HISTOLOGY FULL-TEXT

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

Chapter 18 HAEMOCYTOPOIESIS

Continuous formation of the cells, corpuscles, and platelets of the blood is necessary to keep their numbers relatively constant as they wear out or are lost from the body. The formation is called haemocytopoiesis or haemopoiesis for short.

A DIVISIONS OF HAEMOPOIESIS

l Myelopoiesis - formation of granular leucocytes (granulopoiesis), monocytes (monopoiesis), erythrocytes (erythropoiesis), and platelets (thrombopoiesis).
2 Lymphopoiesis - formation of lymphocytes and plasma cells. (Plasma cells are not normally seen in the blood.)

B SITES OF MYELOPOIESIS

l Embryonic: mesenchyme gives rise to:

(a) blood islands in the yolk sac;
(b) fetal liver haemopoietic tissue;
(c) bone marrow in cavities of the developing bones;
(d) marrow in the spleen and lymph nodes.

2 Adult

(a) Red/myeloid marrow remains mostly in the axial skeleton - skull diploë, ribs, sternum, parts of the vertebrae and pelvis.
(b) Ectopic/extramedullary myelopoiesis may be seen in adults, usually in disease, e.g., fibrosis of marrow, and at the sites of myelopoiesis in the fetus.

C THEORETICAL CONSIDERATIONS OF HAEMOPOIESIS

l Granular leucocytes and RBCs are specialized end products in being unable to divide, and living for only a few weeks. Since their numbers in the blood stay constant, new cells must be forming from less specialized ones.
2 Bone marrow, stained as for a blood smear, has cells, construed from their granularity, eosinophilia, nuclear morphology, etc, as members of developmental sequences, apparently starting with a large, undistinguished weakly basophil, primitive cell, and ending as one of the clear-cut specialized kinds.
3 If all the primitive marrow cells multiplied and then turned into blood cells, when the blood cells were spent, no primitive ones would exist to replace them. Thus, the primitive cells must act as stem cells able to divide, and with two possible fates: some to stay as primitive stem cells, others to differentiate into special forms.
4 Since there are several specialized blood cells, are there separate, but histologically indistinguishable, stem cells: one for each blood cell type? - The polyphyletic theory of committed progenitors for each lineage. Yes, but the monophyletic theory also survives, because rare multipotent/pluripotent stem cells exist, and can replenish the restricted stem cells, e.g., those for erythropoiesis.
5 CFU-S denotes the pluripotent cell in mouse, and forms the basis for naming progenitor cells in humans. Colony-forming unit - spleen/CFU-S was the cell that could give rise to an island/colony of complete haemopoiesis in the spleen of the mouse, after splenic and other sites of haemopoiesis had been totally destroyed by irradiation. Where, then, did the rescuing cell come from to form the colony? The CFU-S was obtained from infant mice and injected just after the irradiation. (A convenient human source for equivalent stem cells is blood from the umbilical cord.)
6 All cell divisions and differentiations need controlling growth factors (cytokines), not only to maintain the stem cell population, but to persuade some of them to fill precisely the ranks of the various blood cells.
7 After a stem cell becomes a committed precursor/progenitor for a certain cell line, a period elapses when histology, without immunostaining, cannot identify the line. Later, perceptible morphological changes make the cell a recognizable precursor, say a pro-erythroblast. Thereafter, the development of the cell is divided into named stages, each based on a significant change in appearance from the previous stage.
The potential for confusion exists, since workers have differed in the number of stages chosen, e.g., omitting pre-stages, and their names for a given cell type, e.g., rubriblast/normoblast for erythroblast.
8 The ability of the few stem cells to divide does not preclude proliferation by committed precursors, and by cells at later, recognizable, stages of development, for continued amplification of cell numbers.
9 Fig. 8 omits: how the early elements shown match the committed precursor-recognizable precursor classification; typical population figures for each cell kind; how recognizable the kinds are; and the controlling factors for proliferation and differentiation (G below).

Fig. 8 Pathways of blood cell differentiation - Stained marrow-smear view

                                                                                
                           . Haemocytoblast/Pluripotent stem cell                     
                        .~     /     |        \                 \                      
H                    .~      /       |          \                 \                       
A                 .~       /         |            \                 \                 
E              .~        /           |              \                 \               
M   Lymphoblast     Monoblast     Myeloblast       Pro-erythroblast   Megakaryoblast  
O        |             |              |                   |                 |         
P        |             |              |                   |                 |         
O        |             |              |                   |                 |         
I        |             |              |                Basophil             |         
E        |             |         Pro-myelocyte        Erythroblast          |         
T        |             |              |                   |                 |         
T        |             |              |                   |                 |         
I        |             |              |                   |                 |  
C        |             |              |              Polychromatic          | 
T        |             |          Myelocyte          Erythroblast           |  
         |             |              |                   |                 |        
T        |             |              |                   |                 |
I        |             |              |                   |                 |
S        |             |              |                   |                 |
S        |             |         Metamyelocyte       Orthochromatic         |
U        |             |              |               Erythroblast          |
E        |             |              |                   |                 |
S        |             |       Band granulocyte      Reticulocyte     Megakaryocyte
- - - - -| - - - - - - | - - - - - - -| - - - - - - - - - | - - - - - - - - | - - - - -
B        |             |              |                   |                 |
L        |             |              |                   |                 |
O   Lymphocyte      Monocyte     Granulocyte         Erythrocyte        Platelets
O        |             |              |
D        |             |              |
- - - - -| - - - - - - | - - - - - - -| - - - - - - - - - - -  - - -  - - - - - - - -
C        |             |           Tissue
T   Plasma cell    Macrophage    Granulocyte

D CHANGES IN DEVELOPING BLOOD CELLS

l Erythrocytes
l Large, weakly basophilic pro-erythroblast increases the free ribosomes in its cytoplasm to become a basophil erythroblast.
2 Cell size decreases, and organelles are lost.
3 Nucleus, initially large and pale, with nucleoli, gets smaller and stains more darkly.
4 Cytoplasm acquires haemoglobin at the expense of ribosomal ribonucleprotein (RNP) - thus its staining affinity changes from basophilia to acidophilia; the mixed-hued halfway stage is the polychromic/polychromatophil erythroblast.
5 Small cell, with orange cytoplasm and a round dark nucleus, is the orthochromic erythroblast/normoblast.
6 Nucleus, in a little cytoplasm, is extruded for phagocytosis.
7 Reticulocyte/polychromatophil erythrocyte is an RBC that is released into the blood still with RNP in its cytoplasm. Supravital staining with brilliant cresyl blue causes this material to clump as a blue network (reticulum) in around 2 per cent of the RBCs of normal blood.

2 Granulocyte
l Myeloblast/granuloblast develops into a
2 promyelocyte synthesises non-specific azurophil granules (lysosomes) in the cytoplasm, and with its nucleus getting smaller and darker.
3 Myelocyte, after a pause, then makes additional granules specific for one of the three kinds of granulocyte in their staining affinity.
4 Nucleus elongates and indents, and chromatin becomes coarser, giving the metamyelocyte (now unable to divide).
5 More granules form and the nucleus becomes sausage-shaped - band/juvenile granulocyte. Then the nucleus starts segmenting, as the cell becomes the mature granulocyte.

3 Platelets
l Haemocytoblast enlarges to become a megakaryoblast.
2 The nucleus experiences several rounds of DNA replication, but each time with reassembly of a single nuclear envelope and no segregation into separate nuclei. Thus the nucleus takes on a distinctive lumpy, polyploid form. (The single, large, lumpy nucleus is the criterion for distinguishing megakaryocytes from nearby osteoclasts in bone sections.)
3 Fine cytoplasmic azurophil granules accumulate as the cell becomes a very large granular megakaryocyte.
4 Many paired membranes of smooth ER (demarcation membranes) appear and contribute plasmalemma to the formation of
5 pseudopodia, which are extended into the lumen of a sinusoid, where they cast off in the blood as platelets.
6 Megakaryocyte cytoplasm might also serve as a transcellular migration pathway for some new leucocytes passing from the marrow into the blood.

4 Agranular leucocytes
l In developing, they do not become so strikingly different from their stem cells as do granulocytes and RBCs.
2 Monocytes form from monoblast/pre-monocyte precursors in bone marrow.
3 Lymphocytes develop from lymphoblasts in bone marrow and lymphoid organs.
4 Some circulating lymphocytes appropriately stimulated can also become lymphoblasts.

E BONE MARROW

l The naked-eye appearance of fresh, unstained marrow may be red from many developing RBCs, or yellow from mainly fat cells.

2 Red marrow has many elements, see Powerpoint:

(a) Blood sinusoids are lined by endothelial cells on an incomplete BL. Collagen fibrils (reticular fibres) support these, and
(b) adventitial stromal/reticular cells, similar to fibroblasts, but extending processes between, and greatly influencing, the haemopoietic cells.
(c) Macrophages cleanse blood, and detect and destroy worn-out RBCs and other elements. The iron recovered is stored, combined with protein as ferritin granules, before release to the labile pool and reuse.
(d) Blood cells develop extravascularly, are stored, then released through the sinusoidal wall into the circulation.
(e) Megakaryocytes form and release platelets.
(f) Fat cells are present, large and empty of fat in embedded sections.
(g) Bone surface cells act as an enclosing sac for the marrow.

3 Microscopic methods for marrow include sections, and smears of aspirated sternal marrow stained with a blood stain.

F CELL COMPARTMENTS, POPULATIONS AND KINETICS

l Although the marrow smear picture appears static, it represents part of a dynamic system of cell populations. For example an RBC may be: (a) in the marrow developing, (b) in the marrow being stored, or (c) circulating in the blood. The population of each compartment at any time represents the balance between the numbers entering and leaving.
2 Most cells of the RBC sequence/erythroid complex/erythron are circulating; whereas the granular leucocyte system has most of the cells developing or being stored in the marrow, and only a minority in circulation. Many of these leucocytes in the vessels hug the wall as a barely moving, marginated reserve.

3 Some factors affecting blood cell formation
l Bacterial infection increases the number of circulating granulocytes (a leucocytosis) and their rate of formation.
2 Erythropoietin is a humoral factor, released from the kidney in response to hypoxia, that increases RBC production. Thrombopoietin controls platelet formation, but has multiple sources, including liver and kidney.
3 RBC formation requires adequate dietary elements, e.g., folic acid, iron, vitamin B₁₂.
4 Androgenic steroid hormones stimulate erythropoiesis.
5 Stromal cells release cytokines and, with the matrix, create a microenvironment favourable for haemopoiesis.

G . A LITTLE CLINICAL HAEMATOPOIESIS

The scheme below depicts the cell types and early events in haemopoiesis, detected mostly by cell-surface antigens. The usual histological chart telescopes these events and, instead, concentrates on later visible changes, which lie on the right side of this Figure, and are under the control of the last-acting specific factors such as EPO and IL-5. (Growth factors causing progress in a stage of a lineage are in italics or a symbol, e.g., *.)

 Fig. 9 Haematopoiesis

                   LP - - - - - - - - - - - - - - - -  T lymphocytes
                  /
               LPC
              /   \
            /      BP - - - - - - - - - - - - - - - -  B lymphocytes
          /         
PSC----CSC                  Meg-CSF            TPO
^  |    #\      CFU-Meg - - - - - - MegaK - - - - - - Platelets
|__|     @\       /
          *\   **/              **              EPO
            \   / - - - BFU-E - - - - - CFU-E - - - - - Red blood cells
             MPC    **
              ~ \  
               ~ \                             M-CSF
                ~ \                  CFU-M - - - - - - Monocytes
                 ~  \**             /
                  ~   \         ** /
                    ~   \         /   **          G-CSF
                      ~   \CFU-GM - - - - CFU-G - - - - - - Neutrophils       
                       ~          \  
                        ~          \
                     IL-3~       ** \
Key                        ~         \          IL-5
#  SCF                        ~       \ CFU-Eo - - - - - Eosinophils
@  IL-6                         ~
*  IL-1                            ~
** IL-3 &                             ~                IL-3
   GM-CSF                                ~ CFU-Mast - - - - - -Basophils
EPO Erythropoietin
TPO Thrombopoietin

PSC Pluripotent stem cell
CSC Committed stem cell
MPC Myeloid progenitor cell; LPC Lymphoid progenitor cell
CFU Colony-forming unit; BFU-E Burst-forming unit-erythroid
CFU-GM Colony-forming unit-Granulocytes & Monocytes;
CFU-M Colony-forming unit-Monocyte, etc.

The above diagram is modified from Kenneth Kaushansky's Fig. 1 in Proteins1992;12:1-9, with the kind permission of John Wiley & Sons, Inc, New York, holders of the 1992 copyright ©, and of the author

[ The ideas and terminology of the diagram are up-to-date. A full 1998 version would include only more growth factors and separate lineages for natural killer and dendritic cells.]

1 Tasks & solutions

To keep the process going - stem cells
To arrive at precise numbers of special cell types - proceed in steps that can be separately controlled, and give precursor cells some options of what they are going to turn into.
To control the necessary events of cell division, choice, specialization /differentiation, and migration into the blood - cytokines and certain hormones - collectively called haemopoietic growth factors
To make the cells responsive to needs, e.g., bacterial infection (more neutrophils), low O₂ (more RBCs) - use feedback from defensive reactions or in hormonal loops

2 Ideas of haemopoiesis
.. The controlling microenvironment, with stromal cells;
.. self-renewal of a cell population;
.. cell differentiation;
.. restricted versus wide-ranging potential/competence for differentiation;
.. progression through stages of differentiation;
.. lineage or sequence of precursors, as the ancestry of a particular cell type;
.. early versus late events and controls;
.. cascades and combinations of factors (signals);
.. colonies of cells grown in culture, thought to mimic clusters/nests in marrow;
.. the clonal colony derived from one original cell.

3 Abbreviations
Lots of them. Welcome to medicine! Here the difficulties are that:

The same letter means more than one thing, e.g., M - myeloid, monocyte; C - cell, committed, colony; P - pluripotent, progenitor/precursor.
Where a factor causes selective differentiation, the convention is to put the abbreviation of the target cell type first, before the one specifying that it is a growth factor, e.g., M-CSF.
Cell-type abbreviations are combined, where the factor acts on less specialized, i.e., multipotent progenitors, e.g., GM-CSF for a factor stimulating differentiation to a precursor of neutrophils (and eosinophils) and monocytes.
'Unit' (U) is a term for 'cell'.

4 Feasible clinical uses for growth factors
Erythropoietin - to combat anaemia from renal disease
Various CSFs:
.. to boost marrow performance after chemotherapy
.. to help injected stem cells or grafted marrow 'take' and perform
.. to restore PMN numbers in AIDS, certain anaemias, and neutropenia
.. as a differentiation therapy for leukaemia

5 Histological accounts
1 Focus on the very late events, discernible with conventional stains of smears, (nuclear and cell-size changes, acquiring granules, etc).
2 Can be wrong, e.g., too closely relating basophil formation to that of other granulocytes.
3 Leave vague the early events that clinicians need to know for untangling types of leukaemia, stimulating greater numbers of a deficient cell type, and finding stem cells to transplant, instead of marrow.
However, haematologists and pathologists use extensively the knowledge and terms, e.g., reticulocyte, band cell, etc, derived from the simple and available techniques of histology.

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