Buffy coat pooling of platelets Blood Bank AIIMS New Delhi

1. Dr. Kabita Chatterjee (MD)
Faculty-In- Charge
Blood Bank (Main Hospital)
A.I.I.M.S., New Delhi – 110029.

2. 2
Countries where the Buffy Coat production method is
predominantly used for the preparation of platelets
derived from whole blood

3. • 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

4. PC FFP
PRP method
PC
Buffy coat method
PR
P
Plasma
RBC
PRP
RBC
Plasm
a

5. 5

6. Platelets prepared through
buffy method
Activated Platelet
prepared through PRP
method
PLATELET – Buffy coat V/s PRP
Platelet Morphological Scoring

7.  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.

8. 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).

9.  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).

10. • 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

11. • 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).

12. • 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

13. • 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).

14. 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

15. 4 or 6 units of buffy from same group were connected
serially using TSCD.
Connected buffy coats with
TSCD
Connected buffy coats with
TSCD
Pool 4 units and
rinse with plasma
Pool 4 units and
rinse with plasma

16. 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

17. 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

18. 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.

19. ID-NAT screened
ID-NAT screened
ID-NAT screened ID-NAT screened
Pooling
Platelet Concentrate

20. • 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 plateletAfter 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

21. • Platelet yield.
• Platelet yield – Aphaeresis vs BC pooled.
• State of metabolism.
• Sterility test.
• Observational study.
• Platelet increment.

22. • 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.

23. B.C.P.P. kept in platelet
agitator at 22O
C and
samples taken for
evaluation on each day up
to 5 days of storage to
analyse the count and
viability of the platelet.

24. 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

25. We had compared the QC
results of BC pooled PC and
Apheresis PC harvested by
HEMONETICS cell
separator.

26. 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.

27. 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.

28. S
.no.
Parameter evaluated
1 Platelet count
2 pH
3 Lactic acid concentration
4 Glucose concentration
5 Pre and Post filtration WBC count
6 Pre and Post filtration platelet count

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

30. 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 pHNo significant difference in the Platelet count and pH
between SDP & BC pooled platelet . Both are found equal inbetween SDP & BC pooled platelet . Both are found equal in
quality point of view.quality point of view.

31. 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

32. • 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.

33.  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).

34. (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 =

35. Estimated Total Blood Volume Х Platelet Count Increment
No. of Platelets Transfused
PPR(%) =

36. 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

37. 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.

38. Cost effectiveness
• Cost of buffy coat pooled platelet is
less than Rs 3000 /-.
• BC Pooled platelet reduce the cost
of platelet therapy.

39. • 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

40. • 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 your
suggestions