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Red Blood Cells
DEFINITION
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.
IDENTIFICATION
• 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% and 5% (v/v). [Note—If 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 according to the following procedure: centrifuge at 4
a suitable volume of Whole Blood at 5000 g for 5 min. Remove the plasma from the top, taking care not to disturb the pellet of red blood cells at the bottom. Add 0.9% saline to obtain a final volume that is about equal to the volume of Whole Blood. Resuspend the pellet, and centrifuge as above. Repeat the procedure once more. Dilute the red blood cells with 0.9% saline to a suspension to obtain a concentration of red blood cells that is about the same as those of the Control preparations. ]
a suitable volume of Whole Blood at 5000 g for 5 min. Remove the plasma from the top, taking care not to disturb the pellet of red blood cells at the bottom. Add 0.9% saline to obtain a final volume that is about equal to the volume of Whole Blood. Resuspend the pellet, and centrifuge as above. Repeat the procedure once more. Dilute the red blood cells with 0.9% saline to a suspension to obtain a concentration of red blood cells that is about the same as those of the Control preparations. ]
Sample 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.
Analysis
Samples:
Control preparations and Sample preparations
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 Sample 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 Sample preparation were added. [Note—A 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 Sample preparation is hemagglutinated by Anti-A reagent, Anti-B reagent, both reagents, or neither, respectively. The blood group of the Sample 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 Sample 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
Test 1
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.
Sample preparation:
On the day of use, dilute Red Blood Cells with 0.9% saline to obtain a 2% to 5% suspension.
Analysis:
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 Sample 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 are agglutinated as described under Identification test A. If the T well indicates negative results (no hemagglutination), incubate the plate at 37 for 15 min, 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.
for 15 min, 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.
Test 2
Anti-D reagent:
Use an 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.
Sample preparation:
Prepare as directed for Test 1.
Analysis:
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 Sample preparation, mix, and incubate at 37 for 15 to 30 min. Centrifuge the tubes at about 1000 g for 15 to 30 s. 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 1000 g for 1 min, 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 1000 g for 15 to 30 s. 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 Sample 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 adding the Control preparation. Also, for the test to be valid, the cells in the Blank tube must not be agglutinated.
for 15 to 30 min. Centrifuge the tubes at about 1000 g for 15 to 30 s. 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 1000 g for 1 min, 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 1000 g for 15 to 30 s. 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 Sample 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 adding the Control preparation. Also, for the test to be valid, the cells in the Blank tube must not be agglutinated.
SPECIFIC TESTS
• 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.
• Hemoglobin Content
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 and 26.
and 26.
[Caution–Drabkin's reagent and Drabkin's solution contain cyanide and are HIGHLY TOXIC. Do not inhale, 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 of USP Hemoglobin RS, accurately weighed, to a 2-mL volumetric tube. Add 1 mL of water, then 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.
Analysis:
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 min at room temperature. [Note—Red 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/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/mL, of hemoglobin in the Test solution. Multiply the value by 2 to obtain the concentration, in mg/mL, of hemoglobin in Red Blood Cells.
Calculate the total hemoglobin content in the Red Blood Cells unit, in g, using the formula:
Result = Conc. of hemoglobin (mg/mL) × the volume of the Red Blood Cells unit (mL)/103
• Leukocyte Count
Sample preparation:
Pipet 40 µL of a suitable red cell-lysing agent into a clean test tube, and add 100 µL of Red Blood Cells diluted with 0.9% saline, if necessary, such that the hematocrit of Red Blood Cells is NMT 60%. Mix by pipetting up and down several times.
Analysis:
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 10–15 min. Remove the lid, and 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/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 (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. [Note—If the level of hemolysis is more than 3%, continue the wash process, and repeat the test until the color is within acceptable limits. ]
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Collect into an approved container (see Transfusion and Infusion Assemblies and Similar Medical Devices 
161
) containing a sterile, pyrogen-free, approved anticoagulant (see Anticoagulant Citrate Dextrose Solution, Anticoagulant Citrate Phosphate Dextrose, 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 are stored at a temperature between 1 and 6 (see General Notices and Requirements). Frozen Red Blood Cells are stored at 
65 or colder.
161) containing a sterile, pyrogen-free, approved anticoagulant (see Anticoagulant Citrate Dextrose Solution, Anticoagulant Citrate Phosphate Dextrose, 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 are stored at a temperature between 1 and 6 (see General Notices and Requirements). Frozen Red Blood Cells are stored at 65 or colder.
• Expiration Date:
Red Blood Cells in Anticoagulant Citrate Dextrose Solution, in 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 1to 6. Red Blood Cells may be stored in an approved additive solution (AS)1 , for up to 42 day at 1to 6. If the hermetic seal of the container is broken during collection, preparation, or further processing, the expiration date is not later than 24 h after the seal is broken (when blood is stored at 1to 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 are 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 are processed for freezing or for thawing, in an open system, the expiration date for the thawed Red Blood Cells is 24 h after removal from 65 storage, provided it is then stored at the temperature of unfrozen Red Blood Cells.
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 1to 6. Red Blood Cells may be stored in an approved additive solution (AS)1 , for up to 42 day at 1to 6. If the hermetic seal of the container is broken during collection, preparation, or further processing, the expiration date is not later than 24 h after the seal is broken (when blood is stored at 1to 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 are 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 are processed for freezing or for thawing, in an open system, the expiration date for the thawed Red Blood Cells is 24 h 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. [Note—The 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
| Product Name | Method of Preparation |
|---|---|
| Red Blood Cells | Prepared from whole human blood |
| Red Blood Cells, Pheresis | Prepared using automated apheresis systems |
| Red Blood Cells, Leukocyte Reduced | Prepared from Red Blood Cells or Red Blood Cells, Pheresis (total leukocytes count <5 × 106) |
| Red Blood Cells, Frozen | Prepared from Red Blood Cells or Red Blood Cells, Pheresis suspended in cryoprotective solution (glycerol) and frozen at an appropriate temperature |
| Red Blood Cells, Deglycerolyzed | Prepared from Red Blood Cells, Frozen, from which glycerol is removed by washing by an approved procedure |
Label it to indicate the ABO Group/Rh Type, as indicated in Table 2. [Note—Every 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
| ABO Group | Rh Type |
|---|---|
| A | Positive |
| A | Negative |
| B | Positive |
| B | Negative |
| AB | Positive |
| AB | Negative |
| O | Positive |
| O | Negative |
• USP Reference Standards 11
11
USP Hemoglobin RS
USP35
1
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.
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Maura C Kibbey, Ph.D.
Senior Scientific Liaison, Biologics & Biotechnology 1-301-816-6309 |
(BIO22010) Monographs - Biologics and Biotechnology 2 |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 2378
Pharmacopeial Forum: Volume No. 36(6) Page 1501