Red Blood Cell Recovery Research Articles

Effect of centrifugation and subsequent storage on red blood cells.

Blood drawn into CPD solution from 33 normal donors was divided into four groups: (I) centrifuged (at 5,000 g for 7 min) after 7 days of storage, (II) centrifuged after 14 days storage, (III) centrifuged after 21 days storage and (IV) uncentrifuged. After 21 days of storage, aliquots of all units were labeled with chromium-51, reinjected into the donor from which they were drawn and erythrocyte survival was measured. Red blood cell recovery and survival for all four groups was essentially the same; 24-hour recovery was 85%; T 1/2 was 28.2--31.6 days. Our results suggest that blood can be centrifuged and stored at any time during its 21-day shelf life without detrimental effect on erythrocyte survival.

Evaluation of a large-scale frozen blood program.

The characteristics of previously frozen red blood cells, prepared in a large-scale frozen blood program using the Red Cross method were evaluated. The use of the method as originally described resulted in approximately 91 per cent freeze-thaw-wash recovery of red blood cells. When the glycerolization step was modified by adding the partially glycerolized erythrocytes into 300 ml of 6.2M glycerol, freeze-thaw-wash recoveries were decreased. However, gradient addition of glycerol to the red blood cells without the use of stylet, resulted in acceptable in vitro recoveries. Thawing frozen units in waterbath, to which no antiseptic was added, could introduce bacteria into units of previously frozen red blood cells. Therefore, it seems advisable to use dry heat thawing procedures. Previously frozen red blood cells prepared in the large scale maintained normal levels of ATP and 2,3 DPG. Therapeutic transfusions had acceptable 24-hour survival in vivo.

The purification of red cells for transfusion by freeze-preservation and washing. V. Red cell recovery and residual leukocytes after freeze-preservation with high concentrations of glycerol and washing in various systems.

Red blood cells freeze-preserved with 40% W/V glycerol in an ionic or low ionic medium at -80 C were washed in one of four commercially available systems: the Haemonetics Blood Processor 15 using a continuous-flow centrifugation procedure, the Fenwal Elutramatic using a continuous-flow centrifugation procedure, the IBM Blood Processor using an automated serial centrifugation procedure, and the Huggins Cytoglomerator using a dilution/agglomeration procedure. Determinations of red blood cell recovery and leukocyte and platelet removal were made for each of these groups. The dilution/agglomeration procedure produced lower red blood cell recovery values and poorer leukocyte and platelet removal than did any of the three wash systems using sodium chloride solutions. The values obtained with the systems using sodium chloride solutions were slightly but significantly different.

Development and use of pediatric frozen red cell packs.

Repeated transfusion of small increments of blood are frequently required for the sick and premature newborn infant to correct endogenous hypovolemia and/or to replace blood obtained for laboratory monitoring purposes. Previously fresh group and type specific whole blood was used. To eliminate waste of fresh whole blood, maintain fresh red blood cell properties, eliminate the hazards of transfusing plasma and to provide a more efficient system, a pediatric frozen red cell pack (PFRCP) has been developed. Units of group O rr red blood cells are glycerolized using a high glycerol method. The glycerolized red blood cells are separated into three equal aliquots and frozen. When needed, the PFRCP are deglycerolized by a modified procedure using the IBM Cell Processor. During a six month period, 71 infants were given 153 separate transfusions of deglycerolized red blood cells using 102 PFRCP prepared from 34 units of red blood cells. Red blood cell recovery, hematocrit, white blood cell removal, residual glycerol, total protein, and supernatant hemoglobin levels were measured. Clinical response was followed and found to be excellent.

Use and analysis of saline washed red blood cells.

Data collected from 50 saline washed units of red blood cells shows that units washed with one liter of 0.9 per cent NaCl on an IBM cell processor have an average hematocrit of 72.2 per cent, with 84.7 per cent of the white blood cells removed, and only 0.6 per cent of the original total protein remaining. The red blood cell recovery is 85.5 per cent. Other advantages include low extracellular potassium and reduction in supernatant hemoglobin, metabolic waste products, debris, and nonviable cells. Over 1500 washed units have been transfused, with an average of 150 per month. The clinical response has been very favorable with only one suspected reaction reported since the use of washed cells, and no reported cases of hepatitis.

The Purification of Red Cells for Transfusion by Freeze‐Preservation and Washing

Red blood cells frozen on the day of collection showed differences in the residual number of leukocytes depending on the freeze‐preservation technique. Red blood cells preserved with 40 per cent W/V glycerol and stored at — 80 C had four times more leukocytes per gram of hemoglobin than did washed red blood cells preserved with 20 per cent W/V glycerol and stored at ‐150 C. Red blood cell recovery was higher and supernatant hemoglobin lower for the red blood cells frozen with 40 per cent W/V glycerol and stored at ‐80 C, but the differences were small. Although the low glycerol concentration freezing method was more efficient in removing leukocytes, the clinical significance of this observation remains to be determined.

Washing of red blood cells previously frozen in liquid nitrogen. Experience with the Elutramatic Ultra-Flo system.

An automated continuous‐flow washing system, the Elutramatic Ultra‐Flo system, has been tested for the deglycerolization of red blood cells previously frozen in liquid nitrogen. Packed red blood cells frozen with 100 ml of a 55 per cent glycerol solution were, after thawing, diluted with 175 ml 3.7 per cent sodium chloride solution. The standard wash program was used for the processing. Approximately 2.2 liter of saline solution were used for the washing of one unit of blood. The processing time for two units was 27 minutes in the automated cycles. The mean red blood cell recovery was 81 per cent and the mean residual hemolysis in the washed blood was 0.36 per cent. No important changes of ATP, DPG, and intraerythro‐cytic potassium occurred during the processing. The 24‐hour posttransfusion red blood cell survival was 91.6 ± 1.8 per cent. For the trained blood bank technician the Elutramatic Ultra‐Flo system offers a rapid and convenient method for the deglycerolization of red blood cells previously frozen in liquid nitrogen.

Leukocyte contamination of red cells in leukocyte-poor and frozen-deglycerolized units.

Red blood cell recovery was better and removal of white cells equally good when units were centrifuged briefly in an upright position as compared to more prolonged centrifugation in an inverted position. Nylon fiber filtration left more white cells than did differential centrifugation; a combination of the two technics provided the smallest amount of white cell contamination. Red blood cells frozen and deglycerolized by the original Huggins process had as many leukocytes as those prepared by differential centrifugation. The current Huggins process left more white cells than did the original method.Removal of white cells from blood frozen and deglycerolized by the Huggins process was better in a continuous flow centrifuge than in the Cytoglomerator. Platelet removal was less complete than that of white cells by either technic. Preliminary results with CPD blood do not appear significantly different from those with ACD blood.

Studies with dextran 40 in cryopreservation of blood

Whole blood from healthy donors was washed twice with phosphate-buffered saline (PBS) and then resuspended in sufficient PBS to give a final concentration of 2 × 10 9/cells/ml. Aliquots were combined with equal volumes of the required diluents to give final dextran 40 concentrations of 0, 5, 10, 15, and 20% in PBS. Fifty-lambda samples in 50-lambda Micropets (Clay Adams) were frozen in alcohol baths at temperatures ranging from −10 to −80 °C. The specimens were frozen either for 1 min or 16 min, rapidly thawed, and resuspended in PBS or PBS plus dextran. Percentage of hemolysis was determined colorimetrically. Results indicate that concentraitons as low as 5% dextran exert a cryoprotective effect. Increased dextran concentration increases cryoprotection at high subzero bath temperatures (−10 ° and −20 °C). Dextran concentrations beyond 12% have a damaging effect at low subzero bath temperatures (below −30 °C). Based on this a two-factor hypothesis for cryopreservation is proposed. Apparent partial recovery of red blood cells without dextran or with 5% dextran during subzero storage was demonstrated.

The effect of gases on the recovery of human red blood cells

The freezing of blood in several types of small tubes is studied. The gas content of the blood in the tubes was controlled. The damage during freezing while in equilibrium with a given gas does not appear to depend upon the gas itself, but upon the change in volume that the gas produces in the cell. Heat transfer calculations indicate that the damage does not appear to be caused by supercooling or by intracellular ice formation, but by an as yet undetermined sequence of events.

1. The effect of gases on the recovery of human red blood cells: F. C. Wessling, Jr. and P. L. Blackshear, Jr. (University of Minnesota, Minneapolis, Minnesota)

1. The effect of gases on the recovery of human red blood cells: F. C. Wessling, Jr. and P. L. Blackshear, Jr. (University of Minnesota, Minneapolis, Minnesota)

Recovery of glycerolized red blood cells frozen in liquid nitrogen.

Human red blood cells were frozen at a rapid rate in the presence of approximately 20 per cent w/v glycerol and an osmotic supporting agent, and were stored in liquid nitrogen vapor at approximately — 150 C for at least two weeks before thawing, washing, and resuspension. The recovery of red blood cells upon thawing was dependent on the type of freezing container used and the volume of red blood cells frozen. An additional loss of red blood cells during postthaw washing was related to the volume of red blood cells frozen, but was not related to the type of osmotic supporting agent used during the wash. The total amount of supernatant hemoglobin in the washed unit was inversely related to the recovery of the red blood cells upon thawing. For adequate recovery of preserved red blood cells, both the geometry of the container in which the cells had been frozen, and the capacity of the reusable bowl‐washing system employed for removing the glycerol, limited the mass of glycerolized red blood cells to less than 600 g.

Recent Advances in Freeze-Preservation of Red Blood Cells

The methods currently in clinical use for freezing human red blood cells (RBC) require the intracellular additive, glycerol, either in high concentration (approximately 45% W/V) with the slow freeze-thaw technique, or in low concentration (approximately 18% W/V) with the rapid freeze-thaw technique. The major technological problem in the current methods is the removal of the glycerol. Postthaw washing is presently accomplished by serial centrifugation (batch washing), continuous centrifugation, or a dilution procedure with recovery of RBC by agglomeration. Recent advances in the use of continuous centrifugation are extremely encouraging. However, for widespread clinical application of this approach disposable washing material Is urgently needed.