1
Blood Components
& Plasma derivatives
Ahmad Sh. Silmi Msc,FIBMS
Clinical Hematologist &
Immunologist
IUG
2
Introduction
Whole Blood (WB)
• Collected directly from donors into blood
transfusion bag containing anticoagulant
• 500 ml transfusion bag is used (contains
63 ml of anticoagulant + 450 ml blood)
3
Anticoagulants in blood units
1) Acid-Citrate-Dextrose (ACD)
2) Citrate-Phosphate- Dextrose (CPD)
3) Citrate,Phosphate,Dextrose,Adenine (CPDA-1)
4
Anticoagulants Used for WB?
• ACD & CPD preserve the unit for 21 days at 2-6ºC.
• CPDA-1 (anticoagulant/preservative for 35 days).
• C = Citrate→ to prevent clotting
• P = Phosphate→ to maintain pH
• D = Dextrose→ ATP generation
• A = Adenine-1→ substrate from which RBC produce ATP
Anticoagulant ratio is 1.4 ml:10ml blood (63ml / 450ml)
5
Additive solutions
• (SAGM) → Saline-Adenine-Glucose- Manitol
• Purpose of additive solution, to improve
RBCs storage viability till 42 days @ 2-6ºC
* Added only to PRBCs
6
Blood Components
• Human blood consists of
plasma, in which cells are
suspended
• The plasma also contains
other specialised substances,
which are important for blood
clot formation (e.g. clotting
factors)
• Whole blood can be separated
at the blood bank into various
components
7
BLOOD COMPONENTS
• Blood separated into different parts:
1.
2.
3.
4.
5.
6.
7.
•
Packed red cells
Platelets
Fresh frozen plasma
Cryoprecipitate
Granulocytes
Factor IX conc.
Factor VIII conc.
There are more than 20 different products
available
8
Whole blood
Red cells
Granulocytes
Platelets
Plasma
Fractionated
products
(Fresh) frozen
plasma (FFP)
F Vlla
Cryoprecipitate
Stored
Plasma
Immunoglobulins
F Vlll
Albumin
F lX
9
Blood Components
• Refers to a product separated from a single unit
of whole blood
• The term plasma derivative indicates a blood
product separated from a large volume of pooled
plasma by a process called fractionation
10
Blood Components
• Separating WB into components of
blood is necessary to avoid
wasting of units.
11
Blood Components Separation Goals
• Decrease harmful effects of blood transfusion.
• Giving patients specific component needed.
• Allow a longer survival for components.
• More than one patient will use the unit.
12
Centrifugation Types?
There are two types of centrifugation:• Light spin; (2000 rpm at 20ºC for 11 min)
• Heavy spin; (3500 rpm at 20ºC for 11 min)
13
Centrifuged blood
Plasma
Buffy Coat (WBCs & Platelets)
Red Blood Cells
14
Blood Components
• Blood components
– Oxygen carrying components
– Red cell concentrates
(RCC)
– Leukocyte poor blood
– Frozen-thawed red cells
– Platelet products
– Platelet rich plasma (PRP)
– Platelet concentrates (PC)
– Plasma products
–
–
–
–
Fresh frozen plasma (FFP)
Frozen plasma (FP)
Cryoprecipitate
Stored plasma
• Plasma Derivatives
– Coagulation Factor concentrates
• Factor VIII concentrates
• Factor IX complex concentrates &
others
– Oncotic agents
• Albumin
• Plasma protein fraction (PPF)
– Immune serum Globulin
•
•
•
•
Hepatitis B Ig (HBIG)
Varicella-zoster Ig (VZIG)
Rh Ig (RhIG)
Tetanus Ig (TIG)
15
A- Blood components that carry
oxygen
• Increase the oxygen carrying capacity of
the blood by increasing the circulating red
blood cell mass.
• Carry oxygen and nourishment to the
tissues and take away carbon dioxide.
16
1- PRBCs
How to make (PRBCs)?
 RBCs have higher specific gravity than
plasma, it moves to lower portion of the bag
by centrifugation
 WB (Light spin)
Two products:
1) PRBCs
2) Platelet Rich Plasma (PRP)
17
Whole Blood Unit
After centrifugation
WB separates into
plasma & platelets &
PRBCS
18
1
2
3
SAGM
19
1- Red blood cell concentrates
• Prepared by removing
approx. 200 ml of plasma
from whole blood after
centrifugation
• RBCs plus 100 ml of residual
plasma
• In CPD-A can be stored for
35 days at 4oC
20
1- Red blood cell concentrates
Whole Blood
Total Volume
500 ml
Red cell
concentrate
300 ml
Volume of red
cells
Volume of
plasma
Hematocrit
200 ml
200 ml
300 ml
100 ml
40 %
70 %
21
1- Red blood cell concentrates
• High hematocrit → viscous → infuse slowly
• Rate of infusion increased by adding saline
• Other fluids should not used
– Calcium containing fluids (eg. Ringer’s lactate)
should not be added
– May cause clotting
– Glucose solutions
– can cause clumping
• Only saline can be added to blood
22
Expiration date!
– Once the PRBC unit is “opened” it has a
24 hour expiration date!
24
hours
23
2- Leukocyte poor blood
• No viable leukocytes
• WBCs are of no
consequence
• In some patients cause
febrile transfusion reaction
• Should receive leukocytes
poor-blood
• WBCs can be removed by
discarding the buffy coat
(inverted centrifugation)
• Or by washing RBCs or by
using filters
Buffy coat
Red cells
24
Leukocyte
Reduction Filters
(maintains closed system)
http://www.pall.com/39378_39479.asp
Final unit must have less
than 5 x 106 WBCs
25
3- Frozen-thawed red cells
• Red cells can be frozen with use of cryopreservation techniques
• Permit storage for up to 10 years
• Expensive procedure & recommended
only in special circumstances
– e.g. Individuals with rare blood types
– For auto-transfusion
26
3- Frozen-thawed red cells
• The RBC's are first incubated in a 40%
glycerol solution which acts as an
"antifreeze" within the cells.
• The units are then placed in special sterile
containers in a deep freezer at less than
-60 degrees C.
• Cryopreserved units are thawed and
washed free of glycerol prior to use as
saline suspended RBC's.
27
3- Frozen-thawed red cells
• Deglycerolized RBCs
– RBCs that have had the
glycerin removed
– Thawed at 37°C
– A blood cell processor
washes the cells with varying
concentrations of saline
– Considered “open”, expires in
24 hrs.
28
4- Washed RBCs
• Washed RBCs
– Not effective in reducing WBCs
– For patients (with anti-IgA) that may react with
plasma proteins containing IgA
– Reactions may be allergic, febrile, or
anaphylactic
29
5- Irradiated RBCs
• Irradiated RBCs
– Prevents T-cell proliferation that may cause
transfusion-associated graft versus host
disease (GVHD)
– GVHD is fatal in 90% of those affected
– Used for:
•
•
•
•
•
•
•
Donor units from a blood relative
HLA-matched donor unit
Intrauterine transfusion
Immunodeficiency
Premature newborns
Chemotherapy and irradiation
Patients who received marrow or stem cells
30
6- Synthetic oxygen carrying agents
• Synthetic oxygen carrying agents
– Perfluorochemical (e.g. Fluosol-DA )
•
•
•
•
Fluorinated hydrocarbons
Readily dissolve oxygen
Poor soluble in plasma
Side effects:
– Hypotension
– DIC
– Chemically modified hemoglobin
• Free Hb has a very short half life
• Chemically modified to:
– increase intravascular survival
– and to make it more effective in carrying oxygen
31
B- Platelets
• Important in maintaining
hemostasis
• Help stop bleeding and form a
platelet plug (primary hemostasis)
• People who need platelets:
– Cancer patients
– Bone marrow recipients
– Postoperative bleeding
32
How platelets are processed
• REMEMBER!!!
• Requires 2 spins:
– Soft – separates RBCs and WBCs from
plasma and platelets
– Heavy
• platelets in platelet rich plasma (PRP) will
be forced to the bottom of a satellite bag
• 40-60 mL of plasma is expelled into
another satellite bag, while the remaining
bag contains platelet concentrate
33
Preparation of platelet
concentrate
Plasma
RBCs
PRP
Platelet
concentrate
34
B- Platelet Products
• Platelet Rich Plasma (PRP)
– Gentle centrifugation of whole blood
– Supernatant transferred to the 2nd
bag
• Platelet Concentrates
– Prepared from PRP by a 2nd
centrifugation
– Removal of all but 50 ml of plasma
– Contain approx. 6X1010 platelets
– 60 – 80% Plts present in whole
blood unit
– Remain 5 days
– Longer at 22oC with continuous
agitation
35
2
1
Then
36
Whole blood unit
Centrifuge using LIGHT spin
Express Platelets Rich Plasma (PRP) into satellite bag
Take PRP and centrifuge again now using HEAVY spin
Express PPP into satellite bag & freeze at -18ºC
Final products :PRBCs, Platelets concentrate, FFP
37
B- Platelet Products
• Contamination by WBCs &
RBCs is usually small
• But there is enough to induce
alloimmunization
• Plt concentrates from Rh +ve
should not be administered to
Rh –ve women
• Storage at 22oC, therefore care
to prevent contamination
38
C- Plasma Products
• Plt poor plasma can be separated into a
number of products
– Fresh frozen plasma
– Platelet concentrate
– Frozen plasma
– Cryoprecipitate
– Stored plasma
39
1- Fresh frozen plasma (FFP)
• Prepared from whole blood
within 6 hours of collection
• Rapid freezing of plasma
preserves the labile
coagulation factors at
maximum levels
• Don't contain cellular
elements
• 200 ml volume
40
1- Fresh frozen plasma (FFP)
Freeze at -18ºC for 1 year from collection
date.
Or freeze at -70ºC for up to 7 yrs
Cross match is not required, but of coarse
should be ABO compatible.
41
Indications of FFP
• Liver disease
• Severe burns
• Provides coagulation factors for
–
–
–
–
–
–
–
Bleeding
Abnormal clotting due to massive transfusion
Patients on warfarin who are bleeding
Treatment of TTP and HUS
Factor deficiencies
ATIII deficiency
DIC when fibrinogen is <100 mg/dL
42
43
2- Platelets Concentrate (PC)
How to prepare PC?
Platelet Rich Plasma (PRP) centrifuged
using (heavy spin), this will produce:
1) Fresh frozen plasma (FFP)
2) Platelets concentrate (PC)
• PC are stored at room temperature on
platelet agitator (prevent platelets clumping)
• PC stored for 5 days at 20-24°C.
• Each unit should elevate the platelet count by
5000/µL
44
2- Platelets concentrate
• Indications:
1.To prevent bleeding due to
thrombocytopenia or platelet dysfunction
2.To a patient undergoing an operation, if the
platelet count is less than 20,000/µL
45
1
2
3
46
Platelet concentrate
47
3- Frozen Plasma (FP)
• Separated from whole blood within 24
hours of collection
• Contains at least 50 % of original factor
VIII & factor V frozen plasma
• Adequate source for treatment of mild to
moderate coagulation factor deficiencies
• 200 ml volume
• Storage at -30oC for up to 12 months
48
4- Cryoprecipitate
• Produced from freshly separated plasma by
freezing at -70oC followed by thawing at 4oC
• Flocculent precipitate is rich in factor VIII,
fibrinogen and fibronectin
• Once thawed, mixture is centrifuged to sediment
the cryoprecipitate & all but 5 to 10 ml of
supernatant plasma is removed
• Contains 250 mg fibrinogen
• 80 clotting units of factor VIII
• Stored at -30oC for 12 months
49
4- Cryoprecipitate
• Increase of 2% of factor VIII level for each
bag of cryoprecipitate infused
• Supernatant plasma removed is called
stored plasma
– Must be used within 5 weeks if stored at 4oC
– Lasts for 2 years at -30oC
50
Cryo..
Indications:
Hemophilia A
Von Willebrand disease (VWD)
Congenital or acquired fibrinogen
defects (i.e., dysfibrinogenemia)
51
5- Stored plasma
• Plasma separated from whole blood after 24 hours of
storage at 4oC
• Can also be derived from cryoprecipitate production
• Contain reduced levels of labile coagulation factors V
VIII & fibrinogen
• It is indicated for patients requiring volume expansion or
protein replacement when labile clotting factors are not
required
• Plasma products do not require crossmatch prior to use
but should be ABO compatible
52
Summary
Blood Components
Blood
Component
Centrifugation
1) PRBCs
WB Light spin=
2) PC
PRP heavy spin=
3) FFP
53
2000rpm-20ºC -11min.
PRBCs + PRP
3500rpm-20ºC -11min.
PC + FFP
Storage
Indication
Temp Time
2-6ºC +SAGM 42d •Anemia
•Newborn
exchange
transfusion
R.T
-18ºC
-65ºC
3-5 d
•Bleeding
•Operation if
plt. Less than
20000/μl
•Clotting
factor
7 years deficiencies
•Severe burns
1year
Summary
Blood Components
Blood
Comp
Centrifugation
Storage
indication
Temp Time
4) Cryo
a. WB special heavy
spin= 3500rpm at 4ºC -
-30ºC 1 year
11min.
RBC + Plasma
b. Plasma
store at -18 ºC
then thaw at 4 ºC then
heavy spin at 4ºC
54
• Hemophilia A
• Von
Willebrand
disease
Plasma Derivatives
55
Plasma Derivatives
• Certain plasma derivatives can be
obtained by fractionating the fresh frozen
plasma or stored plasma
• Fractionation:
 Allows the processing of large volumes of
pooled plasma
 Pooling of many units increases the risk of
viral transmission to the recipient
56
Plasma protein fractionation
• Plasma proteins are separated according
to differences of each protein.
• Fractionation involves changing the
conditions of the pooled plasma (e.g. the
temperature or the acidity)
• Proteins that are normally dissolved in the
plasma fluid become insoluble, forming
large clumps, called precipitate.
• The insoluble protein can be collected by
centrifugation.
57
Plasma protein fractionation
• One of the very effective ways for carrying out
this process is the addition of alcohol to the
plasma pool while simultaneously cooling the
pool.
• This process is sometimes called cold alcohol
fractionation or ethanol fractionation.
• This procedure is carried out in a series of steps
so that a single pool of plasma yields several
different protein products, such as albumin and
immune globulin.
58
Plasma Derivatives
Plasma Derivatives
Preparation avaliable
Factor VIII concentrates
Coagulation Factors
Factor IX concentrates
Anti-thrombin III
Albumin
Albumin
Plasma protein fraction
Non-specific immune serum globulin (ISG)
Rh immune globulin (RhIG)
Immune globulins
Hepatitis B immune globulin (HBIG)
Varicella-Zoster immune globulin (VZIG)
Tetanus immune globulin (TIG)
59
1- Coagulation Factor Concentrates
• Prepared in a freeze-dried form
• Indicated for patients with congenital
coagulation deficiencies
– Risk of hepatitis is high
• Should not used for mild acquired
coagulation deficiencies
– Should be treated with FP or FFP
60
Factor VIII Concentrate
• Commercially prepared, lyophilized
powder purified from human FFP
• Contain also small amounts of
fibrinogen & other proteins
• Can contain blood group Abs
• Treat patients with hemophilia A
61
Differences of Cryoprecipitate & Factor VIII concentrates
Factor VIII
Cryoprecipitate
concentrates
oC
4
Storage Temp.
-30oC
Short period RT
Risk of
Low
High
Hepatitis
Treatment of
Yes
Yes
hemophilia A
Treatment of
Yes
no
vW disease
62
Factor IX Concentrate
• For the treatment Factor IX deficiency or
Hemophilia B (Christmas Disease).
• Have been used to treat patients with
acquired inhibitors of factor VIII
– Have factor VIII bypassing activity
• Contains also factors II, VII & X in
concentrated form
• Vials containing 500 units of factor IX
63
Factor IX Concentrate & liver
disease
• It is contraindicated in patients with liver
disease
– Have low levels of circulating antithrombin III
– Activation of clotting factors present in some
factor IX concentrates,
– cause DIC
64
Blood products & treatment of specific clotting factor deficiencies
Deficiency
Fibrinogen
Factor V
Factor VII
Factor VIII
Blood product Indicated
Cryoprecipitate
Stored plasma
Fresh frozen plasma
Frozen plasma
Factor IX concentrate
Stored plasma
Factor VIII concentrate
Cryoprecipitate
Cryoprecipitate
Von Willebrand’s Disease
Fresh frozen plasma
Frozen plasma
Factor IX
Factor IX concentrate
Factor X
Stored plasma
Factor XI
Stored plasma
Factor XIII
Stored plasma
65
2- Oncotic Agents
• Albumin: volume expansion
• Other colloids are available for blood
volume expansion
– Dextran
– Gelatin
– Hydroxyethyl starch
– Polyvinylpyrrolidone
66
Albumin
• Albumin is prepared by ethanol
fractionation of pooled plasma
• Available in 5% and 25% concentrations.
• Have physiological sodium content
• No risk of hepatitis, sterilized during
preparation
• No coagulation factors or blood group Abs
67
Albumin
• Used for treatment of hypovolaemia and
hypoalbuminaemia (result from abnormal
synthesis, increased metabolism or loss)
• It maintains capillary osmotic pressure
• Carrier protein for drugs, hormones,
enzymes & metabolites
68
Plasma protein Fraction
• Partially purified albumin
• Contains ≈ 85% albumin & 15% other
plasma proteins
69
3- Immune Globulins
• Contains immune IgG antibodies,
prepared from pools of plasma.
• For disease prophylaxis, hepatitis A,
measles, varicella and rubella.
• For the treatment of hypogammaglobulinemia and agammaglobulinemia.
70
Immune Serum Globulin (ISG)
•
•
•
•
•
Primarily IgG Ab
Prevention of some viral diseases
Hypogammaglobulinemia
Congenital immune deficiency
Given by IM injection (aggregates of IgG)
71
Hepatitis B Immune Globulin
(HBIG)
• Contains Hepatitis B immune antibodies.
• From plasma of donors with high titer of
Ab to HBsAg
• Provides passive immunization for HBV.
• For treatment after exposure to HBsAg.
• For the prevention of maternally
transferred HBV (perinatal exposure).
72
Varicella-Zoster immune globulin (VZIG)
• Derived from patients had recent Herpes
Zoster infections
• Herpes Zoster infections result in severe
fatal infection in immunocompromised
individuals
• Passive administration of VZIG during 72
hours of exposure can prevent or
attenuate infection
73
Rh Immune Globulin (RhIG)
• Derived from Rh -ve individuals
• Contains IgG antibodies to the D antigen on red
blood cells.
• Given during pregnancy and post-natally to Rh
negative mothers to prevent the development of
anti-D and hemolytic disease of the newborn
(HDN) due to anti-D.
• Given prophylacticaly following abortion, or
invasive maternal procedures (e.g.,
amniocentesis).
74
Tetanus Immune Globulin (TIG)
• Prepared from individuals specifically
immunized for tetanus toxoid
• Available for individuals at risk following
injury
75
• Changes in stored blood:
– Certain percent of RBC are destroyed, in good
anticoagulant only 10-20 % of RBC are
destroyed.
– Blood become acidic.
– Elevation in K concentration which is bad for
heart.
– Platelets will die within few hours after
collection. So there will no Plts.
– WBC are deteriorated also (no WBC in old
blood).
– Activity of coagulation factors will be grossly
76
Granulocytes
Lymphocyte
Neutrophils
Monocyte
Eosinophils
Basophils
77
Granulocytes
• Neutrophils are the most numerous,
involved in phagocytosis of bacteria/fungi
• Although rare, it is useful for infants with
bacteremia
• Prepared by hemapheresis
• ≥ 1.0 x 1010
• Maintained at room temp for 24 hours
78
79