Red Blood Cell Recovery Research Articles

Influence of the Cryoprotective Agents Glycerol and Hydroxyethyl Starch on Red Blood Cell ATP and 2,3-Diphosphoglyceric Acid Levels

Because hydroxyethyl starch (HAES) is used for volume replacement therapy and as a cryoprotectant for frozen red blood cells (RBCs), this compound, in contrast to glycerol, does not require labor-intensive removal from thawed cells prior to transfusion. We here report the effect of both glycerol and HAES on the RBC organic phosphates ATP and 2,3-diphosphoglyceric acid (2,3- DPG). The CPD-A(1)-stabilized RBCs of 20 healthy donors (3 females, 17 males) were separately frozen in either 40% glycerol or 6% HAES, of molecular weight 200,000. ATP and 2,3-DPG concentrations were determined in CPD-A! RBCs before addition of cryoprotectant and in cryopreserved thawed RBCs after 24 h storage at -80°C (glycerol) and -196°C (HAES). It appears that HAES, but not glycerol, significantly reduces ATP concentrations whereas both lead to a reduction of 2,3-DPG concentrations: this reduction was more pronounced with glycerol than with HAES. Experiments with the blood of 6 donors demonstrated that HAES affects autohemolysis by 16%, in contrast to glycerol, after which cryoprotectant autohemolysis was affected by 3.1% only. RBC recoveries were comparable using glycerol or HAES as cryoprotectants. A distinct pattern of reduction of 2,3-DPG levels by glycerol and less by HAES, and of ATP levels by HAES but not by glycerol, emerges. Our findings may be of importance if HAES is to be introduced as a convenient cryoprotectant.

Quantitation of Immunoglobulin Associated With Senescent Erythrocytes From the Rabbit

The biochemical processes that determine the lifespan of mammalian erythrocytes are unknown; one prominent theory suggests that antibody binding to the senescent red blood cell identifies it for removal from the circulation. To address this question, we have used a newly developed procedure for the isolation of aged erythrocytes that involves the biotinylation of rabbit red blood cells, in vivo aging of these cells, and the eventual in vitro recovery of the aged red blood cells by their affinity for an avidin support. Erythrocytes isolated with this method were found to have near-normal levels of cell-associated Ig throughout their 60-day lifespan. These data suggest that IgG accumulation is not part of the normal senescence process for erythrocytes in rabbits.

Quantitation of immunoglobulin associated with senescent erythrocytes from the rabbit

The biochemical processes that determine the lifespan of mammalian erythrocytes are unknown; one prominent theory suggests that antibody binding to the senescent red blood cell identifies it for removal from the circulation. To address this question, we have used a newly developed procedure for the isolation of aged erythrocytes that involves the biotinylation of rabbit red blood cells, in vivo aging of these cells, and the eventual in vitro recovery of the aged red blood cells by their affinity for an avidin support. Erythrocytes isolated with this method were found to have near-normal levels of cell- associated Ig throughout their 60-day lifespan. These data suggest that IgG accumulation is not part of the normal senescence process for erythrocytes in rabbits.

Delay in Red Blood Cell Recovery after Major ABO Incompatible Bone Marrow Transplantation

Delay in Red Blood Cell Recovery after Major ABO Incompatible Bone Marrow Transplantation

Anti-Asialo GM1 Antiserum Treatment of Lethally Irradiated Recipients Before Bone Marrow Transplantation: Evidence That Recipient Natural Killer Depletion Enhances Survival, Engraftment, and Hematopoietic Recovery

Abstract Natural killer (NK) cells are reported to have an important role in the resistance of lethally irradiated recipients to bone marrow transplantation (BMT). Therefore, we investigated the effects of recipient NK depletion on survival, chimerism, and hematopoietic reconstitution after lethal irradiation and the transplantation of limiting amounts of T-cell-deficient bone marrow (BM). When administered before BMT, anti-asialo GM1 (ASGM1) antiserum treatment, effective in depleting in vivo NK activity, was associated with a marked increase in survival in 3 of 3 allogeneic combinations (BALB/c into C3H/HeN, C57B1/6, or C3B6F1). This enhanced survival was independent of the susceptibility of each recipient strain to accept BALB/c BM. Moreover, recipient anti-ASGM1 treatment was also effective in increasing survival in recipients of syngeneic BM, suggesting that NK cells can adversely affect engraftment independent of genetically controlled polymorphic cell surface determinants. Analysis of chimerism in surviving animals 2 months post-BMT showed that recipient NK depletion significantly increased the level of donor engraftment when high doses of BM were transplanted. These studies also demonstrated that anti-ASGM1 pretreatment mainly resulted in an increase in extramedullary hematopoiesis in the second and third week after irradiation. Anti-ASGM1 treatment also dramatically accelerated the rate of appearance of donor-derived cells with a higher level of donor- cell engraftment apparent at a time when the differences in survival between NK-depleted and control BMT recipients became significant. Peripheral cell counts were also affected by NK depletion, with significantly enhanced platelet and red blood cell recovery and a moderate increase in granulocyte recovery. The overall favorable influence of anti-ASGM1 recipient treatment on hematopoietic events post-BMT suggests that, in humans, pretransplant regimens aimed toward NK depletion should be evaluated.

Anti-asialo GM1 antiserum treatment of lethally irradiated recipients before bone marrow transplantation: evidence that recipient natural killer depletion enhances survival, engraftment, and hematopoietic recovery

Natural killer (NK) cells are reported to have an important role in the resistance of lethally irradiated recipients to bone marrow transplantation (BMT). Therefore, we investigated the effects of recipient NK depletion on survival, chimerism, and hematopoietic reconstitution after lethal irradiation and the transplantation of limiting amounts of T-cell-deficient bone marrow (BM). When administered before BMT, anti-asialo GM1 (ASGM1) antiserum treatment, effective in depleting in vivo NK activity, was associated with a marked increase in survival in 3 of 3 allogeneic combinations (BALB/c into C3H/HeN, C57B1/6, or C3B6F1). This enhanced survival was independent of the susceptibility of each recipient strain to accept BALB/c BM. Moreover, recipient anti-ASGM1 treatment was also effective in increasing survival in recipients of syngeneic BM, suggesting that NK cells can adversely affect engraftment independent of genetically controlled polymorphic cell surface determinants. Analysis of chimerism in surviving animals 2 months post-BMT showed that recipient NK depletion significantly increased the level of donor engraftment when high doses of BM were transplanted. These studies also demonstrated that anti-ASGM1 pretreatment mainly resulted in an increase in extramedullary hematopoiesis in the second and third week after irradiation. Anti-ASGM1 treatment also dramatically accelerated the rate of appearance of donor-derived cells with a higher level of donor-cell engraftment apparent at a time when the differences in survival between NK-depleted and control BMT recipients became significant. Peripheral cell counts were also affected by NK depletion, with significantly enhanced platelet and red blood cell recovery and a moderate increase in granulocyte recovery. The overall favorable influence of anti-ASGM1 recipient treatment on hematopoietic events post-BMT suggests that, in humans, pretransplant regimens aimed toward NK depletion should be evaluated.

The effect of intraperitoneal administration of sodium tolmetin-hyaluronic acid on the postsurgical cell infiltration in vivo

Intraperitoneal administration of sodium tolmetinhyaluronic acid reduced the formation of adhesions at early postsurgical time points. In addition, at 6, 48, 72, and 96 hr following surgery, there was a significant reduction in the number of red blood cells (RBC) recovered from peritoneal lavage. This effect was not the result of fluid or viscous solution in the peritoneal cavity since intraperitoneal administration of Ringer's lactate or Hyskon (a 32% solution of Dextran 70) did not affect RBC recovery. In contrast, the influx of leukocytes into the peritoneal cavity was elevated at 12 hr after surgery, but suppressed at 96 hr. These data may suggest a mechanism by which sodium tolmetin in hyaluronic acid reduced adhesion formation.

Plasma Separator for Encapsulating Fluorescent Probe in Erythrocytes

Using a plasma separator, it is possible to wash large volumes of whole blood free of plasma proteins in short periods. The novelty, however, is that the same apparatus can be used to reversibly hemolyze and reseal the red blood cells (RBCs) using hypotonic and hypertonic dialysates, respectively. This technique was assessed using the fluorescent exogenous agent uranin (fluorescein sodium). Results indicate that the encapsulation of the RBCs can be completed in under 2 h. The encapsulation efficiency of the RBCs was approximately 80% with a RBC recovery rate of 75%.

Pure Red Cell Aplasia of Long Duration Complicating Major ABO-Incompatible Bone Marrow Transplantation

Abstract In 3 of 15 consecutive patients receiving a human leukocyte antigen (HLA)-identical but major ABO incompatible bone marrow transplant (BMT), pure red cell aplasia (PRA) lasting 5 to 8 months was observed. Titers of the incompatible anti-A agglutinin before infusion of the red blood cell (RBC)-depleted BMT was very high in one, and in the usual range in two patients. Decrease of agglutinin titers during the first 4 weeks after BMT were comparable between PRA patients and those of ABO- incompatible BMT recipients with timely RBC recovery. However, in PRA patients, agglutinin titers rose again and remained elevated for 19 to 28 weeks. RBC engraftment and reticulocyte recovery ultimately occurred spontaneously and coincided with the decrease of agglutinin titers below 16. These observations indicate that PRA is antibody-dependent in this setting. Furthermore, it is conceivable that cyclosporine facilitates recipient-derived antibody synthesis after major ABO- incompatible BMT.

Pure red cell aplasia of long duration complicating major ABO- incompatible bone marrow transplantation [see comments

In 3 of 15 consecutive patients receiving a human leukocyte antigen (HLA)-identical but major ABO incompatible bone marrow transplant (BMT), pure red cell aplasia (PRA) lasting 5 to 8 months was observed. Titers of the incompatible anti-A agglutinin before infusion of the red blood cell (RBC)-depleted BMT was very high in one, and in the usual range in two patients. Decrease of agglutinin titers during the first 4 weeks after BMT were comparable between PRA patients and those of ABO-incompatible BMT recipients with timely RBC recovery. However, in PRA patients, agglutinin titers rose again and remained elevated for 19 to 28 weeks. RBC engraftment and reticulocyte recovery ultimately occurred spontaneously and coincided with the decrease of agglutinin titers below 16. These observations indicate that PRA is antibody-dependent in this setting. Furthermore, it is conceivable that cyclosporine facilitates recipient-derived antibody synthesis after major ABO-incompatible BMT.

Use of Imugard IG500 filters for preparation of leukocyte-poor blood for cancer patients.

In order to determine the efficiency of Imugard IG500 filters in producing leukocyte-poor blood (LPB), 109 units of red cell concentrates were processed using cold (4 degrees C) or room temperature saline solution without the application of pressure to the blood bag. The results obtained in terms of red blood cell recovery, white blood cell removal, residual white blood cells, and free hemoglobin in the supernatant of the filtered units are comparable to those processed with the IBM 2991. LPB processed without pressure application to the blood bag and room temperature saline solution can be rapidly, efficiently and safely prepared in any size blood bank/transfusion service. This technique is definitely cost-saving in terms of equipment, software, space and personnel training.

Use of Imugard IG 500 Filters for Preparation of Leukocyte-Poor Blood for Cancer Patients

In order to determine the efficiency of Imugard IG500 filters in producing leukocyte-poor blood (LPB), 109 units of red cell concentrates were processed using cold (4°C) or room temperature saline solution without the application of pressure to the blood bag. The results obtained in terms of red blood cell recovery, white blood cell removal, residual white blood cells, and free hemoglobin in the supernatant of the filtered units are comparable to those processed with the IBM 2991. LPB processed without pressure application to the blood bag and room temperature saline solution can be rapidly, efficiently and safely prepared in any size blood bank/transfusion service. This technique is definitely cost-saving in terms of equipment, software, space and personnel training.

The Risk- Benefit of Autotransfusion

Recent enthusiasm for intraoperative autotransfusion has overshadowed critical assessment of its potential risks. In this study, adult mongrel dogs underwent controlled intraperitoneal hemorrhage of twice their estimated blood volume over a 4-hour period. The blood was replaced by an equal volume of banked blood (Group I, n = 5), or collected and reinfused via the Sorenson System (Group II, n = 6), or the Haemonetics Cell Washing Device (Group III, n = 6). Acid citrate dextrose was the local anticoagulant for Groups I and II, and heparin for Group III. Pulmonary capillary wedge pressure and cardiac output were maintained at baseline values with crystalloid infusion. Core temperature, pO2, and systemic pH remained normal throughout the 4 hours of evaluation. Red blood cell recovery was efficient in all animals, and the 2,3 DPG levels remained normal in the autotransfused dogs. Thrombocytopenia, however, developed uniformly and was more pronounced after autotransfusion. Platelet numbers decreased nearly 45% in the Sorenson as well as Haemonetics animals. Additionally, platelet dysfunction occurred after one blood volume exchange as evidenced by prolonged bleeding times and loss of the secondary wave on Sonoclot profiles. Coagulation studies revealed progressive consumptive coagulopathy and fibrinolysis in autotransfused dogs. The P.T., P.T.T., and T.T. lengthened, and levels of factors II, V, VIII, and fibrinogen fell. Autotransfusion clearly eliminates the infectious and incompatibility problems of banked homologous blood. Despite advances in technique, however, consumptive coagulopathy, fibrinolysis, and platelet dysfunction occur.(ABSTRACT TRUNCATED AT 250 WORDS)

Simplified method for recovery of autologous red blood cells from transfused patients.

Separation of autologous and transfused red blood cells from recently transfused patients is necessary for the proper identification of any red blood cell alloantibody or autoantibody. We compared two methods of separation: the standard technic of microhematocrit centrifugation with phthalate ester solution, and a simplified method of microhematocrit centrifugation without the use of esters. Autologous red blood cells were concentrated in the top layer of the capillary tube by both methods. Separation efficacy was comparable, as determined by blood group antigen reactivity. Good separation was achieved only in samples drawn three or more days post transfusion. Microhematocrit centrifugation without the use of phthalate esters is a simple method for the recovery of autologous red blood cells from recently transfused patients that can be performed by any standard clinical laboratory.

A new approach to washing red blood cells frozen with a high concentration of glycerol in a special freezing container.

We report here on a new approach to washing red blood cells frozen with a high concentration of glycerol in a special freezing container. The wash solution consists of a 150-ml volume of 12% sodium chloride and 2 liters of 0.9% sodium chloride-0.2% glucose-25 mEq/l disodium phosphate. Both the Haemonetics Blood Processor 115 and the IBM Blood Processor 2991 have been used with this protocol, with similar results. The in vitro recovery of red blood cells frozen with 8.6M glycerol was 89%, and that of red blood cells frozen with 6.2M glycerol was 93%. The 24-hour posttransfusion survival values averaged 88% for eight units of outdated-rejuvenated previously frozen red blood cells washed by this protocol and stored at 4 degrees C for 3 days before autotransfusion.

A New Approach to Washing Red Blood Cells Frozen with a High Concentration of Glycerol in a Special Freezing Container

We report here on a new approach to washing red blood cells frozen with a high concentration of glycerol in a special freezing container. The wash solution consists of a 150-ml volume of 12% sodium chloride and 2 liters of 0.9% sodium chloride-0.2% glucose- 25 mEq/1 disodium phosphate. Both the Haemonetics Blood Processor 115 and the IBM Blood Processor 2991 have been used with this protocol, with similar results. The in vitro recovery of red blood cells frozen with 8.6M glycerol was 89%, and that of red blood cells frozen with 6.2M glycerol was 93%. The 24-hour posttransfusion survival values averaged 88% for eight units of outdated-rejuvenated previously frozen red blood cells washed by this protocol and stored at 4°C for 3 days before autotransfusion.

Freezing in the primary polyvinylchloride plastic collection bag: a new system for preparing and freezing nonrejuvenated and rejuvenated red blood cells.

Red blood cells were stored at 4 C in the primary bag with an integrally attached empty transfer pack so that the red blood cells could be rejuvenated or not, as desired before glycerolization and freezing. The rejuvenation and glycerol solutions were added through ports in the system. After glycerolization, the red blood cells were concentrated by centrifugation to remove the supernatant glycerol before freezing with 40% w/v glycerol in the primary polyvinylchloride (PVC) plastic container at -80 C. After thawing, the red blood cells were washed using either the Haemonetics Blood Processor 115 or the IBM Blood Processor 2991-1 or 2991-2. In each system, 50 ml of 12% sodium chloride and 1.5 to 1.6 liters of 0.9% sodium chloride-0.2% glucose-25 meq/l disodium phosphate were used. Recovery of red blood cells in vitro was 91 per cent. After three days of postwash storage at 4 C, nonrejuvenated red blood cells had a mean 24-hour posttransfusion survival of 88 per cent, and outdated-rejuvenated red blood cells a value of 81 per cent. This new system is simpler and safer than methods previously used in this laboratory, and red blood cell recovery and 24-hour posttransfusion survivals were comparable or better.

Therapeutic effectiveness and safety of outdated human red blood cells rejuvenated to restore oxygen transport function to normal, frozen for 3 to 4 years at -80 C, washed, and stored at 4 C for 24 hours prior to rapid infusion.

Human red blood cell concentrates with hematocrit values of 75 V% were prepared from citrate‐phosphate‐dextrose (CPD) blood, stored at 4 C for 20 to 28 days, and biochemically modified with a solution containing pyruvate, inosine, glucose, phosphate, and adenine (PIGPA Solution A). The rejuvenated red blood cells were frozen with 40% W/V glycerol in a polyolefin plastic bag and were stored at −80 C. After three to four years of frozen storage, the units were thawed, washed, and stored at 4 C in a sodium chloride‐glucose‐phosphate solution for 24 hours prior to transfusion. Red blood cell recovery was 97 per cent after thawing and 90 per cent after washing. An automated differential agglutination procedure (ADA) showed 24‐hour survival values of about 80 per cent, and long‐term survival values of about 85 days depending on the disease state of the recipient. The red blood cells had normal affinity for oxygen on the day of transfusion. Plasma hemoglobin levels measured immediately after transfusion indicated extravascular removal of nonviable donor red blood cells. There was no increase in the uric acid level during the 24‐hour posttransfusion period. A pool of three to ten units of rejuvenated washed previously frozen red blood cells was transfused rapidly to each of 19 anemic elderly patients. The red blood cells which had normal oxygen delivery capacity immediately upon transfusion increased the recipient's red blood cell mass and produced no untoward effects.

Is very fresh blood needed in preparing leukocyte-poor red blood cells by inverted centrifugation?

It is recommended that units selected for inverted centrifugal preparation of leukocyte-poor red blood cells be less than 24 hours old. The reason for this is unclear and the recommendation poses practical problems. We evaluated the effectiveness of inverted centrifugation in reducing the WBC:RBC ratio in 31 units stored up to six days at 4 C. Leukocyte removal was at least as effective with six-day stored units as with less than one day old, averaging 77 per cent reduction with 76 per cent recovery of red blood cells. No reduction in leukocytes occurred with six days storage alone. It appears to be unnecessary to restrict leukocyte removal by inverted centrifugation to units less than 24 hours old.

Optimal Leukocyte Removal from Refrigerated Blood with the IBM 2991 Blood Cell Processor

One hundred forty-six units of two to five-day-old refrigerated blood were washed with the IBM Blood Cell Processor. The ABO and Rh types of the units varied according to available inventory. This study was designed to select the best protocol from the 42 possible combinations utilizing the machine's variable red blood cell override (RCO). A one liter wash program was used. The two parameters selected for study were the per cent red blood cell recovery and the per cent white blood cell removal. The protocol of choice uses a RCO of four seconds in the first and second steps of the program. This protocol yields a washed unit with a mean red blood cell recovery of 82 per cent and a mean white cell removal of 93 per cent. Our studies indicate that the IBM Blood Cell Processor can be used to wash two to five day old refrigerated blood for the preparation of leukocyte-poor blood that would meet the Standards of the AABB.