This document discusses the buffy coat method for preparing blood components. It begins by providing some history on blood transfusions. It then explains that the buffy coat method involves a hard spin to separate red blood cells from plasma and buffy coat, followed by a soft spin of the buffy coat to separate platelets. This results in optimal preservation of platelet and leukocyte function compared to whole blood. The document outlines the specific quality control checks that should be performed for various blood components prepared using the buffy coat method, such as requirements for volume, hematocrit, sterility, and leukocyte reduction. It emphasizes the importance of quality control to ensure proper component function and efficacy.
6. Whole blood is not Whole!
After 24hrs of storage WB essentially becomes red cells
suspended in a protein solution
Changes in Platelets
WB stored at 4°C – platelets rapidly lose viability
Granulocytes & Monocytes – function reduced within 8hrs and
disintegrates within 24hrs
Microaggregates increase in number
FVIII, FV – levels decreases
7. Component Therapy
1. Optimal preservation of in vitro function of blood
2. Efficient utilization of blood donations
8. MALABAR CANCER CENTRE
COMPONENTS STORAGE
TEMPERATURE
Packed red blood cells (PRBC) +2 to 6°C
Platelets (PLTS) +22 to +24°C under
constant agitation
Fresh frozen plasma (FFP) -30°C or below
Cryoprecipitate (CRYO) -30°C or below
13. Platelet Rich Plasma (PRP) method
Whole blood
Soft spin within
(1800 rpm 6 hrs
x 9 min at 22oC)
Red cells Platelet rich plasma (PRP)
Hard spin
(3000 rpm
x 7 min at 22oC)
Plasma Platelet Conc.
(RDP)
PRP
RBC
PRP
PC
FFP
MALABAR CANCER CENTRE
16. Buffy Coat Method
Top and Bottom/ Top and top methods are available to
produce Leuco-reduced blood components
17. Buffy Coat method
Whole blood
Hard spin within
(3000 rpm 6 hrs
x 9 min at 22oC)
Red cells Buffy coat plasma
Soft spin
(1800 rpm x 7 min at 22oC)
Platelet Conc. WBC with few RBC
Plasma
RBC
18. 1st Spin
Hard-spin centrifugation step lead to a buoyant density
equilibrium, with
red cells at the bottom followed by a layer of
Granulocytes
Lymphocytes
Monocytes
Platelets and
Plasma on top
19. Buffy Coat & 2nd spin
Buffy coat had an average volume of 55 ml, and
contained more than 80% of the platelets and 60% of
the leukocytes
After dilution with a small amount of plasma, this buffy
coat of about 110 ml and a haematocrit of
approximately 20% was centrifuged again under
differential centrifugation conditions (soft speed), in
which only the platelets were small enough to be
pushed upwards in the centripetal streaming plasma
20.
21. Automatic Component
Extractor
Two parallel pressure plates, one is stationary and
other is pneumatically driven that simultaneously expel
plasma in an empty satellite bag and red cells into
another bag containing SAGM, an additive solution.
The system is designed to leave a specific volume of
buffy coat with plasma in the original collection pack.
A light transmitter and two photocells control the flow
of red cells and plasma into separate bags by the
means of two automated clamping devices
24. Benefits of Buffy coat method
Reduces the Leucocytes level by 70-80% and sacrifices
20% of the red cells
Platelet yield improved a lot (platelets at the top of bag
will fall to the buffy coat but also that the platelets at
the bottom of bags will rise to the buffy coat. So the
buffy coat is good source of platelets)
RBC contamination drastically decreased
25. During collection of blood
Clean venepuncture with minimum tissue trauma and
free flow of blood
If any unit takes more than 10 minutes to draw, it is not
suitable for preparation of platelet concentrate, fresh
frozen plasma or cryoprecipitate
If platelets are to be harvested, the blood bag should
be kept at room temperature (20-24°C) until platelets
are separated. Platelets should be separated within 6-8
hours from the time of collection of blood
26. In centrifugation
Opposing cups with blood bag and satellite bags must
be equal in weight otherwise excessive eccentric loads
placed on rotor of centrifuge cause irregular wear and
tear and eventual breakage
The bags should be so placed that its broad side faces
the outside wall of the cup.
29. QC in Blood Bags
Quality control of blood components is an integral part
of the quality management systems in the Blood Centre
Ensures
Proper yield,
Functionality
Efficacy of components
30. Indian Standards for
QC of Blood Components
1. Drugs and Cosmetics Act 1940,Rules 1945, (Schedule F, Part XII
B), Government of India
2. Blood Bank Standards of NBTC, Ministry of Health and Family
Welfare Government of India
3. NABH Accreditation standards for Blood Banks
31. Essential Quality Elements
1. Sampling techniques
2. Frequency of QC
Atleast of 1 % of all components per month (if >100 per month
prepared )
Atleast 4 units per month (if < 100 per month prepared )
75 % or more of components must meet specifications
Washed Blood Components – on ALL BAGS
34. Quality control of Red Cells in additive solution
(Prepared from 450 ml / 350 ml Whole Blood)
Parameter Quality Requirement
Volume
( + additive solution)
250 ± 10%
(+ 100 mL AS for 450 ml Bag)
150 ± 10%
(+ 80mL for 350 ml Bag)
PCV (Hct) 50-60%
Sterility
By culture
35. Leukocyte Content of Various Blood
Components
Leukoreduction (LR) labeling requirements
US < 5 x 106/unit
EU < 1 x 106/unit
37. Quality control of Leucocytes-
Reduced red cells
Method of
Preparation
Product Volume HCT
Additional
parameter
Buffy Coat removed
Red cells in additive
solution from 450 ml
WB
250 ml± 10%
(+100 ml additive
solution)
50-60%
Red cells in additive
solution from 350 ml
WB
150 ml ± 10%
(+80 ml additive
solution)
50-60%
38. Quality control of Leucocytes-
Reduced red cells
Method of
Preparation
Product Volume HCT
Additional
parameter
Washed using
Normal saline
Washed Red
cells
Within locally
specified volume
range
• 50-60%
• RBC loss
<20%
Total protein
content
< 0.5 g/ unit
Leuco-filtration
Leuco-filtered
RBC
(Leucocyte
depleted)
250 ml ± 10%
(+100 ml additive
solution)
50-60%
• Hemolysis
<0.8% or 1%
• WBC <5 x106
per unit
Irradiation Irradiated RBC
Within locally
specified volume
range
• Hemolysis
<0.8%
39. Example:
Vol. of Whole blood in a 350 mL QB = 356 mL
WBC count in WB = 7.4 x 103/µL
HCT = 44%
Therefore, WBC count in the bag
= 7.4 x 103 x 356 x 103 1mL=103 µL
= 2634.4 x 106
= 2.63 x 109
40. After leuco-reduction by buffy coat method:
Volume of LR-RBC = 241 mL
WBC count = 1 x 103 /µL
HCT = 58 %
Therefore, WBC count in the bag
= 1 x 103 x 103x 241
= 241 x 106
= 2.41 x 108
• Volume – adequate
• WBC count <5
x108/unit
• 1 log reduction obtained
• HCT – 50-60%
41. Quality control of Platelet Concentrate (RDP)
from 350ml/450 ml of Whole Blood
Parameter
Quality Requirement
Appearance Swirling present, No visual RBC contamination,
Volume
50-70 ml (PRP method)
50-90 ml (Buffy coat method)
Platelet count
≥3.5 x 1010 platelets/unit from 350ml WB – PRP method
≥4.5 x 1010 platelets/unit from 450 ml WB blood – PRP
method
>6X1010 platelets/unit from 450 ml WB blood – BC
method
pH
>6.0 (at the end of permissible storage period)
RBC contamination
Traces to 0.5 ml ( < 5x109 RBCs)
Sterility By culture
42.
43.
44. When should QC be done ?
As per SOP
Platelet product done on expiry date
On Installation and after repair of equipments
If any Modification in procedure
Recruitment of new personnel
45. Conclusion
Component therapy has revolutionized transfusion
medicine
Standardized blood components help to assess the
outcome of the component therapy.
Procedures as leukofiltration, Irradiation of blood &
should be standardized before putting in to use
