BETTER SAFER CRYO

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BETTER SAFER CRYO

• Is it possible?
• Is it cheaper?

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BETTER CRYO

• YES!

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MANY VARIABLES

• Blood group
• Time to freeze/thaw
• Rate of freeze/thaw
• Temperature
• Anticoagulant

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Loss of Factor VIII procoagulant activity over
time
Rock 1983
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The effect of temperature on Factor VIII levels
in whole blood
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• Leucoreduction 24 hr plasma hold
• A trade off
• Logistics vs F VIII recovery

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CRYO

• FREEZE PLASMA
• THAW
• CENTRIFUGE
• CRYOPRECIPITATE
• 4 plasma proteins
• Min 80 U FVIII
• 150 mg fibrinogen

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Factor VIII Activity in cryoprecipitate and
supernatant plasma during 4C thawing
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• Thaw Siphon
• heparin
• Additives
• citrate

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Purification of FVIII 1970s

• Run plasma on column
• Obtain FVII with vWF
• Split FVIII off using CaCl2
• BUT
• What if you never removed citrate?
• chelates calcium
• Use heparin as anticoagulant
• Calcium is part of the FVIII molecule
• ARTIFACT

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Structure of Factor VIII
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• Improved yields of factor VIII from
• Heparinized plasma
• Vox Sang 1979 36 294 300
• Rock G., Cruickshank WH.,
• Tackaberry ES., Palmer DS.
• IP 531 U/L 215 U/L

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  THE EFFECT OF IONIZED CALCIUM LEVELS ON FVIIIC
ACTIVITY      
 
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Elution patterns of citrated and heparinized
plasma following gel chromatography
Citrate
Heparin
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The effect of storage on Factor VIII Activity in
Heparinized Plasma
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Heparin
Citrate
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Comparison of Factor VIII Activity of Plasma and
Cryoprecipitate from Blood Collected into Various
Anticoagulants
SD (pgt0.005) from standard CPD values 250 ml each
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CRYO

• Can be further processed in blood bank to
  intermediate purity
• Products can be sterilized

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IN VIVO

• Cryo ½ life hr
• Heparin 1.14 6.8
• CPD 1.13 6.0

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The effects of heparin on Factor VIII
purification
HEPARIN
primary anticoagulant
added to citrate plasma
OR
enhances cryoprecipitability
AND
permits Clg purification step
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Flow scheme for purification of Factor VIII from
citrated plasma following addition of heparin and
using a cold insoluble globulin precipitate step
Freeze -80C Thaw 4C x 75 min
Spin 7,000 x g, 7 min at 0C
Drain
1 Total FVIII 1,633 units Total Fibrinogen
4,404 MG Total Protein 131,520 MG
Fresh CPD plasma1 made to 1 unit heparin per mL
2 Total FVIII 1,325 units Total Fibrinogen 424
MG Total Protein 4,770 MG
Supernatant of Cryoprecipitate
Cryoprecipitate2
Resolubilize 37C, 2-5 min Add 2.5mL CSH
buffer Incubate 0C x 2hr
Spin 7,000 x g, 7 min at 0C
Drain
Cold Soluble Supernatant
Cold Insoluble Precipitate
Wash 0C
Add 10 mL cold CSH buffer
Drain
Cold Insoluble Precipitate
Resolubilize 37C, 5 min Add 2mL CSH buffer
Spin 3,700 x g, 6 min
Factor VIII3 rich solution
Debris
Represents a pool of 6 units of plasma
3 Total FVIII 1,017 units Total Fibrinogen 384
MG Total Protein 1,220 MG
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• In vivo effectiveness of a high yield Factor VIII
  concentrate prepared in a blood bank. New Engl.
  J. Med.
• 1984 311 310-13.
• Rock et al

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IN VIVO

• Heparin CRC (IP)
• 90 recovery _at_ 15
• ½ life 5.8 hrs
• FPA lt 2.5 ng/ml
• TT 3 sec x 1 hr
• PTT 81 to 49 _at_ 1 hr

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NEJM 1984
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1300 U FVIII/L

• I
• 10 u/ml
• 68
• 52
• 560 U/L 43

• II
• 2 u/ml
• 66
• 45
• 40 523 U/L

PLASMA
CRYO I
CRYO II
SUPER
1.9 U/mg ptn more R.Cof vWF
1.5 U/mg ptn
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STERILIZE

• Product Final stages
• Plasma SDP
• Me blue
• psoralens
• riboflavin

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ASSAYS

• Heparin
• Citrate

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• Yes
• Possible
• Cheaper/relative