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QUALITY CONTROL

INTRODUCTION

Following describes a recommended quality control for units of red blood cells frozen and deglycerolized using this method. The quality control section is divided into 3 separate sections.

a. All Units: All units must be inspected for breakage, wet ports, and observation of the effluent (waste solution) throughout the wash cycle.

b. Monthly Quality Control: For each 90 units of red cells deglycerolized, 7 units must be evaluated for quality control. Enclosure 1 defines the method utilized to determine the number of units to be evaluated for monthly quality control. Quality control must be performed as follows:

1. In addition to the above, each of the units evaluated for monthly quality control must also be weighed.
2. A sample of the unit will be removed and the hematocrit value measured.
3. The volume of the unit will be determined by dividing the weight by density.
4. The red cell volume will be determined to confirm that minimum requirements are met.

c. Student Training and Facility Validation Studies: When new students are being trained to perform this procedure or during facility validation, quality control should include the following: visual inspection for breakage, wet ports, and effluent; weight, hematocrit and red cell volume; and measurements of sterility, residual glycerol, supernatant hemoglobin, and supernatant potassium of the deglycerolized units will be determined. Freeze-thaw-wash recovery values will be calculated. Flow rates of wash solutions will be verified.

Units used for student training and facility validation studies should not be used in vivo, even when results are satisfactory. When personnel are being trained to freeze, thaw and wash red cells, the units should be quality-controlled as described and results recorded on the enclosed Red Blood Cell Deglycerolization and Quality Control Record.

A. ALL UNITS

VISUAL OBSERVATION AND INSPECTION (ALL UNITS)

A. BREAKAGE. Discard any unit that shows evidence of breaks or unintended openings at any point during processing.

1. Check each thawed unit for container breaks by gently compressing the unit against a white disposable towel, wiping the entire unit surface after compression, and examining the towel for blood stains.

2. Visually inspect the wash harness, wash chamber, waste bag and interconnecting tubing for evidence of breaks or leaks before, during, and after the deglycerolization process.

B. WET PORTS. Before opening the port seal to connect the thawed unit to wash harness, visually inspect the port areas for any evidence of residual water droplets. Carefully and thoroughly wipe these areas dry with a clean cloth as necessary.

C. OBSERVATION OF EFFLUENT. Check the appearance of the waste solution through the wash cycle for signs of excessive hemolysis or of red cell spillage. Record on the enclosed Red Blood Cell Deglycerolization and Quality Control Record.

1. Hemolysis. At the beginning of the wash cycle the supernatant manifests a pale pink tinge which fades until it disappears after about 1200 ml of wash solution is used. The color of the waste solution should be less than the number 5 on the scale of the color comparator. If signs of excessive hemolysis persist, the unit must be studied to determine whether the unit is safe for transfusion (see below). Hemolysis results from a freeze-thaw lesion or from mishandling during red cell washing, and the following should be checked:

a. Check freezer temperature charts during the storage period.

b. Check to see if the units that exhibit hemolysis were frozen at the same time or by the same person. Isolate any suspect units and evaluate and discard as necessary.

c. Confirm technician understanding of pre-glycerolization handling, the three glycerolization steps and of the need for proper manual mixing of the glycerol with the red cells during the final addition, i.e., use of the table provided to determine the volume of glycerol to red cell weight.

d. Confirm technician understanding of temperature requirements of the glycerol and red cells.

e. Check accuracy of balance used for glycerolization.

f. Confirm technician understanding of the predilution requirements prior to deglycerolization.

g. In units with poor freeze-thaw and freeze-thaw-wash recovery values, studies should be done to determine whether or not the poor in vitro results were due to the quality of the red blood cells that were frozen. Sickle trait red blood cells (SA), hereditary spherocytosis (HS), paroxysmal nocturnal hemoglobinuria (PNH) red blood cells, and red blood cells with glucose-6-phosphate dehydrogenase deficiency do not tolerate the freeze-thaw and freeze-thaw-wash recovery procedures. Red blood cells with poor freeze-thaw and freeze-thaw-wash recovery values with no apparent reason should be tested for these red blood cell abnormalities.

2. Spillage. When washing is performed using continuous-flow centrifugation, intact red cells can be observed in the effluent waste line. Spillage of intact red cells looks similar to hemolysis except that when intact red cells are present, the effluent appears cloudy red whereas when hemolysis is present, the effluent is transparent with a pink tinge. To detect whether hemolysis or loss of intact red cells is present, the effluent must be inspected against a white background. Spillage of red cells into the waste not only represents a loss of red cells from the unit but may also mask the presence of supernatant hemoglobin in the waste. The principal cause of spillage is the presence of too many red cells in the unit at the time of glycerolization. Red cell spillage can also occur if the proper wash solutions are not used and if the spindle speed of the Haemonetics 115 is too slow. The following action is recommended if red cell spillage occurs:

a. Isolated unit spillage. Gradually lower the brackets supporting the sodium chloride-glucose solution and red cells until spillage ceases; these units are acceptable for transfusion as long as they meet all other criteria. Units in which spillage persists should be studied further to determine whether they are suitable for transfusion.

b. Recurrent and uncontrollable spillage.

(1) Check scale used to weigh units prior to glycerolization;

(2) Confirm technician understanding of glycerolization process;

(3) Check spindle speed of 5800 rpm of the cell washing bowl using a hand-held tachometer;

(4) Check labels and composition of wash solution.

B. MONTHLY QUALITY CONTROL

For each 90 units of red blood cells deglycerolized each month, 7 units must be evaluated for weight, hematocrit and red cell volume as described below.

CONSUMABLES:

1. Sterile docking wafers (Terumo 3NCC987)

2. Alcohol swab, 70% (B-D 6894) (3)

3. Syringe, 20 ml (B-D 5661) with 16 g needle (B-D 5198)

METHODS

1. Weigh the blood in the 600 ml transfer pack.

2. Sterilely dock a transfer pack onto the 600 ml bag containing the deglycerolized red blood cells.

3. Transfer approximately 2 ml of washed red blood cells from the 600 ml bag into the transfer pack.

4. Heat seal the transfer pack and remove.

5. Using a needle and syringe, remove the red blood cells from the transfer pack.

6. Measure the hematocrit value.

Total weight of blood: Weigh the blood in the 600 ml bag following deglycerolization. Subtract the weight
of the empty 600 ml bag (32 gm) to determine the net weight of the red blood cell concentrate.

Hematocrit concentration. Measure the hematocrit value.

Determine the density of the blood as follows:

Post deglycerolization unit = (0.0693 X hct) + 1.005

NOTE: Use hematocrit as a decimal.

Divide the net weight of the red blood cell concentrate by the density to determine the volume of the red blood cell concentrate.

Multiply the total volume of the blood by the hematocrit value (as a decimal) to determine the volume of red blood cells.

All units tested for monthly quality control should have a hematocrit value of >28 V%. This will result in a red cell volume of at least 110 ml with a corresponding total hemoglobin of 36 gm.

C. STUDENT TRAINING AND FACILITY VALIDATION STUDIES

During training and facility validation studies, units should be studied extensively for quality control. Units used for student training and facility validation studies should not be used in vivo, even when the quality control results are satisfactory. When personnel are being trained to freeze, thaw and wash red cells, the units should be quality-controlled as described and results recorded on the enclosed Red Blood Cell Deglycerolization and Quality Control Record.

CONSUMABLES:

1. Sampling site coupler (Fenwal 4C2405)

2. Alcohol swab, 70% (B-D 6894) (3);

3. Blood agar plates (2)

4. BBL Septi-Check system (Fisher RD43231)

5. Syringe, 30 ml (B-D 5662) with 16 gauge needle (B-D 5198)

6. Syringe, 20 ml (B-D 5661) with 16 g needle (B-D 5198)

7. Plastic test tube (Falcon 2059)

8. Plastic test tubes (Falcon 2063) (2)

9. 4 X 4 gauze or Kimwipes

10. Chemistrip 4 The OB (urine) (Boehringer-Manheim 417144)

During student training and facility validation studies, deglycerolized red blood cells must be evaluated for sterility, residual glycerol, supernatant hemoglobin, extracellular potassium and freeze-thaw-wash recovery as described below. These units are not sampled using sterile docking and, therefore, should not be used for transfusion, even if the results are acceptable.

STERILITY

After the unit has been deglycerolized, a sample of the red cells is obtained by inserting a sampling site coupler (Fenwal 4C2405) into one of the entry ports of the 600 ml bag containing the red cells. Aseptically remove a 20.5 ml sample with a 30 ml syringe and 16 gauge needle for testing as follows:

1. Place a drop of red cells on each of two plates in the 4 quadrants of a blood agar plate (aerobic) and tilt the plate to allow each drop to streak each quadrant (0.5 ml sample is required).

2. The remainder of the sample will be put into the BBL Septi-Check system (Fisher #RD43231). This system consists of 2 vials, one containing 70 ml of thioglycollate broth (anaerobic) and the other containing 70 ml of trypticase soy broth (aerobic). Aseptically place 10 ml of blood into each of the vials according to the instructions provided with the culture system.

3. Incubate the blood agar plates at 37 C for 3 days and the broth tubes at 37 C for 7 days; examine daily for growth.

DETERMINATION OF RESIDUAL GLYCEROL (OSMOLALITY),
SUPERNATANT HEMOGLOBIN, AND EXTRACELLULAR POTASSIUM LEVELS

SAMPLE REQUIREMENTS: Aseptically insert an 16-gauge needle of a 20 ml syringe through the sampling site coupler
(previously used for obtaining a sample for sterility testing) and withdraw a 15 ml sample of the deglycerolized red cells from the 600 ml transfer pack. Remove the 16- gauge needle from the syringe and discard. (Follow local guidelines for needle removal and disposal procedures. Transfer a 12 ml sample of deglycerolized red cells into a plastic test tube (Falcon 2059). Transfer the remaining 3 ml sample into a plastic test tube (Falcon 2063).

NOTE: Do not transfer the blood from the syringe through the needle into the test tube. This
may cause hemolysis.

Centrifuge the sample at 2200 X g for 10 minutes in a 22 C refrigerated centrifuge. Transfer the supernatant into another plastic test tube (Falcon 2063) using a transfer pipet. The supernatant is used to determine residual glycerol (osmolality or refractive index/refraction), supernatant hemoglobin, and extracellular potassium levels.

RESIDUAL GLYCEROL

Refractive Index/Refraction

Hand Held Refractometer:

1. For field use, a Cambridge Instruments hand-held refractometer (TS meter, Model 10400A) may be used to estimate the residual level of glycerol. The refractometer contains a liquid prism which is self-temperature correcting. The meter has three scales; urine specific gravity, serum or plasma protein, and refraction. The refraction scale should be used; refraction is a mathematically derived value from the refractive index.

2. Using a disposable transfer pipet, transfer a sample of supernatant solution into the measuring prism, as described in the manufacturer's instruction manual.

3. Hold the instrument up to a light source (e.g., fluorescent light, window). Focus the eyepiece
and determine the refraction value of the sample according to the manufacturer's instructions. The refraction value should be less than 30 to insure that the glycerol level is less than 1 g%.

ABBE Refractometer:

1. Turn on the ABBE refractometer (American Optics Corp. Model 10480) and the constant temperature water bath (Haake Model A80) and allow to equilibrate to 20 C.

2. Calibrate the equipment with a known liquid material (e.g., absolute methanol).

3. Using a disposable transfer pipet, transfer 2 drops of supernatant solution into the refractive prism surface.

4. Close the refractive prism and determine the refractive index of the sample according to the manufacturer's specifications. The refractive index should not exceed 1.3355 to insure a residual glycerol level of less than 1 g%.

Osmolality

1. Calibrate the osmometer (Fiske Model 2400) using the manufacturer's procedure manual.

2. Using a Gilson adjustable volume pipettor and pipet tip, transfer 20 microliters of supernatant solution into an osmometer cuvette and determine the osmolality of the sample. Osmolality should not exceed 400 mOsm/kg H2O to insure a residual glycerol level of less than 1 g%.

EXTRACELLULAR (SUPERNATANT) POTASSIUM

1. Calibrate the IL 943 flame photometer using the flame standard 140 mEq/L Na+/5 mEq/L K+, according to the manufacturer's instructions.

2. Using a Gilson adjustable volume pipettor, add 300 microliters of supernatant into the sample cup of the flame photometer and measure the extracellular potassium level. The extracellular potassium level should not exceed 1.5 mEq/L on the day of washing.

SUPERNATANT HEMOGLOBIN

SPECTROPHOTOMETRIC METHOD

1. Set the Spectronic spectrophotometer at a wavelength of 540 nm.

2. Prepare a standard curve using a total hemoglobin standard kit (Sigma 525-A) according to the manufacturer's instructions. Included in the kit are: Drabkin's reagent, 30% BRIJ-35 solution, and a lyophilized hemoglobin standard (18 g%).

A. Reconstitute the Drabkin's reagent (one
vial) with 1000 ml of distilled water. Add 0.5 ml of the 30% BRIJ-35 solution. The Drabkin's solution may be stored at room temperature (18-26 C) in an amber bottle for up to 6 months.

B. Reconstitute the lyophilized hemoglobin standard with 50 ml of Drabkin's solution to prepare an 18 g% solution.

C. Pipet the following solutions to prepare the standard curve:

TUBE#	HEMOGLOBIN SOLUTION (ml)	DRABKIN'S SOLUTION (ml)	HEMOGLOBIN CONCENTRATION (g%)
1	0.0	6.0	0.0
2	2.0	4.0	6.0
3	4.0	2.0	12.0
4	6.0	0.0	18.0
NOTE: These diluted standards are stable for as long as 6 months when stored tightly capped, in the dark at 4 C.

 

D. Place tube 1 into the spectrophotometer
and zero the absorbance value. Read and record the absorbance values for tubes 2 through 4.

E. Plot a calibration curve (absorbance values vs hemoglobin concentration). The curve should be linear and pass through the origin.

F. Using the standard curve, calculate the extinction coefficient, K, as follows:

Hemoglobin Concentration (g%)=(K)(Absorbance)

G. Calculate the average K for using the three hemoglobin standard solutions (6.0, 12.0, 18.0 g%).

H. Total hemoglobin measurements are performed using 0.02 ml of whole blood diluted with 5.98 ml of Drabkin's reagent (1:251 dilution).

I. Measure the supernatant hemoglobin
concentration using a 0.3 ml sample in 4.7 ml of Drabkin's reagent. The overall increase observed is 18-fold for the supernatant hemoglobin samples. The following formula is used to construct the supernatant hemoglobin standard curve:

Supernatant Hemoglobin = Concentration

(K)(Absorbance)(1000 mg/gram) 18

 

3. Using a Gilson adjustable pipettor, pipet a 0.3 ml sample of supernatant and dilute the sample with 4.7 ml of Drabkin's reagent into a 13X100 mm Kimax glass culture tube. Mix and equilibrate for at least 2 minutes for the reaction to occur. Measure the absorbance value for the sample using the Drabkin's reagent solution as the blank.

4. Refer to the supernatant hemoglobin standard curve and determine the hemoglobin concentration of the sample (mg%). The supernatant hemoglobin concentration of the day of washing should be less than 200 mg%.

CHEMISTRIP METHOD
(Boehringer Mannheim Corp. Chemistrip 4 The OB urine test strip, Cat. No. 417144)

NOTE: The Chemistrip Method is only recommended when no spectrophotometer is available. If a spectrophotometer is available, the Chemistrip Method should not be used in lieu of the spectrophotometric method for measurement of supernatant hemoglobin for quality control testing each month.

1. Prepare the supernatant sample as previously described.

2. Briefly (no longer than 1 second) dip the test strip into the supernatant sample. Ensure that the chemically impregnated patches on the test strip are totally immersed in the sample.

3. Draw the edge of the strip along the rim of the test tube to remove excess sample.

4. Turn the test strip on its side and tap once on a piece of absorbent paper to remove any remaining sample and to prevent the possible mixing of chemicals.

5. Wait 60 seconds, then visually compare the protein color patches on the test strip to the color scale printed on the vial label. The protein visual color scale bears five color patches, ranging from light yellow to dark green:

The first patch, designated as negative, indicates a supernatant hemoglobin value of approximately 16 mg%, measured using the spectrophotometric method. The second patch, designated as trace, indicates a supernatant hemoglobin value of approximately 45 mg%, measured using the spectrophotometric method. The third patch, designated as +30, indicates a supernatant hemoglobin value of approximately 96 mg%, measured using the spectrophotometric method. The fourth patch, designated as ++100, indicates a supernatant hemoglobin value of approximately 221 mg%, measured using the spectrophotometric method. The fifth patch, designated as +++500 mg/dl, indicates supernatant hemoglobin value of approximately 428 mg%, measured using the spectrophotometric method.

6. Note the test result on the quality control worksheet. No further calculations are necessary.

ESTIMATION OF FREEZE-THAW-WASH RECOVERY (%)

Total volume of waste solution. Measure the total volume of waste solution using a graduated cylinder. Usually, the total volume of waste solution is 1,500 ml/unit.

Total volume of unit following deglycerolization. Weight the unit following deglycerolization. Subtract the weight of the 600 ml bag (@32 grams) to determine the net weight of the unit. Divide the net weight by the density*to obtain the volume of blood following deglycerolization.

Hemoglobin concentration.

1. Waste: Obtain a 5 ml sample from the waste bag and, using the same method described for the measurement of supernatant hemoglobin (mg%), determine the hemoglobin concentration. The hemoglobin concentration in the waste (mg%) multiplied by the total volume of waste (ml), divided by 100,000, will equal the total grams of hemoglobin lost.

2. Post-wash unit: Obtain a 2 ml sample from the unit and determine the hematocrit value and hemoglobin concentration using an automated analyzer. Multiply the hemoglobin concentration by the total volume to determine the total hemoglobin in the washed unit.

Freeze-thaw-wash recovery calculation: Divide the total hemoglobin in the waste by the sum of the total hemoglobin in the washed unit and the waste to determine the percentage of hemoglobin lost in the waste. Subtract this value from 100 to determine the % freeze-thaw-wash recovery value.

Sample calculation

Volume of waste solution = 1500 ml
Hemoglobin concentration in waste = 300 mg/dl
Total hemoglobin in waste = 4.5 gm

Net weight of blood following deglyc = 425 gm
Hematocrit post deglyc = 35%
Density post deglyc = (0.0693 X 0.35)+1.005 = 1.029
Volume of blood post deglyc = 425/1.029 = 413 ml
Hemoglobin concentration in deglyc unit = 10 gm/dl
Total hemoglobin in deglycerolized unit = 41.3 gm

% of hemoglobin lost in waste = [4.5/(41.3+4.5)] X 100 = 9.8%

Freeze-thaw-wash recovery = 100 - 9.8 = 90.2%.

VERIFICATION OF FLOW RATE OF 12% NACL AND 0.9% NACL-0.2% GLUCOSE WASH SOLUTIONS AND DILUTED RED CELLS

The flow rates of the 12% NaCl and 0.9% NaCl-0.2% glucose wash solutions and of the diluted red blood cells should be checked every 6 months. The recommended flow rates are as follows:

Hang the wash solutions and a bag of saline (in lieu of diluted red cells) on the appropriate hooks. Using a stopwatch, time the delivery of the solutions into an empty plastic bag. Adjust the hooks to achieve the proper flow rates as outlined above, if necessary. Make the appropriate corrections in your Standard Operating Procedure Manual.

RED BLOOD CELL GLYCEROLIZATION WORKSHEET
(Printable Version)

RED BLOOD CELL DEGLYCEROLIZATION RECORD
(Printable Version)

RED BLOOD CELL GLYCEROLIZATION/DEGLYCEROLIZATION QUALITY CONTROL RECORD
(Printable Version)

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