A Method for Counting Phytohemagglutinin-Stimulated Lymphocytes

A technic is described for accurately enumerating the number of viable.PHA-stimulated lymphocytes in culture. This technic is both simple and fast.

The technic depends upon the elimination of debris by means of the proteolytic enzyme, pronase, and the removal of cell cytoplasm by cetrimideleaving clean, intact nuclei which can be counted and sized.

Serially determined absolute lymphocyte counts in cultures with phytohemagglutinin are reported. These show that there is a net gain in both cellularity and number of cells incorporating tritiated thymidine and that there islogarithmic growth throughout the 72-hour culturing interval. Quantitativestudies of this lymphocyte response as a function of PHA concentration arein progress.

Submitted on August 10, 1966 Accepted on December 5, 1966     

Disseminated unicellular green algal infection in two sheep in India

Unicellular green algae infected and disseminated in two adult Nellore brown rams in India. At necropsy multiple green lesions were observed in the lungs, liver and kidneys of both sheep. Histological examination revealed necrotizing granulomas that contained numerous spheroidal to elliptical organisms ranging from 5-20 micrometers in diameter. These organisms occurred in several reproductive stages: small (5-9 micrometers) single, undifferentiated cells; intermediate (9-15 micrometers) cells undergoing nuclear and cytoplasmic cleavage; and large, mature, endosporulating cells containing two to six or more endospores. In addition, occasional large (20-35 micrometers), round thick-walled forms were among the typical cells. The cytoplasm of the algal cells contained numerous, large, irregularly-shaped and densely packed granules of varying sizes, which were strongly stained by the Gridley fungus, periodic acid-Schiff, and Gomori methenamine silver procedures. Electron microscopic studies revealed chloroplasts in the cytoplasm of these organisms. The chloroplasts consisted of smoothly contoured electron-lucent or electron-dense granules, 0.5 to 3.0 micrometers in diameter, that were either surrounded by or contiguous to two or more tightly appressed membranes or multidisc bands. Some of the alternating membranes appeared to be spirally twisted or associated with several cytoplasmic granules in one plane of section. This is the first record of algal infections in India.     

Effectiveness of Red Blood Cells Filtered Through Cotton Wool to Prevent Antileukocyte Antibody Production in Multitransfused Patients

Abstract. The effectiveness of red blood cells made leukocyte-free by filtration through cotton wool to prevent the production of antileukocyte antibodies was evaluated in children suffering from Cooley's anemia. Two studies were performed: study I was carried out prospectively in two groups of non transfused patients, one group treated with leukocyte-free filtered red cells, the other with buffy-coat-free packed red cell units. Different types of antileukocyte antibodies were looked for in both groups and the results were compared. In study II the behavior of pre-existing lymphocytotoxic antibodies found in the serum of children previously transfused with standard or buffy-coat-free packed red cell units was followed after the patients had been passed to a program of transfusion with leukocyte-free filtered red cells. Study I showed that none of the patients transfused with leukocyte-free filtered red cell units have produced antileukocyte antibodies, while these could be found in 2/3 of the patients transfused with buffy-coat-free packed red cell units. Study II showed that the repeated transfusion of leukocyte-free filtered red cells to patients who possessed in their serum preformed lymphocytotoxic antibodies did not cause any increase in the potency or spectrum of these antibodies, but was in fact accompanied in some cases by their decrease or disappearance. It is concluded that filtration through cotton wool is an easy and inexpensive means of preparing leukocyte-free red blood cells for transfusion capable of preventing (or reducing) the production of antileukocyte antibodies in multitransfused patients.     

A New Technic for Separation of Human Leukocytes

A simple technic for separation of human leukocytes is described. It utilizesdifferential sedimentation following red cell and platelet agglutination inducedby Polybrene. In this technic, lymphocytes are partially removed and over50 per cent of polymorphonuclears are recovered. The leukocyte/red cell ratiois about 5, if blood samples with normal leukocyte counts are used. By useof blood samples containing higher white cell counts this ratio may be increased. The separated cells proved to be highly viable as tested by phasemicroscopy, supravital staining and phagocytosis. There was no significantchange in oxygen uptake of leukemic cells in the presence of a relatively largePolybrene concentration.

Accepted on October 30, 1961     

The Septate Junction: A Structural Basis for Intercellular Coupling

Electron microscopy, freeze-etching, and optical diffraction show how the structure of the septate junction may provide the basis for the low-resistance pathway between the electrically coupled cells in mussel gill epithelia. Conventional electron microscopy suggests that the septa are pleated sheets that differentiate from and are structurally continuous with the junctional cell membranes. Freeze-etching exposes geometrically arranged rows of 85-A particles within the junctional cell membranes. Diffraction evidence shows that these membrane particles and the alternate vertices of the intercellular septal sheets are congruent and therefore superposable. Together, the membrane particles and septal sheets provide a channel that extends from the cytoplasm of one cell through the septate junction to the cytoplasm of the adjacent cell.     

Detection of glucose-6-phosphate dehydrogenase deficient heterozygotes

Heterozygotes for the Mediterranean type of severe G-6-PD deficiencywere investigated by a variety of tests.

The methemoglobin reduction test was most successful in detecting heterozygotes (about 80 per cent). Enumeration of methemoglobin containingcells on blood films (Kleihauer-Betke technic) did not improve these results.Quantitation of enzyme level was less successful (65 per cent), and determination of decolorization time by the BCB technic was least sensitivein heterozygote detection.

Methemoglobin reduction technics reflect a more indirect effect of themutant gene than enzyme assay. The superiority of these technics in heterozygote detection is probably caused by the genetically determined presenceof both normal and enzyme-deficient cells in G-6-PD deficient heterozygotes.Since methemoglobin reduction is carried out by individual cells, the population of enzyme deficient cells does not reduce methemoglobin, and thereforeeven a minority of deficient cells leads to abnormal test results. In contrast,enzyme assay is less successful for heterozygote detection, since measurementof enzyme level is carried out on hemolyzed red cells, where cellular mosaicismno longer exists.

An additional source of variation of enzyme levels in heterozygotes iscaused by the existence of genetically determined control of normal enzymelevel. Possession of a high capacity allele for G-6-PD activity may placea heterozygote in the normal range of enzyme activity.

The various tests were also applied to subjects with the mild Greek typeof G-6-PD deficiency. Males with this mutation had enzyme levels varyingbetween 12-45 per cent of the mean of normal males. Methemoglobin reduction test results were considerably less abnormal in hemizygotes with themild type of Mediterranean deficiency than in heterozygotes with the severedeficiency. Fewer heterozygotes with the mild deficiency were detected.

Submitted on February 4, 1966 Accepted on May 31, 1966     

Malaria chemoprophylaxis with chloroquine in young Nigerian children. IV. Its effect on haematological measurements

Haematological measurements were made in 198 Nigerian children aged three months to two years who received weekly malaria chemoprophylaxis with chloroquine from shortly after birth until the age of one or two years and in 185 age-matched control children. Children protected against malaria had a higher mean haemoglobin level and a higher packed cell volume than control children, and they showed fewer abnormalities of their red cells. Total and differential white blood cell counts, mean plasma folate and mean serum ferritin concentrations were similar in both groups of children. However, the geometric mean red cell folate level of children exposed to malaria was significantly higher than the mean level of control children; and it may be that malaria raises the red cell folate through intracellular synthesis by malaria parasites. Children with malaria parasitaemia had a significantly lower haemoglobin and packed cell volume and a significantly higher geometric mean red cell folate and ferritin level than children without parasitaemia. Serum ferritin is probably an unreliable index of iron status in children with malaria.     

Electron Microscopic Study of Human Basophils

Electron microscope studies were carried out on human basophils obtainedfrom peripheral blood and thoracic duct lymph. Delineation of the cells inthick sections was facilitated by staining with Toluidine Blue, which impartedthe characteristic purple-red color to the granules. The cytoplasm containsmany of the organelles seen in other cells, including a small Golgi apparatus,centrioles, mitochondria, fibrils and microtubules. Ribosomes and rough ERare rarely encountered. Basophil granules are surrounded by a unit membraneand contain particles which are uniform in size within the same granule butwhich vary in size in different granules within the same cell. Some granulesreveal a homogeneous texture and/or "myelin" figures. The appearance of mostbasophil granules differs from the ultrastructure of human mast cell granulesreported to date. It is suggested that the difference in fine structure may reflect a difference in the chemical constituents of the granules.

Submitted on September 20, 1966 Accepted on January 9, 1967     

The flow of blood cell suspensions through 3 μm and 5μm Nuclepore membranes: a comparison of kinetic analysis with scanning electron microscopic examinations

This study was designed to investigate the effect of red and white cells on the flow of dilute suspensions of blood cells through 3 microns and 5 microns Nuclepore membrane filters. The rate of flow of blood cell suspensions through 3 microns or 5 microns membranes declines continually due to occlusion of pores by slow cells. The cells which occlude 3 microns pores exceed the number of white cells about seven-fold. Electron microscopic examination of a used membrane confirms that these slow cells are not white cells but are red cells which are visibly damaged. With 5 microns membranes, the number of slow cells is entirely consistent with them being white cells. Again, electron microscopic examination confirms that 5 microns pores are occluded by white cells. With both types of membrane, the same kinetic analysis is valid and yields information about the behaviour of red cells during the filtration procedure.     

Effect of stasis of blood in varicose veins on erythrocyte fragility,with accompanying studies comparing red cells and other blood elements with cubital vein blood

1. Blood from varicose veins was compared with cubital vein blood in 20 patientsin order to determine whether or not the degree of stasis present in varicose veinswould increase red cell fragility. Corollary studies consisted of comparative determinations of red cells, hemoglobin, packed cell volume, white blood cells,platelets and serum proteins.

2. There was no increase in red cell fragility in the varicose vein specimen, indicating that the theory that minor degrees of intravascular erythrostasis contribute substantially to some of the hemolytic anemias is untenable.

3. There was a small but statistically significant elevation in red cells per cu.mm. in varicose vein blood as compared with blood from cubital veins. There wasa suggestive, but not significant, increase in packed cell volume and serum proteinin the varicose vein samples. The evidence indicates a mild degree of hemoconcentration.

4. White cells, platelets and hemoglobin determinations were found to have thesame values in varicose vein blood as in blood from the cubital vein.

     

Evaluation of Three Procedures for the Preparation of Leukocyte-Poor and Leukocyte-Free Red Blood Cells for Transfusion

Abstract. Three methods of leukocyte deprivation of blood for transfusion have been evaluated. Filtration of relatively fresh packed red cells (up to 5 days old) through Erypur filters appears to be the method of choice for the preparation of leukocyte- free red cells, used to prevent the production of antileukocyte antibodies in nonimmunized, nontransfused patients undergoing repeated blood transfusions. Double dextran sedimentation plus saline washings also yields leukocyte-free red cells, although with a lower frequency, and represents an alternative to Erypur filtration, especially because of the lower cost and no need of special equipment. Imugard filtration appears to be a simple and effective way for preparing leukocyte-poor red blood cells. For all the three procedures, results are definitely better when buffy coat-free packed red cell units are processed. In a given hospital the choice of the technique(s) for the preparation of leukocyte-poor (free) red blood cells for transfusion depends on a number of factors, such as type of patients to be transfused (immunized or nonimmunized, small children or boys and adults), aim of the procedure (prevention of the febrile transfusion reaction or prevention of antileukocyte antibody production), continuous availability of materials, cost, length and easiness of the procedure; these three latter parameters are in turn related to a number of local situations and facilities.     

Preparation of leucocyte-and platelet-poor red cell concentrates

Summary The technique described by Schneider and Stützle [9] for preparing leucocyte- and platelet-poor red cell concentrates removes more than 98% of leucocytes and platelets of whole blood. These special red cell concentrates also can be prepared from packed cells by reconstitution with 200 ml phosphate buffer solution to whole blood volume. The reduction of leucocytes and platelets in this preparation is equal to filtered blood or frozen red cells.     

Rate of lateral diffusion of intramembrane particles: measurement by electrophoretic displacement and rerandomization.

A method combining electrophoresis and freeze-fracture electron microscopy is described; the method was used to determine the lateral diffusion coefficient of intramembrane particles (integral proteins) in the mitochondrial inner membrane. An electric current was passed through microsuspensions of purified, spherical inner membranes at pH 7.4, which caused an electrophoretic migration of intramembrane particles in the membrane plane into a single, crowded patch facing the positive electrode. The membrane microsuspensions were quick-frozen at specified times after the packed particles were released from the electrophoretic force and while the particles were diffusing back to a random distribution. Observed concentration gradients of intramembrane particles during this time were quantitatively compared with and found to follow a mathematical model for Fickian diffusion of particles on a spherical membrane. The results determine the kinetics of free diffusion of integral proteins at the resolution of individual proteins. The diffusion coefficient of the integral proteins in the mitochondrial inner membrane was determined to be 8.3 X 10(-10) cm2/sec at 20 degrees C, from which a root-mean-square displacement of 57 nm in 10 msec is predicted.     

Purification without Loss of Viability of Neoplastic Plasma-Cells from the Ascites Passaged Mouse Myeloma, X5563

A simple scheme for purification of murine myeloma cells from ascitesfluid without loss of viability is presented. This is accomplished by repeateddifferential centrifugations followed by passage through a glass bead column.The cells so obtained are 99.9 per cent pure and 99 per cent viable in a largenumber of repetitions of this technic.

Submitted on July 21, 1966 Accepted on September 20, 1966     

A Comparison of Normal Red Cell ATP Levels as Measured by the Firefly System and the Hexokinase System

Widely divergent normal red cell ATP levels have been reported by investigators using different methods. In order to clarify the cause of thesediscrepancies and to establish correct normal values for red cell ATP, the firefly technic for measuring ATP levels was compared with other methods. TheATP content of TCA filtrates of red cells was the same when determined bythe firefly method as by the hexokinase-linked technic. Relatively low concentrations of protein were found to stimulate light output when lyophilyzedfirefly extract, but not freshly prepared firefly extract, was used. Thus, falsely,high values were obtained when red cell extracts were examined, unless protein was also added to the standard. Storage of heparinized blood for as littleas 1 hour resulted in a substantial decrease in red cell ATP levels. The losswith ACD blood was less, and could be obviated entirely by using an ACDsolution with a pH adjusted to between 3.5 and 4.0. Removing the buffy coator washing cells in saline resulted in no further loss of red cell ATP. Extraction of washed red cells with TCA resulted in an average loss of 4.6 per cent ofATP, while extraction of whole blood with TCA resulted in a 14 per cent lossof ATP. In contrast, perchloric acid extraction resulted in no ATP loss. If ATPdeterminations are carried out using the firefly method, protein should beadded to the standard. If red cells must be stored for any period of time priorto extraction of ATP, an ACD solution with a pH of 3.5 to 4.0 should be used.If extracts of red cells are made, perchloric acid appears significantly superiorto trichloroacetic acid.

Submitted on January 10, 1967 Accepted on March 1, 1967     

The effect of donor age on the flow properties of blood. Part I: Plasma and whole blood viscosity in adult males

The relationship between blood viscosity and age was studied using heparinized blood samples obtained from 50 normal male blood donors between the ages of 20 and 65 years. There was a slight but significant decline in packed cell volume (hematocrit) with age. Plasma viscosity showed no significant variation with donor age, but the viscosity of blood samples standardized to a packed cell volume of 45 ml/dl showed an increase as the age of the donor rose. The age-related trend to a higher viscosity was present at shear rates below 46 s-1, but not at higher shear rates. This shows that with age there is a rising trend in the red cell contribution to blood viscosity, which is not dependent on the packed red cell volume; this component increases across a wide age range in early and middle adult life, and is not a characteristic appearing exclusively in later age periods. The tendency for the viscosity trend to be greater at lower shear rates indicates increased shear thinning in blood obtained from older subjects, the cause of which may be either diminished red cell deformability in these subjects, or an increased tendency to form aggregates at low shear rates.     

The hSK4 (KCNN4) isoform is the Ca2+-activated K+ channel (Gardos channel) in human red blood cells

The question is, does the isoform hSK4, also designated KCNN4, represent the small conductance, Ca2+-activated K+ channel (Gardos channel) in human red blood cells? We have analyzed human reticulocyte RNA by RT-PCR, and, of the four isoforms of SK channels known, only SK4 was found. Northern blot analysis of purified and synchronously growing human erythroid progenitor cells, differentiating from erythroblasts to reticulocytes, again showed only the presence of SK4. Western blot analysis, with an anti-SK4 antibody, showed that human erythroid progenitor cells and, importantly, mature human red blood cell ghost membranes, both expressed the SK4 protein. The Gardos channel is known to turn on, given inside Ca2+, in the presence but not the absence of external Ko+ and remains refractory to Ko+ added after exposure to inside Ca2+. Heterologously expressed SK4, but not SK3, also shows this behavior. In inside-out patches of red cell membranes, the open probability (Po) of the Gardos channel is markedly reduced when the temperature is raised from 27 to 37 degrees C. Net K+ efflux of intact red cells is also reduced by increasing temperature, as are the Po values of inside-out patches of Chinese hamster ovary cells expressing SK4 (but not SK3). Thus the envelope of evidence indicates that SK4 is the gene that codes for the Gardos channel in human red blood cells. This channel is important pathophysiologically, because it represents the major pathway for cell shrinkage via KCl and water loss that occurs in sickle cell disease.     

Bovine Erythrocyte Membrane Composition: Association with the FY and other Glycoprotein Systems

Abstract. The bovine blood group F, FV, and V phenotypic classes were found to differ greatly in amounts of red cell neuraminic acid, ghost material per ml packed red cells, phenol-extracted mucoid per ml red cells, and pronase-released mucoprotein per ml red cells. In all of these characteristics, F values were highest, FV intermediate, and V lowest. Phaseolus vulgaris nonagglutinators also had higher values for red cell neuraminic acid, ghost quantity per ml red cells, and phenol-extracted mucoid quantity per ml red cells than agglutinators. At least three, and possibly four, groups of animals with respect to neuraminic acid values were observed. Phenol-extracted mucoids from red cells of different FV phenotypes were nearly identical in carbohydrate composition, although mucoids from F cells may contain more glucose than those from FV or V. The F antigen was seen to be associated with a higher red cell N-acetyl neuraminic acid/total neuraminic acid ratio than the V antigen.     

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     

Folate Concentration in Top,Middle and Bottom Layer of Packed Red Cells in Patients with Vitamin B12 Deficiency: Relation to Treatment

In response to specific treatment of vitamin B₁₂ deficient, anaemic patients there is an influx of folate into the young, circulating red cells. To separate new and old cells, capillary tubes filled with whole blood were centrifuged and the packed red cell column divided into top (T), middle (M) and bottom (B) layer. The newest cells are found in the T layer. The increase in red cell folate (RCF) concentration starts before, during or after the reticulocyte response, and is therefore not directly related to folate metabolism in the red precursor cells in the marrow. The low RCF concentration at the peak of the reticulocyte response in some of the cases demonstrates that the folate material, which may have been accumulated in the red precursor cells in the marrow, may be lost by the time the red cells enter the peripheral blood. The influx of folate into the young, circulating red cells is rapidly followed by an efflux of folate, suggesting that much of the folate material is still in the monoglutamate form. A new influx of folate is noted after a time lapse of from 5 to 10 d. Iron deficiency seems to prevent the uptake of folate by the circulating red cells.