Cells In Blood Smears Research Articles

Identification and characterization of a novel kind of nuclear protein occurring free in the nucleoplasm and in ribonucleoprotein structures of the ″speckle″ type

We have identified, by cDNA cloning and immunodetection, a novel type of constitutive nuclear protein which occurs in diverse vertebrate species, from Xenopus laevis to man, in the form of two different gene products (79.1 kDa and 82.1 kDa in Xenopus, 81.6 kDa and 84.6 kDa in man), remarkably differing in pI (5.4-7.2). This type of protein is characterized by a carboxyterminal domain extremely rich in hydroxyamino acid residues, notably Ser (S), and tetrapeptide repeats of the type XSRS, and hence is termed "domain rich in serines" (DRS) protein. It has been immunolocalized exclusively in the cell nucleus such as in blood cell smears, cultured cells of very different origins and tissue sections, and has also been identified in Xenopus oocyte nuclei, both in sections and by biochemical methods in manually isolated nuclei. In many cell types the protein appears in two different physical states: (i) nuclear granules, identified as ribonucleoprotein (RNP) structures of the "speckle" category by colocalization and cofractionation with certain splicing factors and Sm-proteins, and (ii) in molecules diffusible throughout the nucleoplasm. During mitosis and also in meiosis (Xenopus eggs) the protein is transiently dispersed throughout the cytoplasm but rapidly reaccumulates into the reforming daughter-nuclei. In agreement with this, biochemical experiments have shown that during meiosis (eggs) the protein is recovered in a approximately 11-13S complex of the fraction of soluble cell components. We discuss general constitutive nuclear functions of this apparently ubiquitous and evolutionarily conserved protein.

Influence of flumequine on in vivo mitogen responses of European eel ( Anguilla anguilla L., 1758) lymphoid cells

The influence of flumequine on mitogen induced lymphoid cell proliferation in European eels ( Anguilla anguilla L., 1758) was studied. For this purpose an in vivo test, using peroral drug administration followed by successive intraperitoneal injections with concanavalin A (ConA) or bacterial lipopolysaccharides (LPS) and 5-bromo-2′-deoxyuridine, was applied. Direct counting of proliferated cells in blood smears revealed that flumequine possesses mitogenic properties. A synergistic and an antagonistic effect of the drug was observed after LPS and ConA stimulation, respectively. Flow cytometric analysis of peripheral blood lymphoid cells showed a significant reduction of the mean proportion surface immunoglobulin positive cells in the flumequine-treated animals. It is concluded that flumequine enhances proliferation of lymphoid cells (probably surface immunoglobulin negative cells) in eel under the present experimental conditions.

Formation and microscopical application of a fluorescent 1,10-phenanthroline derivative of ruthenium red.

In this work we describe the formation and microscopical application of a fluorescent derivative of Ruthenium Red (RR) obtained by heating the dye in the presence of 1,10-phenanthroline (OP). The RR-OP reaction product showed absorption maxima at 416 and 444 nm and intense fluorescence emission at 578 nm under 440 nm exciting light. Neither RR nor OP solutions alone were fluorescent when excited at 440 nm. Using fluorescence microscopy, chicken blood cell smears stained 5 min with the RR-OP derivative showed the chromatin of erythrocyte nuclei with a bright orange fluorescence under violet-blue (436 nm) exciting light.

White blood cells in infants with congenital toxoplasmosis: transient appearance of cALL antigen on reactive marrow lymphocytes.

Existing serological methods for diagnosing congenital toxoplasmosis are inadequate. We have therefore characterized the features of peripheral blood cell counts and smears in a prospective study of 8 affected infants during their first year of life. Enlarged, vacuolated lymphocytes were observed in all patients. Six patients had periods of neutropenia; in 1 patient the neutropenia was severe and prolonged. A bone marrow aspirate from this patient showed enhanced myelopoiesis, and after stimulation with hydrocortisone, but not with adrenaline, his neutrophil count normalized. Phenotyping of his bone marrow cells revealed that approximately 55% of both the immature and mature B lymphocytes were positive for the cALL antigen. Of the 8 patients, 6 had mild eosinophilia, 4 had mild monocytosis and 3 had increased numbers of plasma cells in the peripheral blood. These findings may reflect the activity of the congenital toxoplasma infection and may thus contribute to the diagnosis, particularly in patients with silent serology.

Failure to diagnose anemia in medical inpatients: Is the traditional diagnosis of anemia a dying art?

The diagnosis and treatment of anemia in medical inpatients were studied. Anemia was detected from the admitting complete blood cell count, the medical charts were perused for management of anemia by the physicians, and the cases of anemia were classified by the investigators, using the complete blood cell count and peripheral blood smear. Anemia was seldom diagnosed in the traditional manner. In about 25 percent of cases, it was not even recognized. In about 20 percent, it was recognized but not evaluated or treated. In the 55 percent of cases adequately diagnosed and/or treated, the diagnosis was often more “situational” than analytic, the treatment more empiric than specific. Physicians ignored or misused information from the complete blood cell count and smear description and examined the smear themselves in fewer than one in 10 anemic patients. Not all of the missed cases of anemia were mild, expected from the patient's illness, or unimportant to the patient's care.

Differentiation of granulocytes in pappenheim stained blood cell smears using standardized cytophotometric analysis

The well-known problems of the low reproducibility of peripheral blood smear analysis have for some time stimulated endeavours to automate blood cell classification. In the cytophotometric standardized color measurement and analysis, the computed color characteristics for the first time refer to an internationally accepted color system, allowing not only an international comparison of the computer color measurements but an unproblematic mutual interchange of color information between man and machine based on both the human visual color impressions and the conventional morphological color attributes of the white blood cells. The discriminatory power of the method is demonstrated by differentiating the cytoplasm granulations in basophil, eosinophil and neutrophil granulocytes.

Plasma levels of human granulocytic elastase α1-proteinase inhibitor complex (E-α1-PI) in leukemia

There is evidence that polymorphonuclear granulocytes release neutral proteinases such as elastase (E) and cathepsin G in the course of acute leukemia. These proteinases may inactivate clotting factors by unspecific degradation before they are eliminated via complex formation with endogenous inhibitors, e.g. the alpha 1-proteinase inhibitor (alpha 1-PI). In this study it was attempted to correlate plasma levels of the E-alpha 1-PI complex with factor XIII and antithrombin III in acute leukemia. Using a newly developed, sensitive enzyme-linked immunoassay the concentration of E-alpha 1-PI in patients with various types of leukemia, malignant lymphoma or multiple myeloma was determined. Only patients with acute myelocytic or promyelocytic leukemia (AML, APL) and chronic myelocytic leukemia with and without blastic transformation (CML) showed moderate to high levels of E-alpha 1-PI (2- to 20-fold of normal). However, coagulation factor concentration observed in the different types of leukemia seemed to be independent of elastase liberation. Most of the AML-patients with elevated E-alpha 1-PI levels showed peroxidase positive blood cell smears.

Human ferritin: Effects of antigen source and fixation on leucocyte staining by immunoperoxidase technique

The localization of ferritin was studied in peripheral blood cells and variously fixed tissues with the antibodies against ferritins isolated from human heart and spleen. The unlabelled antibody enzyme method (PAP) was used to detect the binding sites of antibodies. In peripheral blood cell smears both antisera gave rise to strong staining of polymorphonuclear (PMN) cell cytoplasm, whereas the monocytes stained relatively weakly. There were no staining differences between the two antisera. In human spleen sections the spleen ferritin antiserum stained the PMN cells and sinusoidal lining cells, whereas the heart ferritin antiserum stained only PMN cells. Neither of the two antisera stained monocytes in the spleen sections. This finding was observed in specimens fixed in Bouin's fixative, Baker's fixative and neutral formalin. However, the immunoreactivity of ferritin was totally destroyed by some other fixatives (Carnoy's fixative, formol sucrose and glutaraldehyde). These results suggest that ferritin is more readily released from monocytes than from PMN cells, and that mature spleen macrophages contain antigenic determinants of ferritin that are recognized only by anti-spleen ferritin antiserum.

Use of carbon labeling to demonstrate the role of blood monocytes as precursors of the ‘ameboid cells’ present in the corpus callosum of postnatal rats

Cells with features suggestive of ameboid motion and phagocytic properties are observed in the rat corpus callosum during the first few days of life. These cells, hereafter referred to as 'ameboid cells', have been investigated in several ways. An electron microscopic study of the corpus callosum in 5- to 7-day-old rats indicated that most 'ameboid cells' were typical macrophages, but some displayed features of monocytes, while others appeared to be transitional between the two types. These observations raised the possibility that blood monocytes were the precursors of 'ameboid cells'. This possibility was tested by injecting a suspension of carbon particles into the circulation of rats of various ages to label and trace monocytes. Within 15 minutes after injection, carbon particles were seen between cells in blood smears as well as in the lumen of capillaries, but not between cells and axons in corpus callosum. By a half hour, a few of the circulationg monocytes, and with time, up to half of them, contained carbon particles. Five days after injection, carbon particles were observed in cells of the corpus callosum identified as 'ameboid cells' of the monocytic and macrophagic type. Such carbon-containing cells were seen in many of the animals injected at the age of 0-1 day, in few of those injected at 3-5 days, and in none of the older animals. Since free carbon had not been observed in corpus callosum spaces, it was concluded that 'ameboid cells' did not pick up carbon locally. The alternative was that blood monocytes, after ingesting carbon particles in the circulation, migrated to the corpus callosum and settled as 'ameboid cells'. In the hope of obtaining a direct confirmation of this conclusion, blood cells obtained from carbon-injected Lewis rats were centrifuged in a Percoll gradient to obtain a fraction which contained 70-80% monocytes, less than 2% granulocytes, and 20-30% lymphocytes. Carbon was present in up to half of the monocytes and 1% of the granulocytes, but not in the lymphocytes; and it was calculated that over 99% of the carbon-labeled cells were monocytes. The cell fraction was then introduced into the blood circulation of 2- to 3-day-old syngeneic Lewis rats, and the animals were sacrificed 5 days later. Occasional carbon-labeled cells appeared not only in liver, spleen and connective tissue, but also in the corpus callosum, where they were identified as 'ameboid cells' of the monocytic and macrophagic type. Even though such cells were infrequent, their presence conclusively demonstrated that at least some 'ameboid cells' of the corpus callosum were derived from circulating blood monocytes.

Estimation of kinetic parameters of neutrophilic, eosinophilic, and basophilic granulocytes in human blood

Two hematologically normal patients with glioblastoma and six patients with chronic lymphocytic leukemia received continuous 3H-thymidine infusions for 3--10 days. In autoradiographs of blood cell smears taken for 25 days or more after the beginning of 3H-thymidine administration the labeling index and the labeling intensity of granulocytes were determined. A sufficiently high labeling intensity, i.e. a sufficiently long autoradiographic exposure time was found to be critical for obtaining valid and reproducible results. On the basis of certain assumptions discussed in detail, complete labeling of cells with 3H-thymidine followed by autoradiographic evaluation and mathematical analysis of the labeling patterns seems to be a suitable method for estimation of kinetic parameters of postmitotic granulocytes in vivo. The mean intramedullary maturation and storage time was observed to be 115 +/- 7 h or neutrophils, 103 +/- 4 h for eosinophils and 103 +/- 11 h for basophils. The mean relative inflow rate into the blood (or relative turnover rate in the blood) was found to be 4.2 +/- 0.4/h for neutrophils, 4.0 +/- 0.4%/h for eosinophils and 1.2 +/- 0.3%/h for basophils. The mean blood transit time (or blood sojourn time) was estimated to be 25 +/- 2 h or neutrophils, 26 +/- 3 h for eosinophils and 89 +/- 21 h for basophils. Accordingly the half lifes (T 1/2) of granulocytes in the blood were 17.3 +/- 1.4 h for neutrophils, 18.0 +/- 2.1 for eosinophils and 62 +/- 15 h for basophils. Under the quasi steady state conditions of this study the kinetics of granulocytes in the present CLL patients appeared to be normal, despite a marked lymphocytic infiltration of the bone marrow. The apparent discrepancy between these findings and the data obtained with autotransfusion of DFP-labeled granulocytes is discussed.

Enumeration of human B lymphocytes in stained blood smears by their binding of bacteria

Human B lymphocytes were enumerated in stained blood smears by two methods. In one method, the surface Ig of B cells was detected by the antibody-mediated binding of anti-light chain antibody-coated Escherichia coli (anti-Ig Ab- E. coli). By using only anti-κ or anti-λ antibody-coated E. coli or a mixture of both, the number of κ-bearing, λ-bearing, and total Ig-bearing lymphocytes was determined. Lymphocytes were found to bear only κ or λ light chains (but not both), indicating that B cells do not normally have passively adsorbed Ig on their surface. The second method was based on the property of a strain of Brucella melitensis (Bm) to bind to human B lymphocytes. This was shown by the fact that Bm and anti-Ig Ab- E. coli bind to the same cells, i.e., B cells. Competition experiments suggested that the binding of Bm to the B cells was not via the surface Ig of the B cells. The number of B cells in the peripheral blood of several normal donors and several patients with chronic lymphocytic leukemia is presented. The use of bacteria to label the B cells in blood smears allows for the morphological identification of lymphocytes and eliminates the need to purify the lymphocytes, thereby also eliminating the problem of the selective loss of T or B cells during the purification. By using this method the enumeration of the κ-bearing, λ-bearing, and total number of human B cells may become a routine diagnostic procedure.

Simultaneous identification of T and B cells in blood smears using antibody coated bacteria

A new method is described which allows separate or simultaneous identification of T and B cells in stained blood smears using as markers morphologically distinguishable antibody-coated bacteria. Glutaraldehyde was used to coat Escherichia coli and itGaffkya tetragena with purified antibody, namely anti-b5Ig allotype (Ifk chains) or rabbit anti-guinea pig IgG, both made in b4 rabbits. Blood was collected from b5 rabbits, the cells were washed, treated with guinea pig anti-rabbit T cell antiserum and washed again. E. coli coated with anti-GpIg and G. tetragena coated with anti-b5Ig were added individually or together to the washed blood cells and centrifuged to promote binding of E. coli to T cells and G. tetragena to B cells. The cells were smeared and stained with Wright's stain. We found that: a) the binding of bacteria to T and B cells depended only on the reaction between the antibody molecules on bacteria and the antigen (surface Ig or guinea pig anti-T cell Ig) on the cell membrane; b) lymphocytes were surrounded by either E. coli or G. tetragena, but never by both; c) of the total blood lymphocytes, 43–48% were T cells and 49–51% were b5Ig bearing B cells; d) about 6% of the lymphocytes were unlabeled (probably B cells bearing λ chain Ig); and e) all monocytes and basophils had surface Ig (cytophilic). Thus, differential counting of T and B cells can be easily performed in stained blood smears using antibody-coated bacteria.

F-Cells in the Adult: Normal Values and Levels in Individuals With Hereditary and Acquired Elevations of Hb F

Specific antibodies to human fetal hemoglobin were prepared and, after conjugation with a fluorescent dye, were used to determine the distribution of Hb F-containing cells in blood smears from normal adults and individuals with hereditary and acquired conditions associated with abnormal levels of Hb F. The mean proportion of F-cells in normal persons was 2.7% +/- 1.4%, with a range of 0.5%-7.0%. Proportions of F-cells were increased in persons with several acquired and inherited disorders that are associated with an increased percentage of Hb F in hemolysates. A strong linear correlation between the amount of Hb F and proportion of F-cells was observed. This technique may prove useful in studies of a variety of disorders associated with Hb F elevations and also in investigations of the mechanisms controlling the transition from fetal to adult hemoglobin during the course of human development.

F-cells in the adult: normal values and levels in individuals with hereditary and acquired elevations of Hb F

Specific antibodies to human fetal hemoglobin were prepared and, after conjugation with a fluorescent dye, were used to determine the distribution of Hb F-containing cells in blood smears from normal adults and individuals with hereditary and acquired conditions associated with abnormal levels of Hb F. The mean proportion of F-cells in normal persons was 2.7% +/- 1.4%, with a range of 0.5%-7.0%. Proportions of F-cells were increased in persons with several acquired and inherited disorders that are associated with an increased percentage of Hb F in hemolysates. A strong linear correlation between the amount of Hb F and proportion of F-cells was observed. This technique may prove useful in studies of a variety of disorders associated with Hb F elevations and also in investigations of the mechanisms controlling the transition from fetal to adult hemoglobin during the course of human development.

The haemoglobins of healthy and anaemic Xenopus laevis.

ABSTRACT The haemoglobins of Xenopus laevis have been studied by carboxymethyl-cellulose column chromatography and by polyacrylamide gel disk electrophoresis. In Xenopus tadpoles, 2 haemoglobins are found (Xenopuj-HbF1 and Aer/opiiS-HbF2). Both haemoglobins persist throughout tadpole life: Aenopur-HbF1 is the main tadpole haemoglobin; Xenopus-HbF2 is present in rather smaller amounts. The proportion of Xenopus-HbF 1 to Xenopus-HbF2 increases as the tadpoles age. Almost all the tadpole haemoglobin disappears soon after metamorphosis, although traces persist throughout adult life. Two adult haemoglobins occur in Xenopus: they first appear during the later tadpole stages. Xenoput-HbA1 comprises the majority of the adult haemoglobin. Between one-twentieth and one-tenth of the adult haemoglobin consists of Xenopus- HbA2, which is not a polymer of Xenopus-HbA1. Immunoglobulins respectively specific to pooled -Xenopus-HbF1 and Xenopus-HbF2, and to pooled Aeno/wr-HbA1 and Aenopui-HbA2 were raised. Samples of these antibodies were conjugated with fluorescein isothiocyanate. Adult Xenopus were made anaemic by bleeding or by injection of phenylhydrazine. Fourteen days after the induction of anaemia it was found, using both chromatographic and electrophoretic techniques, that most anaemic toads had started to resynthesize Aenopur-HbF1. In some animals up to 11 % of the haemoglobin present was found to be Xenopus-FLbF1 and precipitin lines were obtained when the pooled haemoglobins from these animals were tested with a specific anti-Xenopnr-HbF immunoglobulin. During recovery from anaemia greatly enhanced amounts of Aenopur-HbA2 (up to 58 % of the total haemoglobin) were present in the blood. The amounts of tadpole haemoglobins present in such recovering toads were not noticeably greater than in healthy animals. Between 42 and 120 days after the induction of anaemia the haemoglobin profile of the toads regained normality. Blood cell smears were made from anaemic toads which were known to possess abnormal amounts of Xenopus-HbF1 14 days after the induction of anaemia. The smears were treated with specific anti--Xenopus-HbF immunoglobulin conjugated with fluorescein isothiocyanate. Although up to 11 % of the haemoglobin present in the animals was Xenopus -HbF1, the number of fluorescing cells was no greater than in similarly treated smears from healthy control adults possessing less than 1 % Xenopus -HbFl. It is concluded that during anaemia Xenopus starts to resynthesize one of its tadpole haemoglobins. This resynthesis occurs in many or most of the circulating red cells, and is not confined to a small population of cells. During recovery from anaemia enhanced quantities of Xenopus- HbA2 are made.

Significance of Transplacental Hemorrhage in the Induction of Specific Maternal Unresponsiveness<xref ref-type="fn" rid="FN2">2</xref>

Transplacental hemorrhage is reportedly detectable by erythrocytes containing fetal hemoglobin-F in the maternal circulation. The procedure in the present study is based on the premise that fetal hemoglobin is not eluted from red cells in blood smears by an acid buffer, whereas adult hemoglobin is removed. Erythrocytes containing hemoglobin-F were observed in blood smears of both virgin and pregnant BALB/c mice, but the relative number of these cells in the maternal blood increased during pregnancy, reached a peak around the time of parturition, and decreased thereafter. In BALB/c females pregnant by DBA/2 males, the relatively large numbers of acid-resistant erythrocytes in maternal blood were related to the maternal unresponsiveness to DBA/2 allografts. The fetal derivation of at least a fraction of these cells was suggested by the apparent activity of fetal immunogens in the maternal circulation. The blood of peripartum BALB/c mice multiparous by DBA/2 males was injected into normal, nonimmune BALB/c mice. The blood recipients were later challenged with an allotransplantable DBA/2 tumor. A low degree of immunity was indicated by a decreased size of tumor implants. Attempts to demonstrate vertical transmission of immune suppression from mothers to progeny were negative. These findings indicate that transplacental hemorrhage is a potential source of antigens involved in the induction of specific maternal unresponsiveness.

Primordial germ cells in blood smears from chick embryos.

The feasibility of studying avian primordial germ cells in blood smears has been demonstrated. Blood smears prepared from chick embryos of stages 13 to 15 (48 to 55 hours) contained primordial germ cells, which were revealed by the periodic acidSchiff reaction. The presence of glycogen in the cytoplasm of the primordial germ cells facilitated their selective identification.

Compensatory Changes Following Splenectomy in Bartonella Free Rats

In rats that were carriers of Bartonella muris, certain changes were observed, following splenectomy and recovery from the anemia, in the hemocytoblastic tissue and in the reticular and endothelial elements. These changes appeared 3 to 5 months after splenectomy and were associated with immunity to further infection with Bartonella muris. The changes consisted primarily of hyperplasia of hemolymph tissue, hyperplasia of reticular and endothelial elements of lymph nodes, the formation of lymphoblastic foci periportally in the liver and peribronchially and perivascularly in the lung, regeneration of all elements of the thymus and marked hyperplasia of all elements of bone marrow (increased hematopoiesis). In view of the presence of coincident latent infection in the rat, the compensatory changes described following splenectomy in rats of Bartonella carrier stock were difficult to evaluate. What was due to the absence of the spleen and what to previous Bartonella muris anemia remained uncertain. Studies were therefore made in rats in which the exacerbations of latent infections following splenectomy could be excluded. Six Bartonella-free rats, 3 months of age, of Wistar stock, were splenectomized and killed 6 months later. Six unsplenectomized rats of the same age and stock were killed at the same time. The presence of Bartonella infection was excluded by red blood cell counts and smears of peripheral blood at frequent intervals. The anatomical changes observed were similar to those found in splenectomized Bartonella-carrier rats but less extensive. The liver showed little if any perivascular lymphocytic infiltration. The Kupffer cells were not enlarged, nor were they more prominent in phagocytic activity than in normal rats.