Blood Coagulation and Vascular Integrity: Effects of Heparin

The administration of large and repeated doses of heparin to normal ratsresulted in increased output of red blood cells into the lymph, comparable tothat associated with x-ray induced thrombocytopenia. A considerable lapse oftime was observed between the inhibition of blood coagulation and the increase of vascular permeability to red blood cells. The latter was not decreasedby return of the blood clotting to normal.

Submitted on March 21, 1962 Accepted on July 4, 1962     

Blood cell phosphogluconolactonase: assay and properties

6-Phosphogluconolactonase (6-PGL) catalyses the second reaction of the hexosemonophosphate pathway. Although the delta-lactone of 6-phosphogluconic acid is the natural substrate for this enzyme, the more stable gamma-lactone may also be used. We prepared the gamma-lactone of 6-phosphogluconic acid from 6-phosphogluconate. When stored in dimethylsulfoxide, this material was found to be stable in liquid nitrogen for several months. A method for measuring 6-phosphogluconolactonase (6-PGL) using the gamma-lactone as substrate has been developed, after defining conditions under which spontaneous hydrolysis of the lactone is relatively slow and the enzymatic velocity is relatively rapid. The enzyme had no divalent cation requirement and was not significantly inhibited by a 50-fold excess of gluconolactone. It was distinct, therefore, from gluconolactonase. At 25 degrees C normal human red cells were found to contain approximately 50 IU of 6-phosphogluconolactonase/g Hb. The activity of the enzyme was independent of red cell age. Based on protein content, human lymphocytes, monocytes, granulocytes and platelets, contained approximately 10 times the activity of red blood cells. The activity of 6-PGL was stable for at least 6 d in red cells stored at 22 degrees C and for at least 20 d in red cells stored at 4 degrees C.     

Studies on Lymphocytes. I. Lymphopenia Produced by Prolonged Extracorporeal Irradiation of Circulating Blood

1. A method for extracorporeal irradiation of the circulating blood hasbeen developed.

2. Extracorporeal irradiation of the blood will produce a lymphopeniapromptly which persists for weeks.

3. Heparin in high doses in the calf produces a lymphocytosis and neutrophilic leukocytosis.

Submitted on March 8, 1962 Accepted on April 11, 1962     

Microbiologic Determination of Folic Acid Derivatives in Blood

A microbiological method for the determination of folic acid activity(F.A.A.) in blood, and serum with three organisms (L. casei, S. faecalis andP. cerevisiae), is described. The S. faecalis No. 9 strain employed shows a considerably higher sensitivity than the standard ATCC No. 8043 strain, whilstthe specificity towards the various F.A. derivatives tested is similar. Thesensitivity of the P. cerevisiae (L. citrovorum) assay is also increased by adding thymidine, which spares the requirement for folinic acid. Finally, the procedure of extraction of the F.A.A. from blood has been modified to assurerelease and stabilization of the compounds assayed.

F.A.A. in blood and serum of healthy adults and infants are given.

Submitted on April 20, 1962 Accepted on July 12, 1962     

Alpha-chain Thalassemia and Hydrops Fetalis in Malaya: Report of Five Cases

Five cases of severe hydrops and erythroblastosis fetalis in association witha large amount of Hb "Bart’s," all of Chinese origin, are described. The following characteristic clinical and hematologic symptoms were found. Therewere generalized hydrops, ascites and gross enlargement of the liver. Thespleen, however, was not always enlarged. The placenta was large and friable.Severe erythroblastosis of the blood was always found, with reticulocytosis,many target cells and thin cells. The MCV of the red cells was very high.The cells showed an interesting sickling phenomenon. No evidence of isoimmunization was found. In eight parents examined, no abnormal hemoglobinwas detected, and alkali-resistant hemoglobin and hemoglobin A₂ were notfound to be increased. Their blood showed microcytosis of the red cellsexcept in one father and one mother. In this mother, however, the blood wasexamined after a blood transfusion. It is thought probable that these werecases of homozygous -chain thalassemia.

Submitted on April 1, 1962 Accepted on July 30, 1962     

The Effects of Concentrated Eluted Anti-Red Blood Cell Antibodies on the in Vivo Survival of Normal Red Blood Cells

1. A method has been described for the preparation and sterilization of aconcentrated eluate from human red cell stroma.

2. Red cells sensitized by such an eluate prepared from normal controlred cells showed entirely normal in vivo survival, as did cells sensitized byeluate from anti-H coated cells.

3. Sensitization of red cells by concentrated eluates from a patient withCoombs-negative acquired hemolytic anemia and from a patient with Coombs-positive acquired hemolytic anemia did not cause significant alteration in thein vivo survival of the red cells.

4. Red cells sensitized by the concentrated eluate from anti-D sensitizedcells disappeared from the recipient’s circulation very rapidly and were sequestered in the spleen, indicating preservation of the physiologic propertiesof the antibody throughout the elution, concentration and sterilization procedures.

Submitted on June 22, 1959 Accepted on September 27, 1959     

Use of Preserved Autologous Red Blood Cells to Absorb Warm Autoantibodies from the Serum of Patients Receiving Blood Transfusion Therapy

This paper describes two practical methods for the preservation of pretransfusion patient red blood cells for antigen typing and autoabsorption during a course of transfusion therapy. Blood samples from patients who had serum warm autoantibodies and a positive direct antiglobulin test were collected, the serum frozen, and the red cell aliquots separately preserved by PVP-methanol or formaldehyde fixation. After storage and recovery, the IgG antibodies were dissociated and the cells used for absorption of the warm autoantibodies. The preserved red cells removed the warm autoantibodies as effectively as fresh red blood cells from the same patient. Preservation of autologous red cells prior to the onset of transfusion therapy provides an extension of the autoabsorption procedure and a simple alternative to differential absorption.     

Removal of Leucocytes from Whole Blood and Erythrocyte Suspensions By Filtration Through Cotton Wool

Abstract. (1) Blood was stored in polyvinyl-chloride bags containing citrate-phosphate-dextrose (CPD) with adenine in a final concentration of 0.25 mM. (2) Red cell ATP was well maintained (>70% of original) for 4 weeks in whole blood as well as in red cell concentrate (PCV 85 ± 2%). After 5 weeks the ATP level was about 70% in whole blood and about 40% in red cell concentrate. (3) Red cell 2,3-diphosphoglycerate (DPG) was about 60% of the original after 2 weeks and about 30% after 3 weeks of storage when stored both as whole blood and as red cell concentrate. (4) The red cell 24-hour post-transfusion viability was about 80% after 4 weeks of storage both as whole blood and as red cell concentrate. After 5 weeks of storage the 24-hour viability was 78.7 ± 3.5% in whole blood and 76.5 ± 6.7% in red cell concentrate. (5) 820 patients received 3,238 units of CPD-adenine blood, and 761 patients serving as controls received 2,807 units of acid-citrate-dextrose (ACD) blood. The frequency of transfusion reactions was 3.5% for patients receiving CPD-adenine blood and 4.1% for the control group. (6) The maximum storage time was set at 5 weeks for the CPD-adenine blood and 3 weeks for the ACD blood. The longer preservation time decreased out-dating by at least 50%.     

Red blood cell function and blood storage

Red blood cells are ideal vehicles for delivering oxygen to tissues, but their functions deteriorate during liquid preservation. In this article, we review the role of red blood cells in oxygen delivery and methods to evaluate the effectiveness of red blood cell transfusion. Quantitative estimation of transfusion effects could avoid unnecessary transfusion and reduce the risk of transfusion-associated disorders. We also describe the benefits of transfusion of red blood cells having a higher oxygen-delivering capacity. Phosphoenolpyruvate is a promising component to prepare red blood cells having a higher oxygen-delivering capacity.     

Simplified Determination of Blood Adenosine Triphosphate Using the Firefly System

A simplified method is described for the determination of red cell ATPusing the firefly lantern extract method. Variables investigated include theeffect of the time of reading, dilution of firefly extract and the effective rangeof the method. Excellent recoveries were obtained. Optimal extraction of ATPfrom red cells was achieved with a hypotonic buffer at pH 9.2. The methodcould be used with acid-citrate-dextrose, heparin or EDTA as an anticoagulant. The method was found to be highly specific when the nucleotides foundin normal human blood were investigated; only adenosine diphosphate andguanosine triphosphate gave slight readings, neither of which would significantly affect ATP determinations of human blood. Normal human valueswere found to be 5.45 µmoles of ATP/Gm. of hemoglobin or 1.83 µmoles/ml.red cells in heparinized blood samples. This method is believed to be morerapid, more reproducible and more accurate than any previously describedmethod of ATP determination.

Submitted on October 1, 1963 Accepted on December 3, 1963     

Evidence for Stem Cells in the Peripheral Blood of Mice

Leukocytes from the peripheral blood of normal F₁ hybrid mice have beensuccessfully used to promote survival of lethally irradiated parent andclosely related homologous animals. Identification of donor-type red cellsand leukocytes has been established on samples of blood taken from severallong-term survivors. Histologic data and serologic typing of cells from lymphnodes, bone marrow, and spleens of chimeras killed early after irradiation andtreatment established that injected peripheral leukocytes had transplantedand were proliferating into lymphocytes, granulocytes, and erythrocytes. Fe⁵⁹was taken up by red cells and spleens of leukocyte-injected mice but not bytissues of radiation control animals. The newly formed, Fe⁵⁹-labeled erythrocytes were hemolyzed by immune serum specific for donor-type antigens.

Submitted on November 10, 1961 Accepted on March 6, 1962     

Red Cell Preservation: Further Studies with Adenine

Supplementation of the ACD-preservative with small amounts of adenine(0.5 µM per ml. amounting to 37 mg. of the base or 56 mg. of adenine sulfateper 550 ml. unit of blood) preserved satisfactory viability (post-transfusionsurvival greater than 70 per cent) of stored human red cells for 5 to 6 weeks.In the concentrations used, the addition of guanine, cytidine or uridine, aloneor in combination with adenine, had little or no effect in extending viability.Hypoxanthine, even in large amounts, did not appear to be toxic to thestored cells. Preservation of viability after 6 weeks of refrigerated storagemay be somewhat improved by storage in certain plastic containers as compared with glass.

Submitted on April 16, 1962 Accepted on June 22, 1962     

Successful 0 degree C liquid preservation of red blood cells.

In an effort to determine the lowest acceptable temperature for liquid preservation of red blood cells, the latter were stored at 5, 0, -2 and -5 degrees C. When polyvinylchloride (PVC) bags plasticized with di(2-ethylhexyl)phthalate (DEHP) were used as containers, hemolysis of red cells stored for 42 days at 0 degree C was lower than at the other three temperatures. Adenosine triphosphate (ATP) levels, morphology scores, and deformability indices were also best maintained at 0 degree C, and erythrocyte osmotic fragility did not increase at that temperature. On the other hand, when blood was stored in glass bottles at 0 degree C, hemolysis and erythrocyte osmotic fragility increased. The addition of DEHP to red cells stored in glass bottles at 0 degree C decreased hemolysis and erythrocyte osmotic fragility to levels equal to those in PVC bags. Thus, storage of red cells can be done at temperatures as low as 0 degree C, if PVC bags are used as the container. Previous negative assessments of storage temperatures below 4 degrees C, may be due to the use of glass bottles as containers for blood preservation.     

Glutathione Instability in Normal Blood

Studies have been carried out in vitro to determine whether normal erythrocytes may be made to appear glutathione unstable. Results demonstrate thatconstant agitation in air or oxygen will cause a drop in reduced glutathioneupon incubation of normal blood with an oxidant drug in concentrations whichhave no significant effect during quiet incubation.

The results are consistent with the fact that drug-induced hemolysis maybe seen in nonsensitive individuals and that sensitive individuals may have amild hemolytic state without exogenous drug.

Submitted on January 31, 1962 Accepted on April 27, 1962     

Fetal Hemoglobin-containing Erythrocytes I. Counts of Cells Stained by the Acid Elution Method Compared with Alkali Denaturation Measurements

The quantitative study of peripheral smears for red cells containing fetalhemoglobin ("F-containing cells") by the acid elution method is a usefuland dependable screening test. Findings with this approach were comparedwith fetal hemoglobin assay by alkali-denaturation in a series of 19 normalsubjects and 63 subjects with an assortment of blood disturbances. The resultsof the two methods had a correlation of +.79, suggesting a reliable butfar from perfect identity between the methods. The acid elution methodtended to give higher readings, and seemed more sensitive for recognizingindividuals with slight increases in red cell fetal hemoglobin as occurs inthalassemia minor.

Accepted on December 11, 1962     

Erythrocyte Endogenous Proteinase Activity during Blood Bank Storage

We studied proteolytic alterations of membrane proteins in ghosts derived from human red blood cells, preserved up to 35 days in the liquid state either as whole blood or with additive solution. The study was carried out by performing sodium dodecyl sulfate polyacrylamide gel electrophoresis of stromal proteins from erythrocytes, either previously treated with proteinase inhibitors or previously incubated in conditions promoting proteolysis. To differentiate the effect of erythrocyte from granulocyte proteinases, the investigation was also carried out in leukocyte-free red cell preparations. The results show: (1) the effects of endogenous proteinases on membrane proteins derived from red cells stored under blood bank conditions; (2) a decrease of proteolytic effects in ghosts derived from red cells which have been submitted to a longer storage; (3) a relevant influence of the red cell resuspending medium before lysis on the time-dependent onset and exhaustion of proteolysis in ghosts. The presence of increased proteolysis in ghosts could be regarded as a marker of molecular lesions induced in red cells by storage under blood bank conditions.     

Lymphocyte Response to Blood Transfusion in Man: a Comparison of Different Preparations of Blood

The appearance of atypical lymphocytes in post-transfusion blood, their incorporation of tritiated thymidine in tissue culture and the elimination of cytotoxic antibody production, have been used as markers to show that frozen red cells are the least immunogenic when compared with dextran sedimented blood and whole blood donations. The absence of atypical lymphocytes and failure to produce lympho-cytotoxic antibodies after transfusion of frozen cells is highly significant ( P <0.001) when compared with whole blood donations.     

Improved Methods for the Measurement of Acetaldehyde Concentrations in Plasma and Red Blood Cells

Research on the toxic effects of acetaldehyde (Ach) is hampered by analytical difficulties which have been overcome by two new methods suitable for the measurement of Ach in plasma and red blood cells. The first procedure involves rapid separation of plasma, deproteinization, and Ach derivatization with 2,4-dinitrophenylhydrazine (DNP). After extraction with isooctane, the Ach-DNP complex is separated by reverse-phase high performance liquid chromatography. Red blood cell Ach is measured by a modification of the semicarbazide method. The red blood cell hemolysate is mixed with the semicarbazide solution and the extract is injected into the head-space gas chromatograph. The procedure minimizes the artifactual Ach formation which interferes with the direct hemolysis method when human blood is used. After 0.3 g/kg of ethanol, Ach values of 1.5 +/- 0.2 and 9.9 +/- 2.3 microM were detected in plasma and red blood cells of healthy volunteers. These data indicate that an important fraction of Ach circulates in the red blood cells and can be missed or underestimated when only plasma Ach is measured.     

Effects of Local Irradiation (Co60 Teletherapy) on the Peripheral Blood and Bone Marrow

1. Local Co⁶⁰ teletherapy caused a reduction in leukocytes and lymphocytesin the peripheral blood.

2. The bone marrow demonstrated no morphologic change in nonirradiatedcontrol sites.

3. Local irradiation produced a pronounced and persistent hypoplasia inthe treated sites during and after irradiation, with a great reduction in thenumbers of megakaryocytes and precursors of red and white cells. Duringthe period of greatest radiation effect the persistent cells were chiefly plasmacells, "mononuclear cells," and lymphocytes.

4. Even after "cancerocidal" radiotherapy, irradiated bone marrow showssome capacity to regenerate as evidenced by appearance of precursors ofvarious cell series and their ability to incorporate tritium-labeled thymidine.

5. Hemosiderin increased in varying degrees in irradiated sites but showedno change in the control sites.

6. Satisfactory marrow samples can be aspirated from the pubic bone.

Submitted on November 1, 1962 Accepted on December 27, 1962     

Characterization of the Increased Binding of Acetaldehyde to Red Blood Cells in Alcoholics

Using equilibrium dialysis, we found that acetaldehyde, at the levels commonly occurring after ethanol ingestion, did not bind detectably to plasma proteins, but there was significant binding to red blood cells, more in alcoholics than in nonalcoholics. The binding to red blood cells was inhibited by pyridoxal phosphate and N-ethylmaleimide, suggesting adduction to amino and thiol groups. Binding kinetics were consistent with at least two sites. The one with the highest affinity for acetaldehyde corresponded to hemoglobin. Its affinity and Bmax were not changed in alcoholics, but these binding sites accounted for only 44% of the sites available in the red blood cells of alcoholics and 80% of those in controls. Moreover, this binding was not inhibited by N-ethylmaleimide. There was no detectable binding to red cell ghosts. Nonprotein binding was then assessed by changes in NADH produced by the addition of protein-free fractions of the cells to an alcohol dehydrogenase system in equilibrium; this revealed a second binder of lower affinity, larger capacity and with sensitivity to both inhibitors. This binding (possibly due to thiazolidine formation with cysteine) was enhanced in alcoholics, whose red blood cell cysteine content was doubled. Levels of red blood cell cysteine and acetaldehyde remained high for 2 weeks after withdrawal. Because of the prolonged persistence after withdrawal, these changes may provide new markers of alcoholism.