Automated suspension of washed erythrocytes in fresh-frozen plasma for exchange transfusion. Obtaining a desired hematocrit

Stored red blood cells may be automatically saline washed and resuspended in fresh-frozen plasma to yield a final product with any hematocrit that is desired. The technique provides for rapid processing and issue of blood and minimal risk of contamination of the unit. Red blood cells are more readily available than whole blood, and their use, with fresh-frozen plasma, in exchange transfusion, provides similar advantages, including low potassium content and normal content of procoagulants and bilirubin binding capacity. The method of reconstruction is based on calculations that use empirically determined estimates of average hematocrit and specific gravity of red blood cells and the volumetric flow characteristics of a programmable IBM 2991 Blood Processor. Only the weight of the stored red blood cell unit is required as a measurement at the time of preparing the resuspended cells. Quality-control studies of the final hematocrit indicates a 2.4 per cent standard deviation from a target value of 55 per cent.     

Neonatal mortality following transfusion of red cells with high plasma potassium levels

The death of a neonatal infant following cardiac surgery and the transfusion of packed red cells (RBCs) with high plasma potassium levels is reported. The patient had been diagnosed at 2 weeks of age as having multiple cardiac malformations. During cardiopulmonary bypass surgery, multiple units of packed RBCs less than 5 days old were transfused. In response to a "stat" order and after depletion of stock units prepared for neonatal usage, a 32-day-old unit of packed RBCs was issued for transfusion. After approximately 60 mL was rapidly transfused from this unit, the patient experienced cardiac arrest. Serum potassium concentration after transfusion and before death was 8.9 mmol per L. Plasma potassium concentration in the remainder of the transfused packed RBC unit was approximately 60 mmol per L. A model was created to calculate the posttransfusion plasma potassium concentration, and close correlation was found between the model and the observed potassium concentration, which assumes that the potassium load had not yet been distributed to the extravascular and intracellular fluid compartments. It is concluded that the transfusion of relatively large volumes of RBCs be limited to fresh packed RBCs or to packed RBCs that have been saline washed, to minimize the complications of electrolyte disturbances.     

Relationship Between Osmotic Fragility and in Vivo Survival of Autologous Deglycerolized Resuspended Red Blood Cells

The relationship between the osmotic fragility as determined with a "fragiligraph," and the 24 hour posttransfusion survival of autologous, deglycerolized chromium-labeled erythrocytes was studied in 62 units of blood. Thirty-two units of deglycerolized red cells were resuspended in autologous plasma, and thirty units were resuspended in a 5% albumin medium. Fourteen of the units resuspended in autologous plasma, and thirteen of those resuspended in the 5%' albumin medium were stored at 4C for varying periods (5–14 days), and then washed with isotonic saline to reduce the supernatant hemoglobin prior to transfusion. The in vivo survival of the erythrocytes could not be accurately predicted from the analysis of the osmotic fragility curve. However, approximately 65 per cent survival 24 hours after transfusion could be predicted if 6.25% and 12.5% hemolysis occurred at sodium chloride concentrations of 0.69% or less, and 0.52% or less respectively. These findings indicate that an osmotic fragility test may be helpful in the evaluation of the suitability for transfusion of deglycerolized, resuspended erythrocytes.     

The In Vivo Survival, Mode of Removal of the Non-Viable Cells, and the Total Amount of Supernatant Hemoglobin in Deglycerolized, Resuspended Erythrocytes

The data presented indicate that with the use of the Cohn fractionator to add and remove the intracellular additive, glycerol, and using the slow freeze technic with storage in the frozen state at —80 C or —120 C, the viability of erythrocytes can be maintained for at least two months. The thawed, deglycerolized erythrocytes can then be resuspended in either autologous plasma or in an artificial 5% Hyland albumin resuspension medium, stored up to 8 days at 4C, and transfused to the original donor with acceptable red blood cell survival. ACD blood can be stored for up to seven days at 4 C prior to glycerolization with acceptable 24-hour posttransfusion survival. The mode of removal of the non-viable erythrocytes is primarily through an extravascular mechanism without detectable hemoglobinemia. A limiting factor in the clinical acceptability of deglycerolized, resuspended erythrocytes is the amount of free supernatant hemoglobin present in the unit on the day of transfusion. Washing units of deglycerolized erythrocytes resuspended in autologous plasma and stored for up to 12 days at 4 C is effective in reducing the total amount of supernatant hemoglobin, and the saline washed erythrocytes show acceptable posttransfusion survival. However, deglycerolized red blood cells resuspended initially in the 5% Hyland albumin medium and stored at 4 C for five days or longer do not tolerate saline washing; 24-hour survival of less than 70 per cent was observed and intravascular liberation of hemoglobin was noted in this group only.
This paper reports the total in vitro loss of hemoglobin associated with preservation and with the saline wash used to remove the excessive supernatant hemoglobin which accumulates during the post-thaw period at 4C.     

Prevention of adverse reactions to blood transfusion by the administration of saline-washed red blood cells

We prospectively compared the incidence of complications following saline-washed versus packed red blood cell transfusions, to determine whether routine use of washed red blood cells could reduce significantly the incidence of transfusion reactions. Clinical reports of reactions were evaluated carefully to confirm whether the reaction was caused by transfusion. In 3,799 washed red blood cell transfusions, there were eight confirmed reactions (0.21%). Of 6,359 packed red blood cell transfusions, 31 reactions occurred (0.49%). The difference in incidence of confirmed complications was statistically significant (p less than 0.03). Administration of washed red blood cells to all patients requiring transfusions can thus be seen to reduce significantly the incidence of adverse reactions. This is likely the result of the removal of leukocytes and plasma achieved by the washing process. The increased safety of washed red blood cells must be weighed against their extra expense to determine their cost-effectiveness in transfusion therapy.     

Serum potassium changes following packed red cell transfusions in newborn infants

We studied the effect of packed red cell transfusions on serum potassium levels in sick newborn infants. In 11 transfusions the infants' serum potassium levels fell from a mean of 5.1 +/- 1.2 mEq per 1 (SD) to 4.9 +/- 1.2 mEq per 1 within 1 hour of the transfusion. Plasma potassium in stored packed red cells rose markedly within 48 hours of drawing. In spite of this, the transfusion of 10 ml per kg of packed red cells did not affect the serum potassium concentration in the newborn infant.     

Intravascular [correction of intracellular] hemolysis and renal failure in a patient with T polyagglutination

A patient with postoperative intravascular hemolysis aggravated by transfusion with fresh whole blood and fresh plasma products had acute renal failure. Screening with Arachis hypogaea and Glycine soja lectins showed that his red cells were T-transformed. Only washed red cells were transfused subsequently, and no further fresh plasma products were given. All hemolysis ceased, the renal function returned to normal, and the T-polyagglutination as measured by lectin tests disappeared 4 months after surgery. Early diagnosis of polyagglutination using lectin screening tests is simple to perform, and will facilitate the immediate choice of the correct transfusion therapy, which in this case may have been life-saving.     

Analysis of saline-washed red cells for transfusion to neonatal patients

A protocol was developed under which washed red cells were used to provide small-volume transfusions for neonatal patients (under 4 months of age). Two pediatric units, each containing approximately 135 ml of red cells, were prepared from a freshly collected unit of group O Rh-negative whole blood. Units not more than 6 days old were washed with 1l of 0.9 percent NaCl in a blood cell processor. Aliquots of the washed red cells were drawn into labeled syringes for transfusion to neonatal patients. Data collected from 40 units of washed red cells prepared according to this protocol showed 83 percent red cell recovery, and satisfactory reduction in white cells, plasma proteins, extracellular potassium, red cell metabolic waste products, and anticoagulant. Over 5 years, a total of 2085 different neonatal patients received 7875 separate transfusions prepared from 1860 units of washed red cells. The clinical response was excellent, and no suspected reactions were reported.     

IgA content of frozen-thawed-washed red blood cells and blood products measured by radioimmunoassay

The IgA concentration of various blood products was examined by a radioimmunoassay, and the lowest IgA concentrations were seen in frozen- thawed-washed red blood cells. The routine Regional Blood Transfusion Centre preparation contained a mean IgA concentration of 0.117 mg/l (n = 21) in the frozen-thawed-washed red blood cell supernatant fluid. Further washing of frozen-thawed-washed red blood cells with saline reduced supernatant IgA concentrations to below the limit of IgA detection (less than 0.003 mg/l). Ordinary washed red blood cells and other blood plasma fractions contained substantial quantitites of IgA, supporting the view that frozen-thawed-washed red blood cells should be used for transfusion of patients at risk of developing reactions to IgA, and the need for caution in the use of other blood products in such patients.     

The Effects of Continuous—Flow Washing on Stored Red Blood Cells

Whole blood and packed cells stored up to 21 days prior to being washed by a continuous-flow (CF) technic or by batch washing (BW) were found to have an average posttransfusion survival of greater than 70 per cent. CF-washed red blood cells stored in 5 per cent dextrose and 0.9 per cent saline (5% D&S) wash solution for 24 hours had an average survival of 86 per cent. An average plasma protein dilution of 1:25,000 was obtained with the CF technic as compared to 1:600 with the conventional BW technic. A series of bloods were inoculated either with plasma containing hepatitis-associated antigen (HAA), cytomegalovirus (CMV) or poliovirus (PVS) and then washed. CMV could not be detected by plaque assay in the washed red blood cells nor could HAA by immunodiffusion, counterimmunoelectrophoresis or radio-immune assay. The poliovirus-inoculated washed red blood cells had a marked reduction of the virus when washed by the CF technic as compared to the BW cells. Red blood cells washed by the CF technic did not induce a rise in an AHF inhibitor when transfused into a hemophilic patient with a circulating inhibitor.     

Massive blood transfusion in the elective surgical setting.

Massive haemorrhage in elective surgery can be either anticipated (e.g. organ transplantation) or unexpected. Management requires early recognition, securing haemostasis and maintenance of normovolaemia. Transfusion management involves the transfusion of packed red cells, platelet concentrates and plasma (fresh frozen plasma and cryoprecipitate). Blood product support should be based on clinical judgment and be guided by repeated laboratory tests of coagulation. Although coagulation tests may not provide a true representation of in vivo haemostasis, they do assist in management of haemostatic factors. Below critical levels (prothrombin time or activated partial thromboplastin time >1.8; fibrinogen <1.0 g/l; platelet count < 80 x 10(9) 1(-1)) it is difficult to achieve haemostasis. Despite seemingly adequate blood component therapy there remain situations where haemorrhage is uncontrollable. In this setting, alternative approaches must be considered. These include the use of other blood products (e.g. prothrombin complex concentrates; fresh whole blood; fibrin glue) and pharmacological agents (e.g. aprotinin). Complications of massive transfusion result in significant morbidity and mortality. These may be secondary to the storage lesion of the transfused blood products, disseminated intravascular coagulation, hypothermia or hypovolaemic shock. The use of fresh blood products and leucocyte-reduced packed red cells and platelets, may minimise some of the adverse clinical sequelae.     

Transfusion of red cells is associated with increased incidence of bacterial infection after colorectal surgery: a prospective study

BACKGROUND: Several studies suggest that perioperative blood transfusion is a major independent risk factor for postoperative bacterial infections. Transfusion-induced immunosuppression is thought to mediate this effect. STUDY DESIGN AND METHODS: In a randomized clinical trial comprising 697 patients with colorectal cancer, the relationship between two types of red cell components (buffy coat- depleted packed red cells and white cell-reduced [filtered] packed red cells) and postoperative bacterial infections was analyzed. RESULTS: Both types of red cells appeared to be associated with a greater incidence of postoperative infection than was no transfusion (39 vs. 24%, p < 0.01). A dose-response relationship could be demonstrated: the corrected relative risk was 1.6 for 1 to 3 units of red cells and 3.6 for more than 3 units. Multivariate analyses identified the transfusion of red cells and tumor location as the only significant independent risk factors for postoperative bacterial infection. CONCLUSION: Because allogeneic white cells, plasma, microaggregates, citrate, and platelets could be ruled out as risk factors for transfusion-associated postoperative infections, it is hypothesized that the transfusion of red cells is a potentially detrimental factor that transiently impairs the clearance of bacteria by phagocytic cells.     

Therapeutic Effectiveness of Homologous Erythrocyte Transfusions Following Frozen Storage at ‐80 C for up to Seven Years

Cohn‐processed red blood cells that had been stored for as long as seven years at ‐80 C., washed by the ADL procedure and then stored at 4 C for up to 48 hours, showed approximately 90 per cent 24‐hour recovery in vivo by an automated differential agglutination (ADA) technic, recovery in vitro of approximately 90 per cent, and an index of therapeutic effectiveness of approximately 80 per cent. Washing Huggins‐preserved red blood cells with EDTA by the Huggins process produced a significant deterioration (decreased 24‐hour posttransfusion survival and decreased recovery in vitro ) following storage at ‐80 C for as long as three years. In two of seven patients studied the Huggins‐processed red blood cells that had been stored at ‐80 C for 1.8 years and longer and washed by the Huggins procedure showed intravascular destruction of the compatible nonviable red blood cells. Huggins‐preserved red blood cells with EDTA that had been stored at ‐80 C up to 1.6 years showed, following washing with an electrolyte solution in the ADL bowl, a somewhat better 24‐hour ADA survival, better recovery of the preserved red blood cells, lower supernatant hemoglobin concentrations, and higher intracellular potassium levels on the day of washing and resuspension. These findings suggest that Hugginspreserved red blood cells following storage at ‐80 C for one and one half years or more should not be washed by the Huggins dilution/agglomeration procedure.     

Rapid admixture blood warming

A new technique of admixture blood warming allows rapid warming of packed red blood cells by the addition of heated saline. Each 4 degrees C unit of packed red blood cells is mixed with 250 ml of 70 degrees C saline. Diluted blood at 37 degrees C is ready for transfusion in less than 30 seconds.     

The effect of solvent/detergent-treated plasma on red cells stored in vitro

BACKGROUND: The potential use of solvent/detergent-treated plasma (S/D plasma) in transfusion practice raises concerns about the cytolytic effects that any residual solvent and detergent in the virally inactivated blood component might have on units of red cells in vitro, if the two components are mixed during preparation. STUDY DESIGN AND METHODS: S/D plasma was mixed with variously processed units of stored red cells, in vitro, to evaluate the effect the residual solvent and detergent would have on cell membrane integrity. A paired protocol design was used in which half-units of red cells were exposed to S/D plasma (test), and the matched half-units were exposed to either the supernatant additive solution from the original red cell unit or standard fresh-frozen plasma (FFP) (control). After incubation for up to 5 days, the units were evaluated for evidence of hemolysis or changes in other red cell storage assays. RESULTS: This study showed that, for fresh additive solution red cells (AS-1), the 5-day storage plasma hemoglobin levels were comparable in the red cells exposed to S/D plasma (21 mg/dL) and in the paired half-units stored in the original AS-1 supernatant (31 mg/dL) (p > 0.05). Similar findings were recorded for stored AS-1 red cells (S/D plasma; 111 mg/dL vs. AS-1 supernatant, 147 mg/dL; p > 0.05); stored CPDA-1 red cells (S/D plasma, 133 mg/dL vs. FFP, 103 mg/dL; p > 0.05); frozen red cells (S/D plasma, 28 mg/dL vs. FFP, 18 mg/dL; p > 0.017); and stored irradiated AS-1 red cells (S/D plasma, 608 mg/dL vs. AS-1 supernatant, 726 mg/dL; p > 0.05). Comparable results were found for other assays, including levels of plasma potassium, osmotic fragility, and red cell antigen titer. CONCLUSION: These data show that S/D plasma does not induce red cell lysis even after 5 days of in vitro storage. These results are consistent with previous findings by this laboratory that platelets are not harmed by storage in S/D plasma. Red cells resuspended in S/D plasma and stored for up to 5 days maintain in vitro storage characteristics that are acceptable for the use of the cells in clinical transfusion practice.     

Preparation and in vitro function of granulocyte concentrates for transfusion to neonates using the IBM 2991 blood processor

Clinical studies have suggested that granulocyte transfusions may be of value in the treatment of septic neonatal patients who present with severe granulocytopenia. We have developed a protocol for the preparation of granulocyte concentrates from freshly collected units of whole blood, using an automated blood cell processor. The red cells were washed with saline. Then, the buffy coats were collected from the washed red cells and studied for their suitability as granulocyte concentrates for neonatal transfusion. The mean number of granulocytes per concentrate was 1.6 × 10(9) in a mean volume of 25 ml. Studies of granulocyte function, including viability, random mobility, chemotaxis, phagocytosis and nitro-blue tetrazolium reduction, demonstrated that the granulocytes were functionally unimpaired following preparation of the concentrates. These studies suggest that concentrates of functional granulocytes, suitable for transfusion to neonatal patients, can be prepared from fresh units of whole blood, using a cell processor. This procedure is more cost-effective than leukapheresis and allows for delivery of granulocytes for transfusion in a more timely fashion.     

Hypokalemia and massive transfusion

To investigate the etiology of the hypokalemia that is more commonly associated with massive transfusions than hyperkalemia, we determined the plasma potassium concentrations of 26 stored packed cell preparations and reviewed a series of 15 patients who received massive transfusions with whole blood or packed cells. Our results confirm that stored packed cell preparations, like stored whole blood preparations, are hyperkalemic, and suggest that metabolic alkalosis, catecholamine release, and hemorrhagic shock are important factors in the development of hypokalemia associated with massive blood transfusions. In view of the association of hypokalemia and hyperkalemia with massive transfusion, plasma potassium levels should be carefully monitored in patients receiving massive transfusions.     

Factors Influencing the 24-Hour Posttransfusion Survival and the Oxygen Transport Function of Previously Frozen Red Cells Preserved with 40 Per Cent W/V Glycerol and Frozen at —80 C

A 6.2 M glycerol solution was added directly to concentrated red blood cells before storage at — 80 C for at least two and one-half years. The glycerol was removed from the thawed red blood cells by one of four different washing procedures, and the washed cells were stored at 4 C for an additional 24 hours before transfusion. Recovery in vitro was about 90 per cent, and the posttransfusion survival was about 85 per cent. The CPD anticoagulant maintained the oxygen transport much better than ACD during storage of the red blood cells at 4 C for one week prior to freezing. Results were similar whether glycerolized red blood cells were washed in reusable stainless steel bowls, disposable polycarbonate bowls, or collapsible disposable polyvinylchloride plastic bags. The composition of the wash solution had no significant effect on the posttransfusion survival or oxygen transport function. When the washed red blood cells were stored in a sodium chloride-glucose-phosphate solution at 4 C for 24 hours before transfusion, the 24-hour posttransfusion survival and oxygen transport function was satisfactory. Freeze-preservation of red blood cells with hematocrits of about 40 V per cent and postthaw storage at 4 C for 24 hours resulted in an accumulation of supernatant hemoglobin and extracellular potassium. At the time of transfusion, the red blood cells were concentrated by centrifugation, the supernatant medium was removed, and the hematocrit adjusted to 70 V per cent.     

White cell-associated procoagulant activity induced by ABO incompatibility

BACKGROUND: Disseminated intravascular coagulation is an established complication of acute hemolytic transfusion reactions, particularly those involving the ABO red cell (RBC) antigen system. In addition, peripheral blood white cells, particularly monocytes, have demonstrated expression of procoagulant activity (PCA) in response to inflammatory stimuli. To better define the activation of coagulation in immune hemolysis, in vitro experiments were conducted to investigate the expression of PCA by peripheral blood white cells in ABO RBC incompatibility. STUDY DESIGN AND METHODS: Fresh group O heparinized whole blood was incubated with washed, packed group A or O RBCs. White cells were separated, washed, and lysed before assay of PCA, which was measured by a one-stage recalcified clotting time assay. Units of activity were calculated on the basis of a rabbit brain thromboplastin standard curve. Mechanisms of coagulation activation were investigated by using specific coagulation factor-deficient plasmas, blocking antibodies to tissue factor, and anti-CD11b. RESULTS: Significant levels of white cell-associated PCA were found at 2 to 6 hours in response to incompatible (group A) RBCs, but not in response to compatible (group O) RBCs. PCA was not correlated with numbers of platelets in whole blood. Nonimmune lysis of compatible RBCs did not induce PCA. When whole blood reconstituted from washed cells and heat- inactivated plasma was incubated with incompatible RBCs, PCA and hemolysis were abrogated, which suggests that complement is a required intermediate. Protein synthesis inhibition by the addition of cycloheximide (5 mg/mL) to whole blood incubated with RBCs prevented the expression of PCA. Substitution of factor VIII-deficient plasma for normal plasma in the recalcified clotting time assay had no effect, whereas PCA was reduced by 68 percent with factor VII-deficient plasma and was unmeasurable with factor X-deficient plasma. PCA was restored by a 1-to-1 mix of normal and factor VII-deficient plasma. Incubation of samples in the PCA assay with tissue factor antibodies resulted in up to 86-percent inhibition of measured PCA. Titration of the response to the amount of tissue factor antibodies added demonstrated that maximal inhibition occurred with 0.45 mg per mL, above which no further inhibition took place. However, the addition of anti-CD11b (0.75 mg/mL) concomitantly with anti-tissue factor abolished measurable activity. This effect was independent of the amount of added protein, and anti- CD11b alone had no effect on measured activity. The addition to whole blood concomitantly with RBCs of polyclonal antibodies to tumor necrosis factor, sufficient to neutralize 2000 pg per mL, did not alter PCA expression. CONCLUSION: These results indicate that white cell- associated PCA is generated in whole blood in response to ABO RBC incompatibility and may contribute to disseminated intravascular coagulation in acute hemolytic transfusion reactions. Two possible cellular mechanisms are suggested, which involve tissue factor expression and the activation of factor × by a CD11b-dependent mechanism.     

Preparation of IgA-deficient platelets

The IgA-deficient patient presents a difficult transfusion problem, particularly if he or she has previously been immunized and has formed IgA antibodies. Blood components from IgA-deficient donors may be used safely but are often not accessible to transfusion facilities. A satisfactory red cell component may be obtained by standard freezing and washing methods. There is no comparable platelet concentrate that uniformly affords protection against anti-IgA-mediated reactions. A method has been developed for the preparation of an IgA-deficient platelet concentrate by washing with citrate-buffered saline. Platelets prepared in this manner were transfused successfully to an IgA-deficient patient with a history of anaphylaxis related to transfusion. It was further demonstrated that the removal of IgA is enhanced if platelets are washed within 48 hours of collection. When platelets were stored for more than 48 hours, similar washing techniques resulted in significantly higher levels of IgA in the platelets washed with either buffered (p = 0.004) or unbuffered (p = 0.0002) saline. Platelets washed with unbuffered saline (pH 4.5-5.5) contained substantially more IgA than did platelets washed in a similar manner with citrate-buffered saline (pH 6.5-6.8) (p = 0.001).