Francesca Carozzi

HPV LABORATORIO
FRANCESCA CAROZZI
S.C. Laboratorio Prevenzione Oncologica
Responsabile Programma Regionale HPV in Toscana
Istituto per lo Studio e La prevenzione Oncologica (ISPO)
Firenze
F. Carozzi Workshop Pre-congruessuale GISCi 2015
1"
F. Carozzi Workshop Pre-congruessuale GISCi 2015
Lo"Screening"Cervicale"in"Italia"
"
Età
Screening
Intervallo
Da 25y a 30-35 y
Pap-test con HPV di triage
per ASC-US
3 anni
Da > 30-35y a 64
HPV con Pap di Traige
5 anni
25-64
HPV test nel the follow-up di donne :
- Trattate per lesioni CIN2
- Con alterazioni citologiche e negative alla
colposcopia
F. Carozzi Workshop Pre-congruessuale GISCi 2015
• Primary
Screening
with HPV test in
Italy
HPV test used
in HPV
screening
programs
in Italy
• Hc2
• Trento, new
tender
Veneto TP,Roche
Piemonte TP HC2
• Emilia Romagna: TP,
Roche
• HC2
Liguria pap conv + STM, HC2
Cobas Roche
Toscana:
Pap conv+HC2
TP+ HC2
Abruzzo TP, Hc2
HPV test and citology
Triage centralized in
1-2 Labs for Regions
• HPV Regional Programs
• HPV Locals Programs
• Roma G pap
conv, STMAbbott
• Latina ,
surepath
Pap conv
+stm
Hc2+?
Basilicata Pap Conv+STM, Hc2
• HPV Programs in the activation phase
F. Carozzi Workshop Pre-congruessuale GISCi 2015
HPV"Laboratorio:"""
• 
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• 
• 
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• 
• 
I"test"valida:"sono"tu<"uguali?"Hanno"le"stesse"problema:che"o"pun:"di"forza?"
Quali"da:""anali:ci"raccogliere"nei"sistemi"informa:vi"?""
Come"refertare"il"test"HPV"di"screening?"(vedi"tool"box,"vedi"WHO,"vedi"raccomandazioni"
Gisci)"
I"controlli"interni""Come"raccogliere"i"da:"dei"controlli"?""
Quali"VEQ?"È"opportuno"fare"un"progeMo"Gisci"in"questa"fase"cruciale?""
Ges:one"e""della"qualità"del"nuovo"percorso"di"centralizzazione,"qualità"assurance","
valutazione"in"i:nere"di"performance"e"impaMo"
Quali"criteri"per"la"centralizzazione""?"
Qual"è"il"workflow"delle"della"strumentazione"disponibile"sul"mercato?"
Qual"è"il"carico"di"lavoro?""
Il"test"HPV"viene"dalla"ricerca,"abbiamo"finito?"Quale"ricerca"implemntare"?"Biomarctori?"Altri"
test?""
Quale"test"HPV"nelle"vaccinate?""
Quali"le"problema:che"che"si""incontrano"tu<"i"giorni?"
Quale"formazione?"Come"validare"l’entrata"nella"rou:ne?"Sono"u:li"le"check"list?""
Le"modalità"di"risposta"dei"non"valutabili"post"anali:ci"e"preXanali:ci"(tuMo"chiaro?)"
F. Carozzi Workshop Pre-congruessuale GISCi 2015
5"
Il"percorso"di"aMuazione"del"cambiamento:"la"centralizzazione"
Il"ruolo"complesso"del"laboratorio"!
"
• 
L’"introduzione"del"codice"a"barre"come"sistema"iden:fi"ca:vo"del"prelievo"della"donna":"
• 
Adeguamento"dei"servizi"di"acceMazione"campioni"locali"e"del"lab"centrale"
"
• 
Gli"aspe<"logis:ci"lega:"alla"centralizzazione:""
• 
– 
– 
– 
CoXesistenza"di"due"percorsi"(PAP"primario"e"HPV"primario"
Ges:one"di"più":pologie"di"campioni"HPV"(screening,"Triage,"FollowXup"con"cito"o"senza"cito)"
Definizione"flussi"e"comunicazioni"per"non"conformità"in"fase"di"presa"in"carico"
–  impostazione"dei"flussi"di"trasporto"dei"campioni"dalle"unità"di"prelievo"dai"centri"di"riferimento,"con"
eventuali"centri"di"raccolta"all’interno"dela"singola"Azienda""
–  RispeMo"delle"norme"per"il"trasporto"su"strada"(contenitore"triplo)"
L’""alles:mento"del""laboratorio"regionale"di"riferimento""
–  aumento"del"numero"di"test"molecolari"e"il"passaggio"da"ricerca""ad"a<vità"clinica"con"
numeri"eleva:"
–  creazione"di"nuove"procedure"per"la"ges:one"di"tuMo"il"fl"usso"di"lavoro."
–  "creazione"check"list"di"lavoro""
–  Formazione"ed"Addestramento"del"personale"tecnico"da"colorazione"a"test"molecolari""
–  "Alles:mento"di"controlli"di"qualità"molecolari"interni"ed"esterni"
"
Oltre"la"VEQ"e"il"CI,"Ges:one"del"monitoraggio,"qualità"assurance"e"valutazione"di"performance"e"impaMo"con:nuo,""
"
F. Carozzi Workshop Pre-congruessuale GISCi 2015
………….."
."
" screening
Diagramma di flusso
ACCESSO AL PROGRAMMA
REALIZZAZIONE TEST
PERCORSO DIAGNOSTICO
FOLLOW UP
localizzata
Laboratorio centrale
Positivi al test
Potenziale alto rischio
Convocazione
Accettazione
accoglienza
Esecuzione
test
Valutazione
Valutazione
diagnostica
diagnostica
Esito positivo
Ulteriore
Ulteriore
accertamento
accertamento
diagnostico
diagnostico
Intervento
chirurgico/
trattamento
terapeutico
Richiamo precoce
Negativi al test
Rischio basso
Esito negativo
F. Carozzi Workshop Pre-congruessuale GISCi 2015
Follow up
CRITERI"per"la"centralizzazione"
•  Il"report"di"HTA"non"prevede"una"valutazione"di"costo"efficacia"
in"senso"streMo"(per"la"definizione"dei"criteri"di"
centralizzazione"),"ma"fa"una"previsione"degli"scenari"fino"agli"
80.000"test/anno:"
–  "l’obie<vo"era"dimostrare"l’en:tà"della"riduzione"dei"cos:"
all’aumentare"del"numero"di"esami"fa<"
–  "non"stabilire"un"teMo"massimo"o<male"
F. Carozzi Workshop Pre-congruessuale GISCi 2015
8"
Quali criteri per la centralizzazione ? Come definiamo un
laboratorio di grandi dimensioni?
•  Organizzazione dello screening (logistica del territorio,
modalità di trasporto dei campioni)
•  Grado di automazione e workload del sistema
•  Tipologia di prelievo (fase liquida, quali sono i criteri di
sicurezza da rispettare?)
•  Modalità e tempi di processazione dei campioni ed
esecuzione dei test
!  Volume minimo di campioni vs volume max: determinato
da cosa?
! Spazi?
! Altro?
!  Gestione regionale
F. Carozzi Workshop Pre-congruessuale GISCi 2015
CDC , Atlanta USA
F. Carozzi Workshop Pre-congruessuale GISCi 2015
Variations in Cervical Samples
"  Collection Device
- Brush, broom, spatula, swab, tampon
"  Collection Media
- STM, PreservCyt, SurePath, alcohol, paper
"  Collection Method
-  Clinician collected
- Self-collected
F. Carozzi Workshop Pre-congruessuale GISCi 2015
Which high-risk HPV assays fulfil criteria for use in primary cervical cancer screening?
Marc Arbyn, Peter J.F. Snijders, Chris J.L.M. Meijer, Hans Berkhof, Kate Cuschieri, Bostjan J. Kocjan, Mario Poljak
Clinical Microbiology and Infection
27 March 2015 21 April 2015 23 April 2015
The cobas 4800 HPV Test and RealTime High Risk HPV test were consistently validated in
"  two
and three studies, respectively,
PapilloCheck HPV-screening test, BD Onclarity HPV assay and the HPV Risk Assay were
"  validated
each in one study.
tests which partially fulfil the 2009-guidelines are:
"  Other Cervista
HPV HR Test, PCR-LMNX,
•  a homebrew
E6/E7 RT qPCR and MALDI-TOF.
• 
The APTIMA HPV assay targeting E6/E7 mRNA of hrHPV was also fully validated.
"  However,
the cross-sectional equivalency criteria of the 2009-guidelines were set up for HPV
DNA assays. Demonstration of a low risk of CIN3+ after a negative APTIMA test over a
longer period is waited for to inform about its utility in cervical cancer screening at five year
or longer intervals.
F. Carozzi Workshop Pre-congruessuale GISCi 2015
12"
The"HORIZON"Study"
Four HPV assays – four different technologies
• Cobas®HPV test Real-time PCR assay, with co-detection of HPV HR and 16 and
18
• HC2® Hybridization assay with HR HPV detection
• APTIMA® RNA assay with HR HPV detection
• CLART® PCR-Microarray assay with simultaneous detection of 35 genotypes
QiaSymphon
y"RCS"HC2®"
work"flow"
Genomica!
Roche cobas®4800 Gen-Probe
CLART®HPV2
workflow
PANTHER®
APTIMA workflow
F. Carozzi Workshop Pre-congruessuale GISCi 2015
"
F. Carozzi Workshop Pre-congruessuale GISCi 2015
Sensi:vity"for"CIN"3+"
38 CIN 3+ in 2,869 women screened at age 30-65 years*
Screening test
Cytology
Hybrid Capture 2
cobas
CLART
APTIMA
CIN 3+
Detection rate per
100 screened
women
Sensitivity
31
1.1% -- 1 (ref)
82%
34
1.2% -- +10%
89%
36
1.3% -- +16%
95%
36
1.3% -- +16%
95%
33
1.2% -- +6%
87%
* Histological follow-up will be complete by end of 2013
F. Carozzi Workshop Pre-congruessuale GISCi 2015 [Rebolj
et al., in preparation]
HPV e rischio cancerogeno: 2011
Gruppo 1 (cancerogeni per l uomo): 16, 18, 31, 33, 35, 39, 45, 51,
52, 56, 58 and 59.
Gruppo 2A (probabilmente cancerogeni per l uomo): 68.
Gruppo 2B (possono essere cancerogeni per l uomo): 26, 53, 66,
67, 70, 73, 82.
Gruppo 2B (possono essere cancerogeni per l uomo su base
filogenetica): 30, 34, 69, 85 and 97.
Gruppo 3 (non classificabili per la loro cancerogenicità
nell uomo): 6 and 11
Fonte: Monografia 100B IARC 2011
F. Carozzi Workshop Pre-congruessuale GISCi 2015
Elenco test hrHPV clinicamente validati
• 
• 
• 
• 
• 
Come mantenerlo aggiornato?
Chi lo fa?
Con quale frequenza?
Come renderlo disponibile?
Come valutare i dati che saranno pubblicati?
F. Carozzi Workshop Pre-congruessuale GISCi 2015
Considerations before implementing an assay
•  •"What"type"of"assay"is"it?"Can"the"laboratory"provide"the"
infrastructure"and"environment"to"deliver"the"assay"robustly.""
•  •Is"the"laboratory"accredited"to"perform"clinical"microbiology"
tes:ng"or"to"perform"HPV"test"within"screening"program?"
•  •InXhouse"valida:on,"to"show"assay"performs"to"expected"
standard"with"local"operators"and"systems""
•  •Training"and"demonstra:on"of"competence"of"single""
specialist""or"techinician""which"is"monitored"through"regular"
competency"assessment""
•  •Set"up"of"quality"system"and"framework"to"monitor"
performance"longitudinally""
F. Carozzi Workshop Pre-congruessuale GISCi 2015
Example of quality framework for HPV Testing
within screening program
"
"  Accreditation through Regional Audit ; HPV tests within screening program
listed in their repertoire of services
"  Participation in accredited external quality assurance scheme for HPV and good
performance
"  Each member of staff undertaking HPV testing must first pass company training
procedures, with training recorded in individual s training logs
"  Competency of staff/laboratory should be demonstrated through testing of an
HPV Validation Panel
"  Competency should be assessed regularly for as long as staff has responsibility
for the function
"  Laboratories undertaking HPV testing must include in every run known positive
and negative IQC samples validated by repeated testing, in addition to required
kit controls
" 
"  Laboratories undertaking HPV testing should check regularly (eg weekly; 1% of
samples) for systemic errors and perform environmental testing of the
laboratory areas used.
F. Carozzi Workshop Pre-congruessuale GISCi 2015
Quality control
endogenous, within-sample controls
""""""To control for cellularity
or PCR reaction
inhibition?
- Common targets, human gene (beta globin , GAPDH)
- Detected in human cells, not just epithelial cells
- What cellular content is adequate/sufficient for HPV testing, what type
of cellular content?
- Primer sequences can be differentially affected by inhibition*""
"
Does this become an increasingly pertinant question
for HPV primary screening?
*Lefevre et al Journal of Virological Methods 114 (2003) 135–144
F. Carozzi Workshop Pre-congruessuale GISCi 2015
Which high-risk HPV assays fulfil criteria for use in primary cervical cancer screening?
Marc Arbyn, Peter J.F. Snijders, Chris J.L.M. Meijer, Hans Berkhof, Kate Cuschieri, Bostjan J. Kocjan, Mario Poljak
Clinical Microbiology and Infection
27 March 2015 21 April 2015 23 April 2015
F. Carozzi Workshop Pre-congruessuale GISCi 2015
F. Carozzi Workshop Pre-congruessuale GISCi 2015
Assurance quality in programme changes
"  We need to ensure that any change to screening methodology is
at least as good as the current method and for HPV the quality
remains at least as good as in research setting
"  HPV testing procedure has to be monitored to continuous and
rigorous quality assurance to avoid sub-optimal, potentially
harmful practices:
"  Quality control program also for Triage Test (PAP-Test) and
for each phase of the protocol
F. Carozzi
2015
F. Carozzi Workshop
Pre-congruessuale
GISCi 2015
Quality Assurance, Internal Quality Control
and External Quality Assurance
-  Quality Assurance (QA): is essential to guarantee precise and accurate analysis to
support optimal patient care.
-  ensures the right result for the right test, in the right time, the right sample for the right patient and
interpreted with the correct reference data
□  using detailed check lists
-  Internal Quality Control (IQC):
-  verifies the precision of the investigations in the single lab,
-  system precision and stability
-  shall be repeated in every analytical session
-  External Quality Assessment (EQA): data accuracy, comparison with external labs
-  can detect differences between centers that measure the same analyte
-  allows to verify single lab accuracy (Bias) compared to the medium value obteined by all participant labs
• 
•  Since the determination is periodic and retrospective, is commonly used the term "assessment" rather
than "control".
F. Carozzi Workshop Pre-congruessuale GISCi 2015
Implementation of appropriate quality control
procedures for HPV test in a screening setting
  The laboratory must prepare a SOP (Standard Operating
Procedure) with the definition of alarm and rejection of
analytic series based on the value of the control samples
  All results of Internal QC should be recorded, daily archived
and registered in the form like control charts.
  "out of control" results must be registered
  Definition of corrective action to be taken in the event of nonobservance of quality parameters.
F. Carozzi Workshop Pre-congruessuale GISCi 2015
Registrazione giornaliera dei dati
1 DS
Controllo Interno RLU/CO RCS
2 DS
3DS
1,8
1,7
1,6
1. 
1,5
1,4
1,3
RLU/CO
1,2
Standard Operating Procedure with corrective actions and
management of situations that deviate from the expected
value
1,1
1
Serie1
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
0
50
100
150
F. Carozzi Workshop Pre-congruessuale GISCi 2015
200
HPV screening
Program
inalItaly
% di donne
35-64 POSITIVE
Test HPV women aged 35-64yo
% HPV test positive women
NORD
CENTRO
SUD e ISOLE
14%
12,4%
HPV Test validated for screening
12%
Quality Controls for HPV test within screening programs are
necessary to be sure that difference in HPV positivity depends by
prevalence of infection in the different areas/cities
10%
9,1%
7,8%
8%
6,3%
7,1%
6,6%
5,6%
6%
4,6%
4,7%
4,8%
4,8%
5,0%
5,8%
6,0%
6,0%
5,1%
4,0%
4%
2%
TO
RI
NO
LA
RE
TI
NA
G
G
IO
EM
IL
IA
FI
RE
NZ
E
TE
RA
M
O
LA
N
CI
AN
O
PE
SC
A
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AN
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ZZ
ES
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M
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VI
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0%
F. Carozzi Workshop Pre-congruessuale GISCi 2015
External Quality Assurance Programs
for HR- HPV in a screening setting
•  HPV is a virus and normally in clinical virology, it is important to apply
methods with highest sensitivity for the micro-organism that is
responsible for an infection
"  The ‘HPV test’ applied to the screening program doesnt’ follow this rule
BUT it must to be validated and optimized for clinical sensitivity and
specificity in a screening context
• 
the cut-off used in this contest does not correspond with the minimum
detectable level of the method
•  HPV EQA specific for HPV screening Test
"  So EQA panel applied to HPV test in cervical cancer MUST :
•  Be optimised for screening test
•  Evaluate all 12 HR HPV types in a cycle of EQA panel
F. Carozzi Workshop Pre-congruessuale GISCi 2015
EQA, examples of existing schemes
" WHO HPV LabNet (Proficiency Panel)
Plasmid material. Annual proficiency panel of 46 samples (43 extracted DNA + 3 cell samples to be
extracted). Designed to test genotyping assays
" UK NEQAS VEQ
Pooled material of clinical origin, 4 specimens 3 times a year – scores on qualitative performance
(presence/absence of HR HPV types) In ThinPrep
" www.ukneqas.org.uk
" QCMD VEQ
Cell line material and clinical samples. Annual panel (8-10 samples). 2 kind of samples: CORE and
EDUCATIONAL. Score assigned on the basis of CORE samples result. In Thin Prep
" w.qcmd.com
DicoCare VEQ
" (Italian EQA) Clinical samples. 8 specimens 4 times a year. Designed to test clinical performance
of the methodology used and genotyping assays- lyophilized sample to re-suspend in used medium
" Online.dicocare.org
F. Carozzi Workshop Pre-congruessuale GISCi 2015
HPV"primario"e"qualità"dell’intero"processo:"dalla"
fase"preXanali:ca"a"quella"postXanali:ca""
Prevenire"gli"errori"
•  Il"percorso"dello""screening"è"un"sistema"complesso"in"cui"
interagiscono"molteplici"faMori""
•  come"in"altri"sistemi"complessi,"possono"verificarsi"inciden:"
ed"errori,"la"centralizzazione"genera"efficienza"ma"dobbiamo"
valutare"le"fasi"di"nuova"cri:cità,"non"solo"da"parte"del"
laboratorio"."
•  Vanno"pertanto"progeMa:"specifici"modelli"di"controllo"del"
rischio"clinico,"con"l obie<vo"di"prevenire"il"verificarsi"di"un"
errore"e,"qualora"questo"accada,"contenerne"le"conseguenze."
•  Solo"aMraverso"opportune"analisi"è"possibile"iden:ficare"le"
cause"di"un"errore"e"ridisegnare"i"processi"al"fine"di"ridurre"la"
probabilità"che"esso"si"ripeta."
F. Carozzi Workshop Pre-congruessuale GISCi 2015
La!prevenzione!degli!errori!nel!processo!di!implementazione!del!test!HPV!come!
test!di!screening!primario."
XISPO,"Firenze"""X"Centro"per"la"Ges:one"del"Rischio"Clinico"e"la"Sicurezza"del"
Paziente,"Regione"Toscana"
Val"Camonica,""Roma"G,""IOV"Veneto,"AO"Reggio"Emilia""
"
•  Individuare"le"insufficienze"nel"sistema"e"progeMare"le"idonee"barriere"prote<ve."""
""due"approcci:""
–  proa<vo,"in"cui"l’analisi"parte"dalla"revisione"dei"processi"e"delle"procedure"esisten:,"
iden:ficando,"nelle"varie"fasi,"i"pun:"di"cri:cità;"per"validare"delle"procedure"o"per"
monitorare"la"sicurezza"delle"pra:che"di"lavoro"
–  "rea<vo:"l’analisi"parte"da"un"evento"avverso"e"ricostruisce"a"ritroso"la"sequenza"degli"
avvenimen:"con"lo"scopo"di"iden:ficare"i"faMori"che"hanno"causato"o"contribuito"il"
verificarsi"dell’evento"
Vedi Sessione Poster: G.P. Pompeo
•  L approccio"proa<vo"è"quello"che"può"garan:re"la"realizzazione"di"un"progeMo"
sanitario"più"sicuro"e"per"la"sua"realizzazione"lo"strumento"da"preferire"è"la"
cosiddeMa"FMEA"(Failure(Mode(and(Effect(Cri2caly(Analysis)"
•  Applicazione"di""questo"metodo"nell ambito"della"implementazione"del"
Programma"HPV"della"Regione"Toscana"e"di"altri"programmi"che"sono"par::"o"in"
fase"di"implementazione""
F. Carozzi Workshop Pre-congruessuale GISCi 2015
F. Carozzi Workshop Pre-congruessuale GISCi 2015
!Il#Test#HPV+HR#nei#programmi#di#screening!
•  5.1"CaraMeris:che"del""test#hr+HPV#
•  5.2""Indicazioni"sulle"modalità"di"risposta"del"
test"HrXHPV"nello"screening"
•  5.3"Introduzione"di"idonee"procedure"di"
controllo"di"qualità"dei"test"molecolari""
F. Carozzi Workshop Pre-congruessuale GISCi 2015
F. Carozzi Workshop Pre-congruessuale GISCi 2015
Grazie"per"l aMenzione"!!"
"
[email protected]"
F. Carozzi Workshop Pre-congruessuale GISCi 2015