Red Blood Cells
» Red Blood Cells is the portion of blood that contains hemoglobin and is derived from human whole blood, from which plasma and platelets are removed by centrifugation, sedimentation, or by apheresis. In the case of apheresis, the plasma is automatically removed and returned directly to the donor. Red Blood Cells derived from whole blood may be prepared at any time during the dating period of the whole blood from which it is derived.
Red Blood Cells may be further processed by addition of red cell preservatives, irradiation to inactivate lymphocytes, filtration for removal of leukocytes, washing to remove proteins, freezing and thawing, or rejuvenation using validated and approved procedures. The source blood for Red Blood Cells must be tested for syphilis, hepatitis B virus, hepatitis C virus, Human T-cell Lymphotropic Virus (HTLV) type I and type II, human immunodeficiency virus (HIV) type 1 and type 2, and unexpected antibodies to red cell antigens using FDA-approved and licensed commercially available test kits, the results of which must be below the limits of detection specified in the respective test kits by the manufacturers. In addition, the source blood must also be tested for hepatitis C and HIV using FDA-approved nucleic acid assays, the results of which must be below the approved limits of detection for the method. A unit (dose) of Red Blood Cells contains a minimum of 50 g of hemoglobin in a total volume of approximately 180 to 325 mL. A unit (dose) of Red Blood Cells, Leukocytes Reduced contains a minimum of 42.5 g of hemoglobin in a total volume of approximately 150 to 275 mL, and has a residual leukocyte count of less than 5 × 106. A unit (dose) of Red Blood Cells, Deglycerolized contains a minimum of 40 g of hemoglobin in a total volume of approximately 180 to 325 mL. A unit (dose) of Red Blood Cells, Leukocytes Reduced and Deglycerolized contains a minimum of 34 g of hemoglobin in a total volume of approximately 180 to 325 mL and has a residual leukocyte count of less than 5 × 106. A unit (dose) of Red Blood Cells, Pheresis contains a mean hemoglobin content of 60 g of hemoglobin. A unit (dose) of Red Blood Cells, Pheresis, Leucocytes Reduced contains a mean hemoglobin content of 51 g (or 153-mL packed red cell volume, and has a residual leukocyte count of less than 5 × 106.
Packaging and storage Collect into an approved container (see Transfusion and Infusion Assemblies 161) containing a sterile, pyrogen-free approved anticoagulant (see Anticoagulant Citrate Dextrose Solution, Anticoagulant Citrate Phosphate Dextrose Solution, or Anticoagulant Citrate Phosphate Dextrose Adenine Solution). An approved additive solution may be added after removal of the plasma. Store Red Blood Cells in the original container, or transfer to an equivalent container using a technique that does not compromise sterility. Liquid Red Blood Cells is stored at a temperature between 1 and 6 (see General Notices and Requirements). Frozen Red Blood Cells is stored at 65 or colder.
Expiration date Red Blood Cells in Anticoagulant Citrate Dextrose Solution, Anticoagulant Citrate Phosphate Dextrose Solution, or in Anticoagulant Citrate Phosphate Dextrose-Dextrose Solution may be stored for up to 21 days at 1 to 6 after the blood has been drawn. Red Blood Cells in Anticoagulant Citrate Phosphate Dextrose Adenine Solution may be stored for up to 35 days at 1 to 6. Red Blood Cells may be stored in an approved additive solution (AS),* for up to 42 days at 1 to 6. If the hermetic seal of the container is broken during collection, preparation, or further processing, the expiration date is not later than 24 hours after the seal is broken (when blood is stored at 1 to 6). The expiration date for frozen Red Blood Cells prepared with low glycerol content (20% glycerol) is not later than 10 years from the date of collection when stored at 120 or colder, except when Red Blood Cells is prepared for freezing with high glycerol content (40% glycerol), in which case it may be stored at 65 or colder for no later than 10 years from date of collection. If the frozen Red Blood Cells is processed for freezing or for thawing, in an open system, the expiration date for the thawed Red Blood Cells is 24 hours after removal from 65 storage, provided it is then stored at the temperature of unfrozen Red Blood Cells.
Labeling Label the container to indicate the volume of the whole human blood collected from the donor, the collection date, the donation number or other coding means to uniquely identify the unit and to provide traceability to the donor, and the expiration date. Label it to indicate the type of anticoagulant used to collect whole human blood and any additive solutions added subsequent to collection. Label it also to identify donor status (i.e., volunteer or paid). Label it also with the following statements: See Circular of Information for indications, contraindications, cautions, and methods of infusion.; Properly identify recipient.; and Caution: Rx only. In addition, label it to indicate the product name as indicated in Table 1. [noteThe name is determined by the method of preparation of the Red Blood Cells (derived from whole human blood or from apheresis) and by performing the necessary testing to ensure that the product meets the minimum requirements for the named products, as indicated in Table 1.]
Table 1. Red Blood Cells Preparations
Label it to indicate, the ABO Group/Rh Type, as indicated in Table 2. [noteEvery Red Blood Cell product must have a determination made as to its ABO Group and Rh Type and specificity of unexpected red cell antibodies, if any.]
If an ABO blood group color scheme is used, use the following labeling color: Group A (yellow), Group B (pink), Group O (blue), and Group AB (white).
Label the Red Blood Cells with names of the adventitious agents tested and the results of the tests. If it has been issued prior to determination of the test results, label it also with a warning Donor Untested and to specify Uncrossmatched Blood, when appropriate.
Table 2. Blood Group and Rh Type
A: ABO blood group
Anti-A reagent Use approved commercially available monoclonal or polyclonal anti-A blood grouping reagent, two different lots from the same or different manufacturers. Use in accordance with manufacturer's instructions.
Anti-B reagent Use approved commercially available monoclonal or polyclonal anti-B blood grouping reagent, two different lots from the same or different manufacturers. Use in accordance with manufacturer's instructions.
Anti-AB reagent Use approved commercially available anti-AB blood grouping reagent. Use in accordance with manufacturer's instructions.
Control preparations On the day of use, dilute Blood Group A1 (Control preparation A1) and Blood Group B (Control preparation B) red blood cells, obtained from an approved commercial source or prepared by the testing laboratory, with 0.9% saline to suspensions of approximately the same concentration between 2% to 5% (v/v). [noteIf the Blood Group A1 and Blood Group B red blood cells are prepared in the testing laboratory from whole blood of a known blood group, it must be prepared on the day of use following the procedure for the Test preparation under Whole Blood.]
Test preparation On the day of use, dilute Red Blood Cells with 0.9% saline to a suspension of about the same concentration as the Control preparations.
Procedure On a suitable U-bottomed microtiter plate, place 1 drop of 0.9% saline in each of three different wells in a row (Blank Row). Place 1 drop from one of the lots of Anti-A reagent in each of three different wells in a second row. Place 1 drop from the second lot of Anti-A reagent in each of three different wells in a third row. Repeat the same with two different lots of Anti-B reagent and one lot of Anti-AB reagent in separate rows. To each row, add 1 drop of Control preparation A1, Control preparation B, and the Test preparation in the first, second, and the third well, respectively, of each row. Mix the contents of the wells by gently tapping the sides of the plate. Centrifuge the plate at the appropriate conditions established for the centrifuge. Resuspend the cell buttons by manually tapping the plate, or with the aid of a suitable mechanical shaker. Read the optical densities at different wells using a suitable automated photometric microtiter plate reader. Compare the optical density of each well in the Blank Row with the optical density of the wells to which the corresponding Control preparation A1, Control preparation B, or Test preparation were added. [noteA high optical density comparable to those obtained for the wells in the Blank Row indicates negative results (no hemagglutination), which can be corroborated by visual observation of smooth suspensions. A low optical density indicates positive results (hemagglutination), which can be corroborated by visual observation of formation of clumps.] The blood group of Red Blood Cells is A, B, AB, or O, accordingly, as the Test preparation is hemagglutinated by Anti-A reagent, Anti-B reagent, both reagents, or neither, respectively. The blood group of the Test preparation conforms to the blood group indicated on the label. The test is not valid if Control preparations for Blood Group A and Blood Group B red blood cells are not agglutinated by Anti-A reagent and Anti-B reagent, respectively, or if both Control preparations are not agglutinated by Anti-AB reagent. The test is also not valid if the Test preparation is not agglutinated by Anti-AB reagent but is agglutinated by either Anti-A reagent or Anti-B reagent, or is agglutinated by Anti-AB reagent but not by either Anti-A reagent or Anti-B reagent.
B: Rh type
Anti-D (Rho) reagent Use anti-D (Rho) blood grouping reagent approved for use in microtiter plate tests. Dilute, if necessary, following the manufacturer's instructions.
Test preparation On the day of use, dilute Red Blood Cells with 0.9% saline to obtain a 2% to 5% (v/v) suspension.
Procedure On a suitable U-bottom microtiter plate, place 1 drop each of 0.9% saline and Anti-D (Rho) reagent in two separate wells. Label them as the B well (Blank) and the T well, respectively. Add 1 drop of Test preparation to each well, and mix by gently tapping the side of the plate. Centrifuge the plate at appropriate conditions established for the centrifuge. Resuspend the cell buttons by manually tapping the plate, or with the aid of a suitable mechanical shaker. Read the optical densities of the wells using a suitable automated photometric microtiter plate reader, and determine if the Red Blood Cells in the T well is agglutinated as described under Identification test A. If the T well indicates negative results (no hemagglutination), incubate the plate at 37 for 15 minutes, centrifuge, resuspend the cells, and read the optical densities of the wells as above. Agglutination of cells after immediate-spin or after 37 incubation indicates Rh positive typing of Red Blood Cells. The test is valid if cells in the B well are not agglutinated. If the cells are not agglutinated in the T well, proceed as directed in Test 2.
Anti-D reagent Use anti-D blood grouping reagent approved for use for a weak D blood group test.
Antihuman globulin reagent Use polyspecific or anti-IgG antihuman globulin reagent. Dilute, if necessary, following the manufacturer's instructions.
Control preparation Use IgG-coated red cells approved for use as a control for Rh typing. Dilute with 0.9% saline to obtain a 2% solution.
Test preparation Prepare as directed for Test 1.
Procedure Place 1 drop of 0.9% saline into a suitable test tube and 1 drop of Anti-D reagent in another, and mark them as the Blank and Anti-D, respectively. To each tube add 1 drop of the Test preparation, mix, and incubate at 37 for 15 to 30 minutes. Centrifuge the tubes at about 1000g for 15 to 30 seconds. Gently resuspend the cell buttons, and examine them for hemagglutination (formation of clump) by visual examination. The Rh typing of Red Blood Cells is positive or negative according to whether the cells in Anti-D tube are agglutinated or not. If the cells are not agglutinated, add 1 mL of 0.9% saline to the Anti-D tube, and resuspend the cells. Centrifuge the tube at about 1000g for 1 minute, and remove the saline completely. Repeat the step two to three times more to wash the Red Blood Cells. Add 1 drop of 0.9% saline and 1 to 2 drops of Antihuman globulin reagent to the Anti-D tube. Mix gently, and centrifuge the tube at about 1000g for 15 to 30 seconds. Gently resuspend the cell button, add 1 drop of Control preparation, mix gently, centrifuge as above, and examine for agglutination. The Rh Type of the Test preparation conforms to the Rh Type indicated on the label. The test is not valid if the cells in the Anti-D tube are not agglutinated after addition of the Control preparation. Also, for the test to be valid, the cells in the Blank tube must not be agglutinated.
Visual inspection Inspect visually during storage and immediately prior to use. If the color or physical appearance is abnormal or there is any indication or suspicion of microbial contamination, the unit is unsuitable for transfusion.
Drabkin's solution Dissolve Drabkin's reagent in a suitable volume of water, and add a suitable volume of a 30% (w/v) polyoxyethylene (23) lauryl ether solution such that the final concentrations of potassium cyanide, potassium ferrocyanide, and polyoxyethylene (23) lauryl ether in the solution are approximately 0.75 mM, 0.6 mM, and 0.015%, respectively. Store the solution in the dark between 18 to 26. [CautionDrabkin's reagent and Drabkin's solution contain cyanide and are HIGHLY TOXIC. Do not inhale or swallow or allow contact with skin or with eyes. Wear suitable protective clothing, gloves, and eye and face protection. Do not mix with acids. Contact with acids liberates a very toxic gas. If ingested, perform gastric lavage, and immediately call a physician. ]
Blank solution: water.
Standard solution A Transfer about 300 mg USP Hemoglobin RS, accurately weighed, to a 2-mL volumetric tube, add 1 mL water, dissolve in and dilute with water to volume, and mix.
Standard solution B Mix 50 µL of Standard solution A with 25 µL of water.
Standard solution C Mix 50 µL of Standard solution A with 100 µL of water.
Test solution Mix 50 µL of Red Blood Cells with 50 µL of water.
Procedure Label suitable tubes as B1, B2, SA1, SA2, SB1, SB2, SC1, SC2, T1, and T2. Add 5.0 mL of Drabkin's solution to each tube. Add 20 µL of water to each of B1 and B2, 20 µL of Standard solution A to each of SA1 and SA2, 20 µL of Standard solution B to each of SB1 and SB2, 20 µL of Standard solution C to each of SC1 and SC2, and 20 µL of Test solution to each of T1 and T2, rinsing the pipet tip three to four times with Drabkin's solution, and mix. Allow to stand for at least 15 minutes at room temperature. [noteRed Blood cells with appreciable carboxyhemoglobin content, such as those obtained from smokers, may require longer reaction time. If the donor characteristics are not known, the incubation time should be optimized prior to testing.] Read the absorbances of the solutions against the solution in tube B1 at 540 nm. The absorbance of the solution in tube B2 is recorded at the end. The test is not valid if the absorbance of the solution in tube B2 is not within ±0.005.
Calculations Calculate the concentrations, in mg per mL, of hemoglobin in Standard solutions A, B, and C. Plot a calibration curve of absorbance values against the hemoglobin concentration by drawing a best-fit straight line using the least-square linear regression analysis. From the absorbance value of the Test solution, obtain the concentration, in mg per mL, of hemoglobin in the Test solution. Multiply the value by 2 to obtain the concentration, in mg per mL, of hemoglobin in Red Blood Cells. Calculate the total hemoglobin content in the Red Blood Cells unit, in g, by the formula:
Conc. of hemoglobin (in mg/mL) × the volume of the Red Blood Cells unit (in mL) /103.
Leukocyte count Pipet 40 µL of a suitable red cell-lysing agent into a clean test tube, add 100 µL of Red Blood Cells diluted with 0.9% saline, if necessary, such that the hematocrit of Red Blood Cells is not greater than 60%. Mix by pipetting up and down several times. Add 360 µL of 0.01% (w/v) crystal violet in 15% (v/v) acetic acid into the mixture, and mix thoroughly. Fit a hemocytometer with a 50-µL counting volume and a bright background, with a cover slip, and load the counting chamber with the mixture until the counting area is completely covered, but not overfull. Cover the counting chamber with a suitable moist lid to prevent evaporation, and allow to settle undisturbed for 10 to 15 minutes. Remove the lid, place the chamber on the stage of a light microscope fitted with 10× ocular lens and 20× objective. Count the leukocytes in the entire 50-µL counting volume. Calculate the leukocyte count in Red Blood Cells, expressed in leukocytes per µL, by dividing the observed leukocyte count by 10. Calculate the total number of leukocytes in the Red Blood Cells unit by using the following formula:
Total leukocytes = leukocytes/µL × 103 × the volume of the Red Blood Cells unit in mL.
Adequacy of deglycerolization (for Red Blood Cells, Deglycerolized) Interrupt the last wash cycle of the deglycerolization process at a point where the wash fluid is visible in the clear tubing segment leading to the waste receptacle. Hold the tubing against a well-lighted, white background. Note the coloration of the fluid in the tubing, and compare it to a suitable hemolysis color comparator standard. The color of the fluid should be no stronger than the block indicating 3% hemolysis. [noteIf the level of hemolysis is more than 3%, continue the wash process, and repeat the test until the color is within acceptable limits.]
* AS contains sodium chloride, dextrose, adenine, and other substances that support red cell survival and function. Examples of such solutions are AS-1, AS-3, and AS-5.
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