Lecture 1- Blood and Bone Marrow PDF

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Blood Immune and Infection Module ...

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Lecture 1: The blood and bone marrow:

HEMATOPOIESIS  Haematopoiesis is the process by which mature blood cells are generated from stem cells in the bone marrow. - Because mature blood cells of most types are relatively short- lived, haematopoiesis is required constantly even in unstressed adult life to maintain adequate numbers of blood cells. (neutrophils last 10 to 12 hours) - During stress, such as with blood loss or infection, additional cell growth is required.  Hence the bone marrow needs to produce more cells. - The process of haematopoiesis needs to be regulated with precision since circulating levels of mature cells are maintained within narrow limits of variation, yet cell production can be altered rapidly in response to increased or decreased demands.  Mature cells develop from stem cells (embryonic, adults, pluripotent stem cells) - Bone marrow transplant is now called stem cell transplant  Formation of mature blood cells/platelets from stem cells constantly replaces short-lived blood cells/platelets  ↑hematopoiesis sin response to blood loss/infection - Yellow bone marrow can revert to red bone marrow - Liver/spleen can resume fetal hematopoietic roleextramedullary hematopoiesis = hepatosplenomegaly  Packed cell volume (PCV) = Percentage of red blood cells in circulating blood. - Decreased PCV generally means red blood cell loss from any variety of reasons like cell destruction, blood loss, and failure of bone marrow production. - Increased PCV generally means dehydration or an abnormal increase in red blood cell production.

Fetus

Infant

Yolk sac = 0-2 months Liver/spleen = 2-7 months Bone marrow = 5-9 months All bone marrow

Adult

Axial skeleton Proximal femur

IMPORTANCE OF HEMATOPOIESIS 

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Blood tests important part of management of patients - Diagnosis - Monitoring Understanding function of blood cells - Assists in interpretation of investigations - Important for diagnosis and management of blood disorders - e.g. anaemia, leukaemia (Leukemia has too many white cells)

Plasma/Serum with all the proteins in it White blood cells (Buffy Coat) looks exaggerated in this test tube because it is taken from a leukaemia patient (who produces a lot of white cells)

Red blood cells sink to the bottom

HEMATOPOIETIC TISSUES 

The mature cells in the Peripheral blood are derived from eight main lineages - Erythroid (red cells) - Neutrophil

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Monocyte/macrophage (engulf cellular debris) Eosinophil (release granules in response to allergic reactions and in conditions like asthma and dermatitis) , Basophil, Megakaryocyte (Platelets flakes off the megakaryocytes hence don’t see megakaryocytes in blood but only see platelets) T lymphoid, B lymphoid (give antibodies/Immunoglobulins)

In adults, tissues generating non-lymphoid cells of the peripheral blood are referred to as haematopoietic tissues and are restricted to: - Bone marrow (95%) = Mainly sternum (adults), ribs, sacrum, vertebrae, top of the long bones - Spleen: which plays a minor role (5%)  Myelofibrosis- Bone marrow disorder where the Spleen takes over and makes all of the Haematopoietic stem cells.

HEMATOPOIETIC STEM CELLS  Pluripotent Hematopoietic stem cell (HSCs) are crucial but are rare among bone marrow cells (1 in 100,000) - HSCs express CD34clinical marker used when preparing HSCs for transplant - Sources of HSCs for transplant include bone marrow, cord blood and peripheral blood  HSCs can self-renew and give rise to committed progenitor cells of each lineage  Progenitor cells are restricted in their developmental potential, become more differentiated and lose the capacity for self-renewal/proliferation  Mature cells of each lineage migrate into bone marrow sinusoids to enter circulation - ↑ circulating blasts can occur with blood loss, infection, ALL/AML  HSCs can either Self renew or differentiate. - HSC can differentiate into Colony Forming Unit of Spleen (CFU-S) - CFU-S can develop to become Multipotent Progenitor Cells (MPP) - MPP can differentiate to form:  Lymphoid lineage of blood cells (CLP- Common Lymphoid Progenitor) CLP can differentiate to either: o Pre –B to Form B lymphocyte o Pre-T to form T Lymphocyte  Myeloid Lineage of blood cells (CMP- Common myeloid Progenitor), which differentiates to o GMP – GM-CFC to G-CFC to become Granulocyte - GM-CFC to become Macrophage o MEP – BFU-E to CFU-E to become Erythrocyte - Meg-CFC to Megakaryocyte

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Erythrocyte contain Hb (hence pink) and biconcave disc cause they’ve lost their nucleus Megakaryocyte is broken down to form platelets Neutrophils Basophils are more rare than Eosinophils – If they are raised in the blood, highly likely they are because of a Myeloproliferative neoplasm think about my— Monocytes become Macrophages – filled with fat/ debris (bottom picture) Cant tell B and T lymphocyte apart Plasma cells= Mature B cells make antibody or Immunoglobulin. NK cell- Stops cancer and viruses and is also a lymphoid cell

Origin of Haematopoietic stem cell:

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Cells of Haematopoietic tissue are generated from the mesoderm in blood islands of the yolk sac (to produce transient primitive blood cells) and then definitive cells are produced from the endothelium in the aorta- gonad- mesonephros (AGM) region. The site of haematopoiesis then shifts to the fetal liver and subsequently the bone marrow.

SITES OF HEMATOPOIESIS

 Yolk sac mesoderm forms transient primitive blood cells  Hemangioblasts are derived from the aorta-gonad-mesonephros (AGM) region common precursor of endothelial and hematopoietic cells - Hemogenic endothelium of aorta gives rise to definitive hematopoietic stem cells (HSCs) that migrate to liver, spleen and bone marrow  Liver and spleen are the major sites of fetal hematopoiesis until 7 months gestation - At about 30–40 days, definitive haemopoiesis starts to occur in the fetal liver and definitive erythroid cells are released into the circulation at about 60 days. By 10–12 weeks, haemopoiesis starts to migrate to the bone marrow, where eventually erythropoiesis is established during the last 3 months of fetal life  Bone marrow becomes the major site of hematopoiesis in infants and adults - Progressive fatty replacement occurs in the long bones during childhoodred bone marrow is confined to the central skeleton in adults  HSCs reside in the bone marrow and a small number circulates in the blood - G-CSF mobilizes HSCs for peripheral blood stem cells collection

BONE MARROW  In infancy - All the bone marrow is Haematopoietic (even in the metatarsal)  During childhood: - There is progressive fatty replacement of marrow throughout the long bones so that, in adult life, haematopoietic marrow is confined to the central skeleton not in all

bones  In adult: - Haematopoietic areas, approximately 50% of the marrow consists of fat - Fatty marrow is capable of reversion to haematopoiesis  In some blood disorders there is also expansion of haematopoiesis into the long bones  The spleen and liver can also resume their fetal Haematopoietic roles; socalled ‘extramedullary haematopoiesis’ (outside in the medulla –that is in the bones)  Bone marrow constituents: - Adult bone marrow includes trabecular bone containing fat and haematopoietic tissue in variable quantities - Bone marrow contains fat (~50%) and cellular elements (varies between 30-70%)↑ fat and ↓ cellularity with age  Older adults will have around 30 % cells and 70 % fat - Major cellular elements are the haematopoietic cells and bone marrow stromal cells  HSCs reside in endosteal niches within bone marrow stromaprovides physical support and suitable microenvironment for HSCs  Stromal cells include fibroblasts, macrophages, adipocytes, endothelial cellsproduce ECM matrix, adhesion molecules and growth factors  Function of stromal cells: o Provide physical support and a microenvironment suitable for blood cell production o The microenvironment produces an extracellular matrix, adhesion molecules (HSC don’t stray from the nest) and secretes blood cell growth factors

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Bone Marrow Appearances – Trabecular bone

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Bony trabeculae

Haematopoietic cells Stromal cells Purplish spots: blood cells Red cells – they have no nucleus

Fat spaces

Purplish spot: Blood cells

Red cells (don’t have any nucleus)

HAEMATOPOIETIC STEM CELLS: 

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Haematopoietic stem cells (HSCs) sustain haematopoiesis but constitute a very small fraction (1 in 10,000-100,000) of the total bone marrow blood cell population. They are hard to distinguish from other cells hence require a stem cell marker (antigen CD34) Stem cell properties include: - Self-renewal - Generation of one or more specialised cell types Human HSCs express the antigen CD34 - CD34 is used as a measure of stem cell number, particularly when preparing cells for transplantation (immunotyping cell surface marker) Umbilical cord blood is enriched in HSCs - Therefore used increasingly as a source of stem cells for transplantation, especially in the paediatric population  The umbilical cord blood is only of a small volume hence only enough for a child but not for an adult  Suck them out and put them in a bag (give to those that need the stem cells or will be sent to the cord blood bank)

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Peripheral blood stem cell collection: - The donor’s blood is put through a centrifuge machine where the CD34 HSC cells are extracted and the rest of the blood cells are returned back to the donor. - In order to ensure that the HSC are within the blood (since they are very rare), the donor Is given injections containing Granulocyte colony stimulating factors into his abdomen for 4 days, which makes the haematopoietic stem cells circulate in the blood so they can be extracted.

MATURE BLOOD CELLS:  

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The marrow represents a heterogeneous collection of cells without the stratification seen, for example, in the skin or gut Despite this, a series of maturing haematopoietic blood cells can be recognised for each lineage - For example, in neutrophilic granulocyte development, there are a series of distinct cells that differentiate into the mature granulocyte - The recognisable cells in this lineage (from less to more mature) are termed promyelocytes, myelocytes, metamyelocytes , band neutrophils and segmented neutrophils. - It is only the last cell population (segmented neutrophils only) that is observed in the blood, the earlier development of this lineage normally occurs in the bone marrow. In general, only the most mature cells in each lineage enter the circulation in significant numbers, although small numbers of stem cells and progenitors are present in the circulating blood

REGULATION OF HEMATOPOIESIS  Hematopoiesis is controlled by transcription factors and cytokines (growth factors) - TFs (Runx 1 and GATA-2) are involved in the way the stem cell develops and they activate/inhibit gene expression may be general or lineage specific - Cytokines regulate cell proliferation, differentiation, maturation and apoptosis  Growth factors/cytokines are mainly produced by bone marrow stromal cells except for erythropoietin (kidney) and thrombopoietin (liver). Example of cytokines: - TPO drives megakaryocyte and platelet production. - GM-CSF will drive the Myeloid lineage differentiation - G-CSF to drive neutrophil production so that it can cause neutrophil recovery after chemotherapy and make stem cells circulate in the blood - EPO will drive erythropoiesis (production of red blood cells)

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HSC (self renewing) and becomes CFU-S and MPP MPP can Make CLP = B or t lymphocytes (End cells) MPP can make CMP = Granulocytes, Macrophages, and Erythrocytes and Megakaryocytes (End cells) Erythrocytes last 120 days Turnover rate is high as you descend

ASSESSMENT OF BLOOD AND BONE MARROW: 1.

Peripheral blood cells – full blood count (FBC, CBC) (sometimes called complete blood count) - This is usually automated, giving absolute numbers of different cell types - Examination of blood film to look carefully at morphology is important if abnormal parameters are noted

2. Bone marrow examination - Bone marrow aspirate (5mls) = allows cytological examination of haematopoietic stem cells

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Bone marrow trephine = produces a core biopsy which is good for histological examination of marrow architecture and cellularity

3. Stem cells examination: - Can be assessed indirectly by colony assays, and - By Measurement of CD34 positive cells (immunophenotyping/cell surface markers/ flow cytometry)

CLINICAL NOTES  Petechial haemorrhage: small (1–2 mm) red or purple spot on the skin, caused by a minor bleed from broken capillary blood vessels. - Usually present on the lower limbs  Peripheral blood analysis includes - Full blood count (FBC)provides absolute numbers of different cell types - Peripheral blood film provides information on blood cell morphology  Bone marrow analysis includes - Bone marrow aspiratecytological examination of hematopoietic cells - Bone marrow trephinehistological examination of architecture and cellularity - Sample taken from PSIS/sternum in adults and upper medial tibia in infants/children  Stem cells can be assessed using colony assays and measurement of CD34+ cells  Myelofibrosis: - It is a myeloproliferative neoplasm where the bone marrow is scarred up with collagen and reticular. - is a chronic myeloproliferative neoplasm (MPN) characterized by 1. Bone marrow fibrosis, 2. Extramedullary haemopoiesis = Haematopoiesis in liver and spleen which is outside the bone marrow 3. with splenomegaly and 4. Leukoerythroblastosis = An anaemic condition resulting from spaceoccupying lesions in the bone marrow and characterized by the presence of immature granular leukocytes and nucleated erythrocytes in the circulating blood - Spleen is enlarged and has descended near the umbilicus. - Symptoms: Early satiety due to spleen taking up a large part of the abdominal cavity (is a condition marked by feeling full after you have eaten a small amount of food, or before you finish a normal-sized meal) and weight loss. - Painful cause it can cause infarct of blood supply

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Clinical Scenario  Febrile, lethargy and petechial haemorrhages in his skin  Low BP and high heart rate (compensatory)  Pancytopenia (Pan:All Cyto:cells penia:too few): All the blood cells are too low in the blood. - Hb: 76 g/L (half of normal value) - Platelets low: < 10 x 10 ^9/L - No segmented neutrophils. - Large number of immature cells are seen.  The patient proceeds to a bone marrow examination and the aspirate shows a very hypercellular marrow with a homogeneous (all the same) population of blasts cells and little differentiation to more mature myeloid forms  The diagnosis= is acute myeloid leukemia  Treatment = is with chemotherapy (clear out the blast cell population), transfusion support (platelets) and antibiotics for infection...