Infection, immune & Haem IIH

• Know the types of blood cells and their key role

• Understand haematopoiesis and how it is regulated

• Be familiar with why clinically we need to understand haematopoiesis

  • Use of growth factors

  • Haematological malignancies

• Red cells (erythrocytes) - Oxygen transport

• Platelets - Clotting

• White cells (leukocytes) - Immunity

• Lymphoid cells

  • T cells (directs), B cells (makes Abs) and NK cells (natural killer cells - directly kills)

• Myeloid cells may be either monocytes or granulocytes. The 3 types of granulocytes are:

  • Neutrophils, Eosinophils, Basophils (based in gut & Resp)

Innate immune response:

• Non-specific response, therefore no immunologic memory.

• Response is very fast, Inflammation tends to occur

• Also includes physical and chemical barriers e.g. lysozymes in tears and cilia in airways

• Monocytes, granulocytes (Neut, eosinophils, Basophils)

Adaptive immune response:

• Highly specific, can involve memory cells

• Slower as cells require priming

• Includes clonal expansion and clonal deletion

• Some cells survive as memory cells

• Lymphocytes ( T cells, B cells, NK cells)

• Produced in the bone marrow, regulated by GM-CSF

• Part of innate immune response

• Can migrate from blood > tissue > become macrophages

• Key roles in phagocytosis + cytokine production

  • Engulf and destroy dead host cells + pathogens

  • Produce IL-12 and IFN gamma which are important for intracellular immunity (pathogens within host cell)

Neutrophils (most no)- regulated by G-CSF

• Live in blood for few hrs > migrate to tissue and live 4-5 days. Engulf and destroy (phagocytes) bacteria

• Granules contain lysozyme and myeloperoxidase which are important for pathogen killing

Eosinophils - regulated by IL-5

• Response to parasite infection, not phagocytic, it release granules containing toxic substances.

Basophils - regulated by IL-3

• Associated with allergic diseases

Lymphocytes are part of adaptive immune response - small cells with low granularity.

1. T cells - Early progenitors from BM > circulation > thymus. Here they differentiate and selected to recognise antigen

2. B cells - Develop in BM > exit as antigen naive cells. Once recognise Ag, further differentiate into lymph nodes. Directed by T cells

3. NK cells - develop in BM

> Platelets contain granules that secrete substances to control clotting and break down of blood clot.

> Dont have nucleus!

> Lifespan of 8-12 days - removed by macrophages in the spleen and liver

> Low plt > easy bruising and bleed

Necessary for 2 processes to prevent blood loss post injury

1. Haemostasis - mechanism that leads to cessation of bleeding from a blood vessel.

2. Thrombosis - The formation of blood clots. which are then cleared after these processes.

• Have several progenitors - early one is known as megakaryoblast > megakaryocyte

• Megakaryocyte undergo endomitosis (DNA replication without cell division) - causes them to develop lobulated, polyploid nuclei and an expanded cytoplasm volume > more platelets to be produced. This gives distinct appearance.

• Platelets bud off from megakaryocytes

• Production is regulated by Thrombopoietin (TPO) - a peptide produced mainly by liver = liver dysfunction can cause thrombocytopenia due to lack of TPO

• Thrombopoietic GF is used to stimulate plt when low in pts

• Normal value - 150-450 x 10(9)/L

• RBCs are biconcave (to increase SA) Hbs, for gas exchange

• Originate from normoblasts which extrude their nuclei > reticulocytes.

• Reticulocytes have no mitochondria = sensitive to oxidative damage.

  • Retains their RNA = able to make Hb (not possible in erythrocytes).

  • Very flexible due to ankyrin & spectrin proteins

• RBC has lifespan of 120 days, 5x10(12)/L

Important in immune response, produced by haematopoiesis

• Dendritic cells - Professional antigen presenting cells found in tissues. Detect antigens in the tissues then present it to the innate and adaptive immune system

• Mast cells - Produced in the bone marrow but mature in tissues, these cells are very similar to basophils

• Haematopoiesis = process of blood cell production (10(12)

• Begins 17 days post fertilisation & continues throughout life in BM. Regulated by GF and cytokines

• Blood loss / infection > stress demand > haematopoietic tissues > rapid increase in cell production

Haematopoietic stem cells (HSC)

• These are either in quiescent (resting) or non-dividing state, have capacity to self-renew to maintain HSC no or give rise of specialised differentiated cells.

• HSC are multipotent = ability to differentiate into lineages of all blood cells. Commit to 2 pathways:

  • Myeloid precursor (common myeloid progenitor) > RBC, plt, basophil, eosinophils, Neut and monocytes

  • Lymphoid precursor (CLP) > T cells, B cells, NK cells

• Stem cells + multipotent progenitor cells identified by functional assay, while committed precursor cells & mature cells identified by routine marrow staining

The site of haematopoiesis differs depending on age:

• Foetus - yolk sack, moving to foetal liver

• Infants - Bone marrow (virtually all bones)

• Adults - Bone marrow of Axial skeleton (red marrow) i.e sternum, pelvis, vertebrae

• Erythropoiesis > erythrocytes (RBC)

• Begins in BM with HSC > differentiate > proerythroblasts > commit to erythrocyte lineage > forms smaller erythroblasts after division.

• Down the lineage, nucleus get smaller until extruded to form reticulocytes (still have RNA = can make Hb).

• Reticulocytes spends 1-2 days in BM, then 1-2 days in peripheral blood > mature into red cell (loss of RNA)

Note: reticulocyte (arrow) has no nucleus but stain pink due to increasing Hb as RNA is still present.

• Erythroblasts grow in islands (varying stages)

• Cluster of developing RBCs surrounds central macrophage forming erythroid island.

• Macrophage provides iron to the developing erythroblasts, removes nuclei from them to form reticulocyte then release it into blood stream.

• If the RBC are destroyed in the periphery, erythropoiesis is stimulated > high level of precursors e.g reticulocytes.

• It is regulated by erythropoitin (EPO) - O2 sensitive, produced by kidney + 10% by liver. EPO stimulates cells to divide

• Main stimulus is hypoxia > increase mRNA transcription by peritubular interstitial cells in the kidney > incr EPO secretion.

• EPO is low in renal impairment > unable to upregulate BM production of RBC.

Note: If RBCs are destroyed int he periphery > stimulates erythropoiesis > many precursors i.e Reticulocytes

Blood cancers are result of genetic abnormality - can also caused by viral infection e.g. Burkitt’s lymphoma post EBV

• Leukaemia = cancer of haematopoietic cells which arise from BM and spread to bone / LN / spleen

• Lymphoma = cancer of cells in LN / spleen which spead to BM and blood

Primary classification is by lineage - myeloid vs lymphoid

> Lymphoid cancers further classified as to T cell or B cell

> Leukaemias further classified as to whether it is acute or chronic. Acute leukaemia are rapid onset + more aggressive than chronic.

Leukaemia = cancer of haematopoietic cells. Classified into:

1. Acute - arise due to maturation arrest > block in haematopoiesis > over production of immature cells > crowding of BM

1. chronic - lack of maturation arrest > over production of dysfunctional mature cells = no -ve feedback on haematopoiesis > uncontrolled proliferation

• Blocks B cell development > inablity to make antibodies > rely on life long immunoglobulin replacement = highly susceptible to infection

• TFs are proteins that control gene expression > on / off gene transcription. If certain TFs are absent > blocks blood cell differentiation

In order to develop & differentiate, haematopoietic progenitor cells require:

• Intrinsic factors - i.e. lineage determining TFs and their epigenetic regulators

• Extrinsic regulators - both cellular & soluble GF found in the BM microenvironment. These factors can be up/down regulated according to certain stimuli.

• Early acting ones - stem cell factor, Flt3 ligand, IL-3, TPO

• Late acting - G-CSF, GM-CSF, EPO, TPO

  • EPO is used for patients with anaemia in ESRF, cases of myelodysplasia, pre-autologous blood donation and Jehovah’s witness during blood loss

  • G-CSF - SC injection in neutropenia due to chemo or congenital issue. To mobilise stem cells into blood for harvest and transplant

  • TPO receptor agonists- used in thrombocytopenia due to ITP / myelodysplasia or post chemo or aplastic anaemia. E.g. Romiplostin (SC), Eltrombopag (PO)

Note: RBC transfusion lasts one month, platelet few days, stem cell transplant lasts life time

Cytopenia = reduction of mature blood cells. Caused by:

• Issue with production by BM centrally e.g apastic anaemia

• Destroyed rapidly in peripheral circulation e.g immune thrombocytopenia

• Haematopoiesis occuring in adult life again in liver and spleen

• Occurs in myelofibrosis or intreated thalassaemia where BM capacity is compromised or exceeded

• Measure of FBC

• By carrying out immunophenotyping - look at specific proteins or cell markers found on cell surface or inside. Done in bloods or BM

• Normally, peripheral blood contains only small no of HSC. It may be increased when BM is recovering from damage by cytotoxic drugs or post G-CSF to mobilise HSC into blood fot harvesting for transplants

• To evaluate haematopoiesis, BM cells accessed via needle biopsy from iliac crest. 2 types of assays:

  • Aspirate - semi liquid BM aspirate is extracted via thin needled and smeared onto a slide then looked under light microscope to see cell count + types. Can also do chromosome analysis

  • Trephine biopsy - Core of BM is taken with thick needle to look at cellularity, histology and architecture

• Define anaemia

• Signs / symptoms

• Causes of anaemia

  • Haemetenics deficiency

  • Bone marrow failures

  • Haemolysis / bleeding

RBC have no nucleus, biconcave shape and contains Hb which binds to O2. Hb consits of 4 polypeptie chains (globins) each with haem group > 4 binding sites for O2. Type of globin changes throughout human life.

• Fetal Hb (HbF) - made of 2α + 2γ globins, <1% of adult Hb

• HbA - Made of 2α + 2β globins (sickle cell > mutated β globins), most common form of Hb in adult (>95.5%).

• HbA2 - made of 2α and 2δ, less common varient

• HbS - a mutated variant in sickle cell anaemia, consisting of 2α and mutated 2βs > alternate shape of the RBC

• pH - Low pH reduces oxygen affinity.

• Temperature - High temperature reduces oxygen affinity

• CO2 - High concentration reduces oxygen affinity

• 2,3 DPG - an enzyme that reduces oxygen affinity

These factors enable haemoglobin to release more oxygen at respiring tissues (Bohr effect)