Extending the Shelf Life of Thawed Cryoprecipitate

Extending the shelf life of thawed cryoprecipitate

Parvez M. Lokhandwala, MD, PhD Medical Director, Apheresis Service Assistant Professor, Johns Hopkins University School of Medicine

10/14/18 Disclosure

Coinvestigator - Efficacy of Mirasol-treated Apheresis Platelets in Patients With Hypoproliferative Thrombocytopenia (MIPLATE) Funded by TerumoBCT

www.aabb.org 2 Objectives

1. Review AABB standards regarding shelf life of thawed cryoprecipitate 2. Assess issues with the currently mandated short shelf life 3. Review current data regarding extended storage of thawed cryoprecipitate 4. Evaluate future potential for extending the shelf life

www.aabb.org 3 What is cryoprecipitate?

Cryoprecipitate is the insoluble precipitate formed when FFP is thawed at 1 to 6 °C.

Cryoprecipitate is enriched in FVIII, fibrinogen, vWF, FXIII and fibronectin.

www.aabb.org 4 Why is it used?

Historically, it was used for FVIII replacement, hence it is also known as anti-hemophiliac factor (AHF). Currently, cryoprecipitate is primarily used for fibrinogen replacement in - • acquired hypofibrinogenemia • during major bleeding • control bleeding in patients with uremia

www.aabb.org 5 Cryoprecipitate demand exceeds supply

“Demand for pooled cryoprecipitate exceeds availability and requires multiple days to fully serve demand.” – AABB weekly reports

www.aabb.org 6 Reason for increased demand

• Historically, threshold for fibrinogen replacement was <100 mg/dl. • More recent studies recommend a much higher threshold of <150 mg/dl to 200 mg/dl in trauma, peripartum patients and perioperative settings, including cardiovascular surgery.

Summarized by Levy and Goodnough, 2015 www.aabb.org 7 AABB Standards • AABB standards require each unit of cryoprecipitate contain a minimum of 150 mg of fibrinogen and 80 IU of FVIII. • Pooled cryoprecipitate should contain at least 150 mg of fibrinogen and 80 IU of FVIII multiplied by the number of units in the pool. • Shelf life of thawed cryopreciptate: – 4 hours if pooled in an open system – 6 hours for single units, or if pooled using a closed system

www.aabb.org 8 Issues with short shelf life

• Significant outdating • Thawed ad hoc upon request; delaying dispensing and administration

www.aabb.org 9 Cryoprecipitate outdated at Hopkins

6.6%

7.2%

Unpublished data www.aabb.org 10 Delayed cryoprecipitate administration

• Thawed ad hoc upon request • Hopkins’ MTP: 1st and 2nd coolers (6 pRBC + 5FFP) + 1 PLT 3rd cooler (6 pRBC + 5 FFP) + 1 PLT + 1 CRYO pool

www.aabb.org 11 Early administration of fibrinogen might be needed High quality evidence is lacking. • Cryostat – showed that early administration of cryoprecipitate is feasible. • E-FIT1 (Early Fibrinogen in Trauma) – early delivery of fibrinogen conc. within 45 min was NOT feasible Several studies are ongoing. • Cryostat 2 (Early cryoprecipitate administration in major trauma) • FiiRST (Fibrinogen in the initial Resuscitation of Severe Trauma) • PRooF-iTH (Pilot Randomised trial of Fibrinogen in Trauma Haemorrhage) • FEISTY (The Fibrinogen Early In Severe Trauma study)

www.aabb.org 12 Can the shelf life of cryoprecipitate be extended?

• Currently there are no FDA approved methods to extend the shelf life of cryoprecipitate. • Several groups have addressed this question in a research setting.

www.aabb.org 13 www.aabb.org 14 Thawed cryoprecipitate is stable for up to 24 hrs

Sheffield et al, 2016 www.aabb.org 15 Green et al., 2016 www.aabb.org 16 Coagulation factor levels up to 72 hrs after thawing

Green et al., 2016 www.aabb.org 17 www.aabb.org 18 Coagulation factor levels in cryoprecipitate immediately after thawing

Lokhandwala et. al., 2018 www.aabb.org 19 Coagulation factor levels in cryoprecipitate up to 120 hours after thawing

Lokhandwala et. al., 2018 www.aabb.org 20 Coagulation factor levels in cryoprecipitate up to 120 hours after thawing

Lokhandwala et. al., 2018 www.aabb.org 21 Sterility of thawed cryoprecipitate held at ambient temperature

No organisms were detected when 20 cryoprecipitate pools were cultured at 0 hours, but at 120 hours Staph. epidermidis was identified from one pool, potentially a contaminant introduced during repeated sampling.

Lokhandwala et. al., 2018 www.aabb.org 22 Ongoing culture results

• An additional 121 pools of cryoprecipitate cultured after being held for 72 hours at ambient temperature did not grow any organisms.

Unpublished data www.aabb.org 23 Unpublished data from Col. Andrew Cap’s team • Fibrinogen concentration is not significantly decreased at 35 days after thawing.

www.aabb.org 24 Emory experience

• Dr. John Roback, MD, PhD and team from Emory University School of Medicine provided some of the earliest evidence • Fibrinogen in thawed cryoprecipitate remains stable for 48 hours • Aerobic and anerobic cultures inoculated after 48 hours of ambient storage remain negative

Personal communication with Dr. John Roback; shared with permission www.aabb.org 25 FDA position

Personal communication with Dr. John Roback; shared with permission www.aabb.org 26 Potential ways to ensure sterility

• Pathogen reduction technology • Secondary bacterial culture

www.aabb.org 27 Disclosure – speaker is a coinvestigator on a clinical study using a competitive pathogen-reduction technology

www.aabb.org 28 Cryoprecipitate prepared from plasma treated with Riboflavin + UV contain decreased but acceptable coagulation factors

Ettinger, et al. Transfus Apher Sci. 2012 Apr;46(2):153-8. www.aabb.org 29 Cryoprecipitate prepared from amotasalen-treated plasma

Cid et al. Transfusion. 2013 Mar;53(3):600-5. www.aabb.org 30 Bloch et. al., 2018 www.aabb.org 31 Summary

• Thawed cryoprecipitate has a shelf life of 4–6 hours • Short shelf life causes significant wastage • Short shelf life results in ad hoc thawing of cryoprecipitate, which causes delay in administration • Fibrinogen levels in thawed cryoprecipitate held at ambient temperature remain stable for as long as 120 hours.

www.aabb.org 32 Conclusions • Extended shelf life thawed cryoprecipitate has a stable hemostatic profile. • Similar to platelets, sterility of extended shelf life cryoprecipitate could be potentially ensured by secondary bacterial culture or the use of pathogen- reduction technology.

www.aabb.org 33 Extended Storage of Platelets: cold, frozen and freeze-dried

U.S. Army Institute of Surgical Research COL Andrew P. Cap, MS, MD, PhD, FACP Coagulation & Blood Research Department Disclaimer: The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.

Disclosures: I have no relevant conflicts of interest. I am an active duty officer in the U.S. Army. Disclaimer: The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.

Disclosures: I have no relevant conflicts of interest. I am an active duty officer in the U.S. Army. Current Platelet Product Standard-of-Care: Primary use = prophylaxis

• Room temperature (RT, 22°C) with gentle agitation for up to 5-7 days (7 days if using point-of-release bacterial detection)

Advantages • Longer in vivo platelet circulation times suggest benefit for prophylactic transfusion

Disadvantages Shelf life • Increased risk of microbial contamination driven by • Platelet storage lesion  Loss of functionality bacterial • Short shelf life  Limitation in supply risk… • May be sub-optimal for bleeding patients (especially trauma) as reflected by poor in vitro function Recovery and survival of labeled platelets in healthy volunteers drives current storage practices

Optimized for prophylactic transfusion? Refrigerated stored platelets are cleared from circulation within 2 days

Murphy S, G.F., Effect of storage 0 hour temperature on maintenance of platelet viability - deleterious effect of refrigerated storage. N Engl J Med, 1969(280): p. 1094- 1098

BUT, for treatment 8 hour of hemorrhage, platelet activity may be more important 18 hour than recovery and survival. DAYS Platelets are the critical effectors of hemostasis: best metrics of function?

1. Adhesion  primary hemostatic plug: PLT + VWF on exposed collagen

2. Aggregation  recruitment of PLT, binding to and organization of fibrin into bundles

3. Clot retraction  mechanical hemostasis

4. Thrombin generation  PLT surface (& microparticle) phosphatidyl serine + FVa = catalytic surface (cell-based model of coagulation)

5. Secretion  PLT recruitment, PAI-1/A2AP (anti-fibrinolysis), sCD40L (immune activation), serotonin (vasoconstriction), etc.

Prophylaxis vs. Bleeding? 6. Circulation time  recovery & survival Platelet Dose Study (PLADO): dose-dependent increase In Transfusion-related Adverse Events (TRAEs)

fever

Nov/Dec 2017 BPAC topic: platelet bacterial safety Platelet Dose Study (PLADO): no dose-response effect on bleeding, transfusions

Double the PLT transfused… no change in bleeding or transfusions!

Similar bleeding risk from 10K-80K Platelet Dose Study (PLADO): no dose-response effect on bleeding

Effect of storage duration: Low numbers of “fresh” platelets… PSL trend? PROMMTT: aged, RT stored platelet associated w/ higher risk AEs, sepsis

Older platelet unit  higher risk of complications in trauma patients – especially sepsis

Also: 5-7 day shelf life causes shortages

Inaba J Trauma 2011 Room temperature stored platelets feed bacteria!

No growth at 4C 4 logs!

Presence of live platelets at RT increases bacterial growth in plasma over plasma alone by 4 logs!!! (clinical isolates, Acinetobacter baumanii)

Driven by lactate… Ketter ASH 2017. Current Platelet Product Standard-of-Care: Increased risk for DoD (and trauma patients)

• Room temperature (RT, 22°C) drives shelf life of 5-7 days  unable to maintain inventories

• Impossible to ship from US to deployed units RT = High • Unavailable at forward locations Risk • Forces down-range collections  untested units

• Limited bacterial testing It is (and has been) impossible • Limited donor pools for DoD to provide standard- • Loss of hemostatic function (PSL) may increase of-care platelets bleeding risk to combat casualties. • No evidence that R&S matters in acute hemostasis 50% of US population lives >1hr from a trauma center (i.e., no platelets)

From: National Inventory of Hospital Trauma Centers JAMA. 2003;289(12):1515-1522. doi:10.1001/jama.289.12.1515

Level 1&2 trauma centers

Level 3, 4, 5 trauma centers (mostly rural)

No platelets  higher rural trauma mortality Platelet dysfunction in trauma is associated with mortality • 46% of patients on admission • 91% during ICU stay

Kutcher, et al. J Trauma. 2012;73: 13- 19. PROPPR as a study of giving early platelets – reduced early exsanguination Legal alternative available: cold storage

(d) The volume of original plasma used for resuspension of the platelets shall be determined by the maintenance of a pH of not less than 6.2 during the storage period… ..One of the following storage temperatures shall be used continuously: (1) 20 to 24 °C. (2) 1 to 6 °C. Code of Federal Regulation Title 21 Sec. 640.25 General requirements a)Storage. Immediately after resuspension, Platelets shall be placed in storage at the selected temperature range. If stored at 20 to 24 deg. C, a continuous gentle agitation of the platelet concentrate shall be maintained throughout the storage period.

Agitation is optional if stored at a temperature between 1 and 6 deg. C. Hemostatic function is more important than platelet survival for bleeding patients

Shorter bleeding time in vivo (aspirin & thrombocytopenia)

Cold platelets work and are legal.

84% response rate to bleeding for cold-stored but only 39% RT stored

Becker et al. Transfusion, 1973. The Law on Platelet Storage: updated 29 July 2015 and again…

21 CFR 606.65(e) & 610.53(c) To store apheresis platelets at refrigerator temperature (1-6 C) without agitation for up to 3 days. The cold stored platelets will only be used in the resuscitation of actively bleeding patients. The new storage conditions will be reflected in Circular of Information.

DoD needs: • 1-6C storage Already done, thanks! • apheresis or WB-derived platelets • without agitation

• In PAS or plasma Supported by data Supported by data and needed  For up to 21 days for practical benefit 21-day Cold Stored Platelets (CSP): Not as radical as it sounds…

• Bacterial safety clearly better than RT

• Would allow deployment of fully tested product (true standard of care for TTDs)

• CSP contained in licensed product stored to 35 days (in CPDA WB)

• CSP contained in licensed product stored to 40 days (liquid plasma)

• Efficacy is PRESUMED for RT-PLT and no proof of efficacy required for extension of shelf life from 3  5  7 days (only bacterial safety)

At worst, assuming minimal CSP efficacy, you are transfusing liquid plasma +/- PAS… and in any case, downrange and in most of US, you don’t have platelets anyway! How about a platelet product that really works?

Which platelets would you want if you were bleeding? • 5d room temp stored clots are weak • 4C storage maintains clot strength

3 400 100010

2 10010

200

Stress (Pa) Stress 1

1010 * Clot strength (Pa) strength Clot

0 101 0 1 2 3 101 1010 10010 % 100010 0 Strain % Strain Fresh 5 d RT 5 d 4C 10 d 4C 14 d 4C * Compared to Fresh; n=4, p < 0.05 Nair BJH 2017 in press Aggregation response well preserved in PAS at 4°C

Getz Transfusion 2016 Why would you use RT platelets in bleeding patients?

Getz Transfusion 2016 4C PLT: primed and ready RT PLT: exhausted

Mitochondrial respiration better conserved in 4C storage (Also, mitochondrial gene expression…)

RT mitochondrial failure 4C PLT: alive and kicking RT PLT: on the way out

Apoptosis in RT storage PAS is better than plasma RT caspase activation

RT loss of membrane integrity Cold stored platelets sometimes form aggregates in plasma?

COLD STORED ROOM TEMP STORED

PLATELET COUNT per mL

4°C 4°C 4°C 22°C 22°C 22°C Baseline Day 3 Day 5 Baseline Day 3 Day 5

1145 X 103 827 X 103 854 X 103 1150 X 103 1155 X 103 1155 X 103 +/- 36 SEM +/- 118 SEM +/- 30 SEM +/- 26 SEM +/- 28 SEM +/- 28 SEM

Getz et al. Transfusion, 2016 Plasma vs. PAS cold storage: Reduced aggregates in PAS – who cares?

Cold Stored in 100% Plasma Cold Stored in 65% PAS

4°C Day 5 4°C Day 15

Visible Aggregates

Getz et al. Transfusion, 2016 No Visible Aggregates Cold Storage: Plasma or PAS?

Stolla. Transfusion 2018.

PAS looks better visually, plasma R&S is better plus has fibrinogen – it’s a wash! Cold Platelets: aggregation & count after platelet transfusion in cardiac surgery

• Cold platelet transfusion: better aggregation recovery • ROTEM MCF similar in both cold and RT.

Norwegian Cold Platelet RCT

ClinicalTrials.gov NCT02495506

Apelseth et al. AABB 2016

*Results reported as mean ± SEM. Results include first transfusion episode. Storage to 7 days. Blood product utilization

*Results reported as mean ± SEM. Observation time: from start of surgery until 7 o’clock day 1 after surgery. Blood loss lower in Cold Platelet arm (correlates with better aggregation)

Apelseth. AABB 2017.

No difference in: • Mortality • Thromboembolism • ICU days

*Results reported as mean ± SEM. Observation time: from chest closure until 7 o’clock day 1 after surgery. How about 14 day CSP?!

Strandenes and Hervig. AABB 2018. How about 14 day CSP?! Improved aggregation response!

Strandenes and Hervig. AABB 2018.

Going in the right direction! Even after 14 days! Whole Blood Hemostatic Function: 4C>RT, +/- Mirasol PRT  LTOWB as universal (RDCR)

Pidcoke Transfusion 2013 What about CSP + Intercept? Preliminary data looks promising...

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Pidcoke Transfusion 2013 Cold stored platelets vs. Frozen platelets?

CPP: minimal aggregation but lots of thrombin generation due to PS-pos microparticles.

Johnson Transfusion and Apheresis Science 2015. & Johnson Transfusion 2016. Cryo-preserved platelets: use in MTP associated with decreased mortality

In use or evaluation by:

Netherlands Czech Republic Australia France US

MTP: 4 RBC 3 Plasma 1 CPP

Noorman PLoS One 2016. CPP Summary

• CPP preclinical and clinical data demonstrate hemostatic activity.

• CPP do not aggregate following agonist stimulation.

• CPP catalyze thrombin generation due to high PS expression & MPs.

• CPP accelerate early clot formation as measured by TEG, but form weaker clots than cold or RT stored platelets. Trehalose Lyophilized Platelets (TS*)

 A shrimp larva produces trehalose for protection during dehydration (trehalose, green as determined by FTIR).  The larva are able to revive after rehydration.  Loading platelets with trehalose preserves membrane structure and hemostatic function.

 Could present major advantages in shelf life, stability

Thrombosomes®, TS, Cellphire, Rockville, MD TS Background

• In a thrombocytopenic rabbit ear model, TS infused 15 min. before ear injury resulted in >85% reduction of blood loss.

• Safety and toxicity studies in New Zealand white rabbits, canines, and nonhuman primates resulted in no observable adverse events.

• Limited in vitro/ in vivo studies suggest that while TS are not a platelet substitute, they contribute to clot formation and other hemostatic functions. Aggregation Response

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TS (Calcein stained- Brightfield) Platelets (Calcein stained)

TS (Calcein stained- FITC) • TS have no calcein (viability marker) uptake but do adhere to collagen under flow conditions.

• TS appear to compete for collagen TS (unstained- Brightfield) binding with functional platelets causing less binding compared to platelets alone. TS Adhesion Studies (Bioflux)

Note: no thrombin generation in these assays

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In vivo dose finding considerations: PLT count, thrombin generation, clinical scenario… TS Summary

• TS preclinical in vivo data demonstrate hemostatic activity.

• TS have no mitochondrial function (data not shown).

• TS do not aggregate following agonist stimulation.

• TS catalyze thrombin generation due to high PS expression.

• TS could contribute to early clot formation as measured by ROTEM, but have no impact on clot strength.

• TS do adhere to collagen, but compete with platelet adhesion (dose optimization needed). Promise of CPP and TS

• Logistical advantages of potentially long shelf life compelling

• In vitro functional data for both challenging to interpret

• In vivo data demonstrates hemostatic activity

• Both in early clinical development in US

• Could offer aspects of platelet hemostatic function to bleeding patients essentially anywhere (huge improvement over current situation)

STAY TUNED! Got cold platelets (or WB)?

USE PLATELETS THAT WORK -- NOW! STOP BLEEDING COLD!

Code of Federal Regulation Title 21 Sec. 640.20 Platelets. (b) Source. The source material for Platelets is plasma which may be obtained by whole blood collection or by plateletpheresis. (d) The volume of original plasma used for resuspension of the platelets shall be determined by the maintenance of a pH of not less than 6.2 during the storage period… ..One of the following storage temperatures shall be used continuously: (1) 20 to 24 °C. (2) 1 to 6 °C.

Code of Federal Regulation Title 21 Sec. 640.25 General requirements a)Storage. Immediately after resuspension, Platelets shall be placed in storage at the selected temperature range. If stored at 20 to 24 deg. C, a continuous gentle agitation of the platelet concentrate shall be maintained throughout the storage period.

Agitation is optional if stored at a temperature between 1 and 6 deg. C.

Cold platelets legal in US; used by: DoD, Mayo Clinic. Cold-stored WB used by: DoD, Norwegian Military, Mayo Clinic, U Pitt, UT-Houston, Cooper U Hospital/NJ, others… Cold LTOWB Platelets

Questions? Extending the shelf-life of RBCs and plasma

AABB, Boston, 11 Oct 2018

John R. Hess, MD, MPH, FACP, FAAAS

Professor of Pathology and Medicine Director, Division of Transfusion Medicine Medical Director, Stem Cell Lab U. of Maryland School of Medicine, Baltimore Conflict of interest

I am a U.S. Government inventor of the AS-7 red cell storage solution. I receive patent royalty payments from the government under U.S. law.

I am an inventor, advisor, and stockholder in gel-e Life Sciences, maker of advanced hemostatic materials.

I receive author royalties from UpToDate History of RBC storage solutions

1914 - citrate 1917 - citrate & glucose 1942 - acid citrate & dextrose (ACD) 1957 - citrate phosphate dextrose (CPD) 1979 - CPD Adenine 1984 - Additive solutions (BAGPM, SAG, SAG-M, SAG-CP, Circle-Pac) 1993 - Complex additive solutions (PAGGS-M, ErythroSol) Additive solutions maintain storage volume and RBC viability

100 Cr

51 90 -

RECOVERY % % RECOVERY 60 hr Zuck Tx‘77 Simon Tx’86 24 24 50 WB PC PC PC CPDA-1 CPDA-1 AS-3 AS-3 5 week 5 week 6 week 7 week The storability of red cells in CPD from 28 different donors was very different in a way that is reproducible and inherited. Individual means ranged from 52 to 93% after 3-week storage. Effects of increasing volumes of acidic and alkaline storage solutions. Effect of storage solution volume on RBC ATP

6 AS-1 6 EAS-61 pH 5.6 pH 8.4 5 5

4 4 - - 100mL 3 3 -- 100 mL -  - 200 mL -- 200 mL 2 2 -- 300 mL RBC ATP RBC -- 300 mL µmol/g µmol/g Hgb -- 400 mL

0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 8 9 10 11

WEEKS WEEKS

Hess et al. Transfusion 2000 Aug: 40:1000-1006 Binary Additive Solutions • Manufacturers will need to make additive solutions in two parts, one containing the sugar and the other the alkaline salts so the sugar will not caramelize when Pasteurized. • Pall has done this with AS- 17 • Baxter has done this with ErythroSol Attempting to drive RBC 25.00 storage with increasingly 20.00 CPD at pH 5.7 CPD at pH 6.5 alkaline solutions just leads 15.00 CPD at pH 7.5 CPD at pH 8.7 to 2,3-DPG synthesis, an 10.00 intra-RBC phosphate steal, (umol/g 2,3-DPG HGB) 5.00 and decreased ATP 0.00 0 7 14 21 28 35 42 49 56 63 70 77 84 production Day of Storage

7.5 6.00 7.4 CPD at pH 5.7 5.50 7.3 CPD at pH 5.7 CPD at pH 6.5 CPD at pH 6.5 7.2 5.00 CPD at pH 7.5 CPD at pH 7.5 7.1 CPD at pH 8.7 CPD at pH 8.7 4.50 7.0

pH 4.00 6.9

3.50

6.8 ATP (umol/g HGB)

6.7 3.00

6.6 2.50 6.5 0 7 14 21 28 35 42 49 56 63 70 77 84 2.00 0 7 14 21 28 35 42 49 56 63 70 77 84 Day of Storage Day of Storage Zelda and Irwin Rose

Rose ZB. Methods Enzymol 1975 42:450-454. “Bloody vicious cycle”

Hemorrhage

Coagulopathy Resuscitation

Hemodilution & Hypothermia Component Therapy vs What we bleed

Plt 5.5x1010 50 mL PRBC FFP Hct 55% 80% 335 mL 275 mL 500 mL Hct: 38-44% So Component Therapy Gives You Plt: 150-400K 1U PRBC + 1U PLT + 1U FFP Coags: 100% •Hct 29% 1 g •Plt 87K Fibrinogen •Coag activity 65% •950 mg fibrinogen

•Armand & Hess, Transfusion Med. Rev., 2003 Formulation of EAS-81

EAS-81 Adenine 2 Dextrose 80 Mannitol 55

Na2HPO4 12

NaHCO3 26 mOsm, kg/L 244 mOsm supernatant @ 1h 270 pH (22oC) 8.5

120 50.0 ATP % of initial 45.0 Lactate 100 40.0

80 35.0 30.0 60 25.0 AS-3 (110 mL) 40 20.0 81 (110 mL) 15.0 AS-3 (110 mL) 20 10.0 81 (110 mL) 0 5.0 1hr 1 w k 2 w k 4 w k 6 w k 8 w k 9 w k 10 w k 0.0 1hr 1 w k 2 w k 4 w k 6 w k 8 w k 9 w k 10 w k A first attempt at a 110 mL additive solution

120.0 1.80 Morphology score AS-3 (110 mL) 100.0 1.60 1.40 81 (110 mL) 80.0 1.20

1.00 60.0 0.80 % hemolysis 40.0 0.60

0.40 20.0 0.20

0.0 0.00 1hr 6 w k 10 w k 1hr 1 w k 2 w k 4 w k 6 w k 8 w k 9 w k 10 w k Comparing EAS-81 at 8 weeks to AS-1 at 6 weeks of storage

• AS-1 at 6 weeks (n=10) • EAS-81 at 8 weeks (n=6) Transfusion 40:1011 Transfusion 45:50

• Recovery 84 ± 8 % • Recovery 87 ± 2 % • Hemolysis 0.63 ± .39% • Hemolysis 0.38 ± .23% • ATP 2.86 ± .45 µM/gHb • ATP 2.93 ± .32 µM/gHb • ATP 64 ± 8% • ATP 64 ± 6% • Morphology 71 ± 7% • Morphology 77 ± 11% • Vesicle protein 32 ± 7 • Vesicle protein 12 ± 6 Benefits of Better Storage • Longer storage – Reduction of outdating – Ease seasonal shortages – Aid autologous blood programs • Improved quality of RBCs – More viable cells reduce requirements – Reduction of RBC breakdown products such as procoagulant microvesicles – Faster recovery of 2,3-DPG – Decrease incidence of transfusion-related acute lung injury (TRALI/ARDS)? U.S. Army funded a $4,000,000 development project led by Hemerus Medical, LLC, at JMS, Singapore 100

Cr 24 hr RBC RECOVERY % RECOVERY RBC hr 24 Cr 51 50 CPDA CPDA AS-3 AS-3 AS-7 AS-7 AS-7 WB-8h PC-8h PC-8h PC-8h PC-8h PC-24h PC-8h 5 wk 5 wk 6 wk 7 wk 6 wk 6 wk 8 wk Zuck TX ’77 Simon TX ’86 Dumont & Cancelas TX‘15 Storage Solution & Time Improving buffer range and capacity with a phosphate / bicarbonate buffer system

7.4 AS-1 buffered 7.3 7.2 8.5 mM lactate 7.1 AS-7 buffered 7.0 9.4 mM lactate 6.9 6.8 6.7 6.6 6.5

Buffer range pH in range Buffer 6.4 6.3 0.0 0.5 1.0 1.5 2.0 Buffer Capacity (mEq/ Δ 0.1 pH)

Buffer utilization with 6 weeks of RBC storage at 4°C Improving buffer range and capacity = ‒ with a HPO4 /HCO3 buffer system

7.4 7.3 AS-1 7.2 AS-7 7.1 Possible 7.0 6.9 6.8 6.7 6.6 6.5

Buffer range pH in range Buffer 6.4 6.3 0.0 0.5 1.0 1.5 2.0 Buffer Capacity (mEq/0.1 pH)

Reducing plasma use Decreasing plasma use and wastage

at Harborview Medical Center, Seattle

yr Plasma units / units Plasma Liquid plasma

• AABB Standard 5.1.8A.36 – Liquid plasma stored at 1-6°C expires 5 days after the expiration of whole blood from which it is made. – 26 days for liquid plasma made from whole blood drawn into CPD – 40 days for liquid plasma made from whole blood drawn into CPDA-1 • 21 CFR 610.53c applies – Store at 1-6°C – Dating period - 5 days from end of Whole Blood dating period. FFP Hemotherapy

%change: Day 1 Day 2 Day 3 Day 4 Day 5 Day 1 to 5 p VIII 107 76 66 65 65 41 <.05 II 81 81 81 80 80 1 NS V 79 75 71 68 66 16 NS VII 90 81 76 72 72 20 NS X 85 84 84 82 80 6 NS Fib 225 224 224 224 225 0 NS

PLASMA STORAGE LOSSES – Downes K et al. Transfusion 2001;41:570

Backholer L et al, A paired comparison of thawed and liquid plasma. Transfusion. 2017 Apr;57(4):881-889.

• LP and TP were compared, up to 7 days of storage, with results showing no difference in the rate of change over time for any variable measured.

• When compared to Day 5, LP on Day 7 showed no difference for any factors measured; however, on Day 11 Factor (F)II, FV, FVII, and protein S (activity) were lower.

• Analysis of 119 LP units showed that 26 of 119 (22%) exhibited cold-induced contact activation by Day 28. Better Hemorrhage Control Grade V liver injury 1990 - 76% mortality 2015 - 6% mortality

Damage control surgery Damage control resuscitation Saline/RBC resuscitation Hypotensive management Survivors - open wounds 68% non-operative control Plasma on helicopters saves lives

10 % fewer deaths Sperry JL et al. NEJM 2018 Jul 26: 379:315 Conclusions

• The science allows better and longer RBC storage

• The law allows longer plasma storage

• Both can save blood and lives