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. The colour comes from iron-containing Hb.

> 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

  
  

  Note: Haem groups in Hb molecules are synthesised in mitochondria of developing erythrocyte

• 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)

Sideroblast anaemia

• Due to iron accumulation in the mitochondria of thses cells

• The erythroblasts are referred as sideroblasts

Reduction in the Hb concentration of the blood.

May be accompanied by reduced RBC count and reduced haematocrit (proportion of RBCs in blood)

Normal range varies depends on factors such as:

• Gender (loss in menstruation)

• Pregnancy (increased plasma volume) > apparent anaemia despite normal Hb values

• Extremes of age

• Different labs / testing platforms

• Altitude

• Plasma (55% of volume of which 91% is water)

  • Contains - plasma proteins, electrolytes, hormones, nutrients

• Buffy coat (45% of vol)

  • Platelets, WBC, RBC

Note: Variation in plasma vol may be misinterpreted:

• Reduced PV (dehydration) - mask anaemia by causing normal Hb concentration

• Increased PV (preg) - may cause apparent anaemia despite normal Hb levels.