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.

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 deglycerolization disposable set for evidence of breaks or leaks before, during, and after the deglycerolization process.

B. OBSERVATION OF EFFLUENT. Check the graph printout of the waste solution through the wash cycle for signs of excessive hemolysis or of red cell spillage.

1. Hemolysis. At the beginning of the wash cycle the supernatant manifests a pale pink tinge that fades until it disappears after about 1200 ml of wash solution is used. The color of the waste solution should be less than 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, by the same person or using the same machine. Isolate any suspect units and evaluate and discard as necessary.

c. Confirm technician understanding of pre-glycerolization handling.

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

e. 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.

a. Isolated unit spillage. 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 labels and composition of wash solution.

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 and remove the transfer pack.

5. Using a needle and syringe, remove the red 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 >38 V%. This will result in a red cell volume of at least 110 ml with a corresponding total hemoglobin of 36 gm.

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 (aserobic). 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 a 16-gauge needle of a 20 ml syringe through the sampling site coupler (previously used for obtaining a sample for sterility testings) 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 syringethrough 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

Refraction

1. Palm-Abbe refractometer (Model PA200, Misco Products Division, Cleveland , OH ) may be used to estimate the glycerol concentration in the supernatant of the thawed glycerolized RBC and in the supernatant of the deglycerolized RBC. The 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 greater than 400 for the supernatant glycerol in the thawed glycerolized RBC and refraction value should be less than 40 to insure that the glycerol level is less than 1 g%.

Osmolality:

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

2. The thawed supernatant of the glycerolized RBC is diluted one to ten with distilled water prior to testing for supernatant osmolality.

3. 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. Deglycerolized RBC resuspended in AS-3 solution can be stored at room tempe rature for up to 4 hours prior to concentrating th RBC by centrifugation to isolate the supernatant solution for testing for extracellular potassium.

3. 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 3 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 low, normal and high total hemoglobin standard (J.T. Baker 3074), Drabkin’s solution (Sigma D5941) and Brij 35 solution (Sigma B4184).

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:

*Hemoglobin standard concentration is approximate, use exact concentration reported with each vial.

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 hemoglobinconcentration 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 = (K)(Absorbance)(1000 mg/gram)
Concentration 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 150 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. However, 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 thatthe chemically impregnated patches on the test strip is 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 ona 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 colors 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.

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.

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 = 305 gm
Hematocrit post deglyc = 50%
Density post deglyc = (0.0693 X 0.50)+1.005 = 1.03965
Volume of blood post deglyc = 305/1.03965 = 293 ml
Hemoglobin concentration in deglyc unit = 16 gm/dl
Total hemoglobin in deglycerolized unit = 46.9 gm

% of hemoglobin lost in waste = [4.5/(4.5+46.9)] X 100 = 8.8%

Freeze-thaw-wash recovery = 100 - 8.8 = 91.2%