Dr. Kabita Chatterjee (MD)Faculty-In- Charge

Blood Bank (Main Hospital)A.I.I.M.S., New Delhi – 110029.

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Countries where the Buffy Coat production method is predominantly used for the preparation of platelets derived from whole blood

• A layer of mixed white cells and platelets created by high speed centrifugation of whole blood

• It is neutral or buff in colour

• Contains: most of the white cells and platelets and ~ 10 % of RBC

PC FFP

PRP method

PC

Buffy coat method

PRP

Plasma

RBC

PRP

RBC

Plasma

5

Platelets prepared through buffy method

Activated Platelet prepared through PRP

method

PLATELET – Buffy coat V/s PRP Platelet Morphological Scoring

In recent years, platelet rich buffy coat (BC) has

become an alternative sources for preparation

of Platelet Concentrate, particularly in Europe.

It has been suggested that this method causes

less platelet activation and damage during

platelet preparation.

Platelet preparation by Buffy coat pooling method is practiced in European countries, US, Latin America and in other Asian counties.

A.I.I.M.S. is a reference center and getting platelet (SDP) request to correct thrombocytopenia from different clinical specialties.

These patients are from different social status and most

of them can not afford to purchase costly SDP kit (75% A.I.I.M.S. observation).

Even sometimes it is very difficult to get donor because of stringent selection procedure and people do not have time for the procedure(1 ½ hours to 2 hours ).

For BTS SDP is a routine procedure but for the clinicians sometimes they need urgent SDP to correct thrombocytopenia especially at the odd hours and holidays: Buffy coat pooled platelet is of great help in this situation(managing dengue crisis).

• Establish buffy coat pooling method to harvest platelet equivalent to SDP.

- Improve platelet yield.

- Meet emergency requirement for platelet.

- Reduce cost to poor patients.

- Reduce leukocyte contamination in platelet

• Prepared 600 Pooled platelet units from 2400 units of blood

collected in Top and Bottom bags (Pooling of 4 buffy coat

bags).

• Conducted study on platelet yield, storage parameters and

also on sterility parameters(n=125).

• Observational study conducted on transfused patients (n=

100).

• Platelet yield was compared with Aphaeresis platelet (n =25).

• Collection of blood in Terumo penpol

Quadruple top and bottom bags.

• Process Quadruple top and bottom bags

in TACE.

• Pooling of buffy coat.

• Second separation & Filtration.

• QC analysis.

• Observational study on transfused

patients.

Methods

• The Process of pooling Buffy coats can be performed in 2 different ways

• Train Method: Here the buffy coats are sterile docked to each other. All the buffy coat residues are pooled into one of the buffy coat bag, One unit Plasma is added to the same. Then the pooled unit is sterile docked to a platelet storage container with a leukoreduction filter and centrifuged. Our Study is based on this method.

• BP Kit Method: refer to picture(TERUFLEX).

Blood collected in Terumo Penpol 450ml TOP & BOTTOM bag and kept in room temperature at 22*C for 2 hours.

Blood bags are centrifuged at heavy spin and separated using TACE II

4 or 6 units of buffy from same group were connected serially using TSCD.

Connected buffy coats with TSCDConnected buffy coats with TSCD Pool 4 units and rinse with plasma

Pool 4 units and rinse with plasma

Remove the pooled bag( bottom end) and connect to IMUGARDIII PL filter integrated with platelet storage bag using TSCD.

Pooled bag

IMUGUARD III PL storage bag integrated

with filter

Pooling with BP Kit (TERUFLEX Method)

BP Kit is integrated with Buffy coat pooling arm, Platelet medium arm, Leukocyte filter, Pooling bag

and Platelet storage bag

TTI Screening

All four units for Buffy Coat Pooling are tested for TTI markers by:

1. ELISA (4th Generation) 2. ID-NAT (TMA Technology)

Buffy Coat pooling starts when these two reports are released by 5:00 PM

We prefer to do polling by same blood group.

ID-NAT screenedID-NAT screened

ID-NAT screened ID-NAT screened

Pooling

Platelet Concentrate

• Spin the pooled buffy at 1200 g for 9 minutes.Spin the pooled buffy at 1200 g for 9 minutes.• After spinning express out top layer which contains platelet After spinning express out top layer which contains platelet

to Platelet storage bag through Leukocyte removal filterto Platelet storage bag through Leukocyte removal filter

Final product-Platelets

harvested from pooled

Buffy

• Platelet yield.

• Platelet yield – Aphaeresis vs BC pooled.

• State of metabolism.

• Sterility test.

• Observational study.

• Platelet increment.

• Cell counter (Beckman coulter) – Platelet count and WBC count is measured in Beckman Coulter Cell Counter to study the platelet count.

• Blood gas analyzer (Nova) – PH, pCo2, pO2, glucose and lactate count is also taken during 5 days storage study.

• Bact Alert(Biomerieux) to check Bacterial contamination.

B.C.P.P. kept in platelet

agitator at 22OC and

samples taken for

evaluation on each day up

to 5 days of storage to

analyse the count and

viability of the platelet.

The items shown in the table below were tested in order to confirm the performance level of platelets.

Sl.No

Test Content to be confirmed

1 Platelet Count State of Platelets during preservation

2 pH Preservation environment,State of metabolism

3 pCO2, Partial pressure

4 pO2 Partial pressure

5 Lactic Acid concentration

State of metabolism

6 Glucose concentration

State of metabolism

We had compared the QC

results of BC pooled PC and

Apheresis PC harvested by

HEMONETICS cell

separator.

Platelet count per bag is varying

from 2.5 to 4.4 x 10¹¹ in 10

samples of buffy coat pooled

platelet. There was no

deterioration in the count during

its 6 days storage period and

meets the quality control

requirements of Council of

Europe guidelines for apheresis

platelet.

We have checked the sterility

of pooled platelet prepared

after 5 hours and 24 hours of

preparation using Bact Alert

system. None of the

pooled platelet showed

the evidence of

bacterial

contamination at 5

hours and,24 hours.

S .no.

Parameter evaluated

1 Platelet count2 pH3 Lactic acid concentration4 Glucose concentration5 Pre and Post filtration WBC count6 Pre and Post filtration platelet count

Platelet Count and pH values Of Platelet prepared from Buffy Coat

Platelet count (× 1011 )

Day 1 Day 3 Day 5 Day 6

3.31 3.36 3.37 3.36

pH 7.06 7.09 7.08 7.12

Platelet count was found to meet the Council of European guidelines Platelet count was found to meet the Council of European guidelines requirement No significant change in requirement No significant change in pH during 5 days storageduring 5 days storage

Comparison on platelet prepared from Buffy Coat vs Aphaeresis (Average values)

Platelet count (× 1011 )Buffy coat -PC Aphaeresis-PC

3.31 3.21

pH 7.06 6.95

No significant difference in the Platelet count and pH No significant difference in the Platelet count and pH between SDP & BC pooled platelet . Both are found equal in between SDP & BC pooled platelet . Both are found equal in quality point of view.quality point of view.

Viability & metabolic function of platelets were maintained during 5 days storage as depicted by the glucose & lactate level during storage.

BC Pooled platelets Glucose (mg/dl) and Lactate (mmol/L)Concentration

1St day 3rd Day 5th Day

Glucose lactate Glucose lactate Glucose lactate

353.4 11.25 335.1 12.69 319.1 14.34

• To assess the clinical effect of the buffy coat pooled platelets, permission was obtained from the administrative authorities of A.I.I.M.S. to prepare and issue Buffy-coat Pooled platelets to the patients (The matter is in knowledge of DCGI and TRG of NACO).

• Patients who are receiving buffy coat pooled platelets will have to replace three units of blood.

• By this policy A.I.I.M.S. blood bank indent is on rise.

Acute Leukemia on Chemotherapy , thrombocytopenia (n =10).

Aplastic Anaemia undergoing labour(n=4). Dengue (n=125). GI Surgery ( n=4) Replacement surgery in orthopaedics

(n=10). Misseleneous i.e.. DIC or Sepsis etc( n=5).

(Post-transfusion – Pretransfusion Platelet count)(10⁹/1) Х Body Surface Area (m²) / number of

platelets transfused (10¹¹)

Corrected count increment was calculated by the formula.

CCI =

Estimated Total Blood Volume Х Platelet Count Increment No. of Platelets Transfused

PPR(%) =

Diagnosis CCI -1 After 1-2 hour

PPR % after 1 -2 hour

PPR % AT 12 -24 Hours

Dengue n=125

7.5-8 80-85%

40 -50

Follow up was not done in 50 patients due to time constraints, issue in emergency,short stay etc

AIIMS Clinicians are liking to use B.C.P.P.

Blood Bank is observing following points

• Better result is obtained by BCPP within 24 hours of collection

• Our rigorous efforts to create a pool of voluntary aphaeresis donors has failed to gain momentum and this forced us to explore the usage of buffy coat pooled platelets as an alternative of SDP.

Cost effectiveness

• Cost of buffy coat pooled platelet is less than Rs 3000 /-.

• BC Pooled platelet reduce the cost of platelet therapy.

• Ready to use platelet to meet emergency requirement.

• Reduce the cost of platelet therapy.• High quality platelet.• Leukodepleted platelet prevents

WBC associated transfusion reactions.

• Multiple donor exposure risk is reduced as it is tested with NAT.

Advantages of buffy coat pooling

• We are open for your valuable suggestions to help poor patients in case of their need.

• Can buffy coat pooled platelets be used as an subsidized alternative of SDP?

• Blood Bank A.I.I.M.S., is doing more work on it to improve its usage.

A.I.I.M.S., Blood Bank need yoursuggestions