Blood Components Flashcards
Eight main lineages of peripheral blood cells
Erythroid Neutrophil Monocyte/macrophage Eosiniphil Basophil Megakaryocyte T lymphoid B lymphoid
Where would you find most bone marrow?
Sternum, ribs, sacrum, vertebrae and long bones
Which organ apart from bone marrow is involved in generating non-lymphoid cells?
The spleen (although minor)
Primitive haematopoiesis
Haemangioblasts are generated from mesoderm in blood islands of the yolk sac in the embryo and then give rise to endothelial cells and primitive haematopoietic cells
Haematopoiesis then switches from mainly occurring in the yolk sac to the fetal liver, causing definitive haematopoiesis to begin
Definitive haematopoiesis
A second wave of blood cell production that generates long-term haematopoietic stem cells in the fetal liver and spleen and, towards the end of gestation and continuing as an adult, in bone marrow
Haematopoiesis and age
In infancy, haematopoiesis is present in all bones. With increasing age, it is focused in the proximal bones and the marrow space is increasingly replaced with fat cells. In diseased states, haematopoiesis can revert to the fetal pattern.
Extramedullary haematopoiesis
Resumption of haematopoiesis in the spleen and liver of an adult due to disease
Bone marrow and age
In infancy, all bone marrow is haematopoietic, but during childhood there is progressive fatty replacement of marrow throughout the long bones so that in a normal adult most haematopoiesis will occur in the central skeleton.
Fatty marrow is capable of reversion to haematopoietic marrow.
Where is marrow in the bone?
Past the cortical bone and in the trabeculae of the spongy bone
RBC life span
120 days
Platelet life span
5–6 days
Neutrophil circulation time
5–6 hours
Stem cell properties
Self-renewal
Generation on o-ll types
CD34
Antigen expressed by human haematopoietic stem cells which can be measured and used to identify HSC levels
Sources of HSCs
Bone marrow
Umbilical cord
Peripheral blood
3 key haematopoietic growth factors
EPO
TPO
G-CSF
EPO
Erythropoietin
Stimulates RBC production
TPO
Thrombopoietin
Stimulates platelet production
G-CSF
Granulocyte colony stimulating factor
Stimulates neutrophil production
Full blood count
Gives absolute numbers of different cell types in the peripheral blood
Blood film
A peripheral blood smear that is stained to show morphology of blood cells (done if FBC is abnormal)
Bone marrow examination
Can be done for bone marrow aspirate, which allows cytological examination of HSCs, or trephine biopsy, which allows histological examination of marrow architecture and cellularity
Key features of a normal RBC
7 microns in diameter
Discoid
No nucleus or RNA
What is the role of the discoid RBC shape?
Flexibility through narrow capillaries
Increased area for gas exchange
Oxygen transport
Haemoglobin carriage
What determines the unique shape and deformability of RBCs?
Cytoskeletal and membrane proteins, which allow the flexible discoid shape and therefore transport and oxygen carrying abilities
Hereditary spherocytosis
Genetic disorder in which the RBCs are less discoid and dented and more spherical due to abnormalities in membrane and cytoskeletal proteins. This results in shortened RBC lifespan
How do red blood cells keep haemoglobin in a reduced state?
Glycolytic pathways produce ATP and maintain an osmotic equilibrium
The HMP shunt produces NADPH which keeps haemoglobin reduced and therefore able to bind to oxygen
G6PD enzyme deficiency
Glucose-6-phophate dehydrogenase breaks G6P down into lactone which will go on to produce NADPH. In this deficiency, NADPH is not produced and neither is glutathione as a result of this, which normally functions to clean up free radicals resulting from oxidation. Therefore, people with G6PD enzyme deficiency are at risk of oxidising free radicals causing haemolysis of their red blood cells, leading to anaemia.
How does iron deficiency result in anaemia?
Iron is necessary for haem production. In iron deficiency, there is a reduced production of haem and therefore reduced haemoglobin, causing anaemia.
Thalassaemia
A collection of genetic blood disorders in which the production of globin is impaired, resulting in varied levels of anaemia depending on which and how many globin chains are affected
Steps of mature red blood cell formation
1) Progressive increase in haemoglobin
2) Chromatin clumping
3) Nucleus extrusion
4) RNA loss
Describe the kinetics of erythropoiesis
4 cell cycles/divisions
Process takes 7–10 days
Reticulocytes last 2 days
1 pronormocyte = 16 RBCs
Regulation of erythropoiesis
EPO – a glycoprotein produced in the kidney that responds to low oxygen levels
EPO feedback
EPO produced from peritubular interstitial cells of the cortex of the kidney
EPO influences three stages of erythrocyte development: the burst-forming units, the colony-forming units and the pronormoblasts
Pronormoblasts differentiate into reticulocytes, which become circulating RBCs
RBCs deliver oxygen to the kidney
If oxygen level isn’t high enough, kidney responds by producing more EPO. If it is high enough, kidney responds by stopping EPO production
Effects of EPO
Stimulation of BFU-E and CFU-E Increased haemoglobin synthesis Reduced RBC maturation time Increased reticulocyte release Overall, increased Hb and increased O2 delivery
Role of JAK2 in EPO effects
EPO receptors are monomers that dimerise to allow an EPO molecule to bind
The dimerised transmembrane EPO receptor causes JAK2 (which is located on the intracellular part of the receptor) to become autophosphorylated and activated
STAT5 and MAPK signalling cascades ensue, causing gene activation and transcription of RBC growth regulators
Polycythaemia vera
A condition in which mutated JAK2 kinases cause their EPO receptors to be constantly dimerised even without EPO, resulting in overtranscription of RBC growth regulators and causing thick, sticky blood filled with RBCs
Clinical use for recombinant anaemia
Anaemia of renal failure
Other anaemias e.g. myelodysplastic syndromes
Red blood cell destruction
When RBCs accumulate oxidative damage, they become less deformable and are removed in the liver and the spleen
When broken down, Hb is released, which breaks down into globin chains and haem
Haem breakdown
Haem is broken down into iron which is recycled in the bone marrow and protoporphyrin which is converted to bilirubin and excreted as bile via the liver
Why do patients that are haemolysing appear slightly jaundiced?
Increased unconjugated bilirubin circulating due to haem being broken down into iron and protoporphyrin – the protoporphyrin is converted into bilirubin for excretion
Anaemia
Lower than normal haemoglobin concentration for sex and age of patient
Less than 135 g/L in adult males
Less than 115 g/L in adult females
Less than 140 g/L in neonates
A reduction in Hb is normally (but not always) accompanied by a fall in:
PCV
PCV
Packed cell volume
Also known as haematocrit ratio; ratio of RBC volume to plasma volume
Masked anaemia
Because PCV normally accompanies Hb in decreasing during anaemia, if a person is dehydrated and the plasma volume decreases, it can look like the haemoglobin concentration is higher than it actually is and the ratio will appear normal, therefore dehydration can “mask” anaemia or cause polycythaemia
Key symptoms of anaemia
Shortness of breath
Tiredness
Angina
Key signs of anaemia
Pale conjunctiva
Pale palmar creases
Clinical features of anaemia
Increased cardiac stroke volume
Tachycardia
Right shift in haemoglobin dissociation curve (to make oxygen more readily available for tissues)
Eventually, congestive heart failure
Ways to classify anaemia
Pathogenetic, i.e., reduced production vs. increased loss
Morphological, i.e., microcytic or macrocytic
Normal reticulocyte count
0.5–2.5%
25–125 x 10^9/L
Reticulocyte count in anaemia
Reticulocyte count rises in anaemia secondary to increased EPO levels. After an acute haemorrhage, the reticulocyte count rises within 2–3 days and peaks at 6–10 days. Remains high until Hb returns to normal.
If an anaemic patient does not have a raised reticulocyte count, what does this indicate?
Impaired bone marrow function or lack of EPO stimulus
Factors that impair the normal reticulocyte response
Marrow disease Iron, folate, B12 deficiencies Lack of EPO i.e., renal disease Ineffective erythropoiesis e.g., in thalassaemia and myelodysplastic syndromes Chronic inflammation or malignancy
Pathogenetic/aetiological classification
Based on the cause of the anaemia
Anaemia results from one of three fundamental disturbances: impaired RBC formation by bone marrow, blood loss, or excess haemolysis
Morphological classification
Based on RBC appearances under a microscope, MCV, and MCHC
MCHC
Mean cell Hb concentration
Normocytic anaemia
MCV within normal range, i.e., 76–96 fL
Most are also normochromic, i.e., MCHC 310–350 g/L
Hypochromic normocytic anaemia
MCV reduced i.e., less than 76 fL
MCHC also reduced i.e., less than 310 g/L
Macrocytic anaemia
MCV increased i.e., more than 96 fL
Most are also normochromic, i.e., MCHC 310–350 g/L
Anaemia diagnosis
1) Determination of morphological type of anaemia
2) Determination of cause of anaemia
Causes of impaired production anaemia
Deficiency of substances essential for RBC production
Genetic defect in RBC production
Failure of bone marrow
Causes of reduced survival anaemia
Blood loss; usually acute but can be chronic
Haemolysis; can be environmental or intrinsic problem with RBCs themselves
Causes of microcytic hypochromic anaemia
Iron deficiency Chronic illness (iron block) Genetic
Diagnosis of iron deficiency
Measure serum iron, iron binding capacity, and iron saturation
Measure serum ferritin
Can examine iron stores in bone marrow, but rare
Iron binding capacity
Number of spaces available to transport iron
Also named transferrin, i.e., the main protein that transports iron
Iron saturation
Serum iron as a fraction of transferrin
Anaemia of chronic disorders laboratory findings
Serum ferritin normal (or slightly elevated)
Serum iron low
Iron binding capacity normal
Iron saturation high
4 main causes of iron deficiency
Diet
Malabsorption
Increased demand e.g., pregnancy
Chronic blood loss
Causes of anaemia of chronic disease
Underlying malignancy
Chronic inflammation e.g., rheumatoid arthritis
These will present with mild anaemia, low serum iron, low iron binding capacity, normal iron saturation and normal or slightly raised serum ferritin
Can also be genetic e.g., thalassaemia
Macrocytic anaemia causes
Vitamin B12 deficiency Folate deficiency Liver disease Hypothyroidism Alcoholism
Consequences of B12/folate deficiency
Impaired DNA synthesis, which can result in abnormal WBCs too
Diagnosis of B12/folate deficiency
Measure serum vitamin B12 and folate levels
Determine cause of low vitamin B12/folate
Causes of low vitamin B12
Diet (uncommon)
Malabsoprtion due to gastrectomy or pernicious anaemia
Terminal ileum disease
Thalassaemia
Genetic mutation in globin chain production gene causing decreased oxygen carriage
Looks like iron deficiency
Heterozygotes tend to have mild anaemia, homozygotes much more severe, also dependent on particular mutation
Diagnosis of thalassaemia
Haemoglobinopathy screen
Can also do genetic testing for couples wanting children
Causes of low folate
Diet (most common)
Malabsorption
Increased demand/utilisation
Haemolysis
Haemolytic anaemia
Due to RBC destruction
Presents with pallor, mild jaundice, splenomegaly, raised bilirubin, reduced haptoglobins
Also shows reticulocytosis and damaged RBCs
Classification of haemolytic anaemia
Intrinsic RBS defects, usually hereditary e.g., membrane defect
Environmental, usually acquired e.g., autoimmune
Describe megakaryocyte differentiation
1) MK development in the bone marrow from megakaryoblasts
2) Endomitosis: Synchronous nuclear replication causing cytoplasm enlargement and increase in nuclei number
3) Cytoplasmic maturation: Production of components that constitute the mature platelet, then extensive membrane system with invaginations of the plasma membrane develops
4) Proplatelets develop: Long filopodia extend into marrow capillaries
5) Fragmentation: Proplatelets from megakaryocyte fragment, producing mature platelets
6) Platelets are released from bone marrow to circulate in the blood
Thrombocytopoiesis
Thrombopoietin, along with various cytokines, regulate megakaryocyte and platelet development
Platelet homeostasis
Platelet numbers in the circulation are maintained at a constant level
Approximately 1/3 platelets do not circulate and remain in the spleen
Normal platelet life-span about 7–10 days
Platelets consumed by senescence and utilisation in haemostatic reactions
Platelet structure
Discoid, with intricate system of channels consistent with the plasma membrane
Phospholipids of the open membrane + the plasma membrane = large surface area for selective absorption of plasma coagulation proteins
Glycoproteins on surface coat important for platelet adhesion and aggregation
Submembranous area contains contractile filaments and circumferential skeleton of microtubules that maintain the discoid shape
Electron dense granules in platelet cytoplasm
Ca+2 Mg+2 ATP ADP Serotonin + other vasoactive amines
Alpha granules in platelet cytoplasm
Coagulation factors Platelet-derived growth factor TGF-B Heparin antagonist Fibronectin Albumin
Granule release from platelets
Energy for platelet reactions provided by oxidative phosphorylation in mitochondria and utilisation of glycogen in anaerobic glycolysis. Contents of granules (alpha and dense granule contents) released into open membrane system.
Platelet function
Formation of mechanical plugs during vascular injury
Adhesion, aggregation, secretion, contraction, procoagulation
Platelet adhesion
von Willebrand factor recruited out of blood
VWF binds subendothelial collagen fibres, leading to a conformational change in VWF
Conformational change of VWF allows it to bind to glycoprotein Ib–V–IX on the platelet surface
Binding of VWF allows platelet to express integrin alpha IIbB3, which allows granule release
Factors from granules recruit more platelets
Platelet plug secured with fibrinogen
Platelet aggregation
Fibrinogen binds to receptors on activated platelets and links platelets to each other
Platelets adhere to collagen, causing the platelets to become more spherical and extrude pseudopods which enhance the interaction between platelets
Thromboxane A2, a prostaglandin, is synthesised, which activates platelet aggregation and the release reaction
ADP secreted, thromboxane A2 generation and activation of coagulation = thrombin production
Other platelets recruited
Platelet plug formed
Fibrin clot forms and contracts to seal injury
Platelet secretion
Collagen + thrombin activates platelet prostaglandin synthesis
Thromboxane A2 forms, which lowers platelet cAMP levels
Release reaction takes place, causing secretion fo alpha and dense granule contents
Clopidogrel
A drug that binds the P2Y12 receptor (GPCR) on platelet surface, which is usually bound by ADP and activated, leading to aggregation. Clopidogrel irreversibly inhibits this receptor, preventing aggregation.
Platelet quiescence
NO and prostaglandins released from intact endothelium, signalling to platelets to stop their release reactions
Aspirin
Inhibits thromboxane A2 production by covalent acetylation of cyclo-oxygenase which introduces a permanent defect to the platelet, therefore prevents platelet aggregation and also prevents vasoconstriction
Abciximab
iia/iiib inhibitor
Blocks fibrinogen and stops it knitting together, therefore stops fibrin plugs
DIC
Disseminated intravascular coagulation e.g., in meningococcal sepsis
Blood clots form throughout the body and block small blood vessels, causing petechiae
Decreased platelet production causes
Infection
Drugs
Bone marrow failure
Immune thrombocytopaenia
Increased destruction of platelets therefore bone marrow biopsy likely to show increased megakaryocytes
Myeloproliferative neoplasm
Increased production of platelets
Causes of thrombocytopaenia
EBV
Myelodysplasia (can progress to AML)
Drugs
Glanzmann’s thrombasthenia
Glanzmann’s thrombasthenia
No alpha IIbB3, therefore platelets can’t knit together
Autosomal recessive condition
Isolated thrombocytopaenia
Very low platelet count in absence of other blood disorders
Required repeat testing to rule out lab error e.g. platelet clumping on exposure to EDTA
Rule out viral infection or autoimmune disease
Immune thrombocytopaenia
Autoimmune condition causing antibodies to attack platelets
Massive platelet destruction
Donor platelets rarely make a difference as antibodies attack these too
Treated by prednisone, IV Ig, thrombopoietin mimetic, splenectomy
Thrombopoietin mimetic
Drug that binds C-MpI receptor to prevent immune-mediated platelet destruction
Three major components to prevent blood loss
The vessel wall
Platelets
The coagulation cascade
Haemostasis
A dynamic process involving a balance between the components that favour clot formation and those that prevent clot formation
Prohaemostatic components
Platelets
Activated coagulation proteins
When increased, thrombosis can occur
Antihaemostatic components
Physiological inhibitors
Fibrinolytic proteins
When increased, haemorrhage can occur
Three elements of haemostasis
Changes in the vessel wall
Changes in the components of the blood
Stasis
Primary haemostasis process
Vessel injury
Vasoconstriction = reduced blood flow
Damage to endothelial wall exposes collagen in subendothelial tissue = platelet activation
Platelet plug formation
Development of unstable clot
Platelet adhesion via VWF
Platelet aggregation and release reaction
Vasoconstricting amines released = further platelet activation and aggregation
Fibrin
Protein that forms a web around the platelet plug to form a stable clot
Formed via the coagulation cascade
Thrombin
Protein generated in the coagulation cascade that converts circulation fibrinogen into fibrin
Tissue factor
Following vessel trauma, TF is exposed to the circulation and binds factor VII to start the clotting cascade
TFPI
Tissue factor pathway inhibitor
Present in plasma and platelets and accumulates at site of coagulation due to local platelet activation
Binds factor Xa and TF-VIIa complex to prevent further Xa activation
Fibrinogen
Composed of three strands: alpha, beta and gamma
Thrombin cleaves small fragments of the alpha and beta strands, allowing the chains to polymerise into long fibrin strands which are stabilised by factor XIIIa
Intrinsic pathway
AKA contact activation
Coagulation via contact with a negatively charged surface
Factor XII spontaneously converted to factor XIIa which activates XI and XIa and IX to IXa, initiating coagulation
Forms the basis of the APTT clotting test
APTT clotting test
Allows the observation of the intrinsic pathway
Identifies deficiencies of factors XII, XI, IX and VIII
Contact factors
XII
XI
High molecular weight kininogen
Prekallikrein
Thrombin sensitive factors
Fibrinogen
V
VIII
XIII
Vitamin K dependent factors
II
VII
IX
X
How does vitamin K work?
Vitamin K is a pro-clotting molecule that is necessary for the gamma carboxylation of glutamic acid residues that bind to phospholipids interacting with Gla domains on the vitamin K dependent factors (II, VII, IX, X)
Describe the role of the Protein C/S inhibitory system
Protein C is activated to “activated Protein C” (APC) by thrombin in the presence of the cofactor thrombomodulin. Protein S is a cofactor that enhances the action of Protein C. Activated Protein C circulates and inhibits VIIIa and Va. Both of these actions prevent Xa activation and therefore prevent prothrombin being converted to thrombin and therefore prevent fibrinogen being converted to fibrin
Antithrombin
Protease that inhibits Xa and thrombin, preventing fibrinogen conversion to fibrin. Deficiency is severe; heterozygotes majorly prone to thrombus formation and homozygotes thought to be incompatible with life.
Intrinsic pathway of the clotting cascade
XII ---> XIIa XIIa converts XI ---> XIa XIa converts IX ---> IXa Factor VIII (circulating, bound to VWF, produced in the liver) separates from VWF when injury discovered in blood vessel wall and activates to VIIIa VIIIa + IXa convert X ---> Xa
Extrinsic pathway of the clotting cascade
VII (circulating, produced in liver) activated to VIIa by exposure to TF (in blood vessel walls, only exposed when vessel injured)
VIIa + TF converts X —> Xa
Common pathway of the clotting cascade
V (circulating, produced in liver) binds activated platelets and is activated by thrombin to Va
Xa + Va convert prothrombin —> thrombin
Thrombin converts fibrinogen —> fibrin monomer and XIII to XIIIa
XIIIa converts fibrin monomer to fibrin polymer
Vitamin K deficiency
Most likely to be from inability to absorb fat and soluble vitamins, e.g., due to liver disease (decreased bile production)
Blood group antigen
Glycoprotein or glycolipid present on the surface on the surface of a red blood cell
Two ways that blood group antibodies can occur
Naturally: Occur in the absence of exposure to corresponding red cell antigen e.g. ABO antigens. Develop as an immune response to substances found in the environment with similar antigenic determinants.
Immune: Occur following exposure to corresponding antigens e.g. after transfusion or transplacental haemorrhage.
Naturally occurring antibody characteristics
Usually glycolipid
IgM (sometimes IgG)
Activate complement
Intravascular haemolysis
Immune stimulated antibody characteristics
Usually glycoprotein
IgG
Do not activate complement
Extravascular haemolysis
Terminal sugars of the ABO antigens
A = N acetylgalactoamine B = D galactose O = nothing
Where are ABO antigens found?
Red cells
Platelets
Granulocytes
Epithelial cells
Where are Rh antigens found?
Only on red cells
Haemolytic disease of the newborn
Red cell antibodies form in the mother and cross the placenta resulting in destruction of foetal red cells causing severe anaemia and death if untreated. Most commonly occurs as a consequence of RhD incompatibility but rarely can also occur with ABO. Anti-D immunoglobulin given to all RhD -ve women who give birth to an RhD +ve child.
Why is ABO haemolytic disease of the newborn rarely significant?
1) ABO antigens are poorly expressed in the developing infant
2) ABO antigens are widely distributed in placental tissue. Available antibodies attach to these antigens and in endothelial cells, so low levels ever reach the foetus.
HDFN treatment
Generally, all women blood typed during the first birth. RhD negative women will receive Anti-D immunoglobulin and will be monitored closely. Intrauterine transfusion and photothereapy and exchange transfusion for the newborn after birth can be used.
Blood product vs. blood component
Blood product: Any product derived from human blood
Blood component: Blood product manufactured in local centre
4 strategies to maintain a safe blood supply
1) Voluntary donors
2) Excluding high-risk donors
3) Blood donation testing
4) Physical and chemical methods to destroy pathogens
New Zealand guidelines for prospective blood donors
Must be in good general health
Between 16 and 70
Must complete a questionnaire to identify medical and lifestyle factors that might injure the donor or recipient
Shelf life of blood components
RBCs: 35 days
Platelet concentrates: 5 days
FFP: 2 years (when thawed, 1–5 days)
At what level of anaemia should you transfuse?
Below 70 g/L Hb
Between 70 and 100 g/L Hb, may be appropriate after surgery with significant blood loss or when specific symptoms imply there would be a benefit
Pretransfusion testing steps
1) Correct patient identification, sampling and labelling at bedside
2) Blood typing
3) Antibody screen
4) Selection of appropriate cells
5) Final crossmatch
