Blood component preparation

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BLOOD AND ITS COMPONENTS
 BLOOD IS VITAL TO LIFE ,OR WE CAN SAY BLOOD IS LIFE.
 In INDIA blood transfusion was first started in School Of Tropical medicine.
Calcutta,1939, with out any scope for group matching.
 As this procedure was unscientific a committee was formed for
establishment of blood bank in Calcutta.
 Ultimately a small blood bank was started in the dept of Serology, School Of
Tropical Medicine named as RED CROSS BLOOD BANK.
 Later it was shifted to Maniktala as Central Blood bank.
 Pundit Jawhar Lal Nehru donated blood in Calcutta in 1946.
• In spite of dynamic progress in the field medical science, the
life saving role of blood is yet with out parallel, even in the 21st
century.
• Blood is still the most essential factor in saving a life.
• In INDIA total requirement of blood is approximately
80,00,000.units per year, where as its collection from
voluntary donors does not exceed a total of 50,00,000units
even after almost 60 years of independence.
Contd.
• In a statistical study it is seen that total no
of blood donation in
West Bengal- 713535 (Vol. 85.71%)
Bihar – 47863 (Vol. 22.74%)
Jharkhand --- 73238 (Vol. 33.13%)
Uttar Pradesh- 394699 (Vol. 17.3%)
Maharashtra- 377110 (Vol. 86.36%)
Preservation and storage of
Blood
Since 1978 citrate-phosphate-dextrose with
adenine (CPDA-1) is used as blood
preservative for 35 days at 2-40C.
Action of ingredients of anticoagulant solution.
Citrate
Sodium diphospate
Prevents coagulation by
chelating calcium
Prevents fall in pH
Glucose
Supports ATP generation
by glycolytic pathways
Adenine
Synthesizes ATP, increases
level of ATP, extends the
self life of RBC to 42
days.
Action of ingredients of anticoagulant solution.
- Blood
pH on day of collection is 7.5 and on 35th
day become 6.84.
- A fall in pH in the stored blood results in a
decrease in red cell 2, 3-DPG level, which
results in increase in hemoglobin-oxygen
affinity. CPDA-1 maintains adequate
levels of 2,3-DPG for 10 -14 days.
- During storage Na+ and K+ leak through the
red cell membrane rapidly. K+ loss is
greater than Na+ gain during storage.
Biochemical changes in stored blood
Characteristics
Days of storage
Whole blood
RBC conc.
0
35
0
35
% viable cells (24
hrs after
transfusion)
100
79
100
71
pH (Measured at
370C)
7.55
6.98
7.60
6.71
Biochemical changes in stored blood
Characteristics
Whole blood
RBC
2, 3-DPG (% initial
value)
Plasma K (m.mol/l)
100
<10
100
<10
5.1
27.3
4.2
78.5
Plasma Na (m.mol/l)
169
155
Plasma Hb (mg/l)
78
461
111
82
658
Transfusion Reactions
Reaction
Acute
(within 24
hours)
ImmuneMediated
Haemolytic
Febrile non
hemolytic
Allergic
anaphylactic
TR-acute lung
injury.
Haemolytic
Alloimmunization
Post transfusion
purpura
Graft-Vs-host
disease
Non-Immune
Mediated
Bacterial
contamination
Circulatory
overload
hyperkalemia
Hepatitis B & C
HIV 1&2 Syphilis
Malaria
Iron over load
Delayed
(within days or
month
Inspection of blood
Blood should be inspected before
transfusion for possible bacterial
contamination, haemolysis, visible
clots, brown or red plasma.
Plasma with a green hue should not
to be rejected because this is caused
by exposure of bilirubin pigment to
the light.
Yersinia enterocolitica can grow
at 40C and the blood is
haemolysed.
Importance of component separation
Separation of blood into component allows optimal survival of
each constituents
Component separation allows transfusion of only specific
desired component to the patient
Transfusion of only the specific constituent of the blood avoids
the use of unnecessary component
By using blood components several patient can be treated with
the blood from one donor
Blood Components (cellular & plasma) &
Plasma Derivatives
Cellular components
• Red cell concentrate
• Leucocytes-reduced red cells
• Platelet concentrates
• Leucocytes-reduced platelet concentrates
• Platelet Apheresis
• Granulocytes, Apheresis
Contd.
Plasma Components
•
•
•
•
Fresh frozen plasma
Single donor plasma
Cryoprecipitate
Cryo-poor plasma
Plasma derivatives
•
•
•
•
•
•
Albumin 5% & 25%
Plasma protein fractions
Factor viii concentrate
Immunoglobulin
Fibrinogen
Other coagulation factors
Preparation of blood component is
possible due to
 Multiple Poly Vinyl Chloride (PVC) pack system
 Refrigerated centrifuge
 Different specific gravity of cellular components
◦ Red cells spg. 1.08-1.09
◦ Platelet spg. 1.03-1.04
◦ Plasma spg. 1.02-1.03
Due to different specific gravity of cellular
components, they can be separated by centrifuging
at diff g for diff time.
Centrifugation for blood component
preparation
The components are prepared by
centrifuging at diff relative centrifugal force
at diff time.
Relative Centrifugal Force in g
=118 x10-7 x r x N2
Precautions to be observed in preparing
components
In collection of blood
•
proper selection of donor
•
Clean & aseptic venepuncture site to minimize bacterial contamination
•
Clean venepuncture with minimum tissue trauma and free flow of blood
•
The flow of blood should be uninterrupted and continuous. If any unit takes
more than 8 minutes to draw, it is not suitable for preparation of blood
components.
•
A correct amount of blood proportionate to anti coagulant should be collected in
primary bag that has satellite bags attached with integral tubing.
Contd.
• Monitor the collection of blood with automatic mixer which is used for
collecting the desired amount of blood and mixing the blood with
anticoagulant
• If platelets are to be harvested the blood bag should be kept at room
temperature 20-240C until platelets are separated. Platelets should be
separated within 6 hours from the time of collection of blood.
• Triple packs system with two attached bags makes it possible to make
red cells, platelet concentrate and fresh frozen plasma. While quad
packs system with three attached bags are used for preparing red cells,
platelets concentrate , cryoprecipitate (factor viii) and cryo-poor
plasma. Double bags are used making red cells and Platelet rich
plasma only.
Blood Component Separation
centrifuge
blood
pl.rich plasma
PRBC
centrifuge at 20o
freeze -700c
pl.poor plasma
platelet
frozen RBC leuco poorRBC
FFP cryo
poor plasma
cryoprecipitate
Whole
Whole Blood
Whole blood contains 450+63 ml or
350+49 ml of blood plus anticoagulant
solution. The anticoagulant used is CPDA-1.
Whole blood has a hematocrit of 30-40
percent. Minimum 70% of transfused red
cells should survive in the recipient’s
circulation 24 hrs after transfusion. Stored
blood has no functional platelets and no
labile coagulation factors V and VIII.
Preparation of Red Blood Cell
Concentrates
Red blood cells are prepared by removing most
of the
plasma from a unit of fresh blood.
 Red blood cells preparations are:
◦ Sedimented red cells: They have a PCV of 60-70%, 30
% of plasma & all original leucocytes and platelets.
Kept at 2-60C.
◦ Centrifuged red cells: They have a PCV of 70-80
percent, 15 % of plasma and all original leucocytes
and platelets. Kept at 2-60C.
◦ Red cells with additive (Adsol or SAG-M): They have
PCV of 50-60 Percent, minimum plasma and all
leucocytes and platelets. Usually kept at 2-40C.
Leukocytes-Reduced Blood Components
Leukocytes in blood components can cause:
–
–
–
–
–
–
Non hemolytic febrile transfusion reactions (NHFTR)
Human leukocyte antigen (HLA) alloimmunisation.
Transfusion of Leukotropic viruses eg. CMV, EBV, HTLV1.
Transfusion related GVHD
Transfusion related acute lung injury (TRALI)
Transfusion related immunosuppression.
Contd.
Cytokines are generated by leukocytes, even at 260C but to a much greater extent at 20-240C.
Cytokine level rise in direct proportion to the
number of leukocyte. Hence leuko-reduction
before storage in blood bank is much better than
post storage bed side leuko-reduction .
Reducing the leukocyte content <5x106 in
one unit of RBCs prevents non hemolytic febrile
transfusion reactions (NHFTR) and HLA
alloimmunisation or transmission of CMV.
Methods of Preparation of LeukocytesReduced Red Cells




Centrifugation and removing of Buffy coat
Filtration
Washing of red cells with saline
Freezing and thawing of red cells
leuko-reduction can be done at three diff
points
1.
2.
3.
Prestorage leuko-reduction
Post storage leuko-reduction
Bedside filtration
Impact of pre-storage leuko-reduction
Results from pre-storage
leuko-reduction
◦ Cytokine production is
reduced or eliminated
◦ White cells are removed
before fragmentation
transmission
◦ Tumor metastasis are
immunomodulation
reduced .
Potential patient benefit
Decrease in NHFTRs
Decrease alloimmunization
Decrease virus
Prevent
Preparation of Platelet Rich Plasma &
Platelet Rich Concentrate
Are prepared from
• 450 ml of fresh blood by centrifugation or
Aphaeresis.
• A unit of platelet concentrate prepared
from 450 ml of fresh blood contains:
– Plasma vol.
– Platelet yield
– WBC
– RBC
– pH
40-70ml.
5.5x1010
≥108
traces to 0.5ml.
6.0 or more
Calculation of Platelet Yield
– Number of platelet in blood=
platelet per mm3 x1000 x vol. of blood (ml)
– Number of platelet in PRP =
platelet per mm3 x 1000 x vol. of PRP (ml.)
– Number of platelet in P.C. =
platelet per mm3 x 1000 x vol. of P.C. (ml.)
Calculation:
– % of platelet yield in PRP=
Number of platelet in PRP x100/ Number of platelet in
blood
– % of platelet yield in P.C.=
PRP
Number of platelet in P.C. x100/ Number of platelet in
Precaution and storage
pH should never fall below 6. A decline in pH
causes
◦ Changes in shape of platelets from disc to sphere
◦ Pseudopod formation
◦ Release of platelets granules
The above changes are responsible for low
recovery and poor survival of platelets in vivo.
Agitation during storage helps the
exchange of gases, maintenances of pH, &
reduce formation of platelet aggregates.
Granulocyte Concentrates
Granulocyte concentrates prepared by
 Single donor unit
 Leukapheresis by blood cell separators
As the specific gravity of red cells and
granulocytes is very similar, the separation of
granulocytes by centrifugation is not
satisfactory. Leukapheresis is a better method.
Granulocytes can be stored at 20-240C but
they should be used within 8 hrs. & not later
than 24 hrs from blood collection.
Fresh Frozen Plasma (FFP)
It contains all coagulation factors & great care must be taken during
collection of blood , freezing and thawing to preserve their activity.
Collection of blood :
1.
Blood should be collected le by a clean, single venepuncture.
2.
Flow of blood should be rapid and constant.
3.
Total time taken to collect 450 ml of blood should not be more than
8 minutes.
The most labile coagulation factors are preserved for one yr. if
FFP is kept at -300C or below. If FFP is not used within one yr. it is
redesignated as Single Donor Plasma which can be kept further for 4
yrs at -300C or below.
The FFP should be administered as soon as possible after
thawing, and in any event within 12 hrs. if kept at 2-60C.
Cryoprecipitate
Cryoprecipitate are precipitated proteins of plasma rich in Factor VIII
and fibrinogen, obtained from a single unit of fresh plasma (
approximately 200 ml.) by rapid freezing within 6 hrs of collection.
Factors improve the yield of Factor VIII in Cryoprecipitate
1.
Clean single venepuncture at first attempt
2.
Rapid flow of blood, donation of blood (450ml) obtained in less than
8-10 mins should be used
3.
Adequate mixing of blood and anticoagulant
4.
Rapid freezing of plasma as soon as possible after collection in any
case within 6-8 hrs after collection as done for preparing FFP.
5.
Rapid thaw at 40C in circulating water bath.
Storage and shelf life of Cryoprecipitate: One yr at -300C or
below.
After reconstitution Cryoprecipitate should be kept at 2-60C and
administered within 4 hrs.
Single Donor Plasma
Single donor plasma can be prepared by
separating it from red cells any time up to 5
days after the expiration of the whole blood
unit. When stored at -200C or lower, single
donor plasma may be kept up to 5 yrs.
A prolonged pre separation storage
period increases its contents of potassium &
ammonia.
It has no labile coagulation activity.
Cryoprecipitate Poor Plasma
• It is a by-product of cryoprecipitate
preparation.
• It lacks labile clotting factors V and VIII and
fibrinogen.
• It contains adequate levels of stable clotting
factors II, VII, IX & X.
• It is frozen and stored at -200C or lower
temperature for 5 yrs.
Indications, Contraindications &
Complications of Diff Blood
Components
BLOOD TYPES
Fresh Blood
Whole blood or RBC
concentrates less
than 12-24 hours old
form the time of
collection are
considered as Fresh
Blood
• Whole Blood
• Blood after 24 hours
of collection to 35
days are considered
as Whole Blood
(without platelet and
labile clotting factor)
Fresh blood
In new born
exchange transfusion
open heart surgery
Hyperkalemia
Renal failure
One unit increases
0.8 gm% hemoglobin in adult
Disadvantages of Fresh blood transfusion
1.
Chance of transfusion of cytomegalovirus
virus, Human T- Cell Lymphotropic virus
type I & II, E.B Virus, Treponema pallidum
2.
Non –hemolytic febrile transfusion reaction
3.
Transfusion Related Acute Lung Injury
(TRALI) results pulmonary oedema
Cytomegalovirus (CMV)
CMV is a common human pathogen
and present in sub clinical stage in
90 % adults.
- It is the common cause of
congenital defects eg.
Microcephaly, Intra cerebral
calcification, mental retardation,
unilateral or bilateral hearing loss.
Viruses are shed in most of the body
fluids.
It infect the mononuclear leucocytes.
Whole blood
Blood after 24 hours of collection to 35 days
are considered as Whole Blood (without
platelet and labile clotting factor)
Indication
1. Symptomatic decrease in oxygen – carrying
capacity combined with hypovolemia.
2. More than 30% blood loss in acute
haemorrhage.
3. Anticipated surgical blood loss more than 1
litre .
4. Source of protein with oncotic property.
5. Source of Non-labile coagulation factors.
6. Inoperative blood loss more than 15%.
Whole blood
Disadvantages
1. Less oxygen carrying capacity and more
potassium accumulation.
2. Low level of 2, 3 –di- phosphoglycerate
which is important in premature neonates,
patient with impaired cardiac function,
haemorrhagic shock, respiratory distress
syndrome.
3. Develop C.C.F in severe anemic patient.
4. Contraindicated in multiple transfusion
Whole blood
Complication
1.Dilutional thrombocytopenia
2. C.C.F
Red blood cell concentrate
(packed
Red Cell) are prepared by removing
most of the plasma from a unit of whole blood.
Indication:
1.
2.
3.
4.
5.
6.
Urgent operation with haemoglobin less than 10 gm%
Anaemia associated with cardiac failure.
Haemoglobin less than 6 gm%
Approaching delivery and haemoglobin less than 7 gm%
Liberal guideline in thalassaemia major
Anticipated surgical blood loss more than one litre
RBC concentrate
contra-indication
1. Chronic renal failure
2. Pre-operative transfusion to raise
hemoglobin above 10gm%
3. Nutritional Anaemia
4. To enhance general well being,
promote healing, prevent infection
Platelet Concentrate
(Production of platelets are approx.
40000/microlitre/day)
1 unit platelet increases 7000/microlitre
platelet count in adult, 80000/microlitre
in infants, 20000 /microlitre in Child of
18 kg body wt.
Platelet Concentrate
(Production of platelets are approx. 40000/microlitre/day).
Indication:
1. Count
less than 5000/microlitre regardless clinical
condition
2. Count is around 20000/microlitre with
thrombocytopenic bleeding or increase risk of bleeding
in acute leukaemia or chemotherapy
3. Count is around 60000/microlitre with DIC or before
major surgery.
Administration of ABO incompatible platelet is an
acceptable transfusion practice, but not Rh
incompatible platelet.
Platelet Concentrate
Complication
1.Chill, Fever, Allergic reaction
2.Infusion of Bacteria
3.Alloimmunisation
4.Platelet refractory state
5.Graft vs. host disease
Platelet concentrate
contra-indication
1. ITP
2. TTP
3. Heparin induced thrombocytopenia
4. No role in routine open heart surgery
5. Invasive procedure where count is
more than 50000/microlitre
6. Bleeding unrelated to decrease
platelet number and function.
Fresh frozen plasma
Indication
1. Actively bleeding and multiple
coagulation factor deficiency
2. Liver Diseases
3. DIC
4. Coagulopathy in massive transfusion
5. TTP
6. Von Willebrand disease
Fresh frozen plasma
contra-indication
1. Should not be used as blood volume
expander
2. Hypoproteinaemia
3. When prothrombine time is less than
18 second
4. Source of immunoglobin
Limitations
1.Components must be
prepared within 6 hours from
collection time
2.Needs costly instruments and
infrastructures and specially
trained personnel
Plasma Derivatives & Plasma Substitutes
Plasma protein solutions
Plasma protein solutions are prepared from
pooled plasma after removal of factor viii
conc., fibrinogen & immunoglobulin
– Albumin preparation
•
•
•
Albumin 5% soln.
Albumin 25% soln.
contain 96% alb & 4% globulin
Plasma protein fraction (PPF) 5% soln.
Contain 83% alb. & 17% globulin
Characteristics of Albumin Preparation
 The 5% soln. are osmotically and oncotically
equivalent to plasma, 25 % soln. is five times
that of plasma
 Products are heated and chemically treated to
reduce the risk of viral disease transmission
mainly the viruses that have lipid envelope
eg.HIV1 & 2, Hepatitis B&C, HTLV1&2
 Shelf life depends on the storage
temperature—
◦ room temp----3yrs
Indications
 5% albumin & PPF
◦ Blood volume expansion & colloid replacement
◦ Hypoproteinemia following burn & extensive surgery
◦ The replacement fluid in therapeutic plasma
exchange
◦ Hemorrhagic hypovolemic shock
◦ Retroperitoneal surgery in which large vol. of protein
rich fluid may pool in bowel
◦ 25% albumin
 Severe Hypoproteinemia in acute nephrotic syndrome & acute liver
disease
 Hyperbillirubinemia in the new born
 Toxemia in pregnancy
Adverse Effect & Contraindication
• Adverse effects
– Urticaria and anaphylactoid reactions
– Circulatory overload
– Febrile reactions
– Hypotension due to vasoactive substances in
plasma
– Contraindications
• Hypoproteinemia in malnutrition
• Chronic Nephrotic syndrome
• Cirrhosis of liver
Factor VIII Concentrate
 Preparations available
◦
◦
◦
Factor viii prepared from large pools of
plasma is sterile , lyophilized
Commercially prepared by recombinant DNA
technology
Storage

Freeze dried products are stored at 2-60C

Indications
◦
◦
◦
Hemophilia A
Hemophilia A with low levels of inhibitors of factor viii
Von-Willibrand disease
Immunoglobulin preparation
• Immunoglobulin for IM use:
A concentrated solution of the IgG
component of plasma prepared from large
pools plasma of donors containing
antibodies against infectious agents
• Indication
– Congenital Hypogammaglobinemia
– Persons exposed to diseases like Hepatitis A or
Measles
• Immunoglobulin for IV use:
– Indications
• Idiopathic autoimmune thrombocytopenic purpura
• Treatment of immune deficiency states
• Hypogammaglobinemia
• Myasthenia gravis
• HIV related disease
Contd.
• Hyper immune Globulin
Used for prevention of diseases like Hepatitis B,
Varicella Zoster, Rabies, mumps & others
• Anti-Rh (D) Immunoglobulin (anti-D RHIG)
prepared from plasma containing high level of AntiRh D antibody from previously immunized persons.
Indication: To prevent Rh (D) negative mother from Rh
immunization
who is pregnant with Rh. (D) positive infant.
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