2. Centrifugation for Blood Component Preparation: Refrigerated

2. It is possible to transfuse blood component as per the need of patient. This avoids the use of other unnecessary component, which could harm the patient.

3. By using blood components, several patients can be treated with the blood from one donor, giving optimal use of every unit of donated blood.

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Thus, blood component transfusion achieves a most effective way of optimum, safe and economical use of blood. The various blood components that can be separated and transfused are shown in Table 39.1.

These components have highly specific and regulated preparation and storage requirements. Blood group compatibility between the components and patients is considered during transfusion. Each component carries the same risk of transfusion transmitted diseases as that of the whole blood. In contrast, plasma derivatives (fractions) such as albumin, immune serum globulins, and concentrated coagulation factors, etc. have more flexible storage requirements and are given without regard to ABO compatibility.

These fractions also carry a decreased risk of transfusion transmitted diseases. Plasma derivatives are prepared by biochemical or other manufacturing conditions in well equipped plasma fractionation laboratory.

Preparation of blood components is possible due to

i. Multiple plastic packs system

ii. Refrigerated centrifuge

iii. Different specific gravity of cellular components

Red cells specific gravity 1.08 – 1.09

Platelet specific gravity 1.03 – 1.04

Plasma specific gravity 1.02-1.03

Due to different specific gravity of cellular components, they can be separated by centrifuging at different centrifugal force in g for different time.

Centrifugation for Blood Component Preparation:

Refrigerated centrifuge, rotor speed and duration of spin are critical in preparing components by centrifugation.

Each centrifuge should be calibrated for optimum speeds and times of spin for the preparation of each component; Times given here include only the time of acceleration and its speed, not the deceleration time. The blood components are prepared by centrifuging at different relative centrifugal force in g at different time.

Conversion of relative centrifugal force (RCF) to rpm depends upon the radius of centrifuge rotor. It can be calculated by:

1. Nomogram for computing relative centrifugal force and speed.

2. By any one of the formulae:

(i) Relative centrifugal force in g = 28.38 R (rpm/1000)2

R = radius of centrifuge rotor in inches

(ii) Relative centrifugal force in g = 118 x 10-7 x r x N2

R = radius of centrifuge rotor in cm

N = speed of rotation (rpm)

Relative centrifugal force in g (rcf x g) for preparing components:

Components – Spin (rcf) and Time

Red Cells

Plasma – Heavy spin

Platelet concentrate – 5000 x g for 5 minutes

Croprecipitate

Platelet – Rich Plasma – Light spin

2000 x g for 3 minutes

Calculations:

RCF = 28.38 x R x (RPM/1000)2

Or

RPM = v[RCF/(28.38xR)] x 1000

RCF = Relative centrifugal force (X g)

R = Radius in inches

RPM = Revolutions per minute

Precautions to be observed in preparing components:

In blood collection:

1. Proper donor selection and vein puncture to minimize bacterial contamination and tissue trauma.

2. Correct amount of blood in proper anticoagulant and mixed properly.

3. The blood should be collected in primary bag that has satellite bags attached with integral tubings.

4. 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, platelet concentrate, cryoprecipitate (factor VIII) and cryo-poor plasma. Double bags are used for making red cells and plasma only.

In centrifugation:

1. Opposing cups with blood bag and satellite bags must be equal in weight to avoid irregular wear and tear and eventual breakage. If any abnormal vibration is observed till required speed is attained, stop the centrifuge and check the weight of the opposite cups with bags. Rubber discs should be used for balancing.

2. The bags should be so placed that its broad side faces the outside wall of the cup.

3. Correct speed of centrifugation and time must be maintained.

Centrifuges used for separation are calibrated to produce highest product yield in the shortest time at the lowest possible spin so as to cause the least trauma to each product and at the same time maintaining optimal temperature for component viability.

In a unit of blood, the centrifuged products settle in layers, starting from bottom: red blood cells, white blood cells, and platelet rich plasma.

Whole Blood:

Whole blood contains 450 + 45 ml or 350 + 35 ml of donor blood plus anticoagulant solution. The name of the anticoagulant is used with the name of the product, e.g. CPDA

1. Whole blood:

Whole blood has a haematocrit of 30-40%. Minimum 70% of transfused red cells should survive in the recipient’s circulation 24 hours after transfusion. Stored blood has no functional platelets and no labile coagulation factors V and VIII.

2. Red cells:

The red blood cells can be transfused in following different forms.

a. Packed red blood cells:

When red cells are separated from the plasma and used for transfusion, they are called packed red cells.

There is a need to transfuse packed red blood cells in case of— (i) Decreased bone marrow production like aplastic anaemia and leukaemia, (ii) Decreased red cell survival—like haemolytic anaemia and thalassaemia. (iii) In bleeding patients—surgical or traumatic bleeding. The packed red cell transfusion is specially used in cases with congested cardiac failure to reduce the load on the heart by reducing the volume.

Also the packed red cell transfusion lessens severity and incidence of allergic reactions. It is proved that, packed cells are equally effective as whole blood when used for transfusion in elective surgery, saving the plasma for fractionation.

In case of emergency, or group specific shortage, when O blood group is used, it is advantageous to use packed cells, as the plasma with antibody is removed, minimising the risk of haemolytic reactions due to anti-A and anti-B.

The packed red cells are obtained from whole blood either by centrifugation or by gravitational sedimentation, where it is allowed to sediment during storage.

Sedimentation:

The blood after collection is kept upright in refrigerator at 2- 6°C, the red cells settle down and the clear supernatant plasma is transferred into a satellite bag. The sedimented RBCs have 30% plasma and all original leucocytes and platelets.

Centrifugation:

1. Collect appropriate volume of donor blood in CPDA double or triple bag.

2. Store at 2-6sC till processed.

3. Place bags in the buckets of refrigerated centrifuge and balance the opposite bags accurately.

4. Centrifuge at heavy spin (5000 x g) for 5 minutes at 2-6sC.

5. Express approximately ?th of the plasma into the satellite bag.

6. Double seal the tube between primary and satellite bags with plasma. Separate the satellite bag with plasma and keep at – 30eC or below.

7. Keep the red cells at 2-6°C.

8. This sedimented RBCs have 15% plasma and all original leucocytes and platelets.

The increased viscosity of packed red cells may result in significantly reduced infusion rate and a poor cell survival in storage. To reduce the viscosity and also prolong the red cell shelf life, circle packs with additive systems (SAGM, ADSOL, etc.) or addition of 50-100 ml normal saline just prior to the transfusion may be helpful.

From a single unit of packed cells, the haemoglobin is raised by about 1.0 g/dl and Hct by about 3% of total pre-transfusion Hct value. Provided patient is not bleeding from any site.

b. Saline washed red cells:

The saline washed RBCs are free of almost all traces of plasma, most WBCs and platelets. They are generally given to the patients who have severe reactions to plasma – like severe allergies, paroxysmal nocturnal haemoglobinuria or IgA immunisation.

To prepare this, red cells are first separated without the buffy coat and then washed with saline in order to remove most of the remaining white cells. Washing of the cells can be done manually or with the use of machines (haemonetic cell washing machine). The packed red cell should be diluted and mixed with saline, centrifuged and the saline supernatant is removed.

The procedure is repeated for three times. This procedure removes 70 to 95% of leucocytes and effectively removes plasma proteins and micro- aggregates. The washed red cells have to be used within 24 hours. The process is done with aseptic precautions under laminar flow.

c. Leucocyte poor red cells (WBC depleted RBCs):

The leucocyte poor red cell transfusion is needed in case of multi-transfused and multiparous patients like thalassaemia, leukaemia, aplastic anaemia, and immune-suppressed or immune-deficient patients.

These patients get a febrile reaction with any transfusion of packed cells and hence require a leucocyte poor red cell preparation. This implies that nearly 70% of leucocytes are removed and over 70% of red cells are retained.

This can be prepared by inverted centrifugation, washing or freezing cells and by use of leucocyte filters (of pore size 20-40 micron). These filters can remove approximately 99.99% of WBCs with little loss of RBC. They are indicated for patients who have experienced nonhaemolytic febrile transfusion reactions, or for exchange transfusion.

The inverted centrifugation is also simple and helps to reduce the number of leucocytes by avoiding the buffy coat from the packed cell preparation.

In this method the red cells are centrifuged in an inverted position, so that the red cells can be transferred into a satellite bag, leaving buffy coat, some red cells and plasma in primary bag. This method reduces leucocytes by 70-80% and sacrifices 20% RBCs. It is also laborious method; therefore it is mostly replaced by filters.

The leucoreduction can be done at three different points:

i. Pre-storage leucoreduction.

ii. After storage leucoreduction in blood bank, before issue.

iii. Bed side Alteration.

d. Frozen cells:

Frozen cells improve cell survival and increase shelf life to 5 years and above. They are useful in storage of rare blood group cells and blood for autologous transfusion. Frozen cells prevent alloimmunisation.

The disadvantage is its enormous cost, and the time and labour involved. For freezing, red cells are used within 6 days of collection. The widely used high glycerol technique employs a final glycerol concentration of 40% w/v. To a standard unit of packed cells -100 ml glycerol is added at room temperature with constant mixing.

After 5 minutes equilibration another 200 ml glycerol is added. The unit is kept in metal or cardboard canister and stored at – 65°C or lower. The storage period for routine use is limited to 3 years. For use, the unit is thawed at 37°C in a waterbath with agitation.

12% NaCl solution is added and mixed for 5 minutes, and then NaCl in the descending grades is used for washing cells.

The final cell concentrate is suspended in 0.9% NaCl with 0.2% dextrose. This deglycerolised unit has a shelf life of only 24 hours.

e. Neocytes:

Neocytes are the young red cells. The transfusion of these is desirable in young patients with severe chronic anaemia (e.g. thalassaemia patients) who require repeated transfusion. Each ml of RBC contains about 1 mg of iron, which theoretically can be deposited in tissues and cause haemosiderosis.

If neocytes, with an average 90-day lifespan, are transfused instead of conventional RBC, with an average 60-day lifespan, RBC transfusion requirements and the chance of inducing haemosiderosis may be reduced.

The younger cells are larger and less dense and hence the collection of the upper half of the unit may contain more of younger cells. This can be procured more effectively with the use of a cell separator machine. Because of the high cost of neocyte preparation and the time consuming laborious procedure the initial enthusiasm is now dwindling.

Platelet Transfusion:

Platelets are primarily important to maintain normal haemostasis. The need of platelet transfusion is:

Indications for platelet transfusion when:

i. Platelet count is < 5000/?1 regardless of clinical condition.

ii. Platelet count is 5000-10,000/ ?1, if there is increased risk of bleeding due to haematological malignancies, sepsis, severe aplastic anaemia or patient undergoing bone marrow transplant.

iii. Platelet count is 10,000-20,000 ?1, if thrombocytopenic bleeding is present.

Thrombocytopenic bleeding (microvascular bleeding) is:

– Bleeding from the mucous membrane

– Oozing from the surgical incision

– Bleeding from the venepuncture site

– Scattered petechiae

– Ecchymoses

iv. Chemotherapy for malignancy (decreased production), if platelet count < 20,000/ ?1

v. DIC (increased destruction), if platelet count <_50,000/ ?1

vi. Massive transfusion (platelet dilution), if platelet count < 50,000/ ?1

vii. In major surgery if the platelet count is < 70-80,000/ ?1

viii. In addition, platelets are indicated prophylactically for patients who have platelet count < 20,000 ?1 to prevent bleeding.

Two platelet products are available for transfusion:

Platelets obtained from whole blood, known as Platelets or random donor platelets, and platelets obtained by apheresis, known as Platelets, pheresis or single-donor platelets. Random donor platelets are whole-blood-derived platelets obtained by centrifugation of whole blood within 6-8 hours after collection.

The blood is subjected to a low centrifugal force (light spin – 2000 x g for 3 min.) at room temperature (20 – 22°C) to obtain platelet-rich plasma. This plasma is then separated from the red blood cells in a satellite bag and the unit is recentrifuged at a high centrifugal rate (heavy spin – 5000 x g for 5 min.) to concentrate the platelets.

The majority of the plasma is expressed into a separate bag (platelet poor plasma), leaving a residual volume of approximately 50 ml plasma in which to resuspend the concentrated platelets (platelet rich plasma).

Platelets may also be prepared after centrifugation to create a buffy coat containing the platelets, leukocytes, and some red blood cells, a method used mainly in Europe. The buffy coat is then centrifuged at a low centrifugal force to concentrate the platelets. Store platelets at 20-22°C under constant agitation, in platelet incubator with agitator, till used.

A random donor platelet preparation should contain at least 5.5 x 1010 platelets per 50 ml unit and have a pH of 6.0 or higher. The shelf life of this product is currently 5 days. One unit of platelet concentrate should elevate the platelet count by approximately 5000 to 10,000/ (mu) L in a recipient weighing 70 kg.

Platelet pheresis:

The advent of apheresis instruments has facilitated the collection of a product from a single donor. Platelets pheresis yields a product equivalent to 10 random units.

Platelets collected by pheresis should contain at least 3.0 x 1011 platelets suspended in approximately 300 ml plasma and are stored in the same manner and have the same shelf life as random donor platelets, provided the integrity of the system has not been compromised.

Calculation of Platelet Yield:

Number of platelet in whole blood = Platelet per mm3 x 1000 x volume of whole blood (ml)

Number of platelet in PRP = Platelet per mm3 x 1000 x volume of PRP (ml)

Number of platelet in PC = Platelet per mm3 x 1000 x volume of PC (ml)

Calculation:

% of platelets yield in PRP = Number of platelet in PRP x 100 / Number of platelet in whole blood

% of platelets yield in PC = Number of platelet in PC x 100 / Number of platelet in PRP

3. Fresh frozen plasma (FFP):

Fresh frozen plasma is an unconcentrated source of all clotting factors except platelets. It is prepared from whole blood and is frozen within 6-8 hours of collection.

Indications:

i. Correction of bleeding secondary to factor deficiencies for which specific factor replacements are unavailable,

ii. Multifactor deficiency states (e.g., massive transfusion, disseminated intravascular coagulation [DIC], liver failure) and

iii. Urgent warfarin reversal.

FFP can supplement RBCs when whole blood is unavailable for exchange transfusion. FFP should not be used simply for volume expansion.

Procedure:

To prepare fresh frozen plasma, centrifuge the whole blood collected with anticoagulant at heavy spin (5000 x g for 5 min.) at 4oC. Remove about 2/3 rd volume of plasma into the satellite bag. Seal the tube and separate the bag.

The bag should be rapidly frozen (within 1 hr.) It can be achieved by spreading the plasma in a thin layer (bags laid flat not vertical) in freezer at – 70oC or placing the bags protected by plastic over wrap at – 70oC in ethanol dry ice bath. Once frozen, it can be stored at or below – 30oC and can be used up to one year.

The formation of cryoprecipitate (containing factor VII 1 c, fibrinogen and fibronectin) should be avoided during thawing as it would reduce the expected clotting properties.

Thaw it in a plasma defroster (microwave) or place the bag in a plastic over wrap and put in a 37°C circulating water bath (the entry ports of the bag should remain above the water).

The FFP should be administered as soon as possible after thawing, and in any event within 12 hrs if kept at 2-6oC. The most common dosage of FFP is 10 ml/kg of body weight.

4. 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 hours of collection. It is rich in factor VIII, von-Willebrand factor, fibrinogen (Factor XIII) and fibronectin.

Indications:

i. For haemophilia and von Willebrand’s disease

ii. Cryoprecipitate is currently used as a source of fibrinogen in acute DIC with bleeding

iii. Treatment of uremic bleeding

iv. Cardiothoracic surgery (fibrin glue)

v. Obstetric emergencies such as abruptio placentae and HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome

vi. Rare factor XIII deficiency.

Several factors which improve the yield of factors VIII in cryoprecipitate are:

1. Clean, single vein puncture at the first attempt.

2. Rapid flow of blood, donation of blood (450 ml) obtained in less than 8-10 minutes 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 hours after collection as done for preparing FFP.

5. Rapid thaw at 4°C in circulating water bath.

6. Use of siphon technique which prevents thawed plasma remaining in contact with the cryoprecipitate.

Procedure:

1. Prepare fresh frozen plasma (FFP), as described under FFP, for processing into cryoprecipitate.

2. Freeze the plasma at – 70°C in freezer or in ethanol dry bath.

3. Thaw frozen plasma either at 4°C in a cold room (air thaw) or at 4°C in circulating water bath.

i. If FFP is thawed in cold room, hang the bag in an inverted position with ports lower most and place the second satellite bag on a lower shelf. Observe the pack frequently to make sure the thawed plasma is flowing in to the satellite bag and not accumulating in the primary bag. When 10-15 ml of plasma remain with cryoprecipitate seal the tubing and separate bags.

ii. If FFP is thawed in 4°C water bath, centrifuge the bag when the plasma is slushy at 5000 x g for 5 minutes at 4°C. Then supernatant cryo-poor plasma is siphoned out in the satellite bag, leaving 10-15 ml plasma with cryoprecipitate. Seal the tubing and separate the bags. Label bags.

4. Store the bag with cryoprecipitate at 30°C or Lower and bag with cryo-poor plasma in the second satellite bag is stored at 20°C or below.

Storage and shelf life of cryoprecipitate:

One year at – 30°C or below.

Reconstituting Cryoprecipitate:

(Thawing and issue of cryoprecipitate)

Reconstitute cryoprecipitate before issue by placing in an over wrap in a 30°C water bath until the cryoprecipitate has dissolved. Cryoprecipitate should be re-suspended thoroughly by gentle kneading.

After thawing pool the cryoprecipitate from all thawed bags into one bag under laminar flow by means of bag-to-bag connector. Wash the empty bags with 10 ml of normal saline to dissolve residual cryoprecipitate and add to pooled cryoprecipitate. Once thawed, cryoprecipitate should be kept at 2-6°C and administered within 4 hours. It should not be frozen.

One bag of cryoprecipitate contains on an average 80-120 units of factor VIII in 15-20 ml of plasma.

5. Granulocytes:

Granulocytes may be transfused when sepsis occurs in a patient with profound persistent neutropenia (WBCs < 500 ?l) who is unresponsive to antibiotics. These can be obtained by centrifugal leucopheresis and filtration leucopheresis. These are stored at room temperature (22- 24°C) and should be used within 24 hours of collection.