Blood component separation is defined as the fractionation of
fresh whole blood into plasma and cells and their respective elements based on
sequential centrifugation that take advantage of density differences among the
different blood cell types.
Whole blood can provide the following components: red blood cells,
platelets, leukocytes, plasma, albumin, anti–hemophilic factor, fibrinogen,
gamma globulin, factor II, VII, IX, X concentrate and others.
Component therapy is the use of specific blood product to replace
deficient cellular elements.
Advantages of Component Therapy:
1.
Maximum recovery of blood products
2.
Increased service to a wide variety of
patients
3.
Transfusion of specific components needed by
patients at an adequate dose
4.
Elimination of harmful elements in plasma
Equipment used in the collection of blood and
the preparation of blood components:
1.
Blood pack unit
Sterile and pyrogen free bottles
or bag containing anticoagulant. A pilot tube which may be used for typing and
cross–matching purposes is usually attached to it.
2.
Multiple pack unit or Quadruple
blood pack unit
Composed of one large box for
blood collection and three small bags (satellite) connected to it through
plastic tubings. The satellites are used for transferring smaller amounts of
blood and for the preparation of blood components.
3.
Ion exchange blood pack unit
The blood pack is attached to an
ion exchange column through the blood donation flows and in which calcium is
removed.
4.
Leuko–pak filter
Contains nylon fibers for removal
of leukocytes from freshly collected heparinized blood or collected through ion
exchange resin.
5.
Plasma extractor
Removes plasma without disturbing
the cell pack
6.
Platelet mixer
7.
Refrigerated centrifuge
Especially designed for centrifugation of blood in plastic bags or
bottles for the
preparation of blood components. The number of revolution per
minutes and temperature are both adjustable to suit the need of the components
to be prepared.
8.
Needle (gauge 18)
Components of whole blood
I. Whole Blood
A.
Packed Red Blood Cell
B.
Fresh Plasma
1.
Platelet–rich plasma
2.
Platelet–poor plasma
a.
Cryoprecipitate (Factor VII, Fibrinogen)
b.
Cryosupernate (Factor II, VII, IX, X)
****** PREPARATION OF DIFFERENT BLOOD
COMPONENTS ******
1.
Whole Blood
a.
Indication:
(1) Replacement
transfusion of infants having hemolytic transfusion of the newborn
(2) Transfusion
to patient having massive replacement therapy secondary to either surgical
insult or to total circulatory replacement (liver disease)
b.
Temperature
(1) If stored: 1
– 6oC
(2) If
transported: 1 – 10oC
c.
Shelf life
(1) With CPD: 21 days
(2) With CPDA–1:
35 days
2.
Packed Red Blood Cell
a.
Indication
(1) Chronic
anemia of any etiology
(2) Pre–op and
pre–delivery transfusion
(3) Anemic
patients with congestive heart failure (low sodium content)
(4) Anemic
patients with liver failure (low NH3 content)
(5) Uremic
patients (low potassium content)
(6) Bone marrow
failure
(7) Acute
massive burns
(8) Replacement
of blood loss
b.
Preparation
Spinning the whole blood and
removing 2/3 of its plasma. It has a hematocrit of 60 – 70%.
c.
Speed:
1800 rpm for 4 minutes
d.
Temperature: 20oC
e.
Shelf life:
ACD 21 days
CPD 28 days
2 – 6oC on horizontal
position
3.
Washed Red Blood Cell
a.
Indication
(1) Paroxysmal
nocturnal hemoglobinuria
(2) Autoimmune
hemolytic anemia
(3) Hypersensitivity
to plasma proteins
b.
Preparation
Packed RBC washed three times
with sterile saline solution
c.
Shelf – life: 24
hours
4.
Frozen deglycerolized RBC
A unit of anticoagulated whole
blood less than 5 days old is centrifuged at approximately 4400 rpm for 6 to 7
minutes. The supernatant plasma is removed with a plasma extractor and replaced
with a solution of 45% glycerol. The thoroughly mixed glycerolized red cells
are then transferred to a suitable freezer – adapted plastic bag and a flat set
of stainless canister is placed around the plastic bag. The purpose of the
canister is to contain the blood in this specific geometric configuration to
simplify storage and permit maximum surface area exposure during the freezing
process. The entire unit is then placed in a –80oC storage freezer,
where cooling takes place over a period of several hours.
a.
Shelf–life: 10 years at –65oC
b.
Indications
(1) Alloimmunized
patients who have previously experienced non–hemolytic febrile transfusion
reactions due to leukocytes or platelets.
(2) Patient who
are allergic to constituents of plasma proteins, such as those with IgA
deficiencies.
(3) Neonates or
immunocompromised patients in whom the threat of exposure to cytomegalovirus
exist
(4) Patients who
need or desire autologous transfusion
5.
Single donor plasma
Separated from a unit of whole
blood using a plasma extractor. The separation must occur before the fifth day
after the expiration date of the unit of whole blood.
a.
Shelf–life
(1) CPD at 1 – 6oC
(26 days)
(2) CPDA–1 (40
days)
(3) At –18oC
(5 years)
b.
Indications
(1) Severe burns
(2) Hypovolemic
shock
(3) Coagulation
defects
6.
Fresh frozen plasma
Fluid portion of 1 unit of human
blood that has been centrifuged, separated and frozen at 18oC or
colder within 6 hours of collection
a.
Shelf – life
(1) –18oC within 6 hours after
collection
(2) –30oC within 12 months (optimal
storage)
·
If not used within one year of storage at –18oC
or not transfused after storage at 1–6oC for 24 hours, it maybe
redesignated as SINGLE DONOR PLASMA
b.
Indications
(1) Massive
transfusion
(2) Single or
multiple coagulation protein deficiencies, either prophylactically or in
treatment of bleeding.
(3) Warfarin
reversal
(4) Antithrombin
III deficiency
(5) Treatment of
thrombotic thrombocytopenic purpura and immunodeficiencies
7.
Platelet concentrate
Unit of blood collected in triple
packs are to be processed immediately and not more than 4 hours after
collection without intervening refrigeration.
a.
Preparation
(1) Centrifuge
at 2,500 rpm for 3 minutes in a refrigerated centrifuge
(2) Express
platelet rich plasma into first satellite bag
(3) Seal and
separate red cell container. Label plasma properly before returning red cell
container to the refrigerator.
(4) Centrifuge
platelet rich plasma for 5 minutes at 4,000 rpm.
(5) Express all
but 30 to 50 of the plasma into the second satellite bag.
(6) Check
identification, seal and separate between seals.
b.
Shelf–life
48 – 72 hours with constant
agitation
c.
Indication
(1) Active
bleeding due to thrombocytopenia
(2) Cardiovascular
surgery
(3) Surgery with
deficient platelets
(4) Prophylaxis
against bleeding when platelet count is below 20 x 109/L or below.
8.
Cryoprecipitate
Units of blood must be collected
in triple packs with CPD. The unit is collected within 7 minutes to be suitable
for this procedure.
a.
Preparation
(1) Separate red
cells within 4 hours after collection. Centrifuge at 4,000 rpm for 10 minutes
in a refrigerated centrifuge. Express plasma in satellite bag.
(2) Seal and
separate red cell pack. Label properly before returning into the refrigerator.
(3) Freeze
satellite bag at 80oC for 15 minutes or until ready to proceed.
(4) Thaw at 4oC
for 24 hours.
(5) Centrifuge
for 10 minutes at 4,000 rpm.
(6) Express all
but 10 ml into satellite bag. Label should include ABO group.
(7) Seal twice
and separate
(8) Plasma maybe
stored at 4oC. Cryoprecipitate is stored at –30oC or
lower.
b.
Shelf–life: one year
c.
Contents
Factor VIII and fibrinogen
d.
Indications
(1) Hemophilia A
and von Willebrand’s disease
(2) Hypofibrinogenemia
(3) Hemolytic
anemia
(4) Cardiovascular
surgery
9.
Cryosupernate
Prepared from cryoprecipitate
poor plasma and stored at –30oC
a.
Shelf–life: one year
b.
Contents: Factor II, VII, IX, X
c.
Indications
(1) Bleeding
disorders due to lack of above coagulation factors as in liver disease
(2) Plasma
volume expander
10. Leukocyte poor blood
Preparation of leukocyte poor
blood must be done within 24 hours after donation. Collect blood in twin packs
a.
Preparation:
(1) Centrifuge
blood at 3,000 rpm for 5 minutes (blood pack in inverted position)
(2) Express
about 75% of red cells into satellite pack leaving the plasma and buffy coat in
the original container.
b.
Shelf–life: one day
c.
Indications:
(1) Patients
with leukocyte antibodies
(2) Febrile
reactions
(3) Recipients
of tissue transplants
11. Modified whole blood
Prepared by returning to primary
bag (containing the PRBC) the platelet poor plasma. It is considered the same
as whole blood.
12. Fibrinogen
Sterile, freeze dried fraction of
normal plasma which in solution has the property of being converted to fibrin
upon the addition of thrombin. It contains no preservative and stored at 2–10oC.
It has a shelf life of 5 years. It carries a high risk of transmitting serum
hepatitis but is more effective than whole blood transfusion for the treatment
of chronic hyperfibrinogenemia.
13. Serum globulin
It is sterile solution of
globulin containing those antibodies normally present in adult human blood.
Each lot is derived from serum pool of 100 unites. Store between 2–10oC.
Shelf – life: three years
Hyperimmune globulin available
a.
Tetanus immune globulin
b.
Rho immune globulin
14. Serum albumin
Fractionation of human plasma is
carried in different laboratories. It is prepared by varying the ionic strength
of the supernatant left after the other fractions have been removed by altering
its pH to 4.8. This forms a precipitate known as fraction V. By reworking
fraction V, human serum albumin is prepared. From 3 liters of plasma, 200 ml of
25% human serum albumin can usually be prepared.
a.
Clinical uses
(1) Treatment of
severe burns and shock and in exchange of transfusion to infants to assist in
binding unconjugated bilirubin
b.
Storage
(1) Five to ten
years in a liquid state at 5oC and as such shows no evidence of
deterioration
****** OTHER TRANSFUSION
PRACTICES ******
1.
Pheresis
A procedure in which whole blood
is removed from a donor, separated and a portion retained with the remainder
being returned. The retained product may be plasma, platelets or leukocytes and
the associated process is called plasmaphereis, plateletpheresis and
leukapheresis.
2.
Autologous transfusion
Autologous transfusion refers to
procedures for transfusing blood or blood products derived from the recipient’s
own blood. Many hazards of transfusing blood from homologous donors,
particularly hepatitis transmission are thereby avoided. Using conventional
drawing and storage methods, patients may pre–deposit autologous blood products
which are then available to meet future needs. Autologous transfusion may be
the only suitable manner to supply blood products to patients who react
adversely to all homologous blood, to patients of extremely rare blood types,
or to patients who refuse blood from homologous donors because of religious beliefs.
Autologous blood products may also be obtained through salvage of blood lost
during surgery or following trauma (intra–operative salvage). This blood is
collected from the interior of the body by instrumentation and returned to the
patient after filtration. Under no circumstances should this latter procedure
of blood salvage be instituted if the blood is possibly contaminated.
3.
Massive Transfusion
Massive transfusion can be
defined as transfusion of patients’ blood volume during a twelve hour interval.
Effects of massive blood
transfusion
a.
Antigen–antibody reaction
(1) Formation of
immune antibodies by the patient
(2) Reaction
between plasma and red cells of donor’s blood
b.
Pulmonary edema and cardiac arrest due to
circulatory overload (when a patient’s blood volume could not be bought at
normal within 24 hours).
c.
Changes in hemostatic function due
(1) Depression
of coagulation factors
(2) Abnormalities
of platelet function
d.
Biochemical changes such as hypocalcemia, hyperkalemia
and increase in citrate
e.
Hypothermia
f.
Pulmonary dysfunction may result due to
transfused cellular microaggregates
4.
Pediatric Transfusion
Children who are not actively
bleeding should receive red blood cells for the same reason that red blood
cells are superior to whole bloods for adults. If transfusion of small volumes
of blood are to be administered, one donor unit can be collected into a small
multiple container and divided into small volumes needed.
5.
Universal Donor Transfusion
The practice of routine and
indiscriminate transfusing all recipients with Group O blood regardless of
groups is advocated by almost no one.
There are nevertheless, instances
where use of Group O, Rho negative blood, a universal donor is not
only advisable but necessary. The chief indication of use of Group O Rho
negative blood without regard to the recipient’s typing is in case of massive
hemorrhage with attending shock. There is nevertheless an element of risk. The
safety factor is the presence of low titer anti–A and anti–B agglutinins in
Group O blood.
Blood group specific substances A
and B (Witebsky substances) are also used as an aid in the use of universal
donor. In order to neutralize the anti–A and anti–B agglutinins of the Group O
type, purified Group A and B specific substances derived from hog and horse
stomachs respectively are added to the blood. This produce is available in a 10
ml vial for injection into the blood, react immediately prior to transfusion.
However, not all are complete in
agreement on the advisability of injecting the Witebsky substance. Some
immunologist believe that transfusion with O blood with A and B specific
substances added may result in a high immune anti–A or anti–B antibody titer in
subsequent pregnancies.
*********** RED CELL SUBSTITUTES ************
Characteristics of an ideal blood substitutes
1.
It should be non–toxic, non–pyrogenic, non–allergic,
sterile and easy to store at normal temperatures.
2.
Must be equivalent to human blood in
viscosity and osmotic pressure and retained in the vascular system for a
sufficient time to exert the required therapeutic effect
3.
Must be eliminated by normal metabolism or
excretion and must not affect normal hemostasis
4.
Must be relatively cheap to produce and
easily administered
Synthetic red blood cells surrogates
1.
Emulsions of perfluorocarbons
Perfluorocarbons (PFCs) are large
organic compounds in which all the hydrogen atoms have been replaced by
fluorine atoms. They are chemically inert, immiscible in water and not
metabolized. Oxygen transported by a PFC is carried in solution and has approximately
20 times the solubility for oxygen and carbon dioxide as does water, which is
almost three times the oxygen carrying capacity of blood.
The PFC solution Fluosol–DA 20%
has a circulation half–life of about 13 hours and a tissue half–life of 9 days.
Advantages of PFCs:
a.
Contain no antigens; no typing and
crossmatching necessary.
b.
Easily synthesized from readily available
materials.
c.
Free of infectious diseases.
d.
Do not carry carbon monoxide and could
provide oxygen to a carbon monoxide victim until the patient replaces abnormal
cells.
e.
Small particle size enables the suspension to
penetrate occluded vessels in conditions such as cerebral ischemia or
myocardial infarction.
f.
It may be useful as an adjunct to radiation
therapy in inoperable tumors and in angioplasty procedures.
Disadvantages of PFCs:
a.
Potentially cause oxygen toxicity.
b.
Unstable in–vitro – need to be frozen
c.
Retention by the liver and spleen causes pulmonary
hypertension, bronchospasms and cytotoxicity.
d.
Have not been proven beneficial in severely
anemic patients.
e.
The emulsifying agent Pluronic F–68 may cause
a clinical reaction involving the activation of complement. The stability of
the current emulsion limits the shelf – life, even though it can be stored
frozen for up to 1 year.
2.
Stroma–free hemoglobin
This is prepared by slowly lysing
washed red cells with a buffered solution of water, followed by high–speed
centrifugation and micropore filtration. This procedure separates the
fragmented cell membrane into relatively large pieces that can be removed.
Advantages of SFH
a.
Excellent volume expanders
b.
Potential candidate for an emergency
resuscitive fluid.
Disadvantages of SFH:
a.
Toxicity (this can be improved by coupling
the beta chains of hemoglobin through an organic phosphate).
b.
Can activate the complement system.
c.
Short half–life in the circulation (less than
8 hours).
d.
Significant oncotic effect and unacceptably
high affinity for oxygen.
e.
Must be refrigerated or frozen.
3.
Polymerized hemoglobin
Advantages of Poly SFH–P
a.
Relative simplicity of preparation
b.
Approximates the oxygen carrying capacity of
whole blood and has a half-life of 38 hours.
c.
Can be infused without altering the plasma
oncotic pressure
d.
Near normal plasma hemoglobin
e.
Longer intravascular persistence than any
unpolymerized product.
f.
Tissue oxygenation effect is significantly improved
in comparison to stroma– free hemoglobin.
Disadvantage of PolySFH–P
a.
A higher affinity for oxygen than
erythrocytes
b.
A higher content of non–functional
methemoglobin
c.
Nephrotoxicity
d.
May be immunogenic
4.
Liposome–encapsulated hemoglobin
This can be done with the use of
liposomes which can be prepare from a single phospholipid, a mixture of
phospholipids or mixtures of phospholipids and neutral lipids, for example,
phosphatidylcholine plus cholesterol. A 10 g/dl quantity of hemoglobin
encapsulated in an artificial cell results in an intracellular environment
comparable to that of normal red blood cells.
Another way of encapsulating
hemoglobin is with the use of neohemocytes, which are microcapsules containing
purified human hemoglobin and 2,3– diphosphoglycerate. The size range (0.1 –
1.0 um) is small enough to allow free passage through capillaries. With this
specification, they are qualified as prototype artificial cells.
Specifications for prototype
cells
a.
The microcapsule membrane must be
biodegradable and physiologically compatible
b.
The encapsulation process must avoid
significant hemoglobin degradation.
c.
The oxygen affinity of hemoglobin must be
reduced relative to that of free hemoglobin.
d.
The encapsulated hemoglobin must be
sufficiently concentrated (more than 33% of that in erythrocytes)
e.
There should be no evidence of overt
intravascular coagulopathy
f.
The artificial cells must be small enough to
pass unrestricted through normal capillaries.
Overall clearance process of
neohemocytes
a.
Clearance resulting from irreversible binding
to tissues followed by breakdown
b.
Clearance caused by rapid or early
destruction or breakdown
c.
Clearance from uptake by mononuclear
phagocytic system
Advantages of encapsulated
hemoglobin
a.
Reduced permeability and rate of aggregation
b.
Increased resistance to hydrodynamic shear
and chemical disruption
c.
Apparent thromboresistance
d.
Substantially smaller than erythrocytes
Disadvantage of encapsulated
hemoglobin
a.
Toxicity
b.
Stability
c.
Storage
d.
Questionable cost–effectiveness
Ideal biophysical criteria for
artificial red cells
a.
Adequate oxygen carrying capacity and
appropriate oxygen affinity
b.
Rapid gas exchange
c.
Satisfactory circulating half–life and
thromboresistance
d.
Biologic inertness (low pathogenic potential)
e.
Chemical and physical stability
f.
Satisfactory viscosity
g.
Low immunogenicity
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