Week 2 - A - Haemapoiesis - Definition, Sites, Anatomy of bone marrow, Regulation&Investigations for haemapoeisis

Question 1

What is haemaopoesis defined as?

Answer 1

It is defined as the formation of blood cells

Question 2

What are the types of mature blood cells?

Answer 2

Erythrocytes - red cells Thrombocytes - platelets Leukocytes - White blood cells

Question 3

What is the function of the erythrocytes? What is the function of the platelets?

Answer 3

Erythrocytes - they transport oxygen and carbon dioxide Platelets from a solid part of a clot to help prevent bleeding

Question 4

What are the different types of leukocyte and what is their function?

Answer 4

Leukocyte * Granulocytes * Neutrophils - 1st line defence (phagcoytose) * Eosinophils - parasites and hypersensitivity * Basophils - also hypersensitivty Monocytes - become macrophages in the tissues Lymphocyte * Tcells - cell mediated immunity * Bcells - humoral immunity * Natural killer cells - anti viral/tumour

Question 5

Which of the mature cells have no nucleus?

Answer 5

Erythrocytes and thrombocytes have no nucleus

Question 6

What are the different types of haemaopoeisis now that we know the different types of blood cell?

Answer 6

Haemapoeisis * Erythropoeisis - formation of red blood cells * Thrombopoeisis - formation of platelets * Myelopoeisis - formation of granulcoytes and monocytes * Lymphopoeisis - formation of lymphocytes

Question 7

In the steady state, cell loss is balanced by cell production What is the most common blood cell? What is the lifespan of a red blood cell?

Answer 7

The most common blood cell is the red blood cell It has an approximate life span of 120 days

Question 8

What is the approximate lifespan of platelets and neutrophils? How long do monocytes stay in circulation before going into tissues? What do they become once in the tissue? What type of cell in the blood is this tissue residing cell very similar to? When taken for blood and stored appropiately, what is the shelf life of RBCs, platelets and fresh frozen plasma?

Answer 8

Approximate lifespan of platelets - 7-10days Approximate lifespan of neutrophils - 7-8hours Monocytes remain in circulation for 1-2 days before entering the tissues and becoming macrophages - these are very similar to basophils Shelf Life RBC - 35 days, Platelets - 7 days, Fresh frozen plasma - 3years

Question 9

Red cells as stated are the most common cell in the blood When saying the red cell concentration, it is x10 to the power of what? What is it to the power of for platelets and neutrophis? What is the mean red cell volume measured in?

Answer 9

Red cells measured in x10 to the power 12 per litre Platelets and neutrophils measure in x10 to the power 9 per litre Mean cell volume is measured in femtolitres = x10 to the power of -15 litres (red cells are very small)

Question 10

The mature blood cells come from precursor cells - precursor meaning preceding (the more precursor the more immature/primitive it is) What is the earliest precursor for the lineage of red cells? When is the nucleus of a maturing red cell lost? If there is some DNA nuclear fragment left in the red cell, where is this removed? Failure to remove results in the DNA nucleus fragment being known as what?

Answer 10

Earliest precursor for the lineage of red cells is the pronormoblast (proerythroblast) Nucleus of the red cell is lost in the bone marrow when the red cell is the late normoblast - after nucleation the re cell enters the circulation and is known as a reticulocyte If some DNA nuclear remnant remians -removed by the spleen, if persists - then Howell Jolly body (usually due to hyposplenism or post-splenectomy)

Question 11

What is the early precursor to a neutrophil (or granulcoyte) known as? What is the early precursor to a red cell known as? What is the precursor to platelets? What is the immediate precursor to a mature red cell? What is a myelocyte?

Answer 11

Early precursors to neutrophils - meyloblasts (myeloblasts can be a precursor to any of the granulcoytes/monocytes) Early precurosr to red cells - erythroblasts Precursor to platelets is the megakaryoblast (this is described as being polyploidy) Immediate precursor to a mature red cell is the reticulocyte Myelocyte is the nucleated precursor between a myeloblast and a neutrophil

Question 12

The origins of blood cells stem from haematopoetic stem cells What is the most primitive haematopoietic stem cell? What does this give rise to? What capabilities must a stem cell have for its classification as a stem cell?

Answer 12

The long term haematopoietic stem cell (LT-HSC) is the original cell It gives rise to the short term haematopoetic stem cell (ST-HSC) - this has a greater affinity for proliferation Stem cells must be pluripotent (ie can replenish all types of blood cells) and they must be capable of slef renewal - ie when they divide to differentiate, they also form an exact copy of itself

Question 13

The property of self renewal in stem cells is lost in its descendants What does proiferation mean? What does differentiation mean? What does the ST-HSC differentiate to form?

Answer 13

Proliferation means an increase in numbers Differentiation is when descendants commit to one or more lineages The ST-HSC differentiates to form the Multipotent progenitor (MPP) cell - remember the ST_HSC also self renews

Question 14

What do the MPP cells differentiate to form? (these are not stem cells as they cannot self renew)

Answer 14

They differentiate (descendants commiting to one or more lineages) to form the common myeloid progenitors and the common lymphoid progenitors

Question 15

What is maturation? Which cells do not proliferate any longer? What is apoptosis?

Answer 15

Maturation - the cells acquire functional properties and may stop proliferating Mature cells (erythrocytes, neutrophils etc) do not proliferate any more Apoptosis is the means by which descendent undergo (programmed) cell death

Question 16

What can the term myeloid refer to? - can refer to three things

Answer 16

Myeloid can mean the bone marrow It can mean non-lympoid lineages (encompassing erythrocytes, thrombocytes, granulocytes and monocytes) and it can mean Granulcoytes and their precursors

Question 17

More primitive progenitors (eg stem cells) are quiescent/dormant during steady-state haemopoiesis – stem cells only divide every few weeks Are stem cells easily distinguishable on blood film and in the bone marrow?

Answer 17

Stem cells are not easily distinguishable on film or bone marrow and specific analysis tests are required for their identification

Question 18

Which germ cells are haematopoietic stem cells derived from? Where is the first site of erythroid activity and by which week does this site stop this activity?

Answer 18

HSC are derived from the mesoderm layer just after conception The first site of erythrod activity takes place roughly 5 weeks after conception The yolk sac is where this activity takes place but stops by week 10

Question 19

The original site of haematopoiesis is the yolk sac as mentioned Where does this process occur now and up until birth? What other organ contributes to haematopoeisis from the 3rd to 7th month of gestation?

Answer 19

Yolk sac (from 5till 10 weeks) Liver form the 6th week Bone marrow from the 16th week Haematopoiesis also occurs in the spleen from the 3rd to 7th month of gestation So yolk sac then liver then bone marrow (spleen and liver can continue to help when needed from 3rd month onwards)

Question 20

The number of active sites in the marrow reduces with age but some sites are still retained for haematopoiesis What are the regions of active site in the bone marrow during childhood? What are the regions during adulthood/

Answer 20

During childhood - axial skeleton - so skull, vertebra, ribs, sternum, pelvis, proximal shoulders and legs Adulthood - vertebra, sternum, ribs, skull, pelvis

Question 21

What are the different locations for bone marrow aspirate/biopsy in neonates/adults?

Answer 21

Bone marrow aspirate/biopsy Neonate - performed in the tibia Adult - performed in the posterior iliac crest

Question 22

The bone marrow is a complex organ surrounded by a shell of bone with a neurovascular supply. What is the interface of bone marrow and bone known as? Ie what is the name for the membrane that lines the inner surface of bone that meets the bone marrow What is the membrane that lines the outer surface of bone known as?

Answer 22

This interface where bone marrow meets bone is lined by what is known as the endosteum

The membrane that lines the outer surface of bone is the periosteum

Question 23

What is the outer and innner bone known as?

Answer 23

Outer bone is the cortical (compact bone) Inner bone is the trabecullar (cancellous) spongy bone

Question 24

Under the microscope - Bone marrow Compartments Cellular * Haemopoietic cells * Non-haemopoietic cells (eg adipocytes, ‘fibroblasts’ osteoclasts, osteoblasts) Connective tissue matrix Vascular elements – nourish cells in bone marrow and form the passage for developing cells to leave the bone marrow What are the horizontal & vertical canals for blood known as?

Answer 24

Horizontal canals - these are Volkmann’s canals

Vertical canals - these are the Haversion canals

Question 25

Osteons, roughly cylindrical structures that are typically several millimeters long and around 0.2 mm in diameter. Each osteon consists of concentric layers, or lamellae, of compact bone tissue that surround what central structure? What is the bone cells in the osteon known as? They form once an osteoblast has become embedded What are the spaces in which these cells lie in known as?

Answer 25

The osteon is the cylindrical structure surrounding the central canal - Haversions canal The osteocytes are the cells in the osteon formed once an osteoblast has become embedded and the osteocytes lie in the lacuna of the osteon

Question 26

The bone marrow blood cells drains into into sinusoids What do the sinusoids open up into? How does this reach the circulation?

Answer 26

The bone marrow blood cells will drain into the medullary sinusoids which open up into a larger central venous sinus The central venous sinus drains into the systemic circulation via nutrient and periosteal veins

Question 27

Human marrow produces approximately 500 billion blood cells per day, which join the systemic circulation via permeable vasculature sinusoids within the medullary cavity Remember the sinusoids drain into the central venous sinuses to enter the systemic circulation What is the function of the endothelial cells which line the sinusoids which allow blood cells to enter the sinusoid?

Answer 27

The endothelial cells contain gaps known as fenestrations which allow blood to enter the sinusoids Formed blood cells can pass through fenestrations in endothelial cells to enter circulation Release of red cells is associated with sinusoidal dilatation and increased blood flow

Question 28

Neutrophils actively migrate towards the sinusoid What are the long branches that megakaryocytes extend toward the sinusoids to allow the platelets to enter the sinusodial blood vessels?

Answer 28

The megakaryocytes extend branches known as proplatelets to allow for the platelets to enter the sinusoids and later the circulation

Question 29

What is the difference between red and yellow marrow? Which increases with age? How is the marrow cellularity calculated?

Answer 29

Red marrow is the haematopoietically active marrow versus the yellow marrow which is mainly fatty marrow There is an increase in yellow marrow with age resulting a decrease in bone marrow cellularity Ceullarity% of marrow is said to be calculated by 100-age of person

Question 30

The relationship between the neutrophils and their precursors and the proportion of nucleated red cells in the marrow is described as what?

Answer 30

The myeloid:erythroid ratio

Question 31

The myeloid to erythroid ratio ranges from 1.5:1 to 3.3:1 When can reversal of this occur? - ie when are there more erythroid cells over myeloid (neutrophils and their precursors)

Answer 31

Reversal of the myeloid:erythroid ratio can occur in times such as haemolysis where there bone marrow produces red cells in vast quantities to compensate for the reduction in the peripheral blood

Question 32

What regulates haemapoesis?

Answer 32

The intrinsic properties of cells As well as Signals form immediate surroundings and the periphery (microenvironment)

Question 33

What is the main regulator of erythropoeisis? What is required for the regulation of growth and development of megakaryocytes from their precursors?

Answer 33

Mai regulator of erythropoiesis is erythropoietin (EPO) - produced in the kidneys in response to the kidney cells sensing hypoxia - stimulates the production of more red cells Thrombopoietin is required for the growth and development of megakaryocytes from their precursors

Question 34

What does erythroid maturation occur around?

Answer 34

Erythroid maturation occurs in the bone arround a macrophage in the shape of an island - it is this macrophage which causes enucleation of the red cell nucleus The arrow points to the macrophage in the centre

Question 35

Neutrophils and their precursors growth and development is stimulated by a cytokine known as what?

Answer 35

G-CSF - granulocyte colony stimulating factor

Question 36

How do we assess haemopoiesis? (mature cells) 1. Routinely undertaken- blood count, cell indices (by non-specialists), morphology (blood film) 2. Less common (specialist tests)-bone marrow examination Which cells can be seen on this blood film?

Answer 36

Red cells obbviously Bottom left - neutrophil Top right - eosinophil -can see the very red granules

Question 37

What can be seen on this film? What are the types of mononuclear cells? What are the polynuclear cells?

Answer 37

Right of pic - two neutrophils Left of pic - a lymphocyte Mononuclear cells -monocytes and lymphocytes Polynuclear cells - the granulocytes as they have multi-lobed nuclei

Question 38

Which are monocytes and which are atypical lymphocytes?

Answer 38

The atypical lymphocytes (blue arrows) have a more generous and basophilic cytoplasm than the normal, smaller lymphocytes. They also have a more regular nuclear shape compared to the monocytes (red arrows). The lymphocytes also have a more regular cytoplasmic border without the blebs and pseudopods (Pseudopods are temporary cytoplasmic projections of the cell membrane) present in the monocytes. Vacuolation is present in the cytoplasm of the monocytes, which is not present in the lymphocytes.

Question 39

A bone marrow aspirate is usually required when wanting to look at the precursor cells Blood count and film (morphological appearance) is usually suitable for looking at the mature cells The expression of antigens indicating lineage or stage of development can be studied in/on cells but generally not required. This study of antigen expression using specific antibodies is called what?

Answer 39

The study of antigen expression using specific antibodies is known as immunophenotyping and is done to understand the lineage or stage of development of a blood cell

Question 40

To study rare population of blood cells: Immunophenotyping - allows you to know the type of cell by looking at cell surface antigens Cytochemistry - looks at the enzyme expression in a cell Is immunophenotyping or cytochemistry more commonly used today? Where is the bone marrow aspirate taken from in immunophenotyping?

Answer 40

The bone marrow aspirate/biopsy would be taken from the posterior iliac crests Immunopehnoytyping has largely replace cytochemistry as a diagnostic tool

Question 41

There is different antigens expressed on e.g. T cells compared to stem cells. We can then use antibodies plus tag (flurochrome) (the tag for the Tcell antibody has a different colour than the tag for the stem cell antibody). What is then used to look at the different colours of the tag to identify which cell is more abundant?

Answer 41

Laser software is then used to identify the flurochrome tag to identify which cell type is more abundant and this is how immunophenotyping is carried out using flow-cytometry

Question 42

What is the antigen expressed by T cells and virtually no other cells which make this a good immunophenotypical (immunohistochemical if lymph) marker? What is the antigen expressed by haematopoeitc stem cells which can be used for antibodies to target in immunophenotyping?

Answer 42

CD3 - antigen almost exclusive to Tcells CD34 - antigen almost exclusive to LT&ST -HSC