HISTOLOGY FULL-TEXT

William A Beresford MA, D Phil ©
Professor of Anatomy
Anatomy Department, West Virginia University, Morgantown, USA

Chapter l7 BLOOD

Blood might be classed as a specialized connective tissue because its cells are mesodermal in origin and are separated by plasma.

                         _
            Plasma      |  Red blood corpuscles/Erythrocytes (RBCs)
              +         |
      formed, visible   |
         elements    - -|  White blood cells/Leucocytes (WBCs)
     (46% by volume)    |
                        |
                        |_ Platelets

A MICROSCOPIC TECHNIQUES FOR BLOOD

l A blood drop is smeared across a slide and
2 stained with a Romanowsky-type combined stain - a neutral combination of acidic (eosin) and basic (azure) stains.
3 In the stained smear, a differential count by eye or automated counter gives the proportions of the different varieties of leucocyte.
4 Absolute counts of blood, diluted by a known amount, in a counting chamber give the numbers of the formed elements: RBCs - about 5.2 million mm³ (man), 4-5 million (woman); WBCs - 5000-9000 mm³ (healthy adult); platelets 200000-400000 mm³.
5 EM study of WBCs and platelets in the buffy coat after its centrifugal separation from the RBCs.
6 Light and EM examination of cells in the lumens of blood vessels in sections of imbedded tissues.
7 Phase-microscopy and videorecording of leucocytes alive in fresh blood on a warmed slide under a sealed coverslip.
8 Tagged monoclonal antibodies to recognise cell-surface glycoproteins characteristic for particular subtypes of blood cell. This approach allows a specific cell population to be sorted for culture and study using automated flow cytometry.

B ERYTHROCYTES (gas transport)

Biconcave discs; close to 7.5 µm diameter in a smear.
Comprise a flexible membrane enclosing haemoglobin (iron-porphyrin-protein) in a closely packed state which, with membrane-spectrin-actin interactions, maintains the RBC's optimal shape for gas exchanges involving the haemoglobin.
Osmolarity of the plasma affects the shape of an RBC. Hypertonic solutions in vitro cause crenation and shrinkage; hypotonic, swelling and haemolysis.
Globin is acidophilic, and RBCs stain orange with eosin.
Mature RBCs have no nucleus, Golgi body, ER, ribosomes or mitochondria.
RBCs do have glycolytic enzymes and substrates, and methaemoglobin reductase and carbonic anhydrase for their respiratory function:
- (a) Oxygen binds to ferrous iron of haemoglobin (RBC) for transport:
  air --> lungs -- > blood --> tissues
- (b) Carbon dioxide leaves bicarbonate of the plasma and carbaminohaemoglobin (RBC) for transport:
  tissues --> blood --> lungs --> air
Reticulocyte/polychromatophil erythrocyte. An immature RBC, when stained supravitally with cresyl blue, has a blue condensed network of clumped, residual ribonucleoprotein not yet used for protein (globin) synthesis.
Life in circulation is estimated by ⁵¹Cr labelling at around l20 days, then the RBC is sequestered in the spleen, liver or bone marrow to be phagocytosed by macrophages. The spleen is most responsible.
The volume of RBCs as a percentage of centrifuged whole blood - the haematocrit - is a quick, crude measure of the O²-carrying quality.

C LEUCOCYTES (defence)

These are true cells, divided according to the granularity of their cytoplasm into two groups - granular and agranular.

l Granular leucocytes
All kinds appear round in a smear with a diameter l0-l4 µm.

l Polymorphonuclear neutrophil (neutrophil/PMN/polymorph, for short).

(a) Nucleus has coarse, clumped, deeply staining chromatin, usually in two or more lobes or segments connected by thin chromatin strands. Unlobated band nuclei are in immature cells; older nuclei have several lobes.
(b) Cytoplasm is granular from many, small, weakly staining (neutrophil) granules of two kinds:
.. (i) non-specific azurophil granules that are lysosomes with destructive enzymes; and
.. (ii) numerous specific non-lysosomal granules holding a selectin-type glycoprotein for adhesion to endothelium and ECM, and lysozyme, and other bactericidal substances.
(c) This motile cell is attracted out of vessels into the tissues, where it attacks bacteria and phagocytoses them and immune complexes. The attack on bacteria is two-pronged:
.. (i) with a respiratory burst that generates free radicals; also myeloperoxidase catalyzes the production of hypochlorous acid; and
.. (ii) by proteins, e.g., defensins, and bactericidal permeability-inducing protein (BPI), that damage bacterial cell walls.
(d) PMNs make up 55-65 per cent of the total leucocytes.

2 Eosinophil

(a) Nucleus is darkly staining and bilobed.
(b) Cytoplasm has many large, eosinophil granules 0.5-l µm diameter, and some smaller core-less granules.
(c) Specific granules are a form of lysosome, which in EM have a crystalline core and a fine granular region. Defensive basic/cationic proteins, e.g., major basic protein, give the acidophil reaction.
The enzymes differ somewhat from the neutrophil's, e.g., generating antimicrobial O₂ metabolites differently.
(d) Motile, and enter inflamed tissues, especially at sites of allergies and parasitic infestations. They attack helminths using the basic proteins and oxygen derivatives, and also may dampen mast cell-dependent reactions, e.g., by phagocytosing mast-cell granules.
(e) They comprise 2-3 per cent of leucocytes (but rising for (d)).
(f) In EM, the large granules, like a skunk, have a dark lengthwise central stripe, which helps in identifying the eosinophil.

3 Basophil

(a) Nucleus is bilobed and sometimes twisted, but palely staining and often obscured by
(b) basophilic cytoplasmic granules, containing sulphated proteoglycans, heparin and the vasodilator, histamine.
(c) Basophils are reluctant to enter CTs, where there are mast cells holding the same materials. Thus, the function of basophils is in doubt, but they bind IgE and participate in various hypersensitivities.
(d) They are rare; 0.5 per cent of the leucocytes.

2 Agranular leucocytes
l Lymphocyte

(a) Small spheroid cell about 5-8 µm in diameter.
(b) Large, spheroid, darkly staining nucleus leaves only a
(c) narrow rim of cytoplasm with a few small azurophil granules.
(d) Motile to enter CT and epithelial tissue, but is not phagocytic.
(e) Larger lymphocytes up to l2 µm diameter, with more abundant cytoplasm, may be seen in small numbers. The large granular lymphocyte is the natural killer cell.
(f) Unlike granular leucocytes, small lymphocytes can be stimulated to enlarge and divide by antigens, cytokines, and some plant lectins.
(g) The complex role of lymphocytes in immune defences is outlined in Chapter l9 and Fig. 10.
(h) Lymphocytes circulate in blood and lymph systems and migrate to CT and mucous membranes. Some lymphocytes have a lifespan of months or years.
(i) They amount to 25-35 per cent of the leucocytes.

2 Monocyte

(a) Large, spheroid cell about 12-20 µm in diameter.
(b) Nucleus has fine chromatin not densely stained, and is an indented sphere.
(c) Golgi body and centrioles lie by the nuclear indentation.
(d) Cytoplasm is abundant, with a few granules that are precursors of many larger lysosomes seen in EM when the cell is actively phagocytic.
(e) Motile, to leave the vessels after only a day or so to become the phagocytic macrophages/histiocytes of CT, or other derivatives.
(f) Macrophages/MØs spend months in CTs cooperating with lymphocytes in defensive responses (Chapter l9). Macrophages, by releasing cytokines after activation, coordinate inflammatory and defensive reactions.
(g) They comprise 3-l0 per cent of the leucocytes.

D PLATELETS (clotting and vessel-sealing)

l Rounded or ovoid parts of cells, 2-5 µm diameter.
2 Consist of cytoplasm, organelles and inclusions, bounded by a cell membrane, reflecting their formation as pseudopodia breaking away from extravascular cells - megakaryocytes.
3 The dense central granulomere (organelle zone) has mitochondria, dense bodies and alpha granules; the pale peripheral hyalomere (sol/gel region) is cytoplasm deficient in organelles, except for contractile filaments and a shape-giving ring of microtubules.
4 Platelets adhere to collagen, neutrophils and monocytes, and especially to each other; this platelet aggregation/agglutination is used to seal defects in blood-vessel walls.
5 Apart from several molecules for adhesion, the membrane supplies a phospholipoprotein: one of many factors in the cascade causing blood fibrinogen to form fibrin fibres in clotting. Platelets contract and cause a compacting of the fibrin to which they adhere - clot retraction.
They also release from their granules several factors, e.g., serotonin and cytokines, having vasoconstrictive and other actions.

E BLOOD IN BRIEF

Also available in colour as a series of Powerpoint slides.

Liquid PLASMA + visible FORMED ELEMENTS - supplement, control, & extend
                                          what goes on in the plasma

A GAS TRANSPORT - Erythrocytes/Red blood cells/RBCs |
                                                    |
B DEFENCE against the - Leucocytes/WBCs             |___ Describe:
  bad & the dead                                    |    appearances,
                                                    |    roles, &
C DEFENCE for vessel integrity - Platelets          |    means

Description includes Measurements:

ABSOLUTE COUNT numbers/cubic mm  RBCs - 6 X 10⁶, Platelets - 2 X 10⁵
                                 WBCs - 6 X 10³

DIFFERENTIAL COUNT  individual % proportion of 5 kinds of leucocyte

HAEMATOCRIT   Centrifuge tube
            __________________________________
             |///////////////}................\
            _____________________________|____/
                Plasma       |           |
                           Buffy         | RBCs - 45% by volume
                           coat          a measure of O2-carrying
                           (WBCs)        capacity

ERYTHROCYTE     <--- 7.5µm --->
                Biconvave disc  -  high surface/volume ratio

 Membrane with subplasmalemmal cytoskeleton: holds shape, but is
   deformable
 No organelles; no nucleus; haemoglobin binds O₂

NEUTROPHIL/polymorphonuclear leucocyte/PMN [granular] 55%  lobed nucleus
t   phagocytosis of bacteria & debris   m  destructive acid hydrolases
a   bacterial killing                   e  generation of free radicals
s   adhesion to & migration through     a  proteins, e.g., defensins
k    venule & capillary walls*          n  adhesion molecules* to stick
s                                       s   to 'infected' endothelium
     
EOSINOPHIL [granular] 2%, bilobed nucleus, large granules
t                                m  lysosomal enzymes         } similar 
a  anti-parasites                e  oxygen-radical generation } to PMN's
s  role in allergies             a  anti-parasite proteins, e.g. ECP
k                                n   eosinophil cationic protein
s  adhesion & migration*         n  cytokines
                                 s  adhesion molecules*

LYMPHOCYTE [agranular] 30% small cell, dense round nucleus
t  attack foreign cells & microrganisms    m  immune responses
a  attack foreign materials                e  cytokines
s  instruct other cells - come, stay,      a  cell-surface contacts
k   be quiet, be active, proliferate       n
s  proliferates itself                     s  *
   *

MONOCYTE [agranular] 8%, large cell, with indented nucleus
t  general phagocytosis, e.g. of      m  becoming activated macrophage
a   leftovers of damaged tissues      e  lysosomal enzymes
s  coordination of defence and        a  cytokines
k   repair                            n  antigen presentation
s  *                                  a  *

BASOPHIL [granular] ½% lobed, obscured nucleus
t  back up mast cells' responses     m  histamine
a   in inflammation                  e  leukotrienes
s  affects capillaries & speed &     a  prostaglandins
k   intensity of immune responses    m  cytokines
s   e.g. hypersensitivity            s  proteoglycans
   *                                    *

PLATELETS small cast-off pieces of giant marrow cell - megakaryocyte
t  stick together, and to endothelium   m  von Willebrand factor
a   and vessel collagen                 e  other clotting factors
s  start blood clotting                 a  5-hydroxytryptamine
k  control blood flow                   n  thrombospondin
s                                       s  adhesion molecules

* adhesion to endothelium and the cell-specific means thereto
Note that some lymphocytes , e.g., NK cells, have azurophil lysosomal granules

William A Beresford, Anatomy Department, School of Medicine, West Virginia University, Morgantown, WV 26506-9128, USA - - e-mail: -- wberesfo@wvu.edu -- wberesfo@hotmail.com -- beresfo@wvnvm.wvnet.edu -- fax: 304-293-8159