Blood Histology

Staining of Blood Cells

Blood cells are generally studied in smears or films prepared by spreading a drop of blood in a thin layer on a microscope slide.

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Blood smears are routinely stained with special mixtures of red (acidic) and blue (basic) dyes. These mixtures also contain azures, dyes that are useful in staining some structures of blood cells known as azurophilics (azure + Gr. philein, to love). Some of these special mixtures (eg, Giemsa, Wright's, Leishman's) are named for the investigators who introduced their own modifications into the original mixture.

Erythrocytes (red blood cells), which are anucleate, are packed with the O2-carrying protein hemoglobin. Under normal conditions, these corpuscles never leave the circulatory system.

Most mammalian erythrocytes are biconcave disks without nuclei .When suspended in an isotonic medium, human erythrocytes are 7.5       m in diameter, 2.6       m thick at the rim, and 0.8       m thick in the center. The biconcave shape provides erythrocytes with a large surface-to-volume ratio, thus facilitating gas exchange.

A decreased number of erythrocytes in the blood is usually associated with anemia. An increased number of erythrocytes (erythrocytosis, or polycythemia) may be a physiological adaptation. It is found, for example, in people who live at high altitudes, where O2 tension is low. Polycythemia (Gr. polys, many, + kytos, cell, + haima, blood), which is often associated with diseases of varying degrees of severity, increases blood viscosity; when severe, it can impair circulation of blood through the capillaries. Polycythemia might be better characterized as an increased hematocrit, ie, an increased volume occupied by erythrocytes.

Red cell formation (erythropoiesis). The process of erythropoiesis is directed towards producing a cell devoid of organelles but packed with haemoglobin. 

The first recognisable erythrocyte precursor is known as the proerythroblast, a large cell with numerous cytoplasmic organelles and no haemoglobin. Further stages of differentiation are characterised by three main features: decreasing cell size and nuclear extrusion 

progressive loss of organelles; the presence of numerous ribosomes at early stages accounts for the marked cytoplasmic basophilia (blue staining) which steadily decreases as the number of ribosomes falls 

progressive increase in the cytoplasmic haemoglobin content; this accounts for the increasing eosinophilia (pink staining) of the cytoplasm towards maturity. 

Reticulocytes

Reticulocytes are immature red blood cells which have shed their nucleus, but still retain residual nuclear material. Reticulocytes are the immature form in which erythrocytes are released into the circulation from the bone marrow.

The rate of release of reticulocytes into the circulation generally equals the rate of removal of spent erythrocytes by the spleen and liver. Since the lifespan of circulating erythrocytes is about 120 days, reticulocytes constitute slightly less than 1% of circulating red blood cells. 

When severe erythrocyte loss occurs, such as after haemorrhage or haemolysis, the rate of erythrocyte production in the bone marrow increases and the proportion of reticulocytes in circulating blood rises (reticulocytosis). Clinically, the reticulocyte percentage is a useful indicator of erythropoiesis. In cases of anaemia, an elevated reticulocyte count indicates normal marrow function, while a decreased count may mean impaired erythropoiesis. 

Clinical:

Failure to maintain an adequate haemoglobin concentration is termed anemia. There are several common causes of anaemia, including lack of factors required to make haemoglobin, (e.g. iron, or vitamins B12 and folic acid), excessive loss or inappropriate destruction of erythrocytes, or failure of bone marrow to manufacture enough cells. Erythrocyte morphology may be altered in certain types of anaemia. Lack of iron leads to cells that are smaller than normal (microcytes) while lack of B12 and folate leads to cells that are larger than normal (macrocytes). Abnormally rounded and fragile erythrocytes (spherocytes) may be caused by mutations in genes coding for proteins in the red cell cytoskeleton

LEUKOCYTES

According to the type of granules in their cytoplasm and the shape of their nuclei, leukocytes are divided into two groups: granulocytes (polymorphonuclear leukocytes) and agranulocytes (mononuclear leukocytes). Both granulocytes and agranulocytes are spherical while suspended in blood plasma, but some become ameboid after leaving the blood vessels and invading the tissues. Their estimated sizes mentioned below refer to blood smears, in which the cells are spread and appear larger than they actually are in the blood.

Granulocytes 

Neutrophils 

Eosinophils 

Basophils 

Mononuclear leucocytes 

Lymphocytes 

Monocytes 

Neutrophils with more than five lobes are called hypersegmented and are typically old cells. Although the maturation of the neutrophil parallels the increase in the number of nuclear lobes under normal conditions, in some pathological conditions, young cells appear with five or more lobes.

PUS:

Dead neutrophils, bacteria, semidigested material, and tissue fluid form a viscous, usually yellow collection of fluid called pus.

Metachromasia is a property that enables certain substances to change the color of some basic dyes (eg, toluidine blue), being stained by the changed color (purple, in this example). Basophils can liberate their granule content in response to certain antigens, as can mast cells .

LYMPHOCYTES

Lymphocytes constitute a family of spherical cells with similar morphological characteristics. They can be classified into several groups according to distinctive surface molecules (markers), which can be distinguished by immunocytochemical methods. They also have diverse functional roles, all related to immune reactions in defending against invading microorganisms, foreign macromolecules, and cancer cells

The cytoplasm of the small lymphocyte is scanty, and in blood smears it appears as a thin rim around the nucleus. It is slightly basophilic, assuming a light blue color in stained smears. It may contain a few azurophilic granules. The cytoplasm of the small lymphocyte has a few mitochondria and a small Golgi complex; it contains free polyribosomes 

The amount of cytoplasm depends upon the state of activity of the lymphocyte, and in circulating blood there is a predominance of 'small' inactive lymphocytes (6-9 μm in diameter). 'Large' lymphocytes (9-15 μm in diameter) make up about 3% of lymphocytes in peripheral blood.

Platelets contain a well-developed cytoskeleton. At the periphery of the cell is a marginal band of microtubules which depolymerise at the onset of platelet aggregation. The cytoplasm is rich in the contractile proteins actin and myosin which are involved in the functions of clot retraction and extrusion of granule contents as part of degranulation. 

Located deep to the marginal band of microtubules and also scattered throughout the cytoplasm is the dense tubular system (DTS) consisting of narrow membranous tubules which contain a homogeneous electron-dense substance. This system is believed to be an intracellular store of calcium which is released into the platelet cytosol following signaling from platelet surface receptors and secondary messengers. 

Platelets contain a system of interconnected membrane channels the surface-connected canalicular system (SCCS) which is in continuity with the external environment via external pits. Alpha granules fuse with the SCCS as part of secretion of their contents

Important Points : Nuclear Structures

  Cell Part 2 by MIRZA MUHAMMAD ADNAN

Male Reproductive System (histology)

The seminiferous tubules are lined with a complex stratified epithelium called germinal or seminiferous epithelium. Their outer wall is surrounded by a well-defined basal lamina and a fibrous connective tissue consisting of several layers of fibroblasts The innermost layer, adhering to the basal lamina, consists of flattened myoid cells which have characteristics of smooth muscle. Interstitial (Leydig) cells occupy much of the space between the seminiferous tubules .

SPERMATOGENESIS

Spermatogenesis is the process by which spermatozoids are formed. It begins with a primitive germ cell, the spermatogonium (Gr. sperma + gone, generation), which is a relatively small cell, about 12 m in diameter, situated next to the basal lamina of the epithelium. At sexual maturity, spermatogonia begin dividing by mitosis, producing successive generations of cells.

The newly formed cells can follow one of two paths: they can continue dividing as stem cells, also called type A spermatogonia, or they can differentiate during progressive mitotic cycles to become type B spermatogonia Type B spermatogonia are progenitor cells that will differentiate into primary spermatocytes. The primary spermatocyte has 46 (44 + XY) chromosomes and 4N of DNA.

From this first meiotic division arise smaller cells called secondary spermatocytes with only 23 chromosomes (22 + X or 22 + Y). This decrease in number (from 46 to 23) is accompanied by a reduction in the amount of DNA per cell (from 4N to 2N). Secondary spermatocytes are difficult to observe in sections of the testis because they are short-lived cells that remain in interphase very briefly and quickly enter into the second meiotic division.

Division of each secondary spermatocyte results in two cells that contain 23 chromosomes, the spermatids. Because no S phase (DNA synthesis) occurs between the first and second meiotic divisions of the spermatocytes, the amount of DNA per cell in this second division is reduced by half, forming haploid (1N) cells. The meiotic process therefore results in the formation of cells with a haploid number of chromosomes. With fertilization, the normal diploid number is again attained.

SPERMIOGENESIS

The Golgi Phase

The cytoplasm of spermatids contains a prominent Golgi complex near the nucleus, mitochondria, a pair of centrioles, free ribosomes, and tubules of smooth endoplasmic reticulum .Small periodic acid chiff (PAS)-positive granules called proacrosomal granules accumulate in the Golgi complex. They subsequently coalesce to form a single acrosomal granule within a membrane-limited acrosomal vesicle .The centrioles migrate to a position near the cell surface and opposite the forming acrosome. The flagellar axoneme begins to form, and the centrioles migrate back toward the nucleus, spinning out the axonemal components as they move.

The Acrosomal Phase

The acrosomal vesicle spreads to cover the anterior half of the condensing nucleus and is then known as the acrosome .The acrosome contains several hydrolytic enzymes, such as hyaluronidase, neuraminidase, acid phosphatase, and a protease that has trypsin-like activity. The acrosome thus serves as a specialized type of lysosome. These enzymes are known to dissociate cells of the corona radiata and to digest the zona pellucida, structures that surround the oocytes. When spermatozoa encounter an oocyte, the outer membrane of the acrosome fuses with the plasma membrane of a spermatozoon at several sites, liberating the acrosomal enzymes to the extracellular space. This process, the acrosomal reaction, is one of the first steps in fertilization.

THE MATURATION PHASE

Residual cytoplasm is shed and phagocytosed by Sertoli cells, and the spermatozoa are released into the lumen of the tubule.

Sertoli Cells

The Sertoli cells are important for the function of the testes. These cells are elongated pyramidal cells that partially envelop cells of the spermatogenic lineage. Sertoli cells have several functions:

1:Support, protection, and nutritional regulation of the developing spermatozoa,

2:Phagocytosis.

3:Secretion. Production of the anti-mullerian hormone. Anti- mullerian hormone (AMH, also called mullerian-inhibiting hormone),

4:The blood testis barrier.

5:Production of inhibin B, Inhibin B inhibits the production of FSH by the hypophysis.

INTERSTITIAL, or LEYDIG, CELLS

The connective tissue consists of various cell types, including fibroblasts, undifferentiated connective cells, mast cells, and macrophages. During puberty, an additional cell type becomes apparent; it is either rounded or polygonal in shape and has a central nucleus and an eosinophilic cytoplasm rich in small lipid droplets .These are the interstitial, or Leydig, cells of the testis, and they have the characteristics of steroid-secreting cells.

Electron micrograph of a section of an interstitial cell

The highly coiled ductus epididymidis, sectioned several times. Its wall is made of a pseudostratified columnar epithelium surrounded by connective tissue and smooth muscle. PSH stain. Medium magnification. Inset: Higher magnification of the epithelial cells with their long microvilli (stereocilia).