The Preservation of Human Red Blood Cell Agglutinogens in Liquid Nitrogen: Study of a Technic Suitable for Routine Blood Banking

A simple method for the preservation of red blood cells in liquid nitrogensuitable for routine laboratory use is described. With this method, erythrocyteantigens retain their integrity for at least six months and, after thawing, remainactive for at least two weeks in Alsever’s Solution. A panel of cells preservedin liquid nitrogen is as satisfactory as fresh cells in defining irregular antibodies encountered in patient sera. The advantages of this technic over othermethods of red blood cell preservation is discussed.

Submitted on April 9, 1962 Accepted on April 27, 1962     

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     

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     

Sulfhemoglobinemia and Acute Hemolytic Anemia with Heinz Bodies Following Contact with a Fungicide-- Zinc Ethylene Bisdithiocarbamate--in a Subject with Glucose-6-Phosphate Dehydrogenase Deficiency and Hypocatalasemia

Sulfhemoglobinemia associated with Heinz body formation and acutehemolytic anemia following contact with a fungicide, zinc ethylene bisdithiocarbamate, is described in a Persian Jew whose red blood cells had low glucose-6-phosphate dehydrogenase activity with low and unstable reducedglutathione and low catalase activity.

The fungicide, similarly to acetylphenylhydrazine, was capable of decreasing in vitro the reduced glutathione of the patient’s red blood cells, as wellas of those of other subjects with the same enzymatic defect.

The sulfhemoglobinemia and the hemolytic anemia are considered to havebeen produced independently by the fungicide, the glucose-6-phosphate dehydrogenase deficiency having played a role only in the latter. The possibilitythat the hypocatalasemia was a factor in rendering the patient’s red bloodcells sensitive to the hemolysis- and sulfhemoglobin-producing action of thefungicide is discussed.

The importance of zinc ethylene bisdithiocarbamate as a sulfhemoglobin-producing and hemolytic agent is stressed, in view of the widespread useof this fungicide.

Submitted on August 3, 1962 Accepted on December 11, 1962     

The Natural History of Iron Deficiency Induced by Phlebotomy

1. The sequence of characteristic changes of progressive iron deficiency wasdemonstrated by serial bleeding of normal volunteers and polycythemic patients.

2. After bloodletting, changes occurred in peripheral blood in the followingorder: a) fall in hemoglobin concentration; b) decreased plasma iron; c)reticulocytosis, increased MCV and MCH; d) diminution of MCV and MCH,increased total iron-binding protein; and e) decreased MCHC.

3. Characteristic changes of iron deficiency returned to prephlebotomylevels in the following sequence: a) hemoglobin concentration; b) cellularindices; c) serum iron; d) serum iron binding protein; and e) bone marrowhemosiderin, and finally the increased gastrointestinal absorption of ironreverted to normal.

4. Accelerated production of red cells continued in polycythemic patientsdespite the induction of moderate iron deficiency. Quality was sacrificed forquantity, and thereby a more profound microcytosis occurred than in normalsubjects with a similar degree of iron deficiency.

Submitted on February 16, 1962 Accepted on April 11, 1962     

Anabolic agents and relative polycythaemia

after the administration of testosterone an increase in the haemoglobin concentration may occur, and the difference in haemoglobin concentration between males and females has been ascribed to this hormonal influence. This hypothesis has been confirmed experimentally by Kennedy (1962). An increase in the haemoglobin concentration has also been brought about by the administration of ‘anabolic’ drugs (Booij and Kuypers, 1962; Everse and Van Keep, 1962). Both testosterone and anabolic drugs have induced remissions in congenital and acquired aplastic anaemia (Kennedy and Gilbertson, 1957; Shahidi and Diamond, 1959, 1961; Kennedy, 1962). These authors also reported polycythaemia in some cases following treatment with androgens or anabolic drugs. Gardner and Pringle (1961) have described polycythaemia in a woman suffering from a tumour of the adrenal cortex. Booij and Kuypers (1962) reported two cases of polycythaemia after prolonged administration of Nandrolone-phenylpropionate (Durabolin) at high dosage, and they also demonstrated that this drug had an erythropoietic action in rats. But, like most other workers they based their observations only on measurements on peripheral blood and they made no blood volume determinations. Thus they were unable to distinguish between an increase in haemoglobin concentration due to a decrease of the plasma volume and an absolute increase of the whole red cell mass. Kennedy (1957), however, observed a return to normal values of the red cell mass during treatment of breast cancer with testosterone and even an increase exceeding the normal values in some of the patients. Unfortunately no plasma volume determinations were made. The results in three patients with clinical signs of polycythaemia after prolonged administration of anabolic drugs are reported. Examination of the peripheral blood showed an increase in haemoglobin concentration and packed cell volume. However, determination of the plasma and red cell volume showed that these changes were due to a decrease of the plasma volume.     

Sickle Cell--Hemoglobin C Disease: Quantitative Determination of Iron Kinetics and Hemoglobin Synthesis

Quantitative determinations of iron kinetics and hemoglobin synthesis weremade on five patients with sickle cell-hemoglobin C disease. The anemia wasmild in all patients but one who had the hemoglobin of 10.1 Gm. per cent.All patients were in a steady state during the period of this study.

The ferrokinetic determinations demonstrated a hemolytic process in allcases. The mean erythrocyte life span in these patients was 18, 20, 29, 46and 56 days respectively (normal range, 110-130 days). The hemoglobinsynthesis was increased in all. Reduced to terms of daily hemoglobin production per liter of blood, the values were 2.2 Gm., 2.9 Gm., 3.6 Gm., 5.8 Gm.and 6.6 Gm. The latter figure represents a five fold increase over the normalmean value of daily hemoglobin synthesis of 1.3 Gm. (normal range, 1.0 to1.6 Gm. of hemoglobin per liter of blood per day).

The results of the in vivo organ counts demonstrated a significant degreeof sequestration of red blood cells in the spleen of two patients. The questionof advisability of splenectomy in such patients was discussed.

Accepted on December 3, 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     

Morphology of Normal Erythrocyte and Acanthocyte Using Nomarski Optics and the Scanning Electron Microscope

The morphology of normal erythrocytes and of acanthocytes from five patients with abetalipoproteinemia was studied by two relatively new technics.Interference microscopy with Nomarski optics was used to study the red bloodcells in the living state; the scanning electron microscope was utilized to givea three dimensional appearance of the fixed red blood cells.

Isotonic saline washing of normal red blood cells produces multiple smalluniform projections on the surface of the cell: the crenated red cell. Acanthocytes after saline washings also develop similar projections: the crenatedacanthocyte. Crenation of both the normal red blood cell and the acanthocyteis reversed by restitution of a plasma environment and by chemical fixation byglutaraldehyde or osmic acid but the characteristic shape of the acanthocyte ispreserved.

A comparison was made of the changes in the shape of the normal red bloodcells and acanthocytes induced by alterations in the tonicity of the surroundingmedium.

Submitted on October 4, 1969 Accepted on December 22, 1969     

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     

Cytochemical Localization of an Adenosine Triphosphate Dephosphorylating Enzyme in the Granules of Human Eosinophils

The granules of the eosinophilic myeloid cells of normal human bone marrow exerted a pronounced activity to dephosphorylate adenosine triphosphate.This behavior was not shown by the granules of other myeloid cells.

Submitted on January 8, 1962 Accepted on February 2, 1962     

Evidence of trapped red cells in the brain of rhesus monkey infected with Plasmodium knowlesi

59Fe-labelled normal red cells and 51Cr-labelled P. knowlesi infected red cells were used as tracers for a study on the trapped red cells in the brain of rhesus monkeys infected with P. knowlesi. After instantaneous injection into a common carotid artery, blood was sampled from both of the internal jugular veins at various intervals. Results in 6 experiments on 3 infected monkeys showed that about 17% of infected red cells given was trapped in the capillaries of the brain of the infected monkeys. The remainder of the infected red cells travelled on the same flow rate as the normal red cells. As the volume for the flow of the former was less than that of the latter, the mean transit time of the infected red cells (-ty) was therefore shorter than that of the normal red cells (-th). The mean difference in volume was estimated to be 3.4% in the present study. These findings indicated that some of the P. knowlesi infected red cells were trapped in capillaries of the brains of monkeys infected with P. knowlesi.     

Studies of Human Erythrocyte Catalase by Fluorescent Antibody Technic The Distribution of Catalase in Acatalasia and Hypocatalasia

In order to investigate the cellular distribution of catalase in normal,hypocatalasic and acatalasic red blood cells, the fluorescent antibody labellingtechnic was employed. Sensitive anticatalase sera were produced in rabbitsby immunization with purified catalase extracted from human erythrocytes.Specificity against human erythrocyte catalase was confirmed by Ouchterlony’sdouble diffusion method.

The distribution of catalase is fairly homogeneous in normal and hypocatalasic red cells, but in acatalasic cells fluorescence due to the presence ofcatalase was not observed.

By this method the amount of catalase in hypocatalasic red cells was judgedto be between that of normal and acatalasic red cells.

Submitted on October 1, 1968 Accepted on February 13, 1969     

单采血小板中红细胞混入量超标原因调查分析

目的:探讨单采血小板中红细胞混入量超标的原因。方法:根据所采集的单采血小板是否红细胞混入量超标(有肉眼可见红细胞),分设正常对照组与超标组,正常对照组为红细胞混入量符合标准的献血者80例;超标组为红细胞混入量超标的献血者23例。分别对2组献血者样本进行全血常规及血红蛋白项目检测。结果:超标组中献血者的Hb、Hct、MCV、MCH及血浆总蛋白结果均低于正常对照组献血者,差异有统计学意义(P0.01);而2组献血者RBC计数、MCHC比较差异无统计学意义(P0.05)。结论:单采血小板红细胞混入量超标与外周血的Hb、Hct、MCV、MCH以及血浆总蛋白等参数的水平降低有关;与献血者多次献血或混合献血造成红细胞未完全成熟有关。     

Acquired immune hemolytic anemia associated with IgA erythrocyte coating: investigation of hemolytic mechanisms

We have investigated the hemolytic mechanisms in a patient with acquired immune hemolytic anemia whose red cells appeared to be coated with IgA alone. The clinical course was similar to that of patients with hemolytic anemia mediated by warm-reacting IgG antibody. Splenic sequestration of red cells was demonstrated, and marked reduction of hemolysis occurred after corticosteroid therapy. Antibody was eluted from the patient's red cells and used to sensitize normal red cells in vitro. These sensitized red cells were not lysed by fresh autologous serum, nor did they fix detectable amounts of C3. However, red cells sensitized by eluted antibody were lysed by normal human peripheral blood monocytes in a system designed to demonstrate antibody-dependent cell-mediated cytotoxicity. Monocyte-mediated hemolysis of sensitized red cells was inhibited by the addition of low concentrations of normal serum IgA to the system, but not by IgG. The ability of the eluate to induce monocyte-mediated hemolysis was abolished by its adsorption on Sepharose-bound anti-IgA, but not by preincubation with Sepharose-bound anti-IgG. In addition, normal human monocytes were demonstrated to ingest eluate-sensitized red cells. These data demonstrate an in vitro interaction of IgA-sensitized red cells with leukocytes and suggest a possible mechanism for the patient's hemolysis.     

Red Blood Cell Life Span Using DFP32 as a Cohort Label

In the dog, the erythrocyte uptake of DFP³² was found to be dependentupon the dose of administered DFP³². Effective blocking of red cell uptake of isotopically-labeled DFP was achieved by prior administration ofunlabeled DFP. By allowing a time interval between the administration ofDFP and DFP³² the cohort of red cells synthesized in that interval could belabeled with DFP³². The cohort of erythrocytes synthesized in response to acuteblood loss were so labeled and were found to have a shortened life span witha pattern of random destruction.

Submitted on January 5, 1962 Accepted on February 23, 1962     

Red blood cell size is important for adherence of blood platelets to artery subendothelium

The hematocrit is one of the main factors influencing platelet adherence to the vessel wall. Raising the hematocrit causes an increase of platelet accumulation of about an order of magnitude. Our studies concern the role of red cell size. We have studied this effect using an annular perfusion chamber, according to Baumgartner, with human umbilical arteries and a steady-flow system. Normal human red blood cells (MCV 95 cu mu) increased platelet adherence sevenfold, as the hematocrit increases from 0 to 0.6. Small erythrocytes from goats (MCV 25 cu mu) caused no increment in adherence in the same hematocrit range. Rabbit erythrocytes (MCV 70 cu mu) caused an intermediate increase in adherence. Red blood cells from newborns (MCV 110-130 cu mu) caused a larger increase in platelet adherence than normal red cells at hematocrit 0.4. These results were further confirmed with large red blood cells from two patients. Experiments with small red cells (MCV 70 cu mu) of patients with iron deficiency showed that platelet adherence was similar to normal red cells, provided the red cell diameter was normal. Small red blood cells of a patient with sideroblastic anemia caused decreased adherence. These data indicate that red cell size is of major importance for platelet adherence. Red cell diameter is more important than average volume. However, for size differences in the human range, the hematocrit remains the dominant parameter.     

Erythrocyte Superoxide Dismutase,Catalase and Glutathione Peroxidase Activities in β-Thalassaemia (Major and Minor)

The ‘antioxidant’ enzymes superoxide dismutase (SOD), catalase and glutathione peroxidase (GSH-Px) were found greatly elevated in red blood cells of subjects with β-thalassaemia minor and similar to normal values in red blood cells of subjects with β-thalassaemia major. These findings allows us to speculate that red cells in β-thalassaemia minor react to the increased oxidant threat with augmented antioxidant enzyme activities. The normal levels of antioxidant enzymes in β-thalassaemia major seem to be due to the presence of normal red cells owing to multiple transfusions.     

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     

Report of a Three-Year Study of Red Cell Mobility and the Slowing Factor in Serum of Persons with Lymphoma

1) A variety of pathologic conditions retard the electrophoretic mobilityof the red blood cell.

2) Two retarding factors have been found in the blood serum, one that reduces the mobility of the red cell from normal (1.33µ/cm./sec./volt) to 0.89µand one that reduces it to 1.14µ.

3) The "0.89" factor and the "1.14" factor can be differentiated and identified.

4) The 0.89-factor has been found in the serum of 99 per cent of patientswith cancer and Hodgkin’s disease studied and has been lacking from theserum of all persons with benign tumor.

5) The 0.89-factor has also been found in patients with infectious mononucleosis.

6) A few physical characteristics that enable differentiation of the two slowing factors are described.

7) Clarification of the role played by the 0.89-factor in malignant neoplasmis deemed important.

8) Determination of the presence or absence of the 0.89-factor in the bloodserum may become of value in differential diagnosis of malignant neoplasm.

Submitted on May 4, 1962 Accepted on July 18, 1962