Standard Blood Bank Conditions Research Articles

Use of 8-Methoxypsoralen and Long-Wavelength Ultraviolet Radiation for Decontamination of Platelet Concentrates

AbstractTransmission of viral diseases through blood products remains an unsolved problem in transfusion medicine. We have developed a psoralen photochemical system for decontamination of platelet concentrates in which platelets are treated with long wavelength ultraviolet radiation (UVA, 320-400 nm) in the presence of 8-methoxypsoralen (8-MOP). Bacteria, RNA viruses, and DNA viruses ranging in genome size from 1.2 × 106 to 1010 daltons, encompassing the size range of human pathogens, were inoculated into platelet concentrates and subjected to treatment. This system inactivated 25 to 30 logs/h of bacteria Escherichia coli or Staphylococcus aureus, 6 logs/h of bacteriophage fd, 0.9 log/h of bacteriophage R17 and 1.1 logs/h of feline leukemia virus (FeLV) in platelet concentrates maintained in standard storage bags. Platelet integrity and in vitro function before, immediately following photochemical treatment, and during prolonged storage after treatment, were evaluated by measuring: (1) extracellular pH; (2) platelet yields; (3) extracellular lactate dehydrogenase (LDH) levels; (4) platelet morphology; (5) platelet aggregation responsiveness; (6) thromboxane β-2 (TXB-2) production; (7) dense body secretion; and (8) alpha granule secretion. These assays demonstrated that this photochemical inactivation system inactivated bacteria and viruses in platelet concentrates with minimal adverse effects on the in vitro function of platelets in comparison to untreated control concentrates maintained under current, standard blood bank conditions.

Use of 8-methoxypsoralen and long-wavelength ultraviolet radiation for decontamination of platelet concentrates

Transmission of viral diseases through blood products remains an unsolved problem in transfusion medicine. We have developed a psoralen photochemical system for decontamination of platelet concentrates in which platelets are treated with long wavelength ultraviolet radiation (UVA, 320–400 nm) in the presence of 8-methoxypsoralen (8-MOP). Bacteria, RNA viruses, and DNA viruses ranging in genome size from 1.2 x 10(6) daltons, encompassing the size range of human pathogens, were inoculated into platelet concentrates and subjected to treatment. This system inactivated 25 to 30 logs/h of bacteria Escherichia coli or Staphylococcus aureus, 6 logs/h of bacteriophage fd, 0.9 log/h of bacteriophage R17 and 1.1 logs/h of feline leukemia virus (FeLV) in platelet concentrates maintained in standard storage bags. Platelet integrity and in vitro function before, immediately following photochemical treatment, and during prolonged storage after treatment, were evaluated by measuring: (1) extracellular pH; (2) platelet yields; (3) extracellular lactate dehydrogenase (LDH) levels; (4) platelet morphology; (5) platelet aggregation responsiveness; (6) thromboxane beta-2 (TXB-2) production; (7) dense body secretion; and (8) alpha granule secretion. These assays demonstrated that this photochemical inactivation system inactivated bacteria and viruses in platelet concentrates with minimal adverse effects on the in vitro function of platelets in comparison to untreated control concentrates maintained under current, standard blood bank conditions.

Sterility and quality of blood dispensed in syringes for infants.

A unit-dose system was designed to dispense precise quantities of blood in a form ready for immediate transfusion into neonatal patients. The principles were similar to those used by pharmacies to dispense individual doses of drugs. In the blood center, the precise volume of blood ordered for a neonatal patient was aspirated through a microaggregate filter into a labeled plastic syringe for dispensing to the nursery in a correspondingly labeled zip-lock plastic bag. In the nursery, the premeasured and prefiltered blood was ready for immediate infusion, and the syringe was attached directly to a mechanical infusion pump. Several experiments were performed to ensure sterility and quality of whole blood dispensed by this system. Over 300 aerobic and anaerobic cultures were performed, and it was concluded that the extra handling required to prepare syringes of blood did not lead to bacterial contamination. In addition, the quality of whole blood was maintained, for at least 6 hours, equally well in syringes as it was in blood bags stored under standard blood bank conditions when assessed by hematocrit, blood pH, and measurements of plasma potassium, glucose, lactic dehydrogenase, and free hemoglobin. Thus, unit dose dispensing offers a precise and convenient method to prepare small, accurately measured quantities of filtered, sterile, and quality blood products for neonatal patients.

Mononuclear cell-surface antigens during storage of banked blood.

The surface antigens present on lymphocyte subpopulations and monocytes in whole blood stored under standard blood bank conditions were analyzed with a series of monoclonal antibodies and a fluorescence-activated cell sorter. During the first week of storage, the percentage of viable cells bearing T lymphocyte markers declined from 66% to 29% (P less than 0.001). Within the T cell subset, there was a disproportionate decrease in the percentage of cells reacting with anti-Leu 3a (a helper T cell marker) resulting in a reduction in the measured ratio of helper-to-suppressor T cells (P less than 0.01). The relative percentage of cells bearing B lymphocyte markers increased from 11% to 31% (P less than 0.001). The proportion of HLA-DR-positive cells that were B cells increased during the first week of storage from 38% to 65% (P less than 0.01). The degree of residual antigen expression as measured by the remaining median intensity of fluorescence was significantly greater for B cell and HLA-DR antigens as compared with T cell antigens. The measured changes during storage in the relative proportions of mononuclear cells expressing cell-surface antigens probably result from a combination of differential residual antigen expression and differential survival of mononuclear subpopulations. The presence of adenine in the anticoagulant-preservative solution had no measurable effect. Storage at 4 C, however, was shown to result in a 70% decrease in the proportion of antigen-bearing T cells, even as early as 24 hr of storage. The findings may have bearing on the beneficial effect of blood transfusion in renal transplant recipients, as well as on experimental models of the immune response to transfusion.

Lipid composition of freshly prepared and stored platelet concentrates

To evaluate the possibility that changes in lipid composition might be related to the functional lesion that develops when platelets are stored as concentrates for several days, we measured lipid constituents of platelets in freshly prepared concentrates and in concentrates stored for 72 hr at 4 degrees C or at 20 degrees C under standard blood banking conditions. At 20 degrees C, but not at 4 degrees C, platelets lost about 15% of total cholesterol and 7%--11% of total phospholipid. The distribution of individual phospholipids remained unchanged. This was also true of the fatty acid distribution in total phospholipids and in individual phospholipids except for a statistically significant reduction of linoleic acid (18:2) and an increase in oleic acid (18:1) in phosphatidyl inositol (PI). Platelets collected in citrate-phosphate- dextrose (CPD) anticoagulant did not differ significantly in lipid composition from those collected in acid-citrate-dextrose (ACD) anticoagulant during the period of observation. These findings do not provide a basis to suggest that functional abnormalities developing in stored platelets are related to changes in lipid composition.

Lipid Composition of Freshly Prepared and Stored Platelet Concentrates

To evaluate the possibility that changes in lipid composition might be related to the functional lesion that develops when platelets are stored as concentrates for several days, we measured lipid constituents of platelets in freshly prepared concentrates and in concentrates stored for 72 hr at 4 degrees C or at 20 degrees C under standard blood banking conditions. At 20 degrees C, but not at 4 degrees C, platelets lost about 15% of total cholesterol and 7%--11% of total phospholipid. The distribution of individual phospholipids remained unchanged. This was also true of the fatty acid distribution in total phospholipids and in individual phospholipids except for a statistically significant reduction of linoleic acid (18:2) and an increase in oleic acid (18:1) in phosphatidyl inositol (PI). Platelets collected in citrate-phosphate-dextrose (CPD) anticoagulant did not differ significantly in lipid composition from those collected in acid-citrate-dextrose (ACD) anticoagulant during the period of observation. These findings do not provide a basis to suggest that functional abnormalities developing in stored platelets are related to changes in lipid composition.

Ultrastructure of human platelets processed for transfusion under standard blood bank conditions.

Platelets processed for transfusion are routinely subjected to centrifugation, mechanical agitation and storage at either room temperature or 4 C. To evaluate morphologic changes associated with standard blood bank conditions, platelet units were grouped into four categories: linear agitation at room temperature, agitation at a 90 degrees arc with a Hemolater at room temperature, agitation at a 360 degrees arc with a commercial platelet agitator, and refirgeration at 4 C without agitation. Cold (4 C) preservation resulted in a surprisingly good preservation of platelet ultrastructure, even at 72 hours of storage. By contrast, harsh linear agitation at room temperature brought about marked alterations in platelet ultrastructure beginning at 24 hours. The Hemolator agitator with 90 degrees arc was associated with good preservation of platelet organelles even at 72 hours, whereas marked degenerative changes were observed at this time in platelets processed with a commercial agitator with a 360 degrees arc. These results indicate that platelets stored at 4 C or processed at room temperature with an agitator with 90 degrees arc show the best preservation of ultrastructure.

Plasma Factor VIII, Variables Affecting Stability under Standard Blood Bank Conditions and Correlation with Recovery in Concentrates

Some of the variables affecting factor VIII during storage of plasma at 4 C prior to preparing factor VIII concentrates and cryoprecipitates were investigated. No significant difference in factor VIII levels could be demonstrated between whole blood, platelet-rich plasma or plasma depleted of platelets when stored at 4 C. However, in frozen plasma platelets were shown to have a deleterious effect on factor VIII. No significant difference in factor VIII levels was found in whole blood or plasma stored at 4 C for 6 hours as compared with blood or plasma stored for 18 hours (p = greater than 0.1). There was, however, a highly significant difference (p = less than 0.001) between plasma and whole blood stored at 4 C for 4 hours as compared with storage for 18 hours. There was a significant correlation (correlation coefficient 0.714) between factor VIII levels in the starting material and the factor VIII recovered in cryoprecipitates, but in a small pilot study no correlation between factor VIII levels in the starting material and that recovered in freeze-dried concentrates, could be found. There was no significant difference in factor VIII levels between the Group O and Group A donations used in this study (p = greater than 0.1), and no correlation was found between the drop in pH during storage and the factor VIII decay pattern.

Chemotactic activity of human granulocytes preserved in various anticoagulants

Abstract It has been shown that granulocyte function remains unchanged after 24 hours of storage at 4 ° C. when measured by the bactericidal assay and the quantitative nitroblue tetrazolium test. These assays measure the ability of granulocytes to phagocytize latex particles. However, in granulocyte transfusion therapy, these cells must retain the ability to migrate to sites of infection. Therefore, the chemotactic response of granulocytes was studied after collection and during storage in four currently available anticoagulant preservatives. Granulocytes were collected from 18 different donors by phlebotomy into plastic bags using acid citrate dextrose (ACD), citrate phosphate dextrose (CPD), heparin, and ion exchange anticoagulants, and stored under standard blood bank conditions. Immediately after collection, granulocytes from all anticoagulants had chemotactic responsiveness equal to control granulocytes collected directly from the donor. Following 24 hours of storage, there was a 14 per cent loss of chemotactic response in ACD granulocytes, 12 per cent loss of response in CPD granulocytes, 26 per cent loss in heparin granulocytes, and 24 per cent loss of response in ion exchange granulocytes. Loss of chemotactic response of this mild degree suggests that granulocytes stored for up to 24 hours in ACD or CPD may be satisfactory for transfusion.

Effects of Anticoagulants and Storage on Granulocyte Function in Bank Blood

Abstract Storage of granulocytes for transfusion has not been practical because it has been considered that granulocyte function in bank blood is retained for only a few hours after collection. In the present study, granulocyte function was evaluated using the bactericidal assay and the quantitative nitroblue tetrazolium (NBT) method. Granulocytes from whole blood collected into acid citrate dextrose (ACD), citrate phosphate dextrose (CPD), heparin, ion exchange, and sodium citrate anticoagulants showed no functional impairment after 24 hr of storage at 4°C. With further storage, all granulocytes showed a loss of NBT activity. However, after 48 and 72 hr, granulocytes from whole blood stored in ACD and CPD killed the expected number of bacteria in the bactericidal assay. Thus, when tested in vitro, granulocytes maintain normal function, at least during the first 24 hr after collection when stored in certain anticoagulants under standard blood bank conditions.

EFFECT OF BLOOD-BANK STORAGE ON LEUCOCYTE FUNCTION

The bactericidal activity of polymor-phonuclear neutrophils (P.M.N.s) and the ability of lymphocytes to respond to phytohæmagglutinin (P.H.A.) were studied in blood drawn under standard blood-bank conditions into acid-citrate-dextrose anticoagulant and stored at 4°C. There was no loss of bacteridical activity during the first 24 hours of storage, and bactericidal activity was not completely lost until after 4 days of storage. Lymphocytes lost the ability to respond to P.H.A. after 17 days of storage although morphologically normal lymphocytes were present in blood stored for 21 days. Irradiation with up to 6000 roentgens reduced but did not eliminate the lymphocyte response to P.H.A. and did not affect the bactericidal capacity of the P.M.N.S. These results suggest that functionally active P.M.N.S are present in bank-blood during the first few days of storage or may be separated and stored for periods considerably longer than previously indicated. The presence of morphologically normal lymphocytes and responsiveness to P.H.A. stimulation indicate that any bank-blood within the 21-day shelf-life may be the source of a potential graft-versus-host reaction when transfused to patients with impaired immune responses. Irradiation of the blood apparently does not protect against this hazard.

Effects of adenine on clotting factors in fresh blood, stored blood, and stored fresh frozen plasma.

The addition of small quantities of adenine to whole blood may prolong the useful shelf life of bank blood. The present study was undertaken to evaluate the effects of adenine on the clotting factors in blood containing ACD and CPD. Units of whole blood were collected in ACD, ACD‐adenine, CPD and CPD‐adenine, and each was stored 42 days under standard blood bank conditions. Samples of fresh frozen plasma containing these anticoagulants were stored three to four months at ‐30 C. Assays of Factors V, VIII (AHF), IX (PTC), X (Stuart Factor), fibrinogen, and prothrombin were performed on fresh blood, stored blood and stored fresh frozen plasma. The presence of small quantities of adenine did not appear to produce any appreciable alteration in the activity of the clotting factors in fresh blood. Further, adenine did not appear either to improve or worsen the survival of the procoagulants in whole blood stored 42 days or in fresh frozen plasma stored three to four months. There was significant deterioration of Factors V and VIII in whole blood stored 42 days in ACD, ACD‐adenine, CPD, and CPD‐adenine, but the degree of storage loss was independent of the anticoagulant employed. Factor X, fibrinogen, and prothrombin remained stable in blood stored 42 days regardless of the anticoagulant used, but Factor IX activity increased during storage possibly as the result of contact activation. Fresh frozen plasma stored three to four months showed a uniform slight loss of Factor VIII in all four anticoagulants, but Factors V, IX, X, fibrinogen, and prothrombin remained stable in stored fresh frozen plasma regardless of the anticoagulant employed.

Concentrated antihemophilic factor (AHF) from outdated blood.

The antihemophilic factor (AHF) content of bank blood decreases during storage under standard blood bank conditions. In the present study, an average of 58 per cent of the original activity was present after the usual shelf life of 21 days expired. This material was concentrated by a modification of the Pool cryoprecipitation method. Almost 60 per cent of the residual AHF in the outdated plasma was found in the precipitate. The resulting precipitate was resuspended in about 25 ml of the residual plasma. On a volume basis, this had an average of about four times the activity of fresh human plasma. The cold insoluble material resuspended in residual plasma can be used for therapy without further processing. Following intravenous injection into hemophilic patients, high levels of circulating AHF, in excess of 60 per cent of normal, have been achieved without causing circulatory overload.

Effect of high CO 2 tension on banked ACD blood

Sustained reduction in pCO₂ (hypocapnin) in dogs was associated with a progressive rise in lactic and pyruvic acids which accounted for a progressive bicarbonate deficit. Similar changes, i.e., rise in lactate and reduction in bicarbonate, are known to occur in banked ACD blood. The observed lactate-pCO₂ relationship in vivo is the basis for this study of the effect of variable CO₂ concentration on the metabolism of stored blood. Human blood was stored under standard blood bank conditions (4 C), except that one portion was stored in containers with high pCO2 content. Paired containers were analyzed at 0, 7, 14, and 21 days for pH, CO₂ content, lactate, pyruvate, glucose and electrolytes. The results showed a marked inhibition of glycolysis in the blood stored under conditions of high CO₂. This inhibition was reflected by lower lactate and pyruvate levels, less reduction in bicarbonate and less accumulation of [H⁺] in blood stored under CO₂. The inhibition was not permanent. High pCO₂ was found to be the inhibitory factor in experiments designed to study the independent effects of pCO₂ and pH.