Experimental gerontologic transversal examination of rat blood cells paying special regard to daily variations (author's transl)

Four groups of Sprague-Dawley rats aged 8, 13, 28 and 33 months were allowed to participate in this investigation of RBC, volume distribution of red cells and differential leukocyte count as function of age. Furthermore, the authors examined whether daily variations of these parameters should be taken into account. The RBC decreases and the cell volume increases with age. The differential lymphocyte count (%) declines, whereas the differential neutrophile count (%) increases. Because of the small amplitudes of the erythrocyte parameters daily variations can be ignored. The white blood cell counts show considerable daily variations in young and old rats. These variations do not exert influence on the differential blood cell counts (%) because they are equal in phase and period. Daily variations must be taken into account when absolute values are calculated from these results.     

Steroid-responsive pure red cell aplasia associated with natural killer cell lymphocytosis

A patient with pure red cell aplasia and expansion of the peripheral blood natural killer (NK) cell population is described. Despite normal absolute and differential leukocyte counts, NK cells were increased at diagnosis and at relapse. Furthermore, these cells were not morphologically recognizable on the peripheral blood smear examination. A favorable clinical response to glucocorticoid therapy was accompanied by a decrease in NK cells.     

The Application of Freeze-Cleaving Technics to Studies on Red Blood Cell Fine Structure

A simplified freeze-cleave and replication method of tissue preparation forexamination in the electron microscope is applied to studies on red blood cellfine structure. With this technic, the cytoplasm of red blood cells appearsto have a uniform pattern of packed granularity, with individual particles approximating the dimensions anticipated for replicas of individual hemoglobinmolecules. The cell surface is smooth and partially covered with small particles which may represent antigens, enzymes, or some structural proteins.The possibility that particles seen in cells and on cell membranes may represent an artifact is discussed. Pretreatment of cells prior to freezing influencesthe plane of cleavage through packed cells so that the plane of cleavage canbe preferentially directed either through the cytoplasm or along red cellmembranes. The freeze-cleave technic may be of particular value in applications where extensive areas of membrane must be surveyed, such as searchingfor leukemogenic viruses budding through cell membranes.

Submitted on August 16, 1966 Accepted on November 24, 1966     

Automated counting of white and red blood cells in the cerebrospinal fluid

The objective of this study was to examine to what extent the automated method of the Bayer H*2 instrument could replace the visual counting of white and red blood cells in cerebrospinal fluid. The number of white blood cells as well as the percentage of mononuclear and polymorphonuclear cells were counted in the 'Baso channel' (Research screen 3) whereas the number of red cells were registered as the 'R-count' (Research screen 1). All automated cell counts were compared to visual estimates. The automated count yielded reliable results down to 5 x 106 white blood cells/l and 5 x 108 red blood cells/l. In some samples 'noise' was present in the Baso channel. A correct white blood cell count could then be obtained by counting the cells directly as dots on the screen. It was possible to differentiate between polymorphnuclear cells and mononuclear cells at all WBC concentrations. The automated counting of cerebrospinal fluid can be performed without changing thresholds or sample volumes of the instrument. Thus, in the routine practice it will be possible to alternate between automated counting of whole blood samples and cerebrospinal fluid samples.     

Determination of the packed cell volume using 131I-human serum albumin.

When trace quantities of labelled albumin have been added to blood, the packed cell volume (PCV) may be determined by isotope counting using the relationship: (plasma counts-blood counts)/(plasma counts). This method for measuring the PCV is not affected by plasma trapping between the red cells and the results do not depend upon the centrifugation conditions. The PCV determination with labelled albumin has a high precision (coefficient of variation = 0.9%) and there is very good agreement with the centrifugation PCV using Wintrobe tubes after this measurement has been corrected for plasma trapped between the red cells. No significant compression of the red cells occurred when the blood samples were centrifugated in Wintrobe PCV tubes.     

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     

Are polymorphonuclear leukocytes an abnormal finding in cerebrospinal fluid? Results from 225 normal cerebrospinal fluid specimens

Although it is often claimed that the presence of a single polymorphonuclear leukocyte (PMN) in the cerebrospinal fluid (CSF) is abnormal, recently some have suggested that a few PMNs are occasionally present in cytocentrifuged differential cell counts of normal CSF. We examined 225 consecutive normal CSF specimens to determine how frequently PMNs occur in normal CSF and to identify factors associated with the presence of PMNs. One or more PMNs were present in 73 cases (32%). The number of CSF PMNs was strongly correlated with the degree of CSF blood contamination and the hematologic PMN count. Of the 163 specimens having 25 red blood cells or less per cubic millimeter, only eight (5%) had three or more PMNs, and these outliers had abnormally high hematologic PMN counts. Of the 36 specimens having 100 red blood cells or more per cubic millimeter, 17 (47%) had six or more PMNs. We conclude that the number of PMNs found on cytocentrifuged differential cell counts is highly dependent on the degree of CSF blood contamination and the patient's hematologic PMN count and that even minimal blood contamination can result in the presence of one to two PMNs in normal CSF.     

Urokinase could increase the yield of progenitor cells during red cell depletion in elapsed and anticoagulated cord blood

We assessed whether the urokinase could increase the yield of progenitor cells during processing in elapsed, anticoagulated cord blood (CB) after collection, and we also determined the optimal dose of urokinase. The total nucleated cell (TNC) counts after red cell depletion in 48-hr-elapsed CB were significantly higher in samples treated with 10,000 and 50,000 IU of urokinase/mL than in untreated samples or treated with 5,000 IU of urokinase/mL. The CD34(+) cell counts were significantly higher in samples treated with 10,000 IU of urokinase/mL than in untreated samples and in samples treated with 5,000 or 50,000 IU of urokinase/mL. In 6-, 12-, and 24-hr-elapsed CB, however, there were no significant differences of TNC, CD34(+) cells, or CFU-GM counts between untreated samples and samples treated with 10,000 IU of urokinase/mL. These findings suggest that the addition of 10,000 IU of urokinase/mL before red cell depletion in 48-hr-elapsed, anticoagulated CB could increase the yield of progenitor cells. However, there are no advantages in using urokinase for processing CB prior to 24 hr after collection.     

Evaluation of hematological values obtained with reference automated hematology analyzers of six manufacturers

We evaluated assays of the same fresh blood samples with six different types of reference automated hematology analyzers developed by the following manufacturers: Beckman Coulter, Sysmex, Bayer, Abbott, Nihon Kohden and Horiba. Fresh whole blood samples treated with dipotassium ethylenediaminetetraacetic acid (EDTA K2) were collected from three healthy adult volunteers. The complete blood counts (CBC) including red blood cell count (RBC), hemoglobin (Hgb), hematocrit (Hct), mean corpuscular volume (MCV), white blood cell count (WBC), platelet count (Plt), reticulocyte percentage (Ret) and leukocyte differential counts including % neutrophils (Neu), % lymphocytes (Lym) and % monocytes (Mon) were surveyed with a reference automated hematology analyzer from each manufacturer. The process from sampling to analysis was performed according to procedures in hospital clinical laboratories. RBC, Hgb, Hct and MCV exhibited allowable differences within 5% of mean value among all instruments. Large differences greater than 10% of mean value in WBC, Neu and Lym between Horiba and other manufacturers, and in Plt between Nihon Kohden and other manufacturers, were observed. Ret and Mon exhibited large differences over 10% of mean value among almost all of the instruments tested. This survey suggests that all parameters exhibiting differences greater than 10% of mean value among instruments should be improved for clinical use to ensure good external quality control in blood cell counting and leukocyte differential counting using automated instruments.     

Evaluation of flow cytometric enumeration of foetal erythrocytes in maternal blood

In pregnant women subject to abdominal trauma or other foetomaternal haemorrhage, foetal red blood cells containing haemoglobin-F (HbF) can be found in the circulation. Recently, a monoclonal antibody to HbF has become commercially available, enabling application of a flow cytometric immunofluorescence method for accurately determining the concentration of HbF+ red blood cells. We demonstrate that white blood cells are included in the cluster selected as red blood cells and that these white blood cells exhibit a level of autofluorescence that coincides with the fluorescence signal from HbF+ red blood cells. However, these white blood cells can be excluded from the analysis, thus preventing spuriously increased HbF+ red blood cell counts. We present the results of patient samples containing HbF+ red cells as illustrations of the technique and as a potential interference by HbF-containing cells of nonfoetal origin. Using samples spiked with cord blood, the method is exactly linear with a high coefficient of correlation (r=0.997). Furthermore, the assay has excellent precision (CV < 2.4%), a low limit of detection (0.12% HbF+ RBC), is independent of Rhesus D and can be completed within 1.5 h. This method is suitable for accurate determination of foetomaternal haemorrhage.     

The Production of Hemoglobin C in Sheep Carrying the Gene for Hemoglobin A: Hematologic Aspects

The first part of this study describes the hematologic and physiologicchanges observed in sheep homozygous for the hemoglobin A during severeblood loss anemia. It was found possible thus experimentally to replace Hb Aentirely with a new hemoglobin type, Hb C. The following additional observations were made: (1) Hb C could not be distinguished from Hb A by submitting the appropriate red blood cells to an "acid elution" technic. These twohemoglobin types were found to be more resistant to this treatment than asecond adult hemoglobin type, Hb B, while the fetal hemoglobin of the new-born lamb was found to be highly resistant. In sheep heterozygous for Hb Aand Hb B, both hemoglobin types were equally distributed among all redblood cells. (2) During stages of severe blood loss relatively small quantities ofacid resistant red blood cells of larger size were demonstrable in homozygousHb A sheep; these cells were considered to be reticulocytes. Additional observations regarding variations in red cell parameters are also presented. (3)The oxygen affinities and the Bohr effects of blood samples and red cellhemolysates containing over 90 per cent Hb C are presented and comparedwith those of samples containing only Hb A, Hb B or the hemoglobin of thenewborn lamb.

Attempts to produce Hb C in sheep homozygous for Hb A by means otherthan phlebotomy are described in the second part of this report. Smallamounts of Hb C were demonstrable in a sheep homozygous for Hb A afterrepeated injections of a urinary extract of human origin with high erythropoietic activity. Administration of cobalt and of thyroxin did not result in theformation of significant amounts of Hb C.

Submitted on July 19, 1965 Accepted on December 25, 1965     

STUDIES ON THE PHYSIOLOGY OF THE WHITE BLOOD CELL: THE GLYCOGEN CONTENT OF LEUKOCYTES IN LEUKEMIA AND POLYCYTHEMIA

The technic of determining glycogen in isolated white blood cells was appliedto the study of the different types of leukemia and of polycythemia, in order toobtain information on the physiology of the white blood cell. From this study itis concluded that the granulated leukocyte is the only carrier of glycogen in wholeblood. The "reducing substances" in lymphocytes and blast cells are not consideredas true glycogen.

The glycogen content of wet white blood cells in the rabbit amounts to about1 per cent. In the human being a range of from 0.17 to 0.67 per cent was calculated.In disease higher percentages occur, in polycythemia up to 1.64 per cent and inglycogen storage disease up to 3.05 per cent.

The glycogen concentration of normal white blood cells is within the same rangeas that of the striated muscle.

Note: I acknowledge with gratitude my indebtedness to Dr. William Dameshek for giving me the opportunity of analyzing the blood of some of the patients studied. Miss M. H. Campbell, Miss H. A. Clark,and Miss L. M. Garofalo have aided in carrying out many of the blood counts.

     

Failure to demonstrate beneficial effect of prostacyclin (PGI2) infusion on red blood cell deformability in patients with peripheral vascular disease: a possible role of the leukocytes.

The effect of prostacyclin (PGI2) on red blood cell deformability is still controversial. The authors have evaluated some hemorrheologic parameters during PGI2 infusion to patients with severe obstructive peripheral arterial disease not suitable for surgery and not responsive to other medical treatments. PGI2 infusion resulted in a positive clinical effect in 12 of 16 patients, who experienced a significant improvement of their rest pain lasting from two days to more than seven months. Hematocrit, platelet count, and fibrinogen were not modified during PGI2 infusion whereas leukocyte count rose significantly. The evaluation of red blood cell filterability (VRBC) (volume of RBC filtered in one minute) by a whole blood filtration technique showed no significant changes during PGI2 treatment. However, since whole blood filterability is negatively correlated to leukocyte count, a specific regression line was elaborated to remove the potential negative influence of the increased leukocyte counts. VRBC values adjusted to a standard leukocyte number significantly increased during PGI2 treatment.     

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     

Leukocyte concentrations in discrimination of benign from malignant lung lesions

Malignant lung lesions are associated with significant changes in leukocyte concentrations. However, overlap of values between normal subjects and patients with cancer has limited the clinical utility of this determination. To decrease the overlap, 2,000 cells per differential determination were counted, and replicate automated cell counts were performed in blood samples obtained on different mornings between 7 and 9 A.M. Seventy-five patients who presented with undiagnosed lung lesions were analyzed. Benign and malignant lung lesions could be accurately distinguished by either relative granulocyte or lymphocyte counts. With 65.5 percent granulocytes as the cutoff, 86 percent of patients (38 of 44) proved to have lung cancer had higher values, and 97 percent of patients (30 of 31) with benign lesions had lower values. Likewise, with 28 percent lymphocytes as the cutoff, 87 percent of patients with lung cancer (39 of 44) had lower values, and 94 percent of patients (29 of 31) with benign disease had higher values. Relative monocyte counts were not different. Absolute leukocyte concentrations, although different for each group, had considerable overlap and thus were poor discriminators. These data suggest that precise analytical techniques and the use of relative leukocyte concentrations can improve the clinical utility of the leukocyte count as a discriminator of benign and malignant lung lesions.     

Studies on blood passed through an artificial kidney

1. In vitro blood studies using the artificial kidney are discussed in detail.

2. In vitro experiments in which samples of blood in cellophane tubing arerotated on the drum showed a diminution in the red cell volume, leukocyte andplatelet counts.

Dilution, caused by the passage of the dialyzing fluid through the cellophanemembrane and by the presence of some of the rinsing fluid, and rotation of the drumare factors of importance in this regard.

3. The influence of heparin and the chemotactic role of cellophane on the leukocytes are discussed.

Heparin in concentration of 1 mg./10 cc. of blood causes in vitro agglutinationand disintegration of leukocytes after approximately one hour.

Photographs are shown that demonstrate coating of the cellophane by granulocytes. This is in keeping with the findings of Chambers and Grand that cellophaneexerts a positive chemotactic influence on the granulocytes.

4. Hypersegmentation of the leukocytes was noted in samples of blood rotatedon the drum for fifteen minutes. Exteriorization and slow speed centrifugationprobably account for this phenomenon.

     

Leukocyte Reduction Filters Are Reliable and Economic Source for Natural Killer Cell Preparation

Background: An issue that hinders researchers’ access to Natural Killer (NK) cells is their low proportion in peripheral blood leukocytes. This issue is currently addressed by methods involving a series of differentiation and expansions that are time-consuming and expensive.Objective: We have investigated whether the used leukocyte reduction filters, a by-product in the blood transfusion practice that currently is considered waste, can be utilized as a source of the NK cells.Methods: Following the blood donation of 46 donors based on the Iranian Blood Transfusion Organization’s protocols, a sample of peripheral blood of each donor and the leukocyte reduction filter used in their donation procedure have been obtained. The entrapped cells were flushed back from the leukocyte reduction filters. Both groups of samples were analyzed using an automatic hematological analyzer. NK cell isolation was done by the MACS negative selection method. The samples have been comparatively analyzed utilizing flow cytometry data of NK cells’ subpopulation compositions, viability, degranulation patterns, and cytotoxic capacity against the K562 cell line.Results: Every major leukocyte population was abundant in the samples extracted from the used leukocyte reduction filters. The NK cells extracted from leukocyte reduction filters did not show any statistically meaningful differences (P<0.5) from peripheral blood samples in terms of subpopulation composition, viability, degranulation potency, and cytotoxic capacity.Conclusion: Used leukocyte reduction filters can be considered an economic, easy to obtain, and robust source of abundant research-grade NK cells.     

Artefactually-normal automated platelet counts due to malaria-infected RBC

Protein aggregates, red cell or white cell fragments are known to interfere with platelet counts in automated blood analysers, both by aperture impedance and optical technologies. When a falsely high value is suspected, interference by pseudo-platelet particles can be confirmed by systematic examination of stained blood films. The method that best avoids these sources of interference is the reference, immunological platelet count. We describe a case of treated malaria with a false normal platelet count. The blood smear revealed small red cells, infected by trophozoites of Plasmodium falciparum, that interfered with the platelet count. The Cell Dyn 4000 shows different patterns of interference by infected red cells in its impedance and optical counts, and thrombocytopenia was suspected immediately. This was confirmed by a phase-contrast microscopic platelet count.     

The intriguing contribution of white blood cells to sickle cell disease - a red cell disorder

Sickle cell disease (SCD) is characterized by a point mutation that replaces adenine with thymidine in the sixth codon of the beta-globin gene, a unique morphological abnormality of red blood cells, vaso-occlusion with ischaemic tissue injury, and susceptibility to infections. Vascular lumen obstruction in SCD results from interaction of erythrocytes, leukocytes, platelets, plasma proteins, and the vessel wall. The disease phenotype is a product of various genes and environmental factors acting in concert with the protein lesion underlying the red cell anomaly. The severity of SCD increases with leukocyte count. The biological basis and therapeutic implications of this relationship are discussed. Leukocytes contribute to SCD by adhering to blood vessel walls and obstructing the lumen, aggregating with other blood cells with more effective blockage of the lumen, stimulating the vascular endothelium to increase its expression of ligands for adhesion molecules on blood cells, and causing tissue damage and inflammatory reaction which predispose to vaso-occlusion. Patients with impaired ability of leukocytes to kill microbes are more prone to infections; which precipitate sickle cell crisis. Reduction of leukocyte count ameliorates SCD. Similarly, targeted blockade or reduced synthesis of specific leukocyte adhesion molecules and their ligands might confer clinical benefit in SCD.     

Preparation of Leukocyte-Poor Blood: A Comparison of IBM 2991 Washing and Huggins Freeze-Thawing1

Abstract. Frequently, frozen blood is requested in order to provide a leukocyte-poor red cell transfusion and a reduced level of exposure to hepatitis virus rather than in an effort to find serological compatible red blood cells. However, this usage of frozen blood is expensive and, as recently reported, does not eliminate the risk of transmission of hepatitis. To assess the feasibility of substituting washed cell preparations, we have compared the Huggins frozen blood process with the IBM 2991 cell washing technique and have evaluated the efficiency of leukocyte removal and red cell recovery from homologous pools of fresh blood. The results of these experiments combined with the greatly decreased cost (approximately 1/3) and time required (approximately 1/6) indicate that many of the requests which blood banks receive for frozen blood could, in fact, be better met by using cells which have been washed in the IBM 2991.