1 2 Blood Components & Plasma derivatives Ahmad Sh. Silmi Msc,FIBMS Clinical Hematologist & Immunologist IUG.

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Blood Components

& Plasma derivatives

Ahmad Sh. Silmi Msc,FIBMS

Clinical Hematologist & Immunologist

IUG

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IntroductionWhole 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)

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Anticoagulants in blood units

1) Acid-Citrate-Dextrose (ACD)

2) Citrate-Phosphate- Dextrose (CPD)

3) Citrate,Phosphate,Dextrose,Adenine

(CPDA-1)

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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)

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• (SAGM) → Saline-Adenine-Glucose- Manitol• Purpose of additive solution, to improve

RBCs storage viability till 42 days @ 2-6ºC

* Added only to PRBCs

Additive solutions

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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

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BLOOD COMPONENTS

• Blood separated into different parts:

1. Packed red cells2. Platelets3. Fresh frozen plasma4. Cryoprecipitate5. Granulocytes6. Factor IX conc.7. Factor VIII conc.

• There are more than 20 different products available

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Whole blood

Red cells Plasma Platelets

(Fresh) frozen plasma (FFP)

CryoprecipitateStored Plasma

F lX

Immuno- globulins

Albumin

Fractionated products

F Vlla

F Vlll

Granulocytes

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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

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Blood Components

• Separating WB into components of

blood is necessary to avoid

wasting of units.

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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.

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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)

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Centrifuged blood

Buffy Coat (WBCs & Platelets)

Red Blood Cells

Plasma

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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)

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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.

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1 -PRBCsHow 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)

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Whole Blood Unit

After centrifugation

WB separates into

plasma &

platelets &

PRBCS

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1 2 3

SAGM

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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

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1- Red blood cell concentrates

Whole Blood Red cell concentrate

Total Volume 500 ml 300 ml

Volume of red cells

200 ml 200 ml

Volume of plasma

300 ml 100 ml

Hematocrit 40 % 70 %

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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

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Expiration date!

– Once the PRBC unit is “opened” it has a 24 hour expiration date!

24 hours

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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

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http://www.pall.com/39378_39479.asp

Leukocyte Reduction Filters

(maintains closed system)

Final unit must have less than 5 x 106 WBCs

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3- Frozen-thawed red cells

• Red cells can be frozen with use of cryo-preservation 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

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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.

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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.

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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

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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

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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

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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

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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

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Preparation of platelet concentrate

RBCs PRP

Plasma

Platelet concentrate

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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

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1

2

Then

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Whole blood unitWhole blood unit

Centrifuge using Centrifuge using LIGHTLIGHT spin spin

Express Platelets Rich Plasma (PRP) into satellite bagExpress Platelets Rich Plasma (PRP) into satellite bag

Take PRP and centrifuge again now using Take PRP and centrifuge again now using HEAVYHEAVY spin spin

Express PPP into satellite bag & freeze at -18ºCExpress PPP into satellite bag & freeze at -18ºC

Final products :PRBCs, Platelets concentrate, FFPFinal products :PRBCs, Platelets concentrate, FFP

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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

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C- Plasma Products

• Plt poor plasma can be separated into a

number of products

– Fresh frozen plasma

– Platelet concentrate

– Frozen plasma

– Cryoprecipitate

– Stored plasma

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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

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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.

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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

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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

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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

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1 2 3

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Platelet concentrate

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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

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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

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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

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Cryo..Indications:

Hemophilia A

Von Willebrand disease (VWD)

Congenital or acquired fibrinogen defects (i.e., dysfibrinogenemia)

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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

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SummaryBlood Components

Blood Component

Centrifugation Storage

Temp Time

Indication

1) PRBCs WB Light spin= 2000rpm-20ºC -11min.

PRBCs + PRP

2-6ºC +SAGM 42d •Anemia•Newborn exchange transfusion

2) PC PRP heavy spin= 3500rpm-20ºC -11min.

PC + FFP

R.T 3-5 d •Bleeding •Operation if plt. Less than 20000/μl

3) FFP -18ºC 1year

-65ºC 7 years

•Clotting factor deficiencies•Severe burns

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SummaryBlood Components

Blood Comp

Centrifugation Storage Temp Time

indication

4) Cryo a. WB special heavy spin= 3500rpm at 4ºC -11min. RBC + Plasma

b. Plasma store at -18 ºC then thaw at 4 ºC then heavy spin at 4ºC

-30ºC 1 year • Hemophilia A

• Von Willebrand disease

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Plasma Derivatives

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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

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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.

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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.

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Plasma Derivatives

Plasma Derivatives Preparation avaliable

Coagulation Factors

Factor VIII concentrates

Factor IX concentrates

Anti-thrombin III

AlbuminAlbumin

Plasma protein fraction

Immune globulins

Non-specific immune serum globulin (ISG)

Rh immune globulin (RhIG)

Hepatitis B immune globulin (HBIG)

Varicella-Zoster immune globulin (VZIG)

Tetanus immune globulin (TIG)

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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

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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

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Differences of Cryoprecipitate & Factor VIII concentrates

CryoprecipitateFactor VIII

concentrates

Storage Temp. -30oC4oC

Short period RT

Risk of Hepatitis

Low High

Treatment of hemophilia A

Yes Yes

Treatment of vW disease

Yes no

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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

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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

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Blood products & treatment of specific clotting factor deficiencies

Deficiency Blood product Indicated

FibrinogenCryoprecipitate

Stored plasma

Factor VFresh frozen plasma

Frozen plasma

Factor VIIFactor IX concentrate

Stored plasma

Factor VIIIFactor VIII concentrate

Cryoprecipitate

Von Willebrand’s Disease

Cryoprecipitate

Fresh frozen plasma

Frozen plasma

Factor IX Factor IX concentrate

Factor X Stored plasma

Factor XI Stored plasma

Factor XIII Stored plasma

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2- Oncotic Agents

• Albumin: volume expansion

• Other colloids are available for blood volume expansion– Dextran– Gelatin– Hydroxyethyl starch– Polyvinylpyrrolidone

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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

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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

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Plasma protein Fraction

• Partially purified albumin

• Contains ≈ 85% albumin & 15% other plasma proteins

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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 hypogammaglobulin-emia and agammaglobulinemia.

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Immune Serum Globulin (ISG)

• Primarily IgG Ab

• Prevention of some viral diseases

• Hypogammaglobulinemia

• Congenital immune deficiency

• Given by IM injection (aggregates of IgG)

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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).

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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

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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).

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Tetanus Immune Globulin (TIG)

• Prepared from individuals specifically immunized for tetanus toxoid

• Available for individuals at risk following injury

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• 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 reduced (VIII, V).

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Granulocytes

Lymphocyte Monocyte

Neutrophils Eosinophils Basophils

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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

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