BII Flashcards
What is haematopoiesis?
The process by which mature blood cells are generated from stem cells in the bone marrow
Define pancytopenia
Low levels of all categories of blood cell types
Where does haematopoiesis occur?
Non lymphoid cell generation is completed 95% in the bone marrow of ribs, long bones, sacrum etc., and 5% in the spleen
Describe the development of blood cells
Stem cells generate progenitor cells
Progenitor cells generate morphologically identifiable progeny that progressively mature
As they mature they lose their ability to proliferate and become post-mitotic
What are some of the different cells formed by haematopoiesis?
Neutrophils- life of 5-6 hours then move into tissues T and B lymphocytes Granulocytes Macrophages Erythrocytes- life of 120 days Megakaryocytes- life of 5-6 days
What are stem cell niches?
Endothelial and endosteal types that support the normal activities of stem cells.
Where does haematopoietic tissue come from?
Cells of haemopoietic are generated from the mesoderm in blood islands of the yolk sac, to produce transient primitive blood cells, and then definitive cells emerge from the endothelium in the aorta-gonad mesonephros region. The site of haematopoiesis shifts to the fetal liver, and then to bone marrow.
The primitive haemagnioblasts proce haemopoietic and endothelial cells, whereas the endothelium produces only haemopoietic tissue
How does our bone marrow change as we age?
In infancy, all bone marrow is haematopoietic, but there is progressive fatty replacement of marrow in the long bones, so that in adult life, haematopoietic tissue is only in the axial skeleton, and approx. 50% of this is replaced by fat. This is important as when diagnosing conditions we should be biopsying active marrow only
What can happen to fatty marrow?
It can revert to haematopoietic tissue.
Describe extramedullary haematopoiesis
The spleen and liver resume their fetal haematopoietic roles.
Describe bone marrow in an adult
Includes trabecular bone, which contains fat and haematooietic tissue. Cellularity varies and decreases with age. Major cellular elements are haematopoietic cells and stromal cells, including fibroblasts, macrophages, fat cells and endothelial cells. They provide support and a microenvironment suitable, consisting of extracellular matrix, adhesion molecules and blood cells growth factors.
On slides, the pinkish stuff is bone, white spaces are fats and purple cells are the haematopoietic cells.
What are the properties of haematopoietic stem cells?
Self renewal
Generation of one or more specialised cell types
How do we measure HSCs?
They all express the antigen CD34 which can be used as a proxy
What is the difference between immature and mature blood cells?
Immature are made in the bone marrow, while mostly only the mature ones are seen in circulation
What are the major factors regulating haematopoiesis, and what do they do?
Transcription factors that switch on and off to control cell programming.
Cytokines, which are the most clinically useful. They are growth factors produced by the marrows, for which stem cells have receptors.
Includes EPO- increases RBCs
TPO- increases platelets
G-CSF- Increases neutrophils
What are the main ways of assessing blood and bone marrow?
Peripheral blood count- full blood count. Usually automated, giving absolute numbers of cell types. Blood film can be examined to look at morphology
Bone marrow exam- aspirated at look at the liquid marrow, or trephine produces a core biopsy, good for histological exam of architecture. Usually taken from post iliac crest
Stem cells- assessed indirectly by colony assays and CD34 measurement
Where can we source stem cells from?
Umbilical cord blood. There is 60-100mL of immunologically naive blood remaining in the cord
Describe myelofibrosis
Scarring of marrow tissue, causing few marrow cells and lots of collagenous tissue. Patients develop hepato- and splenomegaly due to reversion to the fetal state, and show extramedullary haemapoiesis. (A similar thing happens in children with untreated thalassemia, but the bone marrow itself also expands).
What are the main three properties of red blood cells?
Unique shape and deformability
No nuclei or mitochondria, but still need energy
Carry haemoglobin
Why is the red blood cell’s shape and deformability important?
Allows gas exchange and movement through small capillaries
This propety is determined by membrane and cytoskeletal proteins. Inherited abnormalities of this membrane can cause decreased red cell lifespan due to haemolysis. It can cause anaemia if Hb drops.
How do red blood cells produce energy?
They have glycolytic pathways, as well as an HMP shunt to produce NADPH and keep Hb reduced. Inherited defects in these pathways lead to increased haemolysis
How do red blood cells carry haemoglobin?
In adults it is mainly done by using HbA. This consists of 2 alpha and 2 beta chains with a haem group. There are also small amounts of HbF and HbA2. Defective production of globin chains is qhat causes thalassemia, while irod deficiency causes reduced Haem production and low Hb.
How do erythroids develop?
They start as part of the myeloid multilineage progenitor, and respond to growth factors including IL-2 and GM-CSF. They have erythroid burst forming units, and then colony forming units. They then reach the morphological stages, where they differentiate- there is progressive increase of Hb and chromatin, clumping, extrusion of the nucleus and loss of RNA
They take 7-10 days to develop, and so this is how long you must wait to see changes after treatment.
As they spend 2 days as reticulocytes, reticulocytes in the blood can be used to measure erythrocyte production- high in blood loss or haemolysis, or low in bone marrow failure
What are the critical requirements of erythropoiesis?
Iron
Folate
B12
What regulates erythropoiesis?
Erythropoietin- a glycoprotein produced in the kidney, especially in response to low oxygen
What does erythropoietin do?
Acts through EPO receptors to stimulate PFU-E and CFU-E Increases haemoglobin synthesis Reduces RBC maturation time Increases reticulocyte release Increases Hb Increase O2 delivery
What can some side effects of EPO be?
Renal failure patients can get low Hb
Can be used in clinics to treat anaemias, but has potential for abuse
How are RBCs broken down?
Cells becomes less deformable and are removed in the spleen
Broken down with release of Hb, which is then broken down into globin chains and haem
Iron is carried by transferrin back to the bone marrow
Protoporhyrin is coverted into bilirubn in the liver, and secreted as bile
What is anaemia?
Reduction in Hb concentration in the blood, usually but not always accompanied by a fall in RBC count and packed cell volume
Alterations in plasma volume (eg dehydration) can also mask anaemia or cause polycythemia.
What are the clinical features of anaemia?
Increased SV and HR
Right shift in the haemoglobin dissociation curve (making O2 more available to tissues)
Can be asymptomatic, but eventually, patients will develop SOB, fatigue, pallor and congestive cardiac failure. Degree of symptoms depends on speed of onset, severity and age of patient.
What are the different categories of anaemia?
Macrocytic
Microcytic
What are the most common URTIs in NZ?
Colds, bronchiolitis, pharyngitis and influenza
Distantly followed by pneumonia and tuberculosis
What is a URT?
Upper respiratory tract- everything above the level of the alveoli. Includes sinusitis, otitis media, pharyngitis, tracheitis, bronchitis and pneumonia.
What are symptoms of a URTI?
The middle ear and sinuses are common sites
The mucosa swells as fluids, antibodies etc filter into them. This can cause difficulty breathing through the nose, blockage of eustachian tube and fluid accumulation in the middle ear
What are the common causes of respiratory infection?
Strep Pyogenes
Strep Pneumoniae
Haemophilus influenzae
Rhinoviruses
Coroaviruses
Resp syncytial viruses
Influenza virus
What is the difference between Strep pyogenes and Strep pneumoniae in terms of presentation?
Pyogenes only causes pharyngitis
Pneumoniae causes infection in the middle ear, maxillary and frontal sinus issues, alveolar issues. While it is the most important cause of sinusitis, bronchitis and pneumonia it is not a cause of pharyngitis
What is pharyngitis?
Same as tonsilitis or sore throat. Can involve tonsils, soft palate, uvula, and cervical adenopathy
What are the common causes of pharyngitis?
S. Pyogenes, rhinoviruses- about 50/50 each,
Then Influenza
Then EBV
How can you differentiate between S. pyogenes and viruses for the causes of sore throat
Pyogenes causes a more severe illness with fever, pain, dysphagia and adenopathy, as well as a focal pharyngeal infection
Viruses oftena ffect nose and throat, with hoarse voice, cough etc. but a less severe illness
Describe S pyogenes as a bacteria
Gram positive
Beta haemolysis
Serogroup A
Describe haemolysis and serogrouping
Haemolysis- a haemolysis will show green blood
b haemolysis willl show clear
g haemolysis will show nothing
Lancefield serogrouping means that antibodies will do different things to different streps
Describe infection with S pyogenes
Asymptomatic colonisation occurs in about 30% of nz population
Does give risk of rheumatic fever
Get sick soon after infection, but can stay as an asymptomatic infection for several months. Can have repeat infections as different types have different M type surface receptors
Describe colds
Nasal discharge from ant and post nose, with nasal congestion
Facial congenstion, fullness, pain and pressure
Reduced or absent smell
Fever
Describe the causes of sinusitis
90-98% caused by viruses- rhinovirus, influenza, parainfluenza
Bacteria cover the rest- strep pneumoniae and haemophilus influenzae. Can only be confirmed by sinus aspiration
Give a general overview of causes and consequences of URTIS
Mostly caused by viruses but may be caused by bacteria- very difficult to distinguish
Minimal benefit from antibiotics, but a very common cause of wasteful antibiotic treatment!
What is cellulitis and how does it present?
An infection of the dermis and subcutaneous tissue, often seen around injury site or deep abscess. Presents and diffuse with small abscesses
Describe impetigo and erysepelas
Impetigo- Pus filled lesions, which crust over. Very contagious
Erysepelas- Acute infection of upper dermis and superficial lymphatics, presents with a clear line between healthy and inflamed skin
Describe the inflammatory response to bacterial infection
Bacteria enters a wound
Platelets release blood clotting proteins at wound site- this is important for containment of infection
Mast cells mediate vasodilation for delivery of blood, plasma and cells to injured area by releasing heparin and histamine
Innate immune mechanisms PAMP and compliment activate residential macrophages
Neutorophils degrade and kill pathogens
Neutrophils and macrophages phagocytose debris
Macrophages secrete cytokines that attact immune cells and activate tissue repair cells
This continues until the foreign material is eliminated and wound repaired
Give an overview of PAMPS
Pathogen associated molecular patterns- recognized by pattern recognition receptors on the surface of macrophages to recognize most common receptors on gram positive and negative bacteria- LPS is most common negative one
Describe diapedesis
Leukocytes travel through blood vessels, which have increased adhesion molecule expression (e. selectin). This slows neutrophils to a roll. The vessels have open endothelial junctions allowing cells to squeeze through, and follow a chemotactic gradient.
What organisms can cause skin/subcutaneous tissue infection?
Strep pyogenes
Staph aureus
Viruses
Some other bacteria and fungi
Describe Streptococcus
Gram positive, catalase negative. Causes SSTI, systemic diseases, pharyngitis and potentially rheumatic fever.
Group A serotype
Found exclusively in humans.
How does S pyogenes interact with the immune system?
it has with MSCRAMMS (microbial surface components recognising adhesive matrix molecuse), which are cell wall attached adnesins that bind to host ECM proteins.
It has a hyaluronic acid capsule to prevent opsonisation and phagocytosis, M proteins (MSCRAMMS), secretes toxins
- Streptolysins lyse immune cells
C5a peptidase prevents cheotaxis
DNAses degrade neutrophil extracellular traps
SpyCEP also prevents chemotaxis
How does S pyogenes spread?
Proteases, lipases, hyaluronidase and streptokinase help it to degrade tissues and move further in
Streptokinase is an anticoagulant that activates plasminogen to cleave fibrin plugs
If it gets deep enough it can cause necrotising fasciitis
How do you diagnose the cause of skin infection?
Swab purulent materia, do a culture
Also do a blood cultures- if positive, can cause further complications
How do you determine the category of bacteria?
Gram positive ar staph and strep
Catalase test shows bubbling of H2O2 with staph, but not with strep
Haemolysis can show part, full or no results
Bacitracin susceptibility determines between groups A B and C strep
How do you treat SSTIs?
Supportive care, rest and elevation, analgesia, antimicrobial drugs (s pyogenes is susceptible, but staph needs to be treated with a b lactamase resistant penicillin
All pyogenes are susceptible, but only 10% of aureus are.
How does penicillin work?
It binds to and prevents functioning of the transpeptidase enzyme, which cross links alternating sugars in the bacterial cell wall- this results in a weak, easily lysed cell wall They are a group of B lactam antibiotics, with each class having different side chains
What are the distinguishing features between bronchitis and pneumonia?
Pneumonia shows focal signs in the lungs as well as rep distress. It causes increased resp rate due to hypoxia and nociception, crackles, consodilation, fevers and chills
They can be differentiated by chests on X ray and tests of sputum
Normal chest X ray will be able to follow the lung margins and follow the heart
There will be shadowing and pleural effusion in pneumonia cases
What are risk factors for pneumonia?
Smoking- damages cilia
Age <2 or >65- larger number of bacteria in the lower lung aspirated from the nasopharynx
hronic lung disease
Immune dysfunction
What are some of the defence mechanisms of the airways?
Ciliated epithelium Nasal turbinates Nasal secretions Saliva Epiglottis Goblet cells Airway lysozymes PAMs Surfactant Neutrophils
What are the most likely causes of pneumonia?
Strep pneumoniae (3/4 of cases)
Haemohilus influenzae
Staph Aureus
Describe streptococcus pneumoniae
Alpha haemolytic streptococcus group
Colonises the nasopharynx in 5-10% of adults 20-40% of children.
prevalence increases in winter
Can cause pericarditis and endocarditis
Describe the virulence factors of strep pneumoniae
Surphase protein A binds to epithelial cells and prevents deposition of C3b
Choline binding protein binds to Ig receptor on epithelial cells, allowing transport into the cell
Pneumolysin lyses neutrophils
Polysaccharide capsule prevents phagocytosis and complement deposition
Pili contribute to colonisation and cytokine production
Surface protein C prevents activation of complement
What is pneumococcal disease?
An infection of spinal fluid by strep pneumoniae
What are the possible tests for pneumonia and describe some
CXR Sputum culture- but easily contaminated Nasopharyngeal swab if admitted Blood cultures if admitted Urite ICT- lacks sensitivity Serology
What is treatment of pneumonia?
Antibiotics required for reduced duration and risk of death
However, penicillin resistance is increasing- unlike MRSA, it can be partially susceptible
Oral dosing may be inadequate, with IV dosing best
How can antibiotics target ribosomes?
Prevents protein synthesis May target transpeptidation (macrodiles) Peptidyl transferase Initiation TRNA binding
Describe macrolides and why they may present issues
They are a broad spectrum antibiotic with limited activity against gram negative bacteria.
Theya re acctive against trep, staph and other causes of pneumonia, as well as chlamydia
Can cause GI upset as they increase peristalsis, as well as sudden death (change electricity of heart) and drug interaction
What is anaemia?
A reduction in the concentration of haemoglobin in the peripheral blood below the normal for the age and sex of the patient.
What are the two different ways of classifying anaemia?
Pathologically- based on the cause of the anaemia. Can be impaired red cell formation, blood loss or excessive haemolysis, or a mix
Morphological classification- based on red cell appearance, mean cell volume, or mean cell haemoglobin concentration
Describe normocytic anaemias
The MCV is within the normal range, also usually normochromic with a normal MCHC (although mild hypochromia may also be present)
Describe hypochromic microcytic anaemia
MCV is reduced, and MCHC is reduced. Often due to iron deficiency
Describe macrocytic anaemias
MCV is increased. Most are normochromic but some mild hypochromia can also occur.
What kind of lab tests can be done to determine anaemia’s presence and cause?
Red cell count PCV MCV MCHC Also helped by blood film appearance, bone marrow biopsy, presence of other blood cells
What are some of the causes of impaired RBC production?
Deficiency of substances needed for production (Iron, B12, folate)
Genetic defect- thalassemia
Failure of bone marrow- infiltration by cancer, radiation or drugs
What are some causes of reduced RBC survival?
Blood loss- can be acute, but also can be slow, chronic loss (ie occult bleeding from the gut)
- Haemolysis increased due to environmental or intrinsic RBC production. This also shows jaundice due to increased bilirubin.
What are the causes of microcytic hypochromic anaemias?
Iron deficiency
Chronic illness causing iron block
Genetics (thalassemia)
How do the causes of microcytic anaemia cause this disorder?
Iron deficiency and chronic inflammation mean iron is reduced in its transport, causing less haem formation, or else not delivered from the stores to the haemoglobin
Thalassemia causes mutation meaning that either the alpha or beta globin chains are insufficient.
How do you diagnose iron deficiency?
Measure the serum iron, binding capacity (transferrin) and iron saturation, as well as serum ferritin (stored form of iron)
Rarely need to examine iron stores
Remember that anaemia is a late consequence of iron deficiency
What can iron studies tell us about potential diagnoses?
In iron deficiency, we will see low ferritin, low serum iron, high iron transport protein, and low iron saturation
In chronic disease anaemia, we will see normal ferritin concentration, low serum iron, low transport protein and normal saturation
In iron overload we will see high serum ferritin, high serum iron, and low iron transport.
What are some potential causes of iron deficiency>
Diet
Malabsorption (loss of proximal small bowel function)
Increased demand (pregnancy)
Chronic occult blood loss (GI or GU tract
What are the most common forms of iron deficiency for different groups, and how do we treat them?
Premenopausal women: Imbalance between intake and menstrual blood loss of iron
Children: Dietary
Males/post menopausal females: Occult blood loss from GI tract
Treated with oral iron replacement therapy
Sometimes can see GI toxicity with constipation and diarrhoea.
Can also give IV infusion
Should see Hb increase 20g/L per 3 weeks
Describe how anaemia of chronic inflammation can cause microcytic anaemia
It results in an iron block. It shows adequate iron storage, but this is retained in macrophages and marrow, rather than being given to erythrocytes. It also causes reduced free iron to prevent bacteria from using it
Hepcidin produced in the liver normally stops iron transport from the macrophages to the serum. It is upregulated by cytokine IL6, and blocks absorption and release from macrophages
Describe how thalassemia can cause microcytic anaemia
It is a mutation in alpha and beta globin. Can be widespread. causes 1 or both alpha or beta chains to not be present
Heterozygotes have mild form, while homozygones have the severe form, necessitating lifelong transfusion.
Untreated presents with hepato and splenomegaly, as well as bone marrow expansion to other sites
Diagnosed by microcytic anaemia with exclusion of iron issue. Can then use haemoglobinopathy, and potentially other genetic testing
What can cause macrocytic anaemia?
B12 deficiency
Folate deficiency
Liver disease, hypothyroid, excess alcohol
What can the consequences of low B12/folate be?
Impaired DNA synthesis
Affects all cell lineages if severe, but anaemia comes first
Diagnosed by serum B12 and folate levels.
What can cause low B12?
Diet (uncommon)
Malabsoprtion due to gastrectomy, or immune issue (pernicious anaemia where antibodies against parietal cells/ intrinsic factor are produced)
(body has stores of 3-4 years, so can take a while to present)
What is haemolytic anaemia?
Anaemia due to increased destruction of red cells
Presents with pallor, jaundice, splenomegaly
Labs find raised bilirubin, reduced haptoglobins (proteins mopping up Hb- will all be taken up)
Also features of increased RBC production (reticulocytosis)
Also damaged red cells
How can haemolytic anaemia be classified?
Intrinsic red cell defects- usually hereditary, eg. membrane defects
Environmental, acquired- usually autoimmune
Shortened survival
What are the different categories of leukocytes?
- Phagocytes- include granulocytes (neutrophils, eosinophils and basophils) plus monocytes. Normally only mature phagocytes are seen in the blood
- Lymphocytes
Describe neutrophils
They circulate in the blood for 6-10 hours before moving into the tissues. They have a dense nucleus with 2-5 lobes and pale cytoplasm ith many pink or violet granules. These are termed primary in the promyelocyte stage, and secondary, appearing in the mature neutrophils. Primary granules contain lysosomal acid, enzymes and acid hydrolases, while secondaries contain lysosyme and acid phosphatase.
What are the precursors of neutrophils?
Earliest recognizable is the myeloblast, which gives rise to promyelocytes, with primary granules, and then myelocytes, which have secondary granules.
These then form metamyelocytes, then a band form of neutrophil, and finally mature neutrophils
Describe monocytes
Larger than other leukocytes with a large central oval indented nucleus + clumped chromatin
Cytoplasm is abundant and stains pale blue with many fine vacuoles. Granules often present. Precursors are difficult to distinguish from myeloblasts and monocytes
Describe eosinophils
Similar to neutrophils but with coarser, redder granules and bilobed nuclei.
Describe how eosinophils function
Function like neutrophils in that they are ameboid in motion and phagocytoze bacteria, particles and antigen-antibody complexes.
Attractic by eosinophil chemotactic factor of anaphylaxis and histamine, released by mast cells and basophils. Also bind to parasites and respond to complement.
Can clamp down allergic/hayfever reactions and so are prominent in these conditions
Describe basophils and mast cells
- Basophils are found in small numbers with deep blue granules overlying the nucleus. Basophils are predominantly in the circulation and mast cells in the tissue
Both have IgE attaachment sites, and when cross linked by an allergen it results in degranulation and histamine release, causing allergy symptoms
Describe lymphocytes
Most are small cells with scanty cytoplasm and a central nucleus with coarse chromatin. Produced in the bone marrow and thymus. Larger lymphocytes can appear in peripheral blood, if challenged by antigens.
How is granulopoiesis regulated?
They arise in the marrow from myeloid cells, take 6-10 days to mature, 6-10 hours to circulate and then move into tissues
Regulated by GCSF particularly, which can be used post-chemotherapy and in congenital neutropenia to reduce the time taken to produce granulocytes.
How are monocytes regulated?
Mature in the bone marrow in 6 days, then circulate for 20-40h. Enter the tissues and become macrophages
Regulated by GM-CSF and M-CSF
What are the different tissue-specific populations of macrophages?
Liver-Kupffer cells
Lung- Alveolar macrophages
Skin- Langerhans cells
Brain- Microglial cells
What are the overall functions of neutrophils?
Act against bacterial infections, performing phagocytosis, chemotaxis and antibody-dependent cell-mediated cytotoxicity
What are the overall functions of monocytes/macrophages?
Act against parasitic, protozoal, fungal infections as well as tumours
Perform immune surveillance, phagocytosis, chemotaxis, cytotoxicity and antigen presentation
Describe chemotaxis
Phagocyte is attracted to the bacterir or site of inflammation by chemotactic substances released by damaged tissues, bacteria, prostaglandins, products of fibrinolytic and kinin genetrating systems, and complement components.
Describe phagocytosis
The foreign material or dead/damaged cells of the host’s body are phagocytosed. Recognition of the foreign particle is aided by opsonisation with Ig or complement, as neutrophils and monocytes have surface receptors for the Fc components of Ig and C3/other complement proteins
Describe cell killing
Occurs by oxygen dependent and independent pathways
Dependent: Superoxide and H2O2 are generated from O2 and NADPH. This reacts with myeloperoxidise and halides to kill the bacteria
Non oxidative involves a fall in pH within phagocytic vacuoles into which lysosomal enzymes are released
What can cause neutrophil leukocytosis?
Refers to an increase in neutrophils, often associated with a shift to the left where there are more immature cells in the blood
Occurs reactively, due to bacterial infection, trauma, inflam, blood loss
What can cause neutropenia?
Low neutrophil count
Caused by congenital syndromes, drugs, immune issue, viral infection, recurrent infection
Rarely it is due to neutrophil functional defects
What can cause eosinophilia?
Caused by allergic and parasitic reactions
What can cause monocytosis?
Chronic bacterial infections
Malignancy
How o lymphocytes form and what are their types?
Lymphoid stem cells go to lymphoblasts go to lymph cells
Can be B, T cells or NK cells
What are the primary lymphoid organs and what occurs here?
Bone marrow (maturation of B cells) and thymus (maturation of T cells) Lymphocytes acquire their repertoire of antigen receptors and learn to discriminate between self and nons elf
What are some secondary lymphoid organs and what occurs here?
Include lymph nodes, spleen, and areas like peyer’s patches
Lymphocytes migrate here. This is where antigens are presented
Describe how the spleen functions in immunity
Spleen contains red pulp for removal of old red cells, and white pulp containing lymphoid tissue. This tissue is arranged around a central arterial, termed the periarteriolar lymphoid sheath (PALS)
T cells are found directly surrounding it while B cells are beyond this.
What can cause lymphocytosis?
Seen in viral infections in adults, malignant issues (chronic lymphocytic leukaemia) or in infants and young children in response to infection
What can cause lymphopenia?
bone marrow failure, steroids, HIV, immunodeficiency syndromes
Assoc. with increased risk of opportunistic infection
What can cause lymphadenopathy>
Viral infection, local bacterial infection, lymphoma or metastatic cancer
How are megakaryocytes produced?
It matures by a process of endomitotic synchronous nuclear replication, forming enlargement of the cytoplasm and increasing the number of nuclei by multiples of 2. Mature megakaryocytes contain 16-32N
They then show cytoplasmic differentiation with production of fibrinogen, factor V, platelent factor 4, von willebrand factor, PDGF and glycoproteins
How do platelets develop from megakaryocytes?
An extensive membrane system called demarcation membranes is formed by invagination of the plasma membrane. THe mature megakaryocyte is located next to bone marrow endothelial cells, and develops filopodia that extend into marrow capillaries. These then fragment to release mature platelets (approx. 4000 per megakaryocyte)
What regulates the production of platelets?
Thrombopoietin (TPO). Some contribution from steel factor, IL-6 and IL-11
How long do platelets last and how are their levels maintained in the body?
7-10 days. About 1/3 is kept in the spleen.
Consumption is by senescence and utilization
Describe the structure of platelets
Discoid cells with an intricate system of channels continuous with the plasma membrane. This large surface area allows selective absorption of plasma coagulation proteins
Glycoproteins on the surface coat allow adhesion and aggregation
Submembranous area contains contractile filaments, and circumferential microtubules maintain the discoid shape.
Describe the platelet cytoplasm
Have 3 distinct types of secretory granules, containing calcium, magnesium, ADP, ADP and vasoactive amines including serotonin
Alpha granules contain coagulation factors, platelet derived growth factor, TGF-B, heparin antagonist, and other proteins
Lysosomes contain hydrolytic enzymes
Energy is provided by oxidative phosphorylation in mitochondria and utilization of platelet glycogen in anaerobic glycolysis
What is primary haemostasis?
The process of forming a platelet plug at the site of vessel injury
What are the three main phases of platelet function?
Blood vessel constriction then aggregation to form a loose platelet plug, then the clotting pathway stabilizes the plug
Describe how platelets adhere to foreign surfaces and aggregate.
They adhere to exposed subendothelial collagen, and need to be supported by von Willebrand factor. VWF adheres to the collagen, causing it to undergo a conformational change, and bind to specific receptor sites called glycoprotein Ib IX V on the platelets.
This rolls the platelet to the vessel wall, causing platelet shape change and activation
The granules are released to activate more platelests
The shape change also exposes integrin a IIb B3 receptors, which sticks to VWF and other platelets
Prostaglandin synthesis causes thromboxane A to be produced, activating coagulation reactions and producing thrombin, recruiting other platelets and forming and obstructive plug
Cofactors like fibrinogen binds these integrin receptors together.
What are the functions of thromboxane A2 and how is it released?
Potentiates platelet aggregation, with powerful vasoactive activity
Inhibited by substances such as prostacyclin, which increases cAMP in platelets, sythesised by vascular endothelial cells. This prevents deposition on normal endothelium
Aspirin inhibits A2 production, allowing increased bleeding to happen
What do ADP, fibrinogen and serotonin do in coagulation?
ADP and fibrinogen result in secondary aggregation by binding to receptors on the platelet surfaces. . serotonin mediates vasoconstriction.
What are some platelet-vessel wall interactions that control haemostasis
Tissue factor initiates coagulation
Prostacyclin and NO cause vasodilation, and hihibit platelet aggregation
VWF causes the platelet collagen adhesion and carries factor VIII
Antithrombin, and protein C inhibit blood coagulation
Tissue plasminogen activator causes fibrinolysis
What are some causes of thrombocytopenia?
Reduced production due to viral infection, drugs or bone marrow failure. Aplastic and leukaemic failure are typically pancytopenic. Carcinoma and megaloblastosis cause it in isolation
Increased destruction due to immune thrombocytic purpura, autoimmune disorders, drugs, DIC, hypersplenism, transfusion or viral infection
What can cause thrombocytosis?
Increased production, either due to infection, inflammation or myeloproliferative disorders
Describe immune thrombocytopenia
Acute history of epistaxis, bruising, no lymphadenopathy or hepatosplenomegaly (showing no chronic issues)
Need to exclude other vauses like viral illness, drugs, bone marrow issues
If severe, treated with prednisone. This is normally enough for 2/3 of the population. If not, or it causes relapse, splenectomy helps in 3/4 of this population. The minority don’t respond to either treatment, and need thrombopoetin receptor agonists- but this is an expensive lifelong treatment
Describe platelet clumping as an artefact
Some people’s platelets rect to the anti-clot factor in test tubes and clump, causing the appearance of thrombocytopenia
Define petichiae, purpura and ecchymoses
Petichiae- pinprick bleeds
Purpura- normal bruising
Ecchymoses- huge bruises
What is innate immunity?
Our physical barriers to infectious agents, our microbicidal factors in body fluids, and our phagocytic cells
What is the difference between effector and regulatory immune responses?
Effector responses involve antibody production, antigen specific cytotoxicity, ADCC and NK cells
Regulatory responses involve cytokines, TH cells and Treg cells
What are MHCs?
Major histocompatibility groups Can be class I, controlled by genes HLA A/B/C inhumans and are found on all nucleated cells Can be class II, controlled by genes HLA DP/DQ/DR. Found on professional antigen presenting cells and B lymphocytes
What promotes phagocytosis by neutrophils?
Bacterial cell wall components (weak)
C3b complement components (high affinity)
Fc region of antibodies (immune mediated opsonisation)
What can the innate immune system recognise?
Pathogen associated molecular patterns recognized by pattern recognition receptors (PAMPs and PRRs)
These recognise
- common cell wall structures including lipopolysaccharides and peptidoglycans
- bacterial metabolic products like N formylmethionine peptides (f-Met-leu-phe, formed by prokaryocytes exclusively)
- heat shock proteins released by stressed cells
These lead to danger cells alerting systems to do something
What are acute phase proteins?
Plasma proteins produced early in infection as a response to alarm mediators like IL-1. Many are produced in the liver. They act to enhance host resistance, minimize tissue injury, promote resolution and repair of lesions, and form part of the complement cascade
Give an overview of the different adaptive immune cells
B cells produce antibodies
NK cells do ADCC to function in early anti viral and tumour immunity
TH1 function in viruses and bacteria, with TH2 in parasites and allergies (intra vs. extracellular)
TH17 does mucosal surfaces and inflammatory processes
Treg down regulates inflammation
90% of these sit in the nodes, spleen and tonsils/adenoids/peyer’s patches
Describe lymphocyte circulation
about 10% circulate at any time
They can migrate from the blood into the tissue through high endothlial venules, or can leaves into the lymph nodes
They can then recirculate through the lymphoid system back to the blood, moving through lymphoid organs where they can contact processed antigens
Not all classes circulate to the same extent
How do antigens meet the adaptive immune system?
Non infectious is treated differently to infectious
Intracellular agents will be treated differently to extracellularly
They are taken up by APCs, leave and enter the lymph system, are transported to lymph nodes and presented to nodal lymphocytes
What are the functions of APCs?
Collection, concentration, processing and presentation of antigens
Co stimulation via cytokines and accessory surface molecules
Induction of tolerance by teaching the immune system to tolerate particular antigens
What is the endogenous pathway of antigen processing?
Done by class I MHC receptors
Cytosolic proteins are degraded into fragments by proteosomes
Peptides are inaccessible to MHC molecules, which are still bound on the ER
Peptides are transported to the ER lumen and inspected by TAP bound MHC
When a peptide binds tightly, the MHC folds around it and is transferred to the cell membrane
What is the exogenous pathway of antigen presentation?
Antigen is taken up from outside the APC into intracellular vesicles. Acidification of vesicles activates proteases to degrade the antigen into peptide fragements
Vesicles containing these fragments fuse with MHCII vesicles, and peptides with an affinity bind
This peptide is transported to the surface by the MHCII
Describe myaesthenia gravis
Autoimmune response against ACh receptors, causing defects at the NMJ- eg. causing diplopia at the eyes
Also presents with fatigue, weakness and dysarthria
Describe the pathway of platelet activation
Vessels vasoconstrict, reducing blood flow to the site of injury. Platelets stick to the exposed collagen via VWF, which attaches the GpIb receptor to the collagen. Aggregation follows, causing a shape change from a disc to a spiney sphere. This also causes release of vasoconstricting amines and adenine ucleotides that cause further activation of platelets, and initiate aggregation.
Aggregation is mediated by fibrinogen, which binds platelets together via integrin aIIb B3.
Desribe the coagulation cascade
The second stage of haemostasis, involving proteins rather than cells
The activation of coagulation causes formation of fibrin, which webs around the platelet plug, stabilising the clot.
The role of the coagulation pathway is in the generation of thrombin, which converts fibrinogen to fibrin. The pathway can be activated by contact activation on a charged surface, or one involving tissue factor (most physiologically relevant)
Define thrombosis
Formation of an abnormal thrombus when vessels wall is intact.
What is the difference between the makeup of venous and arterial thrombi?
Venous are formed of red cells and fibrin
Arterial are formed of platelets
What is the importance of cofactors in the coagulation cascade?
They line up substrates with their proteases, so that the binding sites fit together and can react
Give the steps of coagulation
Initial burst
- Tissue factor is exposed on the subendothelial tissues.
- Factor VII binds to TF, and changes shape to prep for activation
- Activated VII activates X to Xa, IX to IXa.
- Process is rapidly turned off by Tissue Factor Pathway Inhibitor, which accumulates due to platelet activation and binds to Xa and the VIIa complex
Thrombin Feedback
- Factor Xa has already converted a small amount of prothrombin to thrombin, in the presence of phospholipid to help binding, and calcium for shape control
- Thrombin amplifies the coagulation pathway by activating V, VIII (increase speed) and XI
- VIIIa, IXa, Ca and Phospholipid form a complex, which converts more X to Xa
- Xa complexes with Va, Ca and Phospholipid, which rapidly convert prothrombin to thrombin
- Thrombin converts fibrinogen to fibrin, as well as activating XI, which converts IX to IXa. It also activates factor XIII, which stabilises the chains (deficiency causes bleeding due to clot breakdown)
- The fibrin is cross linked together, which forms a stable web.
Describe the intrinsic pathway
Involves spontaneous conversion of XII to XIIa after contact with a negatively charged surface. XIIa activates XI and IX, initiating coagulation as above.
It is linked to the bradykinin pathway
Deificiency of XII doesn’t necessarily cause bleeding because it is mainly relevant for lab tests
However, it can be activated by the presence of central lines
What are the contact factors involved in the coagulation cascade?
XII, XI, and two factors in the intrinsic pathway
What are thrombin sensitive factors in the coagulation cascade?
Fibrinogen, V, VIII, XIII
What are the vitamin K dependent factors in the coagulation cascade?
II (thrombin), VII, IX, X
These all have a Gla domain held in shape by Ca2+
These need to be gamma carboxylated, which needs Vit K
Warfarin inhibits vit K, which increases bleeding risk
The same occurs in liver disease, and newborns, as these proteins are produced in the liver
What are the roles of thrombin?
Activations of Platelets VIII V XI Fibrinogen to fibrin by removing the a / b chains from the end and allowing cross linkage XIII cross linkage of fibrin Protein C
What are the inhibitors of the coagulation cascade?
Antithrombin
Protein C
Protein S
TFPI
Describe the action of antithrombin
Inhibits thrombin by forming a 1:1 complex. Also inhibits factor Xa and IX / XI
Desribe the action of protein S and C
C is activated to form activated protein c (APC) by thrombin in the presence of thrombomodulin (found in endothelial cells). S is a cofactor that enhances its action
These inactivate factors Va and VIIIa
Describe the actions of the fibrinolytic system
Digests the clot so that normal vessels structure can re-emerge
Converts plasminogen to plasmin using tPA and uPA
This causes the clot to turn into D dimers, and fibrinogens into fibrinogen degradation products- seen in high levels in inflammation and clot formation
What inhibits fibrinolysis?
Antiplasmin forms a 1:1 complex with plasmin
Thrombin activated fibrinolysis inhibitor (TAFI). Activated by thrombin in the presence of thrombomodulin, and remvoes lysine from fibrin, preventing plasminogen from binding to the clot
Describe the different haematologic malignancies
Myeloproliferative disorders like polycythaemia- mutation in stem cells causing proliferation of 1 or more types. show elevated RBCs, haematocrit and platelets
Lymphomas- enlarged lymph nodes- can be reactive, immune, cancer or metastasised cancer
Plasma cell disorders like myeloma- cancer of plasma cells causing lytic lesions in bones
Leukaemia
Describe leukaemia
Results from accumulation of abnormal, immature WBCs in the bone marrow, replacing normal elements and spilling over into the blood, resulting in marrow failure
Anaemia, thrombocytopenia neutropenia, leukocytosis and infiltration of other organs
Can be myeloid or lymphoid in origin
What are the different leukaemias?
Acute- rapidly progressive
ALL- due to B or T cell proliferation
AML- subtypes based on genetics, phenotype and morphology
Chronic- initially slow
CML- myeloproliferative disorder
CLL- related to lymphomas. Common in older people. Show palapable nodes and spleen, leukocytosis and smear cells
What are the clinical features of the acute leukaemias?
Bone marrow failure, showing anaemia (pale and lethargic), neutropenia (fever, infection, slow healing) and thrombocytopenia (bruising and bleeding)
Organ failure, showing tender bones, lymphadenopathy if lymphoid, hepato and splenomegaly, gum hypertrophy etc
What are the lab findings of acute leukaemias?
Normochromic anaemia
Increased WBC with blasts
Thrombocytopenia
Hypercellular bone marrow with >20% leukaemic blasts
What are the causes of leukaemia?
Chromosomal issues- philadelphia chromosome (translocation between BCR and ABL on 9 & 22, forming a fusion protein that switches on downstream pathways), acquired mutations causing impaired differentiation and maturation, increased proliferation
Predisposing factors include radiation (including for other tumours), familial issues, viral infections, aldylating agents, down syndrome
Most are idiopathic
Gene discovery can help prognosis and therapy decisions
How are acute leukaemias managed?
Supportive care and cytotoxic therapy
- Red cell transfusions, platelets and antibiotics (need indwelling central catheters)
Cytotoxic drug therapy can be for remission, consolidation (mop up leukaemic cells) or maintenance (if ALL)
ALL usually includes prophylaxis as there is increased relapse- so an easier but longer course
AML is shorter but more intense
Where do you source bone marrow transplants for leukaemic patients?
CCan have autologous- own cells when in remission
Allogenic- donated from sibling or unrelated. Can be from marrow, peripheral blood or cord blood.
What are the three main coagulation factor screening tests?
APTT
PR
TCT
Describe the APTT
Performed by collecting venous blood in citrate, which removes Ca2+ to stop clotting in the tube
Sample is spun and plasma is collected
Addition of an activator, phospholipid and Ca2+
Measure the time taken for a clot to form
What does the APTT measure?
All factors except factor VII, but is most sensitive to XII nad XI
Commonly prolonged in haemophilias due to lack of VIII / IX
What is a 1:1 APTT and what does it indicate?
A repeat of the test done with a mixture of known normal plasma
It will correct with factor deficiency
It will not correct with lupus anticoagulant (possibly due to an antiphospholipid syndrome), acquired factor inhibitors, heparin, or dabigatram
Not measuring natural inhibitors like protein C/S, antithrombin
Describe lupus anticoagulant
An antibody mediated antiphospholipid syndrome that occurs transiently in unwell patients
It doesn’t cause bleeding as they bind to phospholipids and interfere with clotting in the lab only
Describe the PR test
Ratio of prothrombin time to clot divided by normal clotting. It should be between .8-1.2
Performed by adding tissue factor to platelet poor plasmia in the persence of calcium
Sensitive to vitamin K dependent factors, so useful in calculating INR for warfarin monitoring and looking at liver function
Describe the TCT test
Adding thrombin to platelet poor plasma and measuring time for clot formation- normal depends on the conc of thrombin used
Test only measures concentration or function of fibrinogen in the plasma.
Also sensative to heparin, dabigatrain, FDPs
How does heparin cause prolonged APTT?
It upregulates antithrombin, but corrects with a protease in the lab
How does dabigatran cause a prolonged APTT?
It inhibits thrombin and doesn’t react with protamine.
Describe haemophilia A and B
They are X linked conditions with deformities in factor VIII (A) and IX (B).
It severe cases, it can cause arthropathy, muscle bleeds, renal, GI, intracranial bleeding. This causes tissue and nerve damage, deformity, arthritis and joint destruction
How is haemophilia treated?
Replacing the missing factor- almost always recombinants. Prophylactic is given in childhood to promote healthy joint development
Describe Von Willebrand’s disease
A defect of primary haemostasis, resulting from reduced or abnormal VWF. The screening test is platelet function assay. As it circualtes with factor VIII, this may also be reduced.
Shows as mucosal bleeding, and rarely functional defects
Commonly treated with DDAVP, which increases release of these components, or with plasma purified replacements in severe cases
What can be told from PT combined with APTT?
Long APTT with normal PT must be factor VIII and IX deficiency if severe, XI if mild, or XII if asymptomatic
Long PT with normal APTT must be factor VII (but occasionally can see mild deficiency in II, V, X and fibrinogen)
Both long: Factors V, X, fibrinogen and thrombin, possibly multiple defect
Describe multi-factor deficiencies
Due to
- warfarin, or else vitamin K deficiency, causing factor II, VII, IX and X deficiency, with normal fibrinogen
- Massive blood loss if also low fibrinogen
- DIC- widespread coagulation activation causing thrombosis and then bleeding. Shows los factors as they are used up. Eg. meningococcal disease, as bacteria release toxins
- Liver disease due to lack of production of all factors except VIII (as this is from endothelial cells)
Describe virchows triad in relation to thrombi
Clots are caused by three mechanisms
Stasis of blood flow, such as in immobility, pressure from a tumour or increased viscosity
Hypercoagulability, such as in increased procoagulants or decreased inhibits, or changes to the vessel wall, like atherosclerosis, trauma, surgery, or prior thrombus
What is hereditary thrombophilia and how can we test for it?
An inherited predisposition to blood clots- up to 1/2 of spontaneous cases are due to this
Normally due to activated protein C resistance secondary to factor V gene mutation
Factor V Leiden test: Direct DNA analysis. APCr is measured by coagulation based assay
Factor V is resistant to cleavage by APC
Can also be due to prothrombin 20210 mutation in promoter region, leading to excess prothrombin production
Can also be due to antithrombin deficiency
None of these are seen on regular coagulation tests
How do we diagnose VTE/PE?
Include unilateral leg pain, swelling, discolouration and oedema
PE shows shortness of breath, chest pain, tachycardia, tachypnoea and hypoxia
High risk patients are tested further with radiology (ultrasound for DVT, CT for PE)
Low risk patients have a D dimer test (breakdown product of fibrin)- if elevated, then onto imaging
How de we treat VTE?
Anticoagulants including warfarin (reduces vit K factors but takes several days) If INR rises above 4 bleeding is at increased risk
Heparin while we await warfarin’s buildup. Often subcutaneous. Accelerates inhibition of activated thrombin and Xa. Requires antithrombin and heparin binds to it.
Bleeding risk is higher in patients who are old, have renal impairment, prior haemorrhage or stroke, CHF or high alcohol intake
How do we prevent bleeding in patients on warfarin
Warfarin can be easily reversed by IV vitamin K
Also can replace plasma coagulation factors (prothrombinex)
What do dabigatran and rivaroxaban do?
Dabigatran- oral inhibitor or thrombin
Rivaroxaban- inhibits Xa
Would be used as warfarin has a large number of drug interactions
These can be equal to warfarin for VTE and better in atrial fibrillation, with less IC haemorrhage. However, they are excreted renally and so aren’t given to renal failure patients
How do dabigatran and rivaroxaban alter the coagulation tests?
Dabigatran: TCT very long, and log APTT (1+1) and PR
Rivaroxaban: Prolonged PR but APTT less so
How do you reverse DOACs?
There is a monoclonal antibody that binds to dabigatran. Rivaroxaban doesn’t have an antidote but can be helped by prothrombinex
What is osteomyelitis?
Infection and inflammation of bone or bone marrow
Can be caused by skin/soft tissue infections as bacteria can arrive through the blood
