Buffy coat
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Transcript of Buffy coat
Dr. Kabita Chatterjee (MD)Faculty-In- Charge
Blood Bank (Main Hospital)A.I.I.M.S., New Delhi – 110029.
2
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