Efficacy and Safety Metrics in Plasma-Based Products -...

Efficacy and Safety Metrics in Plasma-Based Products

Philip C. Spinella, MD, FCCM Associate Professor of Pediatrics Washington University in St Louis

THOR 2014 RDCR Symposium Solstrand Hotel

10 June 2014 1

Disclosures

•  Consultant –  US Army Blood Research Program –  Norwegian Navy Blood Research Program –  TerumoBCT, Entegrion, Octapharma

•  Research Support –  Haemonetics, Diapharma

•  Financial support: –  DoD/USAMRMC/NHLBI, 3 U01 HL0772268-09S1 –  DoD/USAMRAA, W81XWH-10-1-0023 –  DoD/USAMRAA, W23RYX0216N601-N602 –  NIH/NHLBI, 5R01HL095470-02

2

Objectives

•  Summarize processing differences between plasma-based products

•  Present data on quality measure differences between plasma-based products

3

Plasma Collection Process*

Centrifuge 5000 g, 5 min Centrifuge 1200 g, 2.5 min

FP24 FFP

Platelet-rich plasma

Thaw slowly at 4°C, centrifuge at 4°C

Freeze at −18°C within 24 h

Freeze at −18°C within 8 h

Centrifuge

Thaw at 37°C

Cryoprecipitate

Platelet-poor plasma

Thawed plasma

Platelet concentrate

Red blood cells

Whole blood

*Plasmapheresis also provides FFP and platelets as stand-alone products 4

Available Plasma-Based Products in US

FFP1,2 FP241,2

Prepara+on •  From whole blood or apheresis •  Frozen at ≤-‐18° C within 8 h

•  From whole blood or apheresis •  Stored at 1°-‐6° C < 8 h from collec+on •  Frozen at ≤-‐18° C within 24 h

Characteris+cs •  Contains plasma proteins including all coagula+on factors

•  Normal levels of FV and FVIII

•  Reduced levels of FVIII and protein C •  Levels of FV and other labile proteins variable vs FFP

•  Differences are of UNKNOWN clinical relevance

1. Karam O et al. Transfusion. 2013 Aug 27. [Epub ahead of print] 2. FDA Guidance for Industry: An Acceptable Circular of Informa+on for the Use of Human Blood and Blood Components. www.fda.gov/biologicsbloodvaccines/ guidancecomplianceregulatoryinforma+on/guidances/blood/ucm364565.htm. Accessed 9/1/13.

5

Available Plasma-Based Products in US

Thawed Plasma*1-‐3 Liquid Plasma1-‐3 Solvent Detergent Plasma Prepara+on •  FFP, FP24 derived

•  Stored at 1°-‐6° C for 5 d in the US

•  Derived from whole blood •  Never frozen •  Stored at 1°-‐6° C up to 26 d (if CPD whole blood)

•  Pooled plasma deriva+ve •  Stored frozen •  Used within hours of thaw

Characteris+cs •  Coagula+on factors and endothelial repair ability reduce over +me

•  Unknown clinical relevance

•  Coagula+on factors reduce over +me

•  Unknown clinical relevance

•  Pathogen reduced •  Filtering and extrac+on phase reduces cells, micropar+cles

•  Dilu+on of HLA/HNA Abs •  Reduced TRALI reported •  Unknown clinical relevance on TRIM

*Effec+ve July 2, 2012, FDA allowed infusion of thawed FFP for up to 24 h aier thawing. Aier 24 h, product should be labeled as “thawed plasma.”

6

Types of Plasma Transfused: 2008 & 2011

0

500

1000

1500

2000

2500

3000

FFP FP24 Ped Plasma

Jumbo Plasma

Cryo Reduced Plasma

Thawed Plasma

2008

2011

FFP, fresh frozen plasma; FP24, plasma frozen within 24 hours of phlebotomy

US Department of Health and Human Services. The 2011 National Blood Collection & Utilization Survey Report. ISBN #: 978-1-56395-843-03. www.hhs.gov/ash/bloodsafety/nbcus/. Accessed 8/14/13.

Units transfused

in th

ousand

s

7

Plasma Product Content

•  Pro and anticoagulant factors

•  Immunoglobulins, proteins (Albumin)

•  Thousands of proteins and lipids

–  Many unmeasured, unknown functions

8

FFP Contents

•  Residual1 – WBCs: 50-950 cells/microliter

– RBCs: 600-11,400 cells/microliter – Platelets: 2000-38,000 cells/microliter

•  Microparticles of these cellular elements –  Biologically active

•  Donor variability affects cell and MP counts

1. Lambrecht B. et al. Transfusion. 2009:49;1195-1204.

9

Assessment of Plasma Efficacy and Safety

•  We oversimplify plasma therapeutic effects -  INR reduction is narrow minded assessment

of efficacy •  We misdirect our safety concerns -  TRIM larger concern than TRALI

•  Need to take broader more comprehensive approach to the evaluation of plasma efficacy and safety

10

Optimal Plasma Qualities

•  Efficacy –  Hemostasis –  Endothelial integrity –  Vasoregulation effects

•  Impact on flow and oxygen delivery support is essential

•  Safety –  Immune effects

•  Reduced TRALI and TRIM

11

Thawed Plasma Effect on Thrombin Generation

*FFP stored and aged at 4°C between days 0, 5, and 10

Pa+ S et al. J Trauma. 2010;69(suppl 1):S55-‐S63. Used by permission. 12

Pa+ S et al. J Trauma. 2010;69(suppl 1):S55-‐S63. 13

Methods

•  10 FFP and LP single donor units matched by sex and blood group analyzed

•  FFP thawed for 5 days at 4C •  LP stored for 26 days at 4C •  Analyzed samples over time

–  TEG, TG assay, platelet counts and microparticles, pro and anti-clotting factors

16

Results

Platelet MP phenotypes included: CD 41, P selectin, Tissue Factor, Annexin V

Platelet Count by flow cytometry with CD 41

17

Results

18

PotenLal mechanisms for sustained thrombin generaLon: platelet microparLcles, cold acLvaLon of factors, reduced protein S

Results

19

Study Conclusions

•  LP has similar to better hemostatic effect than TP

•  Freeze-thaw cycle decreases clot strength in TP

•  PLT microparticle generation in LP may maintain hemostatic effect as coagulation factors decrease

•  LP maintains efficacy and improves availability

20

Limitations

•  Are data generalizable ? •  Platelet count in LP is OMG high

–  80,000 cells/µl –  Other reports from FFP is 2,000-38,000 cells/µl –  Our FFP data is at most 2,000-8,000 cells/µl

•  What is the cost of PLT microparticles? –  Immune suppression effects?

21

Efficacy and Safety Metrics in Plasma-Based Products

In vitro study with 10 units each of –  FFP derived thawed plasma

•  Type AB –  Liquid plasma (LP)

•  Type AB –  Solvent Detergent (SD) Plasma

•  Type A (one donor pool) –  Resusix –Spray Dried SD Plasma

•  Type AB (one donor pool)

22

Hypothesis

•  Cellular elements and microparticles are reduced in SD Plasma/Resusix compared to FFP/LP

•  Platelet amount in LP lower than previously reported

•  Thrombin generation would be improved in SD Plasma/Resusix compared to FFP/LP

•  Vasoregulatory properties would be improved in SD Plasma/Resusix compared to FFP/LP

–  Due to removal of RBC derived microparticles

23

Methods

•  Blood Centers of Pacific supplied the FFP and LP

•  Entegrion supplied SD Plasma and Resusix

•  FFP, SD plasma

–  Stored at 4C post thaw

•  LP maintained at 4C

•  Resusix reconstituted then stored at 4C

24

Methods

•  Same plasma units used for all four groups of experiments •  All lab methods performed at Wash U except for thrombin

generation assays done at Entegrion •  Funded by US Office of Naval Research

–  Via Entegrion

25

•  Cell counts for RBC, WBC, and platelets –  Sysmex XN-3000 device –  Flow cytometry –  Hemocytometer

Cell Counts in Plasma - Methods

26

Sysmex XN-3000 Automated Cell Counting Device

•  Light scatter & fluorescence

•  Body Fluid Mode –  Larger volume to account

for rare events

•  RBC •  WBC •  PLT

27

Platelet Counts using Sysmex Platelets

Resusix SD FFP LP

0

1000

2000

500

1500

4000

4500

Plasma Product

PLT

(cel

ls /

uL)

Median=200 Median= 0 Median=800 Median=1900

*significant compared to Resusix p<0.05 *

n=10 n=6 n=10 n=3

28

RBC Counts using Sysmex RBC

Resusix SD FFP LP

0

100

200

20

40

60

80

120

140

160

180

380

400

420

Plasma Product

RB

C (c

ells

/ uL

)


n=10 n=6 n=10 n=3

p<0.05 * *significant compared to Resusix

29

WBC Counts using Sysmex WBC

Resusix SD FFP LP

0

20

5

10

15

25

30

35

80

75

85

Plasma Product

WBC

(cel

ls /

uL)

n=10 n=6 n=10 n=3

30

Median = 5.0 Median = 0.5 Median = 0.5 Median = 1.0

•  Becton Dickinson Accuri C6 Flow Cytometer

•  To identify cells, following monoclonal antibodies:

–  Platelets (CD41a PerCP Cy5.5),

–  RBC (CD235a APC)

–  Leukocytes (CD45 PE Cy7)

Flow Cytometry Methods

31

Platelet Counts using FC Platelets

Resusix SD FFP LP

0

2000

4000

6000

8000

10000

Plasma Product

Pla

tele

ts (c

ells

/uL)


*significant compared to Resusix

p<0.001 *

p<0.05 *

n=10 n=6 n=10 n=3

32

RBC counts using FC RBC

Resusix SD FFP LP

0

250

500

750

1000

Plasma Product

RBC

(cel

ls /

uL)


n=10 n=6 n=10 n=3

33

WBC Counts using FC WBC

Resusix SD FFP LP

0

25

50

75

100

Plasma Product

WBC

(cel

ls /

uL)


n=10 n=6 n=10 n=3

34

•  Microparticle phenotypes include

–  Platelets

–  RBC

–  Leukocyte,

•  Monocyte, neutrophils, T cells

–  Endothelial cells

Microparticle Flow Cytometry Methods

35

Flow Cytometry: Methods

•  The Small Bead Calibration Kit (Bangs Laboratories, Fisher, IN), measuring 0.2, 0.5 and 0.8 µm of beads, is used to establish appropriate instrument settings for the analyses.

–  This beads acquisition allows the setting of cytometer to study MPs within constant region (0.2 µm to 1 µm).

•  BD TruCOUNT tubes are used to quantify the absolute cell counts of the microparticles within different plasma products.

36

•  Data from 50,000 events acquired at slow rate and analyzed, using an FSC-H threshold of 1000 and an SSC threshold of 4000 for microparticles determination.

•  Absolute MP counts were determined by the following formula:

Microparticle Flow Methods

# of events in region containing cell # of events in absolute count bead region

# beads per test* Test volume X

37

Resusix SD FFP LP n=10 n=6 n=10 n=3

CD41a+PS+ (Platelet-derived MP)

D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 28

0

250

500

750

1000

1250

1500

1750

2000

Mic

rop

artic

les/

ul

Median (0.2) (0.4) (0.3) (0.9) (0.9) (140.0) (107.5) (69.3) (935.7) (327.7) (208.0)

(p<0.01)

*

(p<0.01)

* (p<0.002)

#

(p<0.01)

#

PLASMA PRODUCT

* Comparing freshest product to Resusix D 0 # Comparing oldest product to Resusix D 1

38

CD154+PS+ (CD40SL-MP)

D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 28

0

20

40

60

80

100

Mic

ropa

rtic

les/

ul


Median (0.0) (0.0) (0.3) (0.0) (0.6) (0.0) (0.0) (1.1) (0.0) (6.7) (2.0)

PLASMA PRODUCT 39

Platelet Derived – Immune effects 1

1 Horstman, L.L. Keio J Med, 2004. 53(4): p. 210-‐30.


CD235a +PS+ (RBC-derived MP)

D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 28

0

10

20

30

40

50

60

70

80

90

100

Mic

ropa

rtic

les/

ul

Median (0.0) (0.0) (0.3) (0.5) (0.0) (17.9) (43.9) (51.1) (9.0) (35.0) (36.1)

(p<0.02) *

(p<0.001)

#

(p<0.01)

*

(p<0.01)

#

PLASMA PRODUCT 40

Prothrombo+c, impair vasoregula+on and immune modula+on 1-‐5


Spinella, P.C.,Transfusion, 2011. 51(4): p. 894-‐900. Tissot, J.D. Curr Opin Hematol, 2010. 17(6): p. 571-‐7. Sadallah, S. J Leukoc Biol, 2008. 84(5): p. 1316-‐25. Xiong, Z. Transfusion, 2011. 51(3): p. 610-‐21. Belizaire, R.M., J Am Coll Surg, 2012. 214(4): p. 648-‐55.

CD47+PS+

Resusix D 0 Resusix D 1 SD D 0 SD D 3 SD D 5 FFP Day 0 FFP D 3 FFP D 5 LP D 3 LP D 14 LP D 28

0

25

50

75

100

125

150

Plasma Product/ Time Course

Mic

rop

arti

cles

/ ul

p<0.05 *

p<0.05 #

41

(RBC derived)

Immune suppressive – Increased monocyte inhibi+on factor 1 Supports tumor cell growth 1

1. Oldenborg, P.A., CD47: A Cell Surface Glycoprotein Which Regulates MulAple FuncAons of HematopoieAc Cells in Health and Disease. ISRN Hematol, 2013. 2013: p. 614619.



CD45+PS+(Leukocyte-derived MP)

D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 28

0

250

500

750

1000

Mic

rop

arti

cles

/ ul

Median (0.6) (0.8) (0.3) (1.1) (1.1) (5.6) (10.9) (28.9) (11.5) (28.0) (10.7)

(p<0.01)

#

PLASMA PRODUCT

42


Proinflammatory, prothrombo+c, endothelial dysfunc+on 1-‐3

1. Gasser, O Blood, 2004. 104(8): p. 2543-‐8. 2. Mesri, M.. J Biol Chem, 1999. 274(33): p. 23111-‐8. 3. Angelillo-‐Scherrer, A.,. Circ Res, 2012. 110(2): p. 356-‐69.

CD14+PS+ (Monocyte-derived MP)

D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 28

0

20

40

60

80

100

Mic

ropa

rtic

les/

ul


Median (0.0) (0.5) (0.2) (0.9) (1.0) (0.0) (0.8) (2.0) (1.5) (0.0) (1.8)

PLASMA PRODUCT 43

Proinflammatory, promote fibrinolysis 1,2

1. Wang, J.G. Blood, 2011. 118(8): p. 2366-‐74. 2. Lacroix, R. Haematologica, 2012. 97(12): p. 1864-‐72.

CD3+PS+(TCell-derived MP)

Resusix D 0Resusix D 1 SD D 0 SD D 3 SD D 5 FFP D 0 FFP D 3 FFP D 5 LP D 3 LP D 14 LP D 28

0

50

100

150

200


Mic

ropa

rtic

les/

ul

p<0.05 *

44


Impair vasoregula+on 1,2

1. Simak, J. Transfus Med Rev, 2006. 20(1): p. 1-‐26. 2. Mar+n, S. Circula+on, 2004. 109(13): p. 1653-‐9.

CD142+PS+ (Tissue Factor-MP)

Resusix D 0 Resusix D 1 SD D 0 SD D 3 SD D 5 FFP D 0 FFP D 3 FFP D 5 LP D 3 LP D 14 LP D 28

0

10

20

30

40

50


Mic

ropa

rtic

les/

ul

45

Prothrombo+c and proinflammatory 1,2

1. Horstman, L.L. Keio J Med, 2004. 53(4): p. 210-‐30. 2. Simak, J. Transfus Med Rev, 2006. 20(1): p. 1-‐26.


CD106+PS+(VCAM Endothelial-derived MP)

D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 28

0

10

20

30

40

50

Mic

ropa

rtic

les/

ul

Median (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (1.2) (0.0) (0.0) (3.5)

PLASMA PRODUCT 46

endothelial stress and inflamma+on 1

1 Horstman, L.L. Keio J Med, 2004. 53(4): p. 210-‐30.

CD105+PS+(Endothelial-derived MP)


0

20

40

60

80

100


Mic

ropa

rtic

les/

ul

47 1. Simak, J. Transfus Med Rev, 2006. 20(1): p. 1-‐26

Endothelial injury 1

CD144+PS+ (Endothelial-derived MP)


0

20

40

60

80

100


Mic

ropa

rtic

les/

ul

p<0.05 *

p<0.05 #

48


Endothelial injury 1

1. Simak, J. Transfus Med Rev, 2006. 20(1): p. 1-‐26

Coagulation parameters: PT, aPTT, INR and Fibrinogen

•  Plasma level for PT, aPTT, INR and Fibrinogen was performed on STAGO instrumentation (STA satellite)

•  Fibrinogen level was determined by modified Clauss Method.

49

Coagulation parameters: ELISA

•  Native plasma of each of plasma products per time point were collected and stored at -80°C.

•  Plasma were be thawed at room temperature,

–  Plasmin α2-antiplasmin (PAP), Factor V, Factor VIIa, Factor Xa,

–  AT-III, Prothrombin Fragments (F1+2), Peptide A (FPA)

–  Total Protein S, Free Protein S, Protein C

50

aPTT

D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 2810

20

30

40

50

60

70

80

90

100

110

120

(sec

onds

)


PLASMA PRODUCT

Median (42.0) (41.8) (35.4) (37.4) (38.1) (35.9) (40.9) (40.1) (39.1) (43.6) (44.7)

(p<0.002)

* (p<0.0001)

* (p<0.001)

#


51

INR

D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 280.5

1.0

1.5

2.0

2.5

Ra

tio


PLASMA PRODUCT

Median (1.3) (1.3) (1.0) (1.0) (1.0) (1.1) (1.2) (1.2) (1.1) (1.2) (1.2)

(p<0.001)

*

(p<0.001)

* (p<0.001)

#

(p<0.0002)

#


52

Fibrinogen

D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 28100

200

300

400

(mg/

dL)


PLASMA PRODUCT

Median (207) (193) (304) (292) (291) (254) (275) (274) (284) (268) (254)

(p<0.001)

* (p<0.001)

# (p<0.001)

#


53

ATIII

D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 280.0

0.1

0.2

0.3

0.4

(mg/

ml)

Median (0.181) (0.179) (0.165) (0.156) (0.187) (0.165) (0.154) (0.133) (0.157) (0.185) (0.180)


PLASMA PRODUCT

54

Total Protein S

D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 2850

75

100

125

150

(%)

Resusix SD FFP LP n=10 n=4-‐5 n=10 n=3

PLASMA PRODUCT

Free Protein S

D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 2850

75

100

125

150

(%)


PLASMA PRODUCT

p<0.05 *

p<0.05 #


Protein C

D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 280.0

2.5

5.0

7.5

(ug/

L)


p<0.05 #


Median 3.6 3.7 4.5 4.2 4.1 3.0 2.6 2.4 4.1 3.9 3.6

PLASMA PRODUCT

Thrombin Generation Assay - Methods

•  Calibrated Automatic Thrombinoscope (CAT) Device

•  During the measurement, the program compares fluorescence in sample to fluorescence in calibrator and computes thrombin concentration.

•  Each sample was analyzed three times in triplicate and the data were averaged.

•  “Low” in sample name means that thrombin generation reaction was triggered by PPP-Low reagent that produces 1pM TF final concentration in a sample.

58

Resusix

0 10 20 30 40 50

0

50

100

150

200

250

Thro

mbi

n (n

M)

Time (min)

Res-1-30min-PFP-Low Res-2-30min-PFP-Low Res-3-30min-PFP-Low Res-4-30min-PFP-Low Res-5-30min-PFP-Low Res6-30min-PFP-Low- 1 Res7-30min-PFP-Low- 13 Res8-30min-PFP-Low- 1 Res9-30min-PFP-Low- 13 Res10-30min-PFP-Low- 1 Res11-30min-PFP-Low- 13 Res12-30min-PFP-Low- 1 Res13-30min-PFP-Low- 13

0 10 20 30 40 50

0

50

100

150

200

250

Thro

mbi

n (n

M)

Time (min)

Res-1-24h-PFP-Low Res-2-24h-PFP-Low Res-3-24h-PFP-Low Res-4-24h-PFP-Low Res-5-24h-PFP-Low Res6-24h-PFP-Low- 4 Res7-24h-PFP-Low- 16 Res8-24h-PFP-Low- 4 Res9-24h-PFP-Low- 16 Res10-24h-PFP-Low- 4 Res11-24h-PFP-Low- 16 Res12-24h-PFP-Low- 4 Res13-24h-PFP-Low- 16

Time 0 Time 1 day

0 5 10 15 20 25 30 35 40 45 50

0

50

100

150

200

250

Thro

mbi

n (n

M)

Time (min)

FFP-1 FFP-2 FFP-3 FFP-4 FFP-5 FFP-6 FFP-7 FFP-8 FFP-9 FFP-10

0 5 10 15 20 25 30 35 40 45 50

0

50

100

150

200

250

Thro

mbi

n (n

M)

Time (min)


0 5 10 15 20 25 30 35 40 45 50

0

50

100

150

200

250

Thro

mbi

n (n

M)

Time (min)


FFP Time 0 Time 3 day Time day 5

59

Donor Variability for FFP Thrombin generation

0 5 10 15 20 25 30 35 40 45

0

20

40

60

80

100

120

140

Thro

mbi

n (n

M)

Time (min)


0 5 10 15 20 25 30 35 40 45

0

20

40

60

80

100

120

140

Thro

mbi

n (n

M)

Time (min)


0 5 10 15 20 25 30 35 40 45

0

20

40

60

80

100

120

140

Thro

mbi

n (n

M)

Time (min)


Time day 0 Time 3 day Time day 5

60

Day-‐0 Resusix vs Day-‐0 FFP ETP day-0 (median & IQR)

ETP

(nM

x m

in)

FFP

Resus

ix800

1000

1200

1400

1600

Lag Time day-0 (median & IQR)

Lag

Tim

e (m

in)

FFP

Resus

ix3.0

3.5

4.0

4.5

5.0

Peak day-0 (median & IQR)

Pea

k (n

M)

FFP

Resus

ix0

50

100

150

200

250

ttPeak day-0 (median & IQR)

ttPea

k (m

in)

FFP

Resus

ix6

7

8

9

10

11

12

P=0.02 P=0.031

P<0.001 P<0.001

61

day-‐0 FFP vs day-‐5 FFP Lag Time FFP (median & IQR)

Lag

Tim

e (m

in)

Day-0

Day-5

3

4

5

6

7

ETP FFP (median & IQR)

ETP

(nM

x m

in)

Day-0

Day-5

0

500

1000

1500

Peak FFP (median & IQR)

Peak

(nM

)

Day-0

Day-5

0

50

100

150

200

ttPeak FFP (median & IQR)

ttPea

k (m

in)

Day-0

Day-5

8

9

10

11

12

13

14

P=0.05 P<0.0001

P<0.0001 P<0.0002

62

Vasoreactivity Methods

•  Samples: •  Unprocessed plasma •  Platelet Free Plasma

–  (centrifuged 2500 g 15 minutes RT) •  Microparticle Free Plasma

–  (centrifuged 20,000 g 30 minutes 4°C)

•  Assays •  NO trapping or scavenging •  Vascular Rings

•  Hypothesis –  RBC Microparticles increase NO scavenging and

vasoactive

63

NO Trapping Assay

64

Nitric Oxide (NO) Trapping Assay

•  Increased NO trapping or scavenging is indicative of reduced ability to support hypoxic vasodilation –  BAD

•  Optimal for NO scavenging or trapping to be REDUCED.

65

Total NO Scavenged- Baseline

Native Plasma Platelet Free Plasma Microparticle Free Plasma0

2000

4000

6000

8000

10000Resusix (n=10)SD (n = 5)FFP (n = 7-10)LP (n = 3)

Plasma Product

(AUC)

p<0.05 *

66

Total NO Scavenged in Native Plasma

D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 280

2000

4000

6000

8000

10000

(AUC)

Resusix SD FFP LP n=10 n=5 n=7-‐10 n=3

PLASMA PRODUCT 67

Vascular Ring Assay

•  Descending Thoracic aorta dissected from male New Zealand White Rabbit

•  Vascular rings cut (sliced like calamari)

•  Rings mounted in all glass chamber – on a pair of metal wire hooks

•  Chamber filled with Krebs, aerated with 95% oxygen/5% carbon dioxide (for pH), and maintained at 37 degrees)

•  Bottom of lower hook fixed in place, top of the upper hook connected by thread to force transducer

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Force Transducer

Organ Chamber

Rack and Pinion

Vascular Ring Assay

69

Preconditioning - Constriction

•  Initial resting tension held at 2 grams (maintained using rack and pinion assembly)

•  Once resting tension held steady, we perform preconditioning

•  Use alpha 1 adrenergic receptor agonist Phenylepherine to induce vascular ring constriction

•  Vessel constriction = increase in gram tension

70

Preconditioning - Relaxation

•  To check for an intact endothelium, following the addition of PE to induce a steady constriction response, we inject acetylcholine.

•  Induces a relaxation response

•  Vessel relaxation results in a decrease force transducer tension = less gram tension

71

Vascular Reactivity in the Freshest Products

NP MPF NP MPF NP MPF NP MPF0

10

20

30

40

50

FFP (n = 7-10)LP (n = 3)

Resusix (n=10)SD (n = 5)

Native Plasma vs Microparticle-Free Plasma

% c

onst

rictio

n (n

orm

aliz

ed to

50n

M

Hb

cons

trict

ion)

p<0.05 *

p<0.05 *

* Significant when compared to Resusix

72

Vascular Reactivity in Native Plasma

D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 280

20

40

60

80

100

Plasma Product

% c

onst

rictio

n (n

orm

aliz

ed to

50n

M

Hb

cons

trict

ion)


73

Vascular Reactivity in Microparticle-Free Plasma

D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 280

50

100

150

Plasma Product

% c

onst

rictio

n (n

orm

aliz

ed to

50n

M

Hb

cons

trict

ion)


74

Plasma Quality and Safety Metrics - Conclusions

•  Counting cells in plasma products is imprecise –  Liquid plasma results from Houston group not generalizable

•  SD plasma products vs. FFP/LP –  Reduced cellular elements and donor variability –  Reduced microparticles from all sources

•  May reduce immune modulation or TRIM associated with FFP –  Increased thrombin generation

•  Plasma is directly vasoactive –  Not related to platelets or Microparticles

•  Storage time effects quality and safety measures

•  Clinical relevance of efficacy and safety measures are unknown, needs exploration

75

Next steps

•  Ex vivo analysis of immune function effects secondary to microparticle differences between products

•  Mechanistic studies in trials that compare SD treated plasma to thawed FFP/FP24 products

–  Immune

–  Coagulation

–  Endothelial

76

Acknowledgements

•  Washington University in St. Louis –  Allan Doctor Lab

•  Allan Doctor •  Stephen Rogers

–  Spinella Lab •  Ayu Frazier •  Dan Martin •  Ryan McDonough

–  Coagulation Lab •  Dennis Dietzen

•  Entegrion •  Oleg Gorkun

77

•  FDA approved in early 2013 •  Indications

–  Replacement of multiple coagulation factors in patients with acquired deficiencies

•  Due to liver disease •  Undergoing cardiac surgery or liver transplant

–  Plasma exchange in patients with thrombotic thrombocytopenic purpura (TTP)

Solvent Detergent Plasma (Octaplas-LG)

79

•  Extensive donor screening (serology and NAT) –  Screened for non-enveloped viruses and prions

•  HAV, HEV, Parvovirus B19

•  Donors retested 6 months after plasmapheresis

•  Pooling of of 630 to 1500 single units of FFP

•  Filtration at 0.2 microns for cell and debris removal

Solvent Detergent Plasma Manufacturing Process

80

•  Solvent/detergent treatment - tri(n-butyl)phosphate (TNBP) and octoxynol –  Inactivates all lipid enveloped viruses

•  (HIV, HBV, HCV, WNV, EBV, CMV….) –  Does not inactivate non-enveloped viruses

•  (eg, HAV, HEV, parvovirus B19)

•  Lipid extraction –  Removes solvent/detergent –  Removes bioactive lipids, lipoproteins, microparticles

Solvent Detergent Plasma Manufacturing Process

81

•  Reduced viral infection risk •  Reduced TRALI, TA-GVHD and POSSIBLY TRIM

-  Pooling

•  Dilution of anti HLA and HNA antibodies

•  Immune neutralization of potential pathogens

-  Extraction Phase and Filtering •  Removal of microparticles, biologic reactive mediators, WBCs

•  Consistent dosing (less batch to batch variation) -  Pooling

SD Plasma Processing Advantages1-4

1. Flesland O. Intensive Care Med. 2007;33 Suppl 1:S17-‐S21. 2. Krusius T et al. Vox Sang. 2010;99(suppl 1):P-‐1018. 3. Sachs UJ et al. Transfusion. 2005;45(10):1628-‐1631. 4. Sinnou P et al. Eur J Immunogenet. 2004;31(6):271-‐274.

82

Nitric Oxide (NO) Trapping Assay

•  Inject nitric oxide (NO) donor into a vacuum tight reaction cell continuously flushed with Helium - linked to nitric oxide analyzer

•  NO donor (Dipropylenetriamine NONOate): –  spontaneously dissociates in a pH dependent, first order process

(made up in 10mM NaOH and injected into PBS pH 7.4) at 37°C –  half-life of three hours at 37°C, pH 7.4 –  liberate 2 moles of NO per mole of parent compound

•  Once steady NO signal achieved, plasma samples injected and area under curve measured - representing total NO scavenged

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0.8 µm

0.5 µm 0.2 µm

Size Gating

84

CD41a+ Annexin V+ CD41a+ Annexin V-‐

Determining cell type specific MP

85

Beads

Sample inside the gated area

86

•  Briefly, a total of 10 µl platelet free plasma (PFP) from Resusix plasma, S/D plasma, thawed-FFP, or liquid plasma is incubated with 2 µl of lineage-specific monoclonal antibodies, or with and without 2 µl of Annexin V (in the presence of 2.5mM CaCl2) for 30 min at RT in the dark.

•  After incubation, samples will be diluted with stain buffer or Annexin V binding buffer up to a total volume of 300 µl.

Microparticle Flow Methods

87

PT

D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 2810

15

20

25

30

(seconds)


PLASMA PRODUCT

Median (16.2) (16.3) (13.7) (13.6) (13.5) (14.5) (14.8) (14.9) (13.4) (14.7) (14.7)

(p<0.001)

* (p<0.001)

#

(p<0.0002)

*

(p<0.0003)

#


88

Plasmin- anti Plasmin (PAP)

D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 280

200

400

600

800

1000

(ng/

ml)


PLASMA PRODUCT

Median (192) (192) (132) (137) (147) (196) (200) (220) (235) (233) (158)


89

Factor V

D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 2810

20

30

40

50

(ng/

ml)


PLASMA PRODUCT

(p<0.001) *

(p=0.001) #

Median (18.7) (19.0) (17.4) (19.4) (17.0) (27.5) (28.6) (28.0) (17.5) (17.1) (17.5)


90

Factor VIIa

D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 280

50

100

150

200

(ng/

ml)


PLASMA PRODUCT

(p<0.06) *

(p=0.02) *

Median (48.4) (48.6) (30.3) (54.3) (59.1) (29.5) (37.5) (39.1) (30.4) (63.6) (61.7)


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PF1+2

D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 280

100

200

300

400

500(p

mol

/L)


PLASMA PRODUCT

(p=0.001) *

(p=0.001) #

Median (73.2) (71.7) (80.3) (79.1) (75.0) (247.5) (263.1) (264.6) (80.3) (82.3) (84.6)


92

Fibrinopeptide A

D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 280

20

40

60

80

100

(ng/

ml)


PLASMA PRODUCT

(p=0.002) * (p=0.001)

* (p=0.01) *

(p=0.01) # (p=0.001)

# (p=0.01) #

Median (62.0) (62.8) (45.3) (46.1) (48.7) (42.1) (42.2) (41.8) (17.2) (14.5) (15.3)


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Factor Xa

D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 280

20

40

60

80

100

(ng/

ml)


PLASMA PRODUCT

(p<0.01) *

Median (27.6) (28.8) (31.5) (31.4) (31.5) (44.3) (42.2) (44.6) (23.9) (21.8) (21.5)


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Lag

Tim

e (m

in)

Day-3

Day-5

3

4

5

6

7ETP FFP (median & IQR)

ETP

(nM

x m

in)

Day-3

Day-5

600

700

800

900

1000

1100

1200


Peak

(nM

)

Day-3

Day-5

0

50

100

150


ttPea

k (m

in)

Day-3

Day-5

9

10

11

12

13

14

P=0.79 P=0.06

P=0.12 P=0.06

95


Lag

Tim

e (m

in)

Day-0

Day-3

3.0

3.5

4.0

4.5

5.0

5.5

6.0

ETP FFP (median & IQR)

ETP

(nM

x m

in)

Day-0

Day-3

0

500

1000

1500


Peak

(nM

)

Day-0

Day-3

0

50

100

150

200


ttPea

k (m

in)

Day-0

Day-3

8

9

10

11

12

13

P=0.06 P<0.001

P=0.002 P=0.29

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Day-‐0 Resusix vs Day-‐1 Resusix Lag Time Resusix (median & IQR)

Lag

Tim

e (m

in)

Day-0

Day-1

3.0

3.5

4.0

4.5

5.0

ETP Resusix (median & IQR)

ETP

(nM

x m

in)

Day-0

Day-1

900

1000

1100

1200

1300

1400

1500

Peak Resusix (median & IQR)

Pea

k (n

M)

Day-0

Day-1

100

150

200

250

ttPeak Resusix (median & IQR)

ttPea

k (m

in)

Day-0

Day-1

6

7

8

9

10

P=0.06 P=0.07

P=0.002 P<0.0001

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Thrombin generation

Upon endothelial damage, +ssue factor (TF) is exposed to the blood and binds factor VII, which is ac+vated to factor VIIa. The TF:VIIa complex enables subsequent ac+va+on of factor X and prothrombin, aier which small amounts of thrombin ac+vate the factor XI-‐IX feedback loop on the platelet surface. Factor IXa will then ac+vate addi+onal factor X. Simultaneously, the trace amounts of thrombin will then ac+vate factors VIII (cofactor to factor IX) and V (cofactor to factor X), which drama+cally enhances cataly+c ac+vity of factors IX and X. Finally, thrombin (factor IIa) ac+va+on leads to fibrin deposi+on. In parallel, local polyphosphate (polyP) release by ac+vated platelets may addi+onally s+mulate ac+va+on of factor XII, factor V, and FXI and inhibit clot lysis. Adopted from Versteeg et al., Physiol Rev (2013) 93:327-‐358

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Material and Methods

•  Frozen plasma samples were thawed at 37C for 10 minutes and 80µl of plasma was added to each of three sample wells containing tissue factor (TF) and phospholipids (PL) enough to produce final concentrations of 1pM TF and 4µM PL.

•  In addition, 80µl of same plasma was added to each of two calibrator wells. The use of calibrator (solution containing fixed amount thrombin/α2-macroglobulin complex; Calibrator, Stago) added to the sample plasma allows for correction of inner-filter effect and substrate consumption.

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