Background: Immunocytochemistry (ICC) is a widely available and extensively used ancillary method in diagnostic cytopathology with great variability in all test phases and a low level of adequate quality management. The non-standardized ICC landscape is now challenged with the introduction of the new European (EU) In Vitro Diagnostic Medical Devices Regulation (IVDR). According to this regulation, ICC on cytological slides falls under the category of Laboratory-Developed Tests (LDT), which requires rigorous standardization, validation, and thorough quality management. Summary: Complete standardization of pre-analytical and analytical steps in ICC is impossible due to the complexity of the method and the constantly evolving antibodies, detection systems, and platforms. However, similar to the approach in immunohistochemistry, improving and standardizing “best practices” in quality management will result in high-quality, correct, accurate, and reliable ICC results. In this review, the current challenges of ICC in diagnostic cytopathology will be discussed, along with practical insights into ICC standardization and validation. Key Messages: Control slides prepared in the same manner as the patient samples, optimized ICC protocols, and participation in external quality control for ICC are the pillars of good quality management and essential to ensure safe and reliable patient diagnostics.

1.
Cheung
CC
,
D’Arrigo
C
,
Dietel
M
,
Francis
GD
,
Gilks
CB
,
Hall
JA
, et al
.
Evolution of quality assurance for clinical immunohistochemistry in the era of precision medicine: Part 1: fit-for-purpose approach to classification of clinical immunohistochemistry biomarkers
.
Appl Immunohistochem Mol Morphol
.
2017
;
25
(
1
):
4
11
.
2.
Torlakovic
EE
,
Cheung
CC
,
D’Arrigo
C
,
Dietel
M
,
Francis
GD
,
Gilks
CB
, et al
.
Evolution of quality assurance for clinical immunohistochemistry in the era of precision medicine: Part 2: immunohistochemistry test performance characteristics
.
Appl Immunohistochem Mol Morphol
.
2017
;
25
(
2
):
79
85
.
3.
Verbeke
H
,
Van Hecke
D
,
Bauraing
C
,
Dierick
AM
,
Colleye
O
,
Dalle
I
, et al
.
Belgian recommendations for analytical verification and validation of immunohistochemical tests in laboratories of anatomic pathology
.
Appl Immunohistochem Mol Morphol
.
2024
;
32
(
1
):
1
16
.
4.
Bogen
SA
,
Dabbs
DJ
,
Miller
KD
,
Nielsen
S
,
Parry
SC
,
Szabolcs
MJ
, et al
.
A consortium for analytic standardization in immunohistochemistry
.
Arch Pathol Lab Med
.
2022
;
147
(
5
):
584
90
.
5.
Canadian Association of Pathologists-Association canadienne des pathologi
,
stes National Standards Committee
,
Torlakovic
EE
,
Riddell
R
,
Banerjee
D
,
El-Zimaity
H
,
Pilavdzic
D
, et al
.
Canadian Association of Pathologists-Association canadienne des pathologistes National Standards Committee/Immunohistochemistry: best practice recommendations for standardization of immunohistochemistry tests
.
Am J Clin Pathol
.
2010
;
133
(
3
):
354
65
.
6.
Cheung
CC
,
Banerjee
D
,
Barnes
PJ
,
Berendt
RC
,
Butany
J
,
Canil
S
, et al
.
Canadian Association of Pathologists–Association canadienne des pathologistes National Standards Committee for High Complexity Testing/Immunohistochemistry: Guidelines for the Preparation, Release, and Storage of Unstained Archived Diagnostic Tissue Sections for Immunohistochemistry
.
Am J Clin Pathol
.
2014
;
142
(
5
):
629
33
.
7.
Hammond
MEH
,
Hayes
DF
,
Dowsett
M
,
Allred
DC
,
Hagerty
KL
,
Badve
S
, et al
.
American society of clinical oncology/college of American pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer
.
J Clin Oncol
.
2010
;
28
(
16
):
2784
95
.
8.
Torlakovic
EE
,
Cheung
CC
,
D’Arrigo
C
,
Dietel
M
,
Francis
GD
,
Gilks
CB
, et al
.
Evolution of quality assurance for clinical immunohistochemistry in the era of precision medicine. Part 3: technical validation of immunohistochemistry (IHC) assays in clinical IHC laboratories
.
Appl Immunohistochem Mol Morphol
.
2017
;
25
(
3
):
151
9
.
9.
Jain
D
,
Nambirajan
A
,
Borczuk
A
,
Chen
G
,
Minami
Y
,
Moreira
AL
, et al
.
Immunocytochemistry for predictive biomarker testing in lung cancer cytology
.
Cancer Cytopathol
.
2019
;
127
(
5
):
325
39
.
10.
Goldstein
NS
,
Hewitt
SM
,
Taylor
CR
,
Yaziji
H
,
Hicks
DG
,
Members of Ad-Hoc Committee On Immunohistochemistry Standardization
.
Recommendations for improved standardization of immunohistochemistry
.
Appl Immunohistochem Mol Morphol
.
2007
;
15
(
2
):
124
33
.
11.
Fitzgibbons
PL
,
Bradley
LA
,
Fatheree
LA
,
Alsabeh
R
,
Fulton
RS
,
Goldsmith
JD
, et al
.
Principles of analytic validation of immunohistochemical assays: guideline from the college of American pathologists pathology and laboratory quality center
.
Arch Pathol Lab Med
.
2014
;
138
(
11
):
1432
43
.
12.
Goldsmith
JD
,
Troxell
ML
,
Roy-Chowdhuri
S
,
Colasacco
CF
,
Edgerton
ME
,
Fitzgibbons
PL
, et al
.
Principles of analytic validation of immunohistochemical assays: guideline update
.
Arch Pathol Lab Med
.
2024
;
148
(
6
):
e111
53
.
13.
Kirbis
IS
,
Maxwell
P
,
Flezar
MS
,
Miller
K
,
Ibrahim
M
.
External quality control for immunocytochemistry on cytology samples: a review of UK NEQAS ICC (cytology module) results
.
Cytopathology
.
2011
;
22
(
4
):
230
7
.
14.
Srebotnik Kirbiš
I
,
Rodrigues Roque
R
,
Bongiovanni
M
,
Strojan Fležar
M
,
Cochand-Priollet
B
.
Immunocytochemistry practices in European cytopathology laboratories: review of European Federation of Cytology Societies (EFCS) online survey results with best practice recommendations
.
Cancer Cytopathol
.
2020
;
128
(
10
):
757
66
.
15.
Shi
Y
,
Yee-Chang
M
,
Shi
S-R
.
Application of immunohistochemistry in cytology
.
Appl Immunohistochem Mol Morphol
.
2023
;
31
(
7
):
459
66
.
16.
Roy-Chowdhuri
S
.
Immunocytochemistry of cytology specimens for predictive biomarkers in lung cancer
.
Transl Lung Cancer Res
.
2020
;
9
(
3
):
898
905
.
17.
Satturwar
S
,
Malenie
R
,
Sutton
A
,
Dai
D
,
Aly
FZ
.
Validation of immunohistochemical tests performed on cytology cell block material: practical application of the College of American Pathologists’ guidelines
.
CytoJournal
.
2019
;
16
:
6
.
18.
Vlajnic
T
,
Savic
S
,
Barascud
A
,
Baschiera
B
,
Bihl
M
,
Grilli
B
, et al
.
Detection of ROS1-positive non-small cell lung cancer on cytological specimens using immunocytochemistry
.
Cancer Cytopathol
.
2018
;
126
(
6
):
421
9
.
19.
Roy-Chowdhuri
S
.
The updated College of American Pathologists principles of analytic validation of immunohistochemical assays: a step forward for cytopathology
.
Cancer Cytopathol
.
2024
.
20.
International Organization of Standardization
.
Medical laboratories: requirements for quality and competence. ISO Guide 15189
.
Geneva (Switzerland)
:
ISO
;
2022
.
21.
Commission E
.
Regulation (EU) 2017/746 of the European Parliament and of the council_of 5 April 2017 on in vitro diagnostic medical devices and repealing_directive 98/79/EC and commission decision 2010/227/EU
.
2017
. https://eur-lex.europa.eu/eli/reg/2017/746/oj
22.
Shidham
VB
,
Janikowski
B
.
Immunocytochemistry of effusions: processing and commonly used immunomarkers
.
CytoJournal
.
2022
;
19
:
6
.
23.
Krogerus
L
,
Kholová
I
.
Cell block in cytological diagnostics: review of preparatory techniques
.
Acta Cytol
.
2018
;
62
(
4
):
237
43
.
24.
Shidham
VB
.
CellBlockistry: chemistry and art of cell-block making: a detailed review of various historical options with recent advances
.
CytoJournal
.
2019
;
16
:
12
.
25.
Torous
VF
,
Cuda
JM
,
Manucha
V
,
Randolph
ML
,
Shi
Q
,
VandenBussche
CJ
, et al
.
Cell blocks in cytology: review of preparation methods, advantages, and limitations
.
J Am Soc Cytopathol
.
2023
;
12
(
2
):
77
88
.
26.
Srebotnik Kirbis
I
,
Kholova
I
,
Huhtala
H
,
Bongiovanni
M
,
Strojan Flezar
M
,
Hodgson
C
, et al
.
Cell block practices in European cytopathology laboratories
.
Cancer Cytopathol
.
2024
;
132
(
4
):
250
9
.
27.
Sauter
J
,
Grogg
K
,
Vrana
J
,
Law
M
,
Halvorson
J
,
Henry
M
.
Validation of commonly used antibodies in exfoliative cytology for immunohistochemistry on the CellientTM automated cell block system
.
J Am Soc Cytopathology
.
2015
;
4
(
6
):
S62
.
28.
Sauter
JL
,
Grogg
KL
,
Vrana
JA
,
Law
ME
,
Halvorson
JL
,
Henry
MR
.
Young investigator challenge: validation and optimization of immunohistochemistry protocols for use on cellient cell block specimens
.
Cancer Cytopathol
.
2016
;
124
(
2
):
89
100
.
29.
Koomen
BM
,
de Boer
M
,
van Dooijeweert
C
,
van Lindert
ASR
,
Deckers
IAG
,
Voorham
QJM
, et al
.
Nationwide differences in cytology fixation and processing methods and their impact on interlaboratory variation in PD-L1 positivity
.
Virchows Arch
.
2023
;
482
(
4
):
707
20
.
30.
Crapanzano
JP
,
Heymann
JJ
,
Monaco
S
,
Nassar
A
,
Saqi
A
.
The state of cell block variation and satisfaction in the era of molecular diagnostics and personalized medicine
.
CytoJournal
.
2014
;
11
:
7
.
31.
Nambirajan
A
,
Jain
D
.
Cell blocks in cytopathology: an update
.
Cytopathology
.
2018
;
29
(
6
):
505
24
.
32.
Wu
HH
,
Jones
KJ
,
Cramer
HM
.
Immunocytochemistry performed on the cell-transferred direct smears of the fine-needle aspirates: a comparison study with the corresponding formalin-fixed paraffin-embedded tissue
.
Am J Clin Pathol
.
2013
;
139
(
6
):
754
8
.
33.
Harabajsa
S
,
Milutin
L
,
Breški
A
,
Ražnjević
K
,
Šimić
V
,
Branica
BV
, et al
.
Quality of cell blocks prepared from residual pleural effusion and bronchial washing samples for immunocytochemistry
.
Cytopathology
.
2023
;
34
(
3
):
264
70
.
34.
Srebotnik Kirbiš
I
,
Rodrigues Roque
R
,
Bongiovanni
M
,
Strojan Fležar
M
,
Cochand-Priollet
B
.
Immunocytochemistry practices in European cytopathology laboratories-Review of European Federation of Cytology Societies (EFCS) online survey results with best practice recommendations
.
Cancer Cytopathol
.
2020
;
128
(
10
):
757
66
.
35.
Maxwell
P
,
Salto-Tellez
M
.
Validation of immunocytochemistry as a morphomolecular technique
.
Cancer Cytopathol
.
2016
;
124
(
8
):
540
5
.
36.
Kirbis
IS
,
Kholová
I
.
Comments to: nationwide differences in cytology fixation and processing methods and their impact on interlaboratory variation in PD-L1 positivity
.
Virchows Arch
.
2023
;
482
(
4
):
797
8
.
37.
Gorman
BK
,
Kosarac
O
,
Chakraborty
S
,
Schwartz
MR
,
Mody
DR
.
Comparison of breast carcinoma prognostic/predictive biomarkers on cell blocks obtained by various methods: cellient, formalin and thrombin
.
Acta Cytol
.
2012
;
56
(
3
):
289
96
.
38.
Buonocore
DJ
,
Konno
F
,
Jungbluth
AA
,
Frosina
D
,
Fayad
M
,
Edelweiss
M
, et al
.
CytoLyt fixation significantly inhibits MIB1 immunoreactivity whereas alternative Ki-67 clone 30-9 is not susceptible to the inhibition: critical diagnostic implications
.
Cancer Cytopathol
.
2019
;
127
(
10
):
643
9
.
39.
Maleki
S
,
Dorokhova
O
,
Sunkara
J
,
Schlesinger
K
,
Suhrland
M
,
Oktay
MH
.
Estrogen, progesterone, and HER-2 receptor immunostaining in cytology: the effect of varied fixation on human breast cancer cells
.
Diagn Cytopathol
.
2013
;
41
(
10
):
864
70
.
40.
Kirbis
IS
,
Flezar
MS
,
Krasovec
MU
.
MIB-1 immunostaining on cytological samples: a protocol without antigen retrieval
.
Cytopathology
.
2004
;
15
(
3
):
154
9
.
41.
Pinheiro
C
,
Roque
R
,
Adriano
A
,
Mendes
P
,
Praca
M
,
Reis
I
, et al
.
Optimization of immunocytochemistry in cytology: comparison of two protocols for fixation and preservation on cytospin and smear preparations
.
Cytopathology
.
2015
;
26
(
1
):
38
43
.
42.
Srebotnik Kirbiš
I
,
Us Krasovec
M
,
Pogacnik
A
,
Strojan Flezar
M
.
Optimization and validation of immunocytochemical detection of oestrogen receptors on cytospins prepared from fine needle aspiration (FNA) samples of breast cancer
.
Cytopathology
.
2015
;
26
(
2
):
88
98
.
43.
Savic
S
,
Bode
B
,
Diebold
J
,
Tosoni
I
,
Barascud
A
,
Baschiera
B
, et al
.
Detection of ALK-positive non-small-cell lung cancers on cytological specimens: high accuracy of immunocytochemistry with the 5A4 clone
.
J Thorac Oncol
.
2013
;
8
(
8
):
1004
11
.
44.
Zhang
C
,
Randolph
ML
,
Jones
KJ
,
Cramer
HM
,
Cheng
L
,
Wu
HH
.
Anaplastic lymphoma kinase immunocytochemistry on cell-transferred cytologic smears of lung adenocarcinoma
.
Acta Cytol
.
2015
;
59
(
2
):
213
8
.
45.
Lozano
MD
,
Panizo
A
,
Toledo
GR
,
Sola
JJ
,
Pardo-Mindan
J
.
Immunocytochemistry in the differential diagnosis of serous effusions: a comparative evaluation of eight monoclonal antibodies in Papanicolaou stained smears
.
Cancer
.
2001
;
93
(
1
):
68
72
.
46.
Lozano
MD
,
Rodriguez
J
,
Algarra
SM
,
Panizo
A
,
Sola
JJ
,
Pardo
J
.
Fine-needle aspiration cytology and immunocytochemistry in the diagnosis of 24 gastrointestinal stromal tumors: a quick, reliable diagnostic method
.
Diagn Cytopathol
.
2003
;
28
(
3
):
131
5
.
47.
Bank
PCD
,
Jacobs
LHJ
,
van den Berg
SAA
,
van Deutekom
HWM
,
Hamann
D
,
Molenkamp
R
, et al
.
The end of the laboratory developed test as we know it? Recommendations from a national multidisciplinary taskforce of laboratory specialists on the interpretation of the IVDR and its complications
.
Clin Chem Lab Med
.
2020
;
59
(
3
):
491
7
.
48.
Kahles
A
,
Goldschmid
H
,
Volckmar
AL
,
Ploeger
C
,
Kazdal
D
,
Penzel
R
, et al
.
Structure and content of the EU-IVDR : current status and implications for pathology
.
Pathologie
.
2023
;
44
(
Suppl 2
):
73
85
.
49.
Bussolati
G
,
Leonardo
E
.
Technical pitfalls potentially affecting diagnoses in immunohistochemistry
.
J Clin Pathol
.
2008
;
61
(
11
):
1184
92
.
50.
Ramos-Vara
JA
,
Miller
MA
.
When tissue antigens and antibodies get along: revisiting the technical aspects of immunohistochemistry: the red, brown, and blue technique
.
Vet Pathol
.
2014
;
51
(
1
):
42
87
.
51.
Engel
KB
,
Moore
HM
.
Effects of preanalytical variables on the detection of proteins by immunohistochemistry in formalin-fixed, paraffin-embedded tissue
.
Arch Pathol Lab Med
.
2011
;
135
(
5
):
537
43
.
52.
Lin
F
,
Chen
Z
.
Standardization of diagnostic immunohistochemistry: literature review and geisinger experience
.
Arch Pathol Lab Med
.
2014
;
138
(
12
):
1564
77
.
53.
Gong
Y
,
Joseph
T
,
Sneige
N
.
Validation of commonly used immunostains on cell-transferred cytologic specimens
.
Cancer Cytopathology
.
2005
;
105
(
3
):
158
64
.
54.
Dalquen
P
,
Bittel
D
,
Gudat
F
,
von Overbeck
J
,
Heitz
PU
.
Combined immunoreaction and Papanicolaou’s stain on cytological smears
.
Pathol Res Pract
.
1986
;
181
(
1
):
50
4
.
55.
Travis
WD
,
Wold
LE
.
Immunoperoxidase staining of fine needle aspiration specimens previously stained by the Papanicolaou technique
.
Acta Cytol
.
1987
;
31
(
4
):
517
20
.
56.
Pohar-Marinsek
Z
,
Srebotnik-Kirbis
I
.
Desmin detection in FNAB samples of rhabdomyosarcoma: an immunocytochemical study
.
Cytopathology
.
2000
;
11
(
3
):
171
8
.
57.
Abendroth
CS
,
Dabbs
DJ
.
Immunocytochemical staining of unstained versus previously stained cytologic preparations
.
Acta Cytol
.
1995
;
39
(
3
):
379
86
.
58.
Liu
J
,
Farhood
A
.
Immunostaining for thyroid transcription factor-1 on fine-needle aspiration specimens of lung tumors: a comparison of direct smears and cell block preparations
.
Cancer Cytopathology
.
2004
;
102
(
2
):
109
14
.
59.
Kalhor
N
,
Zander
DS
,
Liu
J
.
TTF-1 and p63 for distinguishing pulmonary small-cell carcinoma from poorly differentiated squamous cell carcinoma in previously pap-stained cytologic material
.
Mod Pathol
.
2006
;
19
(
8
):
1117
23
.
60.
Strojan Flezar
M
,
Srebotnik-Kirbis
I
,
Gutnik
H
.
Use of vimentin immunocytochemical staining for evaluation of atypical cells in voided urine samples
.
Diagn Cytopathol
.
2017
;
45
(
2
):
85
90
.
61.
Strojan Fležar
M
,
Gutnik
H
,
Jeruc
J
,
Kirbiš
IS
.
Typing of renal tumors by morphological and immunocytochemical evaluation of fine needle aspirates
.
Virchows Arch
.
2011
;
459
(
6
):
607
14
.
62.
Lozano
MD
,
Abengozar-Muela
M
,
Echeveste
JI
,
Subtil
JC
,
Berto
J
,
Gurpide
A
, et al
.
Programmed death-ligand 1 expression on direct Pap-stained cytology smears from non-small cell lung cancer: comparison with cell blocks and surgical resection specimens
.
Cancer Cytopathol
.
2019
;
127
(
7
):
470
80
.
63.
Noll
B
,
Wang
WL
,
Gong
Y
,
Zhao
J
,
Kalhor
N
,
Prieto
V
, et al
.
Programmed death ligand 1 testing in non-small cell lung carcinoma cytology cell block and aspirate smear preparations
.
Cancer Cytopathol
.
2018
;
126
(
5
):
342
52
.
64.
Stokes
BO
.
Principles of cytocentrifugation
.
Lab Med
.
2003
;
35
(
7
):
434
7
.
65.
Miceska
S
,
Škof
E
,
Novaković
S
,
Stegel
V
,
Jeričević
A
,
Grčar Kuzmanov
B
, et al
.
Cytopathological assessment is an accurate method for identifying immunophenotypic features and BRCA1/2 mutations of high-grade serous carcinoma from ascites
.
Cancer Cytopathol
.
2023
;
131
(
3
):
188
97
.
66.
Strojan Fležar
M
,
Srebotnik Kirbiš
I
.
Identification of carcinoma origin by thyroid transcription factor-1 immunostaining of fine needle aspirates of metastases
.
Cytopathology
.
2009
;
20
(
3
):
176
82
.
67.
Dremelj
A
,
Miceska
S
,
Kuhar
A
,
Nolde
N
,
Kloboves-Prevodnik
V
.
Validation and implementation of OptiView and EnVision FLEX detection systems for immunocytochemical staining protocols of the ten most commonly used diagnostic markers in routine cytopathological practice
.
Diagnostics
.
2024
;
14
(
6
):
657
.
68.
Torlakovic
EE
,
Nielsen
S
,
Francis
G
,
Garratt
J
,
Gilks
B
,
Goldsmith
JD
, et al
.
Standardization of positive controls in diagnostic immunohistochemistry: recommendations from the international ad hoc expert committee
.
Appl Immunohistochem Mol Morphol
.
2015
;
23
(
1
):
1
18
.
69.
Kahles
A
,
Goldschmid
H
,
Volckmar
A-L
,
Ploeger
C
,
Kazdal
D
,
Penzel
R
, et al
.
Regulation (EU) 2017/746 (IVDR): practical implementation of annex I in pathology
.
Die Pathologie
.
2023
;
44
(
Suppl 2
):
86
95
.
70.
Torlakovic
EE
,
Nielsen
S
,
Vyberg
M
,
Taylor
CR
.
Getting controls under control: the time is now for immunohistochemistry
.
J Clin Pathol
.
2015
;
68
(
11
):
879
82
.
71.
Grillo
F
,
Bruzzone
M
,
Pigozzi
S
,
Prosapio
S
,
Migliora
P
,
Fiocca
R
, et al
.
Immunohistochemistry on old archival paraffin blocks: is there an expiry date
.
J Clin Pathol
.
2017
;
70
(
11
):
988
93
.
72.
Grillo
F
,
Pigozzi
S
,
Ceriolo
P
,
Calamaro
P
,
Fiocca
R
,
Mastracci
L
.
Factors affecting immunoreactivity in long-term storage of formalin-fixed paraffin-embedded tissue sections
.
Histochem Cell Biol
.
2015
;
144
(
1
):
93
9
.
73.
Combs
SE
,
Han
G
,
Mani
N
,
Beruti
S
,
Nerenberg
M
,
Rimm
DL
.
Loss of antigenicity with tissue age in breast cancer
.
Lab Invest
.
2016
;
96
(
3
):
264
9
.
74.
Giunchi
F
,
Degiovanni
A
,
Daddi
N
,
Trisolini
R
,
Dell’Amore
A
,
Agostinelli
C
, et al
.
Fading with time of PD-L1 immunoreactivity in non-small cells lung cancer tissues: a methodological study
.
Appl Immunohistochem Mol Morphol
.
2018
;
26
(
7
):
489
94
.
75.
DiVito
KA
,
Charette
LA
,
Rimm
DL
,
Camp
RL
.
Long-term preservation of antigenicity on tissue microarrays
.
Lab Invest
.
2004
;
84
(
8
):
1071
8
.
76.
Jacobs
TW
,
Prioleau
JE
,
Stillman
IE
,
Schnitt
SJ
.
Loss of tumor marker-immunostaining intensity on stored paraffin slides of breast cancer
.
J Natl Cancer Inst
.
1996
;
88
(
15
):
1054
9
.
77.
Bertheau
P
,
Cazals-Hatem
D
,
Meignin
V
,
de Roquancourt
A
,
Verola
O
,
Lesourd
A
, et al
.
Variability of immunohistochemical reactivity on stored paraffin slides
.
J Clin Pathol
.
1998
;
51
(
5
):
370
4
.
78.
Srebotnik Kirbis
I
,
Praça
MJ
,
Roque
RR
,
Košuta
T
,
Saudade
A
,
Strojan Flezar
M
.
Preservation of biomarkers immunoreactivity on cytospins protected with polyethylene glycol
.
Cytopathology
.
2020
;
32
(
1
):
84
91
.
You do not currently have access to this content.