ensi ely ( onsil, placen a), hen 2 weeks fixed
issues, espec i ely. Fo s anda diza ion o he e hanol
fixa ion, he fixa ion ime should be kep as sho as
possible, simila o he o malin fixa ion ime. Pos -fixa ion
wi h he o malin could dec ease he ad e se e ec o
e hanol fixa ion, especially i he an ibody is designed
agains he o malin-fixed and linea ized epi ope.
The e o e, one no able ad an age o mIHC/IF is he
imp o ed accu acy acili a ed by image analysis, which
u ilizes landma k ma ke s o indica e issue a chi ec u e [6,
27]. Ne e heless, in e p e ing mul iplexed s ained samples,
pa icula ly hose using fluo escence, can be challenging. The
use o fluo escence may cause mul iple a ge s o blend,
complica ing esolu ion and po en ially muddling isual
assessmen . Addi ionally, in FFPE issues, he e is he po-
en ial o issue au ofluo escence, u he complica ing isual
in e p e a ion [6]. P e ious s udies examined whe he
s aining wi h a specific an ibody in a mul iplex p o ocol is
quali a i ely compa able o a single s aining and concluded
ha mul iplex s aining can be quali a i ely compa able o
single s aining. Howe e , achie ing his equi es s anda di-
za ion, alida ion, and ca e ul conside a ion o de ec ion
me hods and an ibody cha ac e is ics [31, 32]. In addi ion,
p e ious esea ch has concluded ha mul iplex IHC/ICC can
quan i a i ely eplica e single s aining when ho oughly
alida ed and op imized. I should also be no ed ha he
loca ion o an ibodies in he mul iplex panel a ec s all an-
ibodies in he panel, equi ing ca e ul op imiza ion o a oid
p oblems o an ibody shedding and in e e ence [33–35].
P io o he possible in eg a ion o mIHC/IF ech-
nology in o clinical applica ions, i is c ucial o es ablish a
s anda dized and alida ed wo kflow ha encompasses
he en i e p ocess. This comp ehensi e wo kflow should
be capable o suppo ing mul isi e ials and aligning wi h
he equi emen s o clinical labo a o y p ocedu es and
acc edi a ion equi emen s [24].
In op imizing issue a ailabili y o molecula es ing, i is
ad isable o unde ake a es ic ed diagnos ic
wo kup. Pulmona y cance diagnosis necessi a es a mul-
idisciplina y app oach. The landscape o lung cance
he apy is inc easingly pe sonalized, conside ing indi idual
pa ien ac o s such as his ologic cell ype, sub ypes, and
molecula s a us. The ole and app oach o pa hologis s in
diagnosing lung cance in small biopsies and cy ology
specimens ha e unde gone significan ans o ma ion [29].
Quali y Assu ance
The Eu opean Fede a ion o Cy ology Socie ies (EFCS)
su ey om 245 labo a o ies highligh s he need o s an-
da dized p o ocols and obus quali y assu ance/quali y
con ol (QA/QC) p ac ices in ICC [9]. Simila findings
we e iden ified in a CAP su ey wi h 345 esponden labo-
a o ies [8], as well as in he UK NEQAS su ey [36] and he
me a-analysis al eady pe o med in he yea 2011 [37].
Fu he esea ch e o s a e c ucial o de elop op imal fixa ion
and p ocessing me hods o di e se cy ology samples, es-
ablish alida ed ICC p o ocols applicable o a ious cy ology
p epa a ions, and implemen s anda dized QA/QC measu es,
including posi i e con ols and in e nal and ex e nal quali y
Fig. 4. Mul i- issue sec ion s ained wi h PD-L1 (SP263) clone.
E hanol-fixed issues a e punched wi h a 3 mm punche and o malin-
fixed issues wi h 4 mm, espec i ely. The s aining in ensi y o he PD-
L1 is op imal using o malin-fixed issues and p o ocol ecommen-
da ion by he endo . Placen a and onsil a e good con ol issues o
quali y con olling o PD-L1 s aining. O he issues o he hyb id
con ol a e nega i e con ol o PD-L1 an igen. No e: s aining in ensi y
dec eased in e hanol-fixed issues compa ed o he o malin-fixed
issues. S aining in ensi y dec eased especially in onsil issue. Top ow,
e hanol-fixed issue; bo om ow, o malin-fixed issue. Con ols om
op le . aSkin, 1 week. bTonsil, 2 weeks. cIn es inal epi helium, 2
weeks. dTonsil, 1 week. eSmall in es inal smoo h muscle, 1 week.
Placen a, 1 week. gSmall in es inal smoo h muscle, 2 weeks.
hIn es inal epi helium, 1 week. Con ols om bo om le . iSmall
in es inal smoo h muscle. jIn es inal epi helium. kSkin. lPanc eas.
mTonsil. nPlacen a (×5 magnifica ion).
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