Lecture #13: STORAGE OF BLOOD

The relationship between glucose metabolism and energy production is of prime importance in blood storage. Blood should be preserved in such a way that will provide glucose levels for conversion to ATP and conditions that interfere with or inhibit this glycolytic pathway will be deleterious to cell survival.

Functions of glucose in red cell metabolism

1.      Maintenance of cellular shape and membrane integrity

2.      Transport of ions across the cell membrane

3.      Gaseous exchange

4.      Maintenance of hemoglobin on reduced form

Metabolism of glucose

1.      Embden–Meyerhoff glycolytic pathway – where two moles of ATP and one mole of lactic acid is being obtained from each mole of glucose.

2.      Hexose–monophosphate shunt – metabolizes the remainder of the glucose, converting it to carbon dioxide and generating energy in the form of NADPH.

The anticoagulants routinely used in blood banks include glucose in their composition as well as chelating agents to bind calcium ions, so inhibiting coagulation. The glucose levels are such that the red cells can continue to generate ATP through glycolytic metabolism. Maintenance of ATP levels correlates viability during storage.

An anticoagulant is a substance which acts to prevent clotting of red cells. The ideal anticoagulant is one which can preserve blood elements for a long duration of time and at the same time does not alter its functions.

Types of anticoagulant used in Blood Bank

1.      Acid–citrate–dextrose (ACD)

2.      Citrate–phosphate–dextrose (CPD)

3.      Citrate–phosphate–dextrose–adenine (CPD–A1 and CPD–A2)

4.      Heparin

Anticoagulant–preservative additive solution

1.      Adsol (AS–1 )

2.      Nutricel (AS–2)

I.                   Acid Citrate Dextrose

II.                Citrate Phosphate Dextrose

Composition

a.       Trisodium citrate                                    26.3 grams

b.      Citric acid                                                 3.27 grams

c.       Sodium dihydrogen

Phosphate (monohydrate)                    2.22 grams

d.      Dextrose                                                  25.5 grams

e.       Distilled water                                         1000 ml

Amount of anticoagulant used

per bottle of blood                                        63/450 ml

Volume per 100 ml blood                            14 ml

Shelf–life                                                        28 days

III.             Heparin

For extra corporal and exchange transfusion. It has the advantage of being less toxic

Composition:                         2, 250 units in 30 ml buffered saline

Amount needed:        50 ml / 500 ml blood

Volume per 100 ml blood:   6 ml

Shelf–life:       24 hours. If not used within 24 hours, it may be transferred to ACD

                        Solution by means of a closed system and kept normal storage

                        periods

IV.              EDTA (Ethylene diamine tetraacetic acid)

For preparation of platelet concentrate

Composition:                         3 grams disodium EDTA in 100 ml of 7% saline

Shelf–life:                   24 hours

V.                 Ion exchange resin

Selectively removes Calcium ions from the blood as it is being taken into a container. Recommended for massive blood transfusion

Anticoagulant restriction

            Donor’s weight

            ---------------------          x          450     =          amount of blood to be collected (ml)

                      110

            Amount of blood collected

            ----------------------------------      x          volume per 100 ml   =          amount of

                        100                                                     of blood                      anticoagulant

                                                                                                                               (ml)

Storage of erythrocyte in the liquid state

1.     Criteria

a.       Erythrocytes which are infused into a recipient must be viable and functioning properly.

b.      ATP plays an important role in the glycolytic process of erythrocytes.

c.       2,3 DPG has an important role in erythrocytic capability of releasing oxygen.

2.     Temperatures

The metabolic process of erythrocytes is retarded at low temperatures.

a.       For liquid storage:          1 – 6^oC

b.      For transportation:         1 – 10^oC  

c.       For frozen storage:         –65 to –95^oC for electric freezers

–150 to –196^oC for liquid nitrogen  

Storage of blood in the frozen state

In order to keep erythrocytes on the frozen state, cryoprotective agent must be added to prevent injury from freezing and thawing. These are called Endocellular Cryophylactic Agents (ECA).

There are two types of cryoprotectives:

a.       Intracellular (penetrating)

1.      Glycerol

2.      DMSO (dimethyl sulphoxide)

b.      Extracellular (non–penetrating)

1.      HES (hydroxyethyl starch)

Currently, only glycerol is used to keep erythrocytes in frozen state from hemotherapy. High concentration of glycerol, about 40–47% is required for cells kept in a electric freezer, while low concentrations of glycerol, about 14–17% are required for cells kept in liquid nitrogen.

Labels for blood, components and samples

Basic labels for containers of blood or blood components and stick on labels to be applied after processing or alteration of contents may be designed to meet individual needs. The label must include all the required information. Remember that the prevention of labeling errors is very essential.

            The following color code is used to differentiate the ABO group labels

            Blood Bag’s Color Code

           

                        Blood Group O          =          blue

                        Blood Group A          =          yellow

                        Blood Group B          =          pink

                        Blood Group AB        =          white

            Typing sera reagent color code

                        Anti – A                       =          blue

                        Anti – B                       =          yellow

                        Anti – AB                    =          white  

At the time of collection, the following information shall appear in clear, readable letter on a label firmly attached to the container:

1.      Name of product

2.      Amount of blood collected

3.      Kind and amount of anticoagulant

4.      Donor number

5.      Required storage temperature

6.      Expiration and/or collection date

7.      Name and address of the blood bank

Transportation of Blood

1.      Blood should be shipped in insulated containers that maintain 1–10^oC temperature for at least 18 hours.

2.      Ice or other cooling device should not physically be in contact with blood unit.

3.      A temperature indicator should be placed in the container to ensure that the blood temperature has not exceeded the transportation limits.

Reissue of blood

Blood can be reissued after returning from the ward if the following conditions are met:

1.      The closure must not have been entered in anyway.

2.      The blood must have been kept between 1–10^oC

3.      The pilot tube or sealed segment of the donor tube must still be attached to the container

4.      Records must be available that verify all inspection criteria

Blood storage

1.      Blood bank refrigerator should contain only blood, components, reagents and patient and donor blood samples.

2.      The temperature of the refrigerator must be between 1^o and 6^oC. There should be a fan that circulates cool air to maintain an even temperature throughout the interior.

3.      Separate shelves should be clearly designated and labeled for unprocessed blood, labeled blood, crossmatched blood and outdated or quarantined blood.

4.      A recording thermometer should be present that also has an audible alarm when abnormal temperatures occur.

5.      All units of blood should be inspected daily and prior to issue. Blood should be rejected if the color or other physical appearances are abnormal. Contamination should be suspected if the red cell mass is purple, if hemolyzed, if clots are seen or if plasma is very cloudy or discolored.

Changes in stored blood with anticoagulant

                                                            

 With CPDA–1 with 35 days storage

                                                                        Whole Blood              Red Cells

Plasma hemoglobin (mg/dl)                        46                                658

Plasma potassium (mEq/L)                         27.3                             78.5

2,3 DPG (% of initial value)                          5                                  3

ATP (% of initial value)                                57                                45

Changes in stored blood component

1.     Red Blood Cells

a.       Shape becomes spherical with loss of membrane lipids and increase in cellular rigidity. (Storage at 4^oC halts glycolysis which is responsible for the low production of ATP).

b.      Low 2,3 diphosphoglycerate (DPG) level in the red blood cells. Red blood cells will have a greater affinity for oxygen and require lower PO₂ to release oxygen to the tissues. Transfusion of large volumes of stored with low 2,3 DPG may cause transient hypoxia.

2.     Platelets

Platelets in blood stored at 2^oC have a less satisfactory post transfusion survival but are hemostatically effective. Platelets stored at 22^oC will be viable, but may not resume function on effectivity until after 24 hours after transfusion. In both cases, the shelf life of platelet is 72 hours.

3.     White Blood Cells

a.       Granulocytes survive for 2 days

b.      Lymphocytes survive for 17 – 21 days

c.       Microaggregates of cellular debris from senescent leukocytes can cause transfusion problems.

4.     Coagulation Factors

a.       Factors I, II, VII, IX, X – unaltered

b.      Factor VIII – easily activated by contact and will trigger the intrinsic system to form thromboplastin. When a patient’s liver is not functioning well, clearance of thrombin may not be accomplished and activation of the hemostatic system can occur.

5.     Hyperkalemia

Plasma may contain up to 10 mEq/L of potassium after 10 days, 20 mEq/L after 14 days and 30 mEq/L after 28 days. This is dangerous to infants and adults who already have a high potassium level. Hyperkalemia may cause cardiac arrhythmia.

6.     Contaminations

a.       Di–2–ethylehexylpthalate (DEHF) and acetyl–tri–n–butyl citrate which are components of polyvinyl chloride, the soft plastic used to make blood bags may contaminate blood. DEHF accumulates more in ACD than in CPD.

b.      Organisms that have the capacity to grow at a wide range of temperature (4^oC–37^oC) may contaminate blood bank bloods. The following organisms are the one usually isolated: Pseudomonas, Citrobacter, Achromobacter and various enteric organisms.