Nucleic acid testing (NAT) in Polish blood donors - organization, methodology, results

and significance for transfusion safety

Piotr Grabarczyk Department of Virology

Transfusion medicine in Poland

• Population in 2019: 38,5 mln

• Polish Blood Transfusion Act of 22 August 1997

• 23 blood transfusion centers

• 139 local collection sites

• 1,2 mln donations/year

• 600 hospitals

• Rosiek A, Tomaszewska A, Lachert E, et al. Blood transfusion service in Poland in 2017. J Transfus Med 2018 • Stenholm E. Monitoring of blood transfusion operations in EU countries. Stockholm (Sweden): Karolinska University Hospital; 2015.

26%

74%

gender distribution

females males

33% 67%

donors category distribution

first time repeat

• Voluntary and non-remunerated

<=20 23%

21-30 37%

31-40 22%

41-50 12%

51-60 5%

>60 1%

age distribution

Characteristics of Polish blood donors

(data for 2005-2018)

2010

NAT

Roth WK, Busch MP, Schuller A, et al. International survey on NAT testing of blood donations: expanding implementation

and yield from 1999 to 2009. Vox Sang 2012;102:82-90.

system/test

Individual Donation Testing

markers

Amplicor system/test

MP48 pool

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019

NAT in Poland (2000-2019)

HCV/HIV/HBV markers

IDT

Ultrio Elite Panther

AmpliScreen

Procleix Ultrio Tigris Procleix Ultrio Plus Tigris

s201 MPX

HCV RNA, HIV RNA, HBV DNA HCV RNA, HIV RNA, HBV DNA

cobas 6800 MPX

s201 MPXv2.0

HCV RNA/ HIV RNA

TMA

PCR

MP24 MP6 MP6

HCV RNA

Procleix HCV/HIV-1

NAT methods evolution in Poland

External Quality Control in each RBTC • EDCNet : Every instrument's working-day from 2005- till now

• QCMD: 2/year

- February 2018: Evaluation BioQControl vs QConnect

SCREENING

Regional Blood Transfusion Centers (RBTC)

Mini-pool (MP) testing

•MP 6 - cobas s201 MPX v2 (12-19)

- cobas 6800 MPX (2-2)

MP 4 – Ultrio Elite (1 - 2 Panthers)

Individual Donation Testing (IDT)

•Ultrio Elite (4 - 9 Panthers)

NAT in Poland (2018)

BALTIC SEA

LITHUANIA

RUSSIA

BELARUS

UKRAINE

SLOVAKIA

CZECH

REP.

Kraków

(3)

Kielce

(1)

Szczecin

(2)

Lublin

(1+1)

Gdańsk

(2)

Bydgoszcz

(2)

Białystok

(2)

Poznań

(4)

Kalisz

(1+1)

Racibórz

(1)

Rzeszów

(1)

Warszawa (3) GERMANY

Łódź

(2)

Wałbrzych

(1)

Olsztyn

(2)

Katowice

(2)

Słupsk

(1)

1

10

100

1000

2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017

2,8 2,8 2,8 3 3 3 3 3 3 3 3 3 3 3

2064 2064 2064

504 504

66 66 66 66 66 41 41 41 41 41 41

MP

IDT

Cobas AmpliScreen (MP 24)

Cobas TaqScreen MPX v1 (MP6) Cobas TaqScreen MPX v 2 (MP6)

Procleix Ultrio Procleix Ultrio Plus Procleix Ultrio Elite

HCV 3.0 Elisa, Ortho Architect Anti-HCV, Abbott

Architect Anti-HCV, Abbott Vitros Anti-HCV, Ortho

Architect Anti-HCV, Abbott Vitros Anti-HCV, Ortho Monolisa HCVAg/Ab UltraII, BioRad Elecsys Anti-HCVII (Roche)

rok

Cobas Amplicor(MP48)

Procleix HCV/HIV-1

Analytical sensitivity of HCV RNA screening in Poland

8

Analytical sensitivity of HBV DNA screening in Poland

Uzupełnić czułość

HBsAg+

HBsAg-

/DNA

HBV+

HBsAg+/

DNA

HBV+

IDT1

10

100

1000

2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015

7,5 7,5 10 10 10

2,1 2,1 2,14,3 4,3 4,3

360 360 360

22,8 22,8 22,8 13,8 13,8 13,8 13,8 13,8

syste

m b

ad

an

ia

Czu

łoś

ć b

da

nia

prz

eg

ląd

ow

eg

o-

95

% L

OD

(IU

/ml)

rok

HBsAg (EIA)

Biomerieux/OCD

Abbott Vitros / Abbott

MP

IDT

Analytical sensitivity of HIV RNA screening in Poland

1

10

100

1000

10000

2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017

32,3 32,3 45 45 45 45

27,6 27,6 27,6 18 18 18 18

1469 1469

294 294 294 294 294 302 302 302 302 302 302

syst

em b

adan

ia

Czu

łość

bd

ania

prz

eglą

do

weg

o (

IU/m

l)

rok

MP

Cobas Ampliscreen (MP24) Cobas Taqscreen MPX v1 (MP6)

Cobas Taqscreen MPX v2 (MP6)

Procleix Ultrio półautomatyczny

Procleix Ultrio Tygris Procleix Ultrio Elite*

Biomerieux/OCD

HIV Ab/Ag

IDT/*MP8 i MP4

Procleix Ultrio Plus*

HIV-1 , HIV-1 grupa O, HIV-2

HIV-1 grupa M, HIV-1 grupa O, HIV-2

amplifikacja 2 regionów HIV-1

(LTR i pol)

*2012-2013 4 CKIK (Procleix Ultrio Plus MP8) *2017 1 CKIK (Procleix Ultrio Elite MP4)

Architect

HIV Ab/Ag

Vitros HIV Ab / Architect HIVAg/Ab

Architect HIV Ag/Ab, Abbott

Vitros HIV Ab, Ortho

Genscreen Ultra HIV Ag/Ab,

BioRad

Elecsys HIV combi PT, Roche

Screening and confirmatory testing algorithm - TMA

RNA HIV WB/INNO LIA

Ultrio (ID) Ultrio Plus (MP8) (95% LOD testu dla ID badanej w MP8: 221 IU/ml)

Ultrio (ID)

dHBV

dHCV

dHIV

Ultrio [10 IU/ml]

Ultrio

ID ID ID ID ID ID ID ID

(Umanual: 32,3 UTigris 45 UPlus: 27,6 UElite: 18 IU/mL)

(Umanual: 32,3 IU/mL)

Initial reactive Initial reactive Initial reactive

Reactive

dHBV

dHCV

dHIV

Ultrio or dHIV Reactive

Ultrio and dHIV Non Reactive

dHIV Reactive

Screening and confirmatory testing algorithm - PCR

RNA HIV WB/INNO LIA

MP6 (MPX)

Reactive

HIV Reactive

ID ID ID ID ID ID

HIV Reactive

MP24 (Ampliscreen)

MP6 MP6

Reactive

ID ID ID ID ID ID

MP6 MP6

Reactive

(MPXv1.: 95% LOD dla ID w MP6 = 294 IU/mL) (MPXv2.:95% LOD dla ID w MP6 = 302 IU/mL)

(ID MPXv1= 49 IU/mL)

HIV Reactive

(ID MPXv2= 50,3 IU/mL)

(95% LOD testu dla ID badanej w MP24: 1469 IU/ml)

Screening and confirmatory testing algorithm: HIV Ab -/HIV Ab/Ag -/NAT (HIV) Reactive

Analytical sensitivity (95% LOD) IU/ml

Confirmatory

testing

Procleix Ultrio Cobas Ampliscreen

32.3 IU/ml 61.2 IU/ml

Ultrio Elite 18 IU/ml

Cobas Ampliscreen 61.2 IU/ml

Ultrio Elite 18 IU/ml

GFE Blut v 1.1 89.5 IU/ml

GFE Blut v 1.2 13.7 IU/ml

2005 - 2009 - 2012 - 2014 -

WB / INNO LIA tesing of follow-up i look back samples - follow up: HIV Ab; WB or INNO LIA; RNA HIV

- look back: RNA HIV

Procleix Ultrio (półautomatyczny)

HIV Reactive

dHIV

dHCV

dHBV

Ultrio

HIV Reactive

= RNA HIV in tested donation (infected donor)

Cobas Ampliscreen

RNA HIV

Ultrio

dHIV

HIV Reactive

lub

HIV Reactive

Cobas Ampliscreen

RNA HIV

lub GFE

Non-enveloped viruses screening in Poland Requirements Criteria

for plasma discard Time of screening NAT results

applied to qualification of cellular blood components

domestic Other

HAV not required plasma for fractionation

confirmed infection quality control before shipment to manufacturer

only if available

B19V

plasma dedicated for anti-D, and anti-HBs production, RBC for immunization

plasma for fractionation

VL> 1-1.6x10E5 IU/ml (ID)

quality control before shipment to manufacturer

only if available

HEV not required

plasma dedicated for country with mandatory RNA HEV screening

confirmed infection in paralel to RNA HCV, DNA HBV and RNA HIV screening

obligatory

Real-time PCR screening algorithm LODs (cut-off) for screening and resolution of reactive MPs

Reactive result in MP96

8 subpools testing (MP12)*

12 individual donations testing*

testing sensitivity

105.6 IU/mL

13.2 IU/mL

1.1 IU/mL

cut-off for MP

(calculated for ID):

1.04x10E3 IU/mL

(1x10E5 IU/mL)

testing sensitivity

(calculated for ID)

166.8 IU/mL

13.9 IU/mL*

* Calculated for Cobas DPX based on data from instruction manual

HAV RNA B19 DNA

TMA screening algorithm LODs (cut-off) for screening and resolution of reactive MPs

Reactive result

in MP16

16 individual donation testing*

HAV RNA

16.96 IU/mL

1.06 IU/mL

cut-off for MP

(in respect to ID):

1.0x10E4 IU/mL

(1.6x10E5 IU/mL)

325 IU/mL

HEV RNA

126.24 IU/mL

7.89 IU/mL

*LODs for Procleix Assays run on Procleix Panther based on instruction manual

HAV RNA B19V DNA HEV RNA

Quality control for NAT in Poland

IHTM -- Institute of Haematology and Transfusion Medicine, reference lab in Warsaw RBTC – Regional Blood Transfusion Center, screening lab

Preliminary evaluation of method (in IHTM prior to implementation) • analytical sensitivity: multiple testing of six IS dilutions • clinical sensitivity assessment (polymorphic forms, if available) • proper identification of infected donations in MP testing (n=16-96) including high VL

samples: risk of contamination and false results

Procedure validation (at RBTC, prior to implementation and every 12 months) • several coded positive (including high VL) and negative samples tested in MPs • several coded positive (including low VL) and negative samples tested individually

External quality control program (at RBTC, at least every 12 months) e.g. LabQuality, EDQM, QCMD

Identification of seronegative donations infected with HCV worldwide

Busch MP. 10 Years of NAT testing—what has been achieved and what needs to be done? Workshop on Surveillance and Screening of Blood Borne Pathogens Zagreb, Croatia, 2010, May 16-17

Region/kraj donations number

tested anti-HCV(-) /RNA HCV(+)

NAT yields /1 mln donations

USA 39.000.000 170 1:229.411.

Europe 50.000.000 50 1:1.000.000.

Poland(2008) 7.600.000 83 1:91.566. 1:64516

Detection of early HCV infections (window period) in blood donors in Poland

1 2

4 5

6

12

6

1

5

8

6

9 9

12

7

20

0

2

4

6

8

10

12

14

16

18

20

22

2017 2016 2015 2014 2013 2012 2011 2010 2009 2008 2007 2006 2005 2004 2003 2002

Licz

ba

zaka

żeń

se

ron

ega

tyw

nyc

h

rok

Number of HCV infected blood donors

frequency/1 mln donors (+/-95% CI)

In total min (/rok)

max (/rok)

HCV WP 75 1 12 9.3 (7.4-11.6)

Detection of seronegative HBV infections (occult and window period) in blood donors in Poland

HBV NAT yields (WP)

2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018

year

0

10

20

30

40

50

60

70

80

num

ber

of

HB

V N

AT

yie

lds (

WP

)/1 m

ln d

onors

HBV NAT yields (OBI)

2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018

year

0

10

20

30

40

50

60

70

80

num

ber

of

HB

V N

AT

yie

lds (

OB

I)/1

mln

donors

Number of HBV seronegative infections

frequency /1 mln donors (+/-95% CI)

In total Min (/rok) Max (/rok)

HBV OBI 184 4 26 22.7 (19.7-26.3)

HBV WP 47 0 9 5.8 (4.4-7.7)

Detection of seronegative HIV infections (window period)

in blood donors in Poland

0

1

2

3

4

5

2006 2008 2009 2011 2012 2013 2014 2015 2016 2017 2018

per year

0 1 2 3 4 5 6

Katowice

Warszawa

Łódź

Poznań

Wałbrzych

Kalisz

Bydgoszcz

Kielce

Wrocław

WCKiK In regions

number of seronegative HIV infections

frequency /1 mln donors (+/-95% CI)

In total min (/year)

max (/year)

HIV WP 23 0 4 2.8 (1.9-4.3)

Residual risk for HBV, HCV, HIV - for RBC: MP6 and cobas MPX v2; IDT and UE

cumulated data for period 2005-2015, repeat blood donors; WP ID50=3.16, OBI ID50=316,

0,447

1,5

0,278 0,35 0,345

1,2

0,164 0,196

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

HBV-WP HBV-OBI HCV HIV

Exp

ect

ed

nu

mb

er

of

TTI

s/ 1

mln

RB

C t

ran

sfu

sio

ns

MPX2-MP6 UE-IDT Based on Weusten J, et al., Transfusion 2017 Mar;57(3pt2):841-849. Weusten J et al., Transfusion 2011 Jan;51(1):203-15.

HBV MP6~2 IDT~1,55

Residual risk for HBV (WP)

Frequency of high viral load parvovirus B19 infections in Polish blood donors, 2013-2018

relative risk for 2016 vs 2018 = 10,5 (95% CI: 7.2-15.3), p<<<0.05

donors number

period tested high VL B19V+

2013-18 1.998.451 454

2013 124.425 32

2014 233.452 88

2015 406.673 46

2016 502.513 32

2017 471.536 82

2018 260.164 174

2013 2014 2015 2016 2017 2018

year

0

10

20

30

40

50

60

70

80

nu

mb

er

hig

h v

ira

l lo

ad

B1

9V

do

no

rs/1

00

,00

0

Hepatitis A virus incidence in Polish blood donors

2013-2018

Source: PZH-NIZP IHTM *data reported for donors tested until the end of 2018

**infected donors and surveillance data reported until the end of April 2019

0

1

2

3

4

5

6

7

8

9

10

0

100

200

300

400

500

Jan

-15

Feb

-15

Ma

r-15

Ap

r-15

Ma

y-15

Jun

-15

Jul-

15

Au

g-15

Sep

-15

Oct

-15

No

v-15

Dec

-15

Jan

-16

Feb

-16

Ma

r-16

Ap

r-16

Ma

y-16

Jun

-16

Jul-

16

Au

g-16

Sep

-16

Oct

-16

No

v-16

Dec

-16

Jan

-17

Feb

-17

Ma

r-17

Ap

r-17

Ma

y-17

Jun

-17

Jul-

17

Au

g-17

Sep

-17

Oct

-17

No

v-17

Dec

-17

Jan

-18

Feb

-18

Ma

r-18

Ap

r-18

Ma

y-18

Jun

-18

Jul-

18

Au

g-18

Sep

-18

Oct

-18

No

v-18

Dec

-18

Jan

-19

Feb

-19

Ma

r-19

NU

MB

ER O

F R

NA

HA

V P

OSI

TIV

E D

ON

OR

S

NU

MB

ER O

F H

EPA

TITI

S A

-SU

RV

EILL

AN

CE

DA

TA

survailance data donors

2013 2014 2015 2016 2017 2018

0

1

2

3

4

5

nu

mb

er

of H

AV

RN

A p

ositiv

e d

on

ors

/100

,00

0

frequency (95% CI) in blood donors* number of infected blood donors vs surveillance data**

RNA HAV positive blood components follow-up (1)

Donation collection

• 8-475 days

HAV NAT

• 7-217 days

Referral to doctor

RNA HAV positive blood components follow-up (2)

Total no. transfused

19 (47.5%)

Total no. prepared

40

blood components

15 donations

red blood cells (RBC)

15

13

(86.7%)

platelates (PLT)

10

6

(60%)

plasma

15

0

(0%)

RNA HAV positive blood components follow-up (3)

RECIPIENTS

HAV VL

serological status: IgG/IgM

No. of days

between transfusion and HAV markers testing

in recipient

BLOOD COMPONENTS

HAV VL

serological status: IgG/IgM

TRANSFUSION

RBC

12 IU/mL

neg/neg

48 days

neg

pos/neg

RBC

11,600 IU/mL

pos/pos

43 days

neg

pos/neg

RBC

100 IU/mL

pos/pos

292 days

neg

pos/neg

RBC

172 IU/mL

pos/pos

210 days

neg

pos/neg

RBC

26 IU/mL

neg/neg

390 days

neg

pos/neg

RBC

3,280,000 IU/mL

neg/neg

12 days

2,860,000

pos/neg*

PLT

217,000 IU/mL

neg/neg

53 days

54,600

pos/pos*

Transmission unlikely unlikely indeterminable/indeterminable/indeterminable probable probable

*64 year-old patient /neurological ward, no symptoms of hepatitis A after transfusion **30 year-old oncological patient with bile duct cancer, died due to exacerbation of basic illness, assessment of clinical significance of HAV infection in process

Year

study: 2015

publication: 2018

HEV infections in Polish blood donors

Phylogenetic analysis of HEV isolates from Polish blood donors – identification of genotype 3 (subtypes 3c and 3i)

NAT perspectives

Application

• HEV

• HAV i B19V

• Emerging pathogens

Further improvements

• Widening tests panel in multiplex (+HEV; +HAV)

• Increasing HBV DNA testing sensitivity (?)

Screening centralization