Some background on haematopoesis and related malignancies.

1. Haematopoesis
HDT 2:1 – Semester 2

2. An Overview
• A blood film is a slide containing red blood cells and white cells (leucocyte), which stain purple.
The white cells are split into lymphocytes and granulocytes.
• White cells (leucocytes) are split into lymphoid cells and myeloid cells. Lymphoid cells are split
into T, B and NK cells, whilst myeloid cells are split into monocytes and granulocytes. The
granulocytes are further divided into eosinophils, basophils and neutrophils.
• Platelets have no granules, but do contain granules containing substances that are involved with
clotting and anticoagulation. They have a lifespan of 7 – 10 days, where after they are removed by
the macrophages in the spleen and liver. Low levels leads to haemorrhage and bruising.
• Red blood cells only last 120 days after production (transfer of a unit lasts about 3 weeks),
neutrophils last less than 48 hours, platelets last 7 – 10 days and lymphocytes last years.

3. Myeloid and Lymphoid Cells
• Neutrophils defend against bacterial infection via phagocytosis. Their granules contain lysosome
and myeloperoxidase. They reside in the blood for a few hours, where after they migrate to tissues
and live for 4 – 5 days. A deficiency of neutrophils results in death by bacterial infection.
• Eosinophils are not phagocytic and developed historically to prevent against helminthic and
parasitic infections but now they have a role in allergy and atopy (genetic tendency to develop
allergic diseases). Basophils are the same.
• Monocytes are not granulocyte cells. They migrate from blood into tissues and mature to become
macrophages. They are phagocytic and can engulf and destroy fungi, parasites, bacteria and
dead cells.
• Lymphocytes are divided into T, B and NK cells. They are small cells with a low granularity.
• T cells develop from an early progenitor in the bone marrow but ultimately mature in the thymus. B
cells develop in the bone marrow and exit from the marrow as naïve cells.
• NK cells also develop in the bone marrow and have the role of secreting cytotoxic chemicals in
response to threat.
• Other cells produced by Haematopoesis include dendritic and mast cells. Dendritic cells are
antigen presenting cells, whilst mast cells are produced in bone marrow and mature in tissues.
Mast cells are involved in inflammation, extreme anaphylaxis and shock.

4. Stem Cell Maturation
• Stem cells can divide indefinitely and give rise to specialized, differentiated cells. Heamatopoetic
stem cells (HSCs) are multipotent and found in the bone marrow. Multipotent means they can
produce many cell types within one family.
• Stems cells mature and commit to become progenitor cells. Progenitor cells then commit further
and become precursor cells, which then become mature cells.
• Stem cells and progenitors can only be detected via functional assays, and immunophenotyping,
where cell surface markers are detected by antibodies.
• Precursors and mature cells can be seen under a microscope via routine bone marrow staining.

5. Haematopoetic Development
• Haematopoesis begins in the yolk sac of the foetus, then goes to the foetal liver and then in adults
is found to occur in the bond marrow of adults (axial skeleton only; pelvis and sternum).
• LT-HSCs are long-term haematopoetic stem cells that have multipotency. ST-HSCs are short-term
haematopoetic stem cells. MPP are multipotent progenitors which give rise to cells which have
lineage bias; this is where cells commit to become either myeloid or lymphoid cells.
• HSCs are marked with CD34 molecules (clustered differentiation mark 34) but are largely rare
cells. The more committed cells are found more frequently.
• A mixture of cells from a bone marrow aspirate can be used to identify the commitment via colony
assays, where cells are colonized in agar plates with nutrients and growth factors. Bone marrow
aspirates are where liquid bone marrow is drawn from ileac bone. It is taken from the spongy
bone.
• In vivo models (mice) are needed to assay true HSCs.
• Lineage commitment is governed by transcription factors, which have largely been identified in
mice models.
• In the bone marrow, stem cells are intimately related to other cells and elements which comprise
the stroma, including extracellular matric, fat cells, fibroblasts and endothelial cells. These help the
cell orient itself and interact via growth factors and adhesion molecules.

6. Haematopoetic Development
• Growth factors are cytokines, of which there are many types, which can be split into early acting
and late acting cytokines. Mimetics for TPO exist, whilst EPO, G-CSF and GM-SCF are in clinical
use alone.
Early acting Late acting
Stem cell factor G-CSF (granulocyte colony stimulating factor)
Flt3 ligand GM-CSF (granulocyte/macrophage stimulating factor)
IL-3 (interleukin 3) EPO (erythropoietin)
TPO (thrombopoetin) TPO (thrombopoetin)

7. Bone Marrow Aspirates
• Bone marrow aspirates are taken from the spongy bone using a trephine, to be used in smears,
biopsies and slides. Slides are stained with MGG to reveal many bone cell types.
• Normal slides have heterogeneous cell types, whilst abnormal cell slides can be seen i.e. in
patients with acute myeloid leukemia, where there are far fewer cells and many are stuck in
maturation; they remain blasts and do not commit.
Normal Abnormal

8. Granulocyte Maturation
• As granulocytes mature, they develop more granules, have progressively more condensed
chromatin, and a more lobulated nucleus.
• Mature cells have no mitotic capability, and have no nucleoli, which indicate cell immaturity.
• Myeloblasts mature into promyelocytes, which become myelocytes, which become myelocytes,
and neutrophils.

9. Erythropoiesis
• Starts with normoblasts, which mature eventually into reticultocytes, which retain some RNA and
so can make haemoglobin but cannot replicate. This is because they have no nucleus.
• Erythrocytes cannot even manufacture haemoglobin. Their production is governed by EPO, which
is produced in response to hypoxia in the kidneys and to an extent in the liver.
• Platelets arise from the cytoplasm of megakaryocytes in the bone marrow. Each megakaryocyte
can produce 2000 – 3000 platelets, which is able to occur via continued nuclear division without
cellular division (endomitosis). This means each megakaryocyte can have 4 to 64 nuclei in their
cytoplasm, capable of producing many proteins and platelets.
• Platelet production is regulated by TPO, mainly produced by the liver. The receptor for TPO can
be found in all stages of production.

10. Clinical Use
• Erythrocyte transfusions last 3 weeks to a month. Platelet transfusions last a few days. HSC
transplants should last for life. HSC is taken after donors have received a stimulating factor which
mobilizes the HSC cells into the blood, which is then taken via apheresis, rather than removing
bone marrow under general anesthetic.
• Recombinant EPO is given via subcut injections to improve anaemia to reduce need for
transfusion. It is commonly given in end-stage kidney disease, as endogenous EPO is low. It can
also be used in myelodysplasia (bone marrow abnormalities; pre-leukaemic), where one of the
first things to occur is anaemia. Can also be given to Jehovah’s Witnesses where blood products
are not accepted.
• EPO is also misused in sport to boost RBC production and oxygen carrying capacity. However,
polycythaemia can develop which is overproduction of RBCs, leading to blood clotting and
hypertension.
• Recombinant G-CSF is given via subcut injections for primary or secondary prevention of
infections in neutropenic patients. It is now pegylated to reduce dosing regimen. Neutropenia can
be caused by chemotherapy, but can also be congenital (i.e. Kostman’s syndrome).
• G-CSF can also be used to mobilise HSCs in the blood for collection.
• TPO agonists (AMG 531 and eltrombopag) are licensed for ITP (immune thrombocytopaenia
purpura); this is where immune attack of platelets results in deficiency and bruising. It could also
be used for thrombocytopaenia in myelodysplasia, or post-chemotherapy.

11. Haematological Malignancies
• Leukaemias are malignancies of haematopoetic cells. These cells are found in the bone marrow
and spread to involve the lymph nodes and the spleen. These can spread to the whole body.
• Lymphomas are malignancies of lymphoid cells, arising in secondary lymphoid cells (i.e. in lymph
nodes and spleens). These too can spread to the whole body.
• Myelomas are malignancies of plasma (B cells) in the bone marrow.
• Myeloproliferative diseases involve overproduction of cells and myelodysplasia involves abnormal
production of cells.
• Maturation arrest causes acute leukaemia, an example of which is acute myeloblastic leukaemia
(malignancy of the myeloid cells arising from haematopoetic cells in the bone marrow).
• No maturation arrest leads to over-production, an example of which is chronic myeloid leukaemia
(malignancy of myeloid cells arising from haematopoetic cells in the bone marrow).
• Leukaemias can be classified by first considering their lineage; lymphoid or myeloid cells. If it’s
lymphoid, then it could be AML or chronic myeloid leukaemia (arises from a mature cell – chronic).
If it’s lymphoid, it could be acutre lymphoblastic leukaemia or chronic lymphocytic leukaemia
(arises from a mature cell – chronic condition).