Pathogeninaktivierung - Rotes Kreuz · Pharmakokinetik Toxizität Toxizität Akut Mehrfachanwendung...

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Wolfgang R. Mayr

Pathogeninaktivierung

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Blutsicherheit

� Spenderauswahl

� Spendertestung

� Klinische Indikation

� Produktion

� PI

� QM

� Hämovigilanz

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Infektionsrisken

� Diagnostisches Fenster

� Bekannte Viren ohne Testung

� Unbekannte (emerging) Viren

� Bakterien

� Protozoen

� Jeder Erreger, der während der asymptomatischen Phase der Infektion, im Blut vorkommt, kann via Transfusion übertragen werden

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Testung (BSZ W,N,B)

� 1950 AB0, RH, Ery-Ak, Lues

� 01. 1973 AuSH Ag

� 02. 1979 HBs Ag

� 07. 1985 HIV Ak

� 01. 1989 ALT

� 07. 1990 HCV Ak

� 04. 1995 Neopterin

� 02. 1999 NAT – HBV, HCV, HIV

� 06. 2003 NAT – Parvo B19

� 10. 2003 NAT - HAV

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Restrisiko 2009

� HCV, HIV 1 : 5.000.000

� HBV 1 : 500.000

(okkulte HBV Infektionen)

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Pathogeninaktivierung - PI

� Fensterperiode – Viren mit Test

� Unbekannte Viren

� Bakterien

� Protozoen

� Leukozyten (TA GvHD)

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Leukozyten

� Zell-assoziierte Viren (CMV, EBV, ...)

� Febrile Transfusionsreaktionen NHFTR

� HLA / HNA Alloimmunisierung

� Transfusions-assoziierte Immunmodulation

� TA GvHD

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Pathogeninaktivierung

� Wirksamkeit

� Klinik

� Pharmakokinetik

� Toxizität

Toxizität� Akut

� Mehrfachanwendung

� Subchronisch

� Reproduktiv

� Genotoxizität

� Carcinogenität

� Phototoxizität

� Venenirritation

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Schroeder DD & Mozen MM,Science 1970; 168: 1462-1464

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Prion-removal capacity ...(Thyer J et al, Vox Sang 2006; 91: 292-300)

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PI – stabile Blutprodukte

� Pasteurisierung (60°C, 10 h)

� Erhitzen (trocken, feucht)

� S/D (solvent-detergent)

� Nanofiltration

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Burnouf T, Transf Med Rev 2007; 21, 101-117

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PI - FFP

� Pool S/D inaktiviert „Octaplas“

� Einzelspender Quarantäne

PI durch Amotosalen

PI durch Methylenblau

PI durch Riboflavin

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S/D FFP versus Einzelspender-FFP

FFPFFPFFPFFP S/D FFPS/D FFPS/D FFPS/D FFP QuarantQuarantQuarantQuarantääääne FFPne FFPne FFPne FFP PI Einzelspender PI Einzelspender PI Einzelspender PI Einzelspender FFPFFPFFPFFP

Standardisierung (Volumen, Proteine, Zellgehalt, ...)

Hoch Gering Gering

Umhüllte Viren Sicher Okkulte HBV Sicher

Nicht-umh. VirenB19, HAV

NAT Nicht getestet Relativ sicher

„Emerging“ Viren Pool Einzelspender Relativ sicher

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PI - labile ProdukteThrombocyten

� Hauptproblem: bakterielle Infektion

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Division of Division of Division of Division of Blood Group Blood Group Blood Group Blood Group

Blajchman MA et al, Transfus Med Rev 2005; 19: 259-272


REDUCTION OF SEPTIC TRANSFUSION REACTIONS WITHOUT BACTERIA DETECTION

(Andreu et al, ISBT Science Series 2008; 3: 124-132 )

• Reduction of initial bacterial contamination– Hygiene, skin desinfection– Diversion of the first 35 ml– Postdonation information– Donor selection

• Reduction of bacterial growth– Practice for delivery– Universal leukodepletion– Storage of whole blood before processing (2 – 20 h)

• Improvement of knowledge and practice on bacterial contamination

– Management of possible bacterial infection– Research on bacterial contamination

Bacterial screening of apheresis platelets (ARC 2004-2006)


Eder AE et al, Eder AE et al, Eder AE et al, Eder AE et al, Transfusion Transfusion Transfusion Transfusion 2007; 47: 11342007; 47: 11342007; 47: 11342007; 47: 1134----1142114211421142


te Boekhorst PAW et al, Transfusion 2005; 45: 514-519

Screening platelet concentrates for bacterial contamination: low numbers of bacteria and slow growth in contaminated units mandate

an alternative approach to product safety

Murphy WG et al, Vox Sanguinis 2008; 95: 13-19

Conclusions:Screening platelet concentrates for bacterial contamination using the most sensitive method available has a sensitivity of less than 40% because of the low numbers of bacteria in the initial contamination. Effective resolution of this problem will require a pathogen-inactivation technique.


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PI – labile Blutprodukte:Thrombocyten

� Verhinderung der DNA Replikation: Photosensitizer + Licht

� Verhinderung der DNA Replikation:

UVC (254nm) Behandlung

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Blood Donation Center for Vienna, Lower Austria and Burgenland,

Austrian Red CrossDepartment for Blood Group

Nucleic Acids Must “UnZip”During Pathogen Replication

Strand SeparationDNA / RNA Replication ofNucleic Acids and Pathogen



DNA or RNAof pathogen

Amotosalen Mechanism of Action

Amotosalen

Docking Permanent Crosslinking

UVA Illumination


Austrian Red CrossDepartment for

Pooling Container

Leukodepletion Filter

Whole BloodSeparation

INTERCEPT Blood System for Platelets Buffy Coat Preparation

Pooling and Addition of InterSol Solution

Centrifugationto Pellet Red

Blood Cell

Separationof Platelets

Buffy Coat Platelets

InterSol Solution

1 2 3 4



INTERCEPT Blood SystemAmicus Platelet Preparation

Amicus enables theAmicus enables theautomatic collection ofautomatic collection ofone or more therapeutic dose(s)one or more therapeutic dose(s)of SDP in 35% plasmaof SDP in 35% plasmaand 65% InterSol Solution.and 65% InterSol Solution.



WithinWithinone day

of collectionof collection

INTERCEPT Blood System for Platelets Process Timeline

11 22 33

IlluminateIlluminateStart ProcessingStart Processing Removal & StorageRemoval & Storage

3 J/cm3 J/cm 22

forfor~3-6 minutes

Incubate plateletsIncubate plateletson the CAD for a minimum of on the CAD for a minimum of

4 hours: SDP6 hours: buffy coat platelets

1. UV light only: reversible inactivation

� UV light alone breaks chemical bonds in the

nucleic acids of pathogens

2. UV light + riboflavin: irreversible inactivation

� Riboflavin molecules form complexes with nucleic acids

� UV light from the Mirasol Illuminator activates the

riboflavin molecule in the complex

� Photoactivated riboflavin induces a chemical alteration to

the functional groups (such as guanine bases) of nucleic

acids making pathogens unable to replicate

The Mirasol PRT System inactivates disease-causing agents by

altering their nucleic acids in two primary ways:1

Mirasol PRT System Primary Modes of Action

1 Kumar et al 2004

Reduction of activepathogen load

Typicalperformance

References

VirusesVirusesVirusesViruses(enveloped, non(enveloped, non(enveloped, non(enveloped, non----enveloped; enveloped; enveloped; enveloped; intracellular, extracellular)intracellular, extracellular)intracellular, extracellular)intracellular, extracellular)

~2–6 log(99.0–99.9999%)

Ruane, et al., 2004; Goodrich, et al., 2006a,b;CaridianBCT data on file.

BacteriaBacteriaBacteriaBacteria(Gram +, Gram (Gram +, Gram (Gram +, Gram (Gram +, Gram ----))))

~2–5 log(99.0–99.999%)

Ruane, et al., 2004; Goodrich, et al., 2006b; CaridianBCT data on file.

ParasitesParasitesParasitesParasites ≥ 3.0 to ≥ 5.0(≥ 99.9% to ≥ 99.999%)

Cardo, et al., 2006; Cardo, et al., 2007; Rentas, et al., 2007; Tonnetti, et al., 2007;

Sullivan, et al., 2008.

Mirasol PRT System Performance

Inactivation of whiteblood cells

Typicalperformance

References

White blood cell inactivationWhite blood cell inactivationWhite blood cell inactivationWhite blood cell inactivation ≥ 6.0(>99.9999%)

Fast, et al., 2006a. (in-vitro)

Cytokine production and Cytokine production and Cytokine production and Cytokine production and expressionexpressionexpressionexpression

prevented Fast, et al., 2006a. (in-vitro)

GraftGraftGraftGraft----versusversusversusversus----Host DiseaseHost DiseaseHost DiseaseHost Disease prevented Fast, et al., 2006b. (animal model)

Alloimmunization & Alloimmunization & Alloimmunization & Alloimmunization & transplant rejectiontransplant rejectiontransplant rejectiontransplant rejection

prevented Asano, et al., 2007. (animal study)

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UVC Behandlung (Mohr H et al, Transfusion 2009; 49: 1956-1963)

� Beutel 19 x 38 cm

� Thrombocyten: in 350 ml (109 / ml)

� Bestrahlung mit UVC (254 nm)

� Agitation (100 rpm, horizontal)

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UVC Behandlung

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Mohr et al, Transfusion 2009; 49: 1956-1963

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PI bei Thrombocyten

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Stramer et al, Transfusion 2009; 49: August Suppl 1S – 29S

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Amotosalen: Apherese- und BC-Thrombocyten

� CCI nach 1 und nach 24h geringer als bei nicht behandelten Thrombocyten

� Hämostaseologische Funktion nicht verändert

� Keine unerwünschte Nebenwirkungen

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Amotosalen: Hämovigilanz

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Red Cell Processing

SS--303303

Collected RedCollected RedCells withCells with

Additive SolutionAdditive Solution

MixingMixingContainerContainer

ReactionReactionContainerContainer

FinalFinalStorageStorage

ContainerContainer

CADCAD

Copyright 2001 – Cerus Corporation



Collected red blood Collected red blood cells with additive cells with additive

solution;solution;LeukodepletedLeukodepleted

INTERCEPT Blood System for Red Blood Cells

Proposed Process Timeline

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TreatmentTreatmentStart ProcessingStart Processing Removal andRemoval andStorageStorage

IncubateIncubatered blood cells red blood cells

and Sand S --303303at 19at 19--2525°°C forC for

12 hours

RemoveRemoveresidualresidual

compoundcompoundat 19at 19--2525°°C forC for

8 hours

SS--303303

CADCAD

ReconstituteReconstituteSS--303 and mix303 and mix

with red blood cellswith red blood cells

------- Development of an automated process is on-go ing -------

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PI of RBC (S-303, Inactine, ...)

Treated red cells demonstrated acceptable recovery and comparablTreated red cells demonstrated acceptable recovery and comparablTreated red cells demonstrated acceptable recovery and comparablTreated red cells demonstrated acceptable recovery and comparable e e e lifelifelifelife----span to conventional red cellsspan to conventional red cellsspan to conventional red cellsspan to conventional red cells

Repeated transfusions of autologous treated red cells did not prRepeated transfusions of autologous treated red cells did not prRepeated transfusions of autologous treated red cells did not prRepeated transfusions of autologous treated red cells did not provoke ovoke ovoke ovoke autoautoautoauto----antibody formationantibody formationantibody formationantibody formation

PostPostPostPost----transfusion recovery after repeated transfusions of autologous transfusion recovery after repeated transfusions of autologous transfusion recovery after repeated transfusions of autologous transfusion recovery after repeated transfusions of autologous treated red cells was comparable to that of untreated red cells treated red cells was comparable to that of untreated red cells treated red cells was comparable to that of untreated red cells treated red cells was comparable to that of untreated red cells and to and to and to and to that of single exposures of treated red cellsthat of single exposures of treated red cellsthat of single exposures of treated red cellsthat of single exposures of treated red cells

Full unit transfusions of treated red cells were well toleratedFull unit transfusions of treated red cells were well toleratedFull unit transfusions of treated red cells were well toleratedFull unit transfusions of treated red cells were well tolerated

? Production of antibodies against treated RBC? Production of antibodies against treated RBC? Production of antibodies against treated RBC? Production of antibodies against treated RBC

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BlutspendewesenLublin DM, Transfusion 2000; 40: 1285-1289

Pathogeninaktivierung - Rotes Kreuz · Pharmakokinetik Toxizität Toxizität Akut Mehrfachanwendung...

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