Buffy Coat Component Production

transcript

2009Medical Residents

Outline

• Background on buffy coat production method

• Description of the buffy coat production method

• Clinical implications

Background• “Buffy Coat” refers to a method for preparing

components from whole blood donation that was developed in Europe >25 years ago and is in widespread use around the world.

• In this method whole blood is spun down hard and three distinct layers can be seen. The middle “buff” coloured layer is made of White Cells and Platelets and rests on top of the RBC layer or “coats” the RBC layer

Buffy Coat – Around the World

Comparison of PRP & BC Methods

Methodology PRP Buffy Coat

Centrifugation Soft / hard Hard / Soft

Method of extraction Manual Compomat G4

Time allowance Within 8 hours

Within 24 hours

# of units pooled 5 4

Pooled Where? Hospital CBS

Potential Future Benefits• Platelets prepared by BC method can be

suspended in platelet additive solutions. These solutions improve platelet quality to the level required to extend platelet storage beyond 7 days. This will be investigated by CBS R&D group at a later date.

• Pathogen reduction/inactivation technologies for platelets have been designed only for apheresis or buffy coat platelet products.

Key Benefits – cooling trays

• Increase in time allowed for component production (24 hours vs. 8 hours) through the use of a rapid cooling technique

• improves availability of platelets

Key Benefits – automated extraction

• Instrument-controlled extraction allows for increased process controlbetter quality productmore consistent

recoveries therefore more consistent platelet dose

higher percentage of platelets recovered per donation

Key Benefits – sterile docking

• Hospitals receive a pooled platelet concentrate that has been produced in a closed system.

• Less donor exposure for recipients – 4 donations pooled instead of 5

• Product is pooled in plasma from male donors to reduce the risk of TRALI

Pooling Process

Benefits for CBS = Benefits for Hospitals

• Increased volume of Recovered Plasma– approx. 70-80 mL/donation– reduces Canada’s dependency on open

market IVIG, improving security of supply and reducing overall cost to the health care system

Medical Overview• Key Questions

– Will the Buffy Coat Product work as well as the previous product?

– Will there be an increase in adverse reactions?

– Are there different anti-coagulants and will they effect neonatal practice?

– Will there be changes to Autologous and Directed Components?

– Do I need to change my blood prescription practice?

– Do we need to test pooled buffy coat platelets for hemolysins?

– After buffy coat implementation will all platelets produced by CBS be routinely bacterially tested?

– Are there any differences on the ward?

Yes and No

Maybe for platelets and plasma

No, provided “correct” spiking method is used

Yes - Packed Red Blood Cells now

Maybe – if this is part of your apheresis platelet routine

Hb content of RBC unitsBuffy coat RBC (n=319)*

Non buffy coat RBC (n=311)

Median Hct 0.62 0.53

Min Hb g/L 39.0 40.0

Max Hb g/L 76.0 71.0

Mean Hb g/L 54.3 56.7

Median Hb g/L 54.0 57.0

*QC results from Edmonton (6 months 2008)

Anticoagulant CPD Description of change Impact (s)

Anticoagulant for Whole Blood collections will be CPD (rather than CPDA-1 or CP2D)

1. For most transfusions, no significant impact identified 2. CPD has lower glucose levels, therefore one can expect smaller changes in glucose levels for newborns undergoing massive transfusion with plasma replacement.

Anticoagulant in Whole blood Derived Plasma*

Content CPD (new)

CP2D (previous)

CPDA-1(previous)

Citric acid 327 mg 327 mg 327 mg

Sodium citrate 2.63 g 2.63 g 2.63 g

Sodium acid phosphate 251 mg 222 mg 222 mg

Dextrose 2.55 g 5.09 3.19 g

Adenine 27 mg

Water for injection 100 mL 100 mL 100 mL

* Data provided by bag manufacturer

Discontinuation of CPDA-1 Description of change Impact (s)

There will no longer be collection of whole blood in CPDA-1.

CPDA-1 whole blood or red cells will not be available for pediatric, autologous, directed, or other uses.

RBC Preservative Solution Description of change Impact (s)

RBC preservative solution will be Saline Adenine Glucose Mannitol, (SAGM) rather than AS-3.

SAGM has been in use in Europe and elsewhere since the early 1980s.

1. No significant impact identified

2. For neonates – similar safety with both AS-3 and SAGM i.e. no problem for small volume transfusions; for massive transfusions, the theoretical risks are similar for both

RBC Preservative Solution* (continued)

Substance content (mmol added per unit of red cells)

SAGM(new)

AS-3(previous)

NaCl 15 7

Adenine 0.125 0.22

Glucose 4.5 5.5

Na2HPO4 - 1.9

Na3 citrate - 1.9

Citric Acid - 0.2

Mannitol 2.9 -

Volume (mL) 100 100

Shelf life (days) 42 42

*Vox Sang 1998; 74 (Suppl 2) : 177-187, Preparation and Preservation of Red Cells, C.F. Hogman

Table 6. Formulation of anticoagulant preservation solutions in blood collection containers.

CPDA CPD CPD CP2D CPD

Constituent SAGMAdsol (AS-1)

Nutricel (AS-3)

(AS-5) (Terumo)

Volume (ml)** 63 100 100 100 100Sodium chloride (mg) - 877 900 410 877Dextrose (mg) 2000 900 2200 1100 900Adenine (mg) 17.3 16.9 27 30 30Mannitol (mg) - 525 750 - 525Trisodium citrate (mg) 1660 - - 588 -Citric acid (mg) 206 - - 42 -Sodium phosphate (monobasic) (mg) 140 - - 276 -

** Based on a 450 ml collection

Other Changes

Description of change Impact (s)

Pediatric units will be available with one empty satellite bag attached.

Transfusable plasma products such as cryoprecipitate and frozen plasma will be stored in a slightly larger satellite bag. (2 cm longer)

1. Reduction in the number of attached empty satellites for some Hospitals.

2. Storage boxes will be slightly larger to accommodate.

Leukoreduction of Plasma

Description of Change Impact (s)

Whole blood collections for non-platelet production will continue to be leukoreduced as a by-product of the whole blood filtration.

Whole blood collected for buffy coat production will be leukoreduced by centrifugation.

1. The total number of white cells in plasma remains very low, but is somewhat higher than current FFP (see data on the next slide)

2. Health Canada only requires leukoreduction of cellular blood components. As plasma is a non-cellular component no labeling claims of leukoreduction will be made in frozen plasma products.

Comparison of Residual WBC in Plasma

Previous method Buffy Coat method

(n= 9 units measured)117 94 x 103cells/unit

(n = 9 units measured)647 +/- 263 x 103cells/unit

Frozen plasma from whole blood donations change from 8 to 24 hours hours

Description of change Impact (s)

Transfusable plasma produced from whole blood donations will be Frozen Plasma (frozen within 24 hours after collection rather than 8 hours).

NOTE: This is an extension of a practice already performed by CBS.

1. Questions from clinician users about the differences between the products.2. FFP from whole blood will no longer be available. (still have AFFP)3. Coagulation factor function is retained at a clinically appropriate level (see following slides)

FFP vs BC FP Protein Function (ABO matched, n=20)

0.50 - 1.50 U0.1880.870.1421.03Factor IX0.50 - 1.50 U0.2500.910.3151.26Factor VIII0.50 - 1.50 U0.2070.900.2301.09Factor VII0.50 - 1.50 U0.1921.060.1891.15Factor V0.50 - 1.50 U0.1230.950.1371.12Factor II0.50 - 1.50 U0.1510.940.1611.11Factor XI0.50 - 1.50 U0.1641.070.1641.24Factor X

NL RangeStd DevMeanStd DevMean

FP produced from BC 20-24 hrs after

collection

FFP current method 8 hours after collection

Note: small data sample size may not be reflective of current performance.

FFP vs BC FP Protein Function (ABO matched, n=20)

---------0.0840.960.0841.01Alpha 2 Antiplasmin

>0.50 U0.3771.130.4051.24Von Willebrand

>0.65 U0.1801.030.4721.15Protein S2.00 - 5.00 g/L1.9453.920.4813.01Fibrinogen

>0.70 U0.1221.050.2231.19Protein C>0.75 U0.0531.010.0780.97Antithrombin

NL RangeStd DevMeanStd DevMean

Produced from BC 20-24 hrs after

collection

Produced from PRP within 10 hours of

collection

Note: small data sample size may not be reflective of current performance.

Cryoprecipitate will be produced from FP

Description of change Possible impact (s)

Cryoprecipitate and cryosupernatant plasma will be made from FP rather than FFP.

1. Cryoprecipitate will be relabelled to reflect its modern clinical use, i.e., not for the treatment of Hemophilia or vWD, but as a fibrinogen replacement.

Results*Bag/production scenario

Pall/current Maco Pharma cooling tray

Maco Pharma Insulated cont.

Baxter Cooling tray

Baxter insulated container

Fibrinogen - cryo (mg/bag)

307.9 (176.4-464.5)

354.2 (226-527.7)

415.2 (281.6-537.2)

385.3 (256.4-669.4)

318 (217.9-624.6)

FVIII - cryo (IU/bag)

174.3 (112.5-275.8)

125.7 (72.3-182.3)

120.7 (67-248.1)

141.1 (80.1-189.1)

155.3 (64.8-266.8)

vWF activity - cryo (IU/mL)

4.1 (2.4-7.2)

7.5 (2.7-13.8)

7.1 (4.2-13.3)

8.3 (5.3-11.7)

8.3 (3.6-14.7)

FXIII - cryo (% activity of pooled plasma)

106.8 (12.5-468.2)

355.4 (133.4-578.7)

330.6 (45.9-820.9)

331.6 (91-649.1)

334.8 (115.1-656.9)

ADAMTS-13 -CSP (% of normal pooled plasma)

85.7 (49.8-115.2)

70.3 (55.4-105.5)

77.7 (55.9-108)

80.4 (53.2-112.9)

67.9(41.2-87.5)

vWF multimers – CSP ** (bands 3-6)

2.8 (0.3-7.8)

4.1 (0.6-12.8)

1.0 (0.1-1.9)

2.8 (0.2-12.6)

0.9 (0.3-1.7)

*Results are reported as mean with result in brackets to show range

**No high molecular weight bands seen in any CSP plasma product

Autologous Collections Description of change Possible impact (s)

Autologous blood will be collected in CPD and separated into SAGM RBCs and plasma.

Autologous plasma will be provided only if specifically requested prior to donation, when the appointment is booked

1. Autologous RBCs will have a shelf life of 42 days. 2. Autologous whole blood will not be available.3. Autologous plasma will be available only by special order. 4. Autologous, adjusted anticoagulant Whole Blood will no longer be available. 5. Autologous, low volume SAGM RBCs , will still be available.

Directed Collections

Description of change Possible impact (s)Directed donations will be collected in CPD and separated into SAGM RBCs and plasma only.

Maternal plasma will only be provided if it is compatible and there is a clear medical indication (TRALI prevention)

1. All Directed RBCs will have a shelf life of 42 days. 2. Directed whole blood will not be available.

3. Directed maternal plasma will not be available 4. Platelets from directed whole blood will no longer be available

Buffy Coat PlateletsDescription of change Impact (s)

Platelets will be pooled at the manufacturer (CBS).

Pools will go from 5 units to 4. The male plasma from one of the platelet donors will be used for resuspension of the pool

The bag size will be the same as for apheresis platelets

The platelets will be bacterially tested by CBS

1. No pooling to be done by hospital

2. Change how platelets ordered as only a standard adult dose will be provided.

3. Consideration of same policy for BC platelets as for apheresis when transfusing group O to non-O recipients

4. Assessment of agitator space with change in bag size (fewer but larger units).

Platelet counts in buffy coat platelet pools (pools of 4)

Mean x 1011 SD Median x 1011 Min x 1011 Max. x 1011

At expiry(n=71)

3.05 .56 3.00 2.07 4.76

Council of Europe standard - BC: > 6.0 x 1010/single unit equivalent; 1% of units CSA standard - PRP: > 5.5 x 1010/unit in 75% of units tested

(4 or 5 x 5.5 x 1010 = 2.2 or 2.75 x 1011) - Apheresis units: 3 x 1011/unit in 75% of units tested

*QC results from Edmonton (last 6 months)

Platelet dose for neonates & infants

• Neonatal platelet dose– 10-20 ml per kg– Preterm infants can weigh less than 1 kg

• Pediatric platelet dose– 5-10 ml per kg, or– 1 unit per 10 kg

Platelets for neonates & infants• Three types of platelet concentrates will be available:1. Single units of PRP platelets 2. An aliquot of an apheresis unit that has been

prepared sterilely to allow for: – Storage of the remainder of the apheresis unit– pH must be > 6.2 (6.8 – 7.4)– max platelet concentration 1.5 x 109/ml– information from manufacturer for apheresis units

– conditions are met if remaining volume > 100 ml3. An aliquot from a BC unit

– vendor storage requirements for remainder of bag contents

Spiking…….

Hospital Impacts - Spiking• Lack of awareness of different technique to access

ports and spike. Some hospitals had difficulty spiking and required vendor training sessions during Edmonton pilot (October 2005).

• Changes since Edmonton pilot – Port diameter increased to reduce spiking difficulties– Where difficulties identified with specific transfusion sets,

vendors will provide alternate products – Implementation of ISO standard Pall medical bags has

occurred which require the different technique • Hospitals were notified to assess needs at least 5

months prior to implementation

Trouble Shooting Tips for Spiking

1.Do not over spike. Over-spiking will result in the inability to remove the set.

2.Always insert/remove spike using ¼ turn motions. Pulling the spike in a straight downward motion will result in the tightening of the port on the spike.

Medical Overview• Key Questions

– Will the Buffy Coat Product work as well as the previous product?

– Will there be an increase in adverse reactions?

– Are there different anti-coagulants and will they effect neonatal practice?

– Will there be changes to Autologous and Directed Components?

– Do I need to change my blood prescription practice?

– Do we need to test pooled buffy coat platelets for hemolysins?

– After buffy coat implementation will all platelets produced by CBS be routinely bacterially tested?

– Are there any differences on the ward?

Yes and No

Maybe for platelets and plasma

No, provided “correct” spiking method is used

Yes packed cells now

Maybe – if this is part of your apheresis platelet routine

Additional resources

• CBS Circular of Information• www.transfusionmedicine.ca• www.blood.ca

Buffy Coat Component Production

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