Haematological Pathology Flashcards
What is Haemopoiesis?
- Physiological developmental process that gives rise to cellular components of blood (from stem cells)
- A single multipotent haemopoietic stem cell can divide & differentiate to form diff cell lineages which will populate the blood
- (Haemopoiesis= Haematopiesis= hematopoiesis)
- Stem cells give rise to sufficient n.o.s of committed haemopoetic progeniators to maintain bloods cellular contents throughout individuals lifespan.
- Differentiation; myelopoiesis, lymphopoiesis.
- Mature blood cells have a finite life e.g. an erythrocytes (RBC)- 120 days, neutrophils (WBC)- 6-10 hours in bloodstream.
- Serial divisions & prolifs that end in specialised blood cells
What are the properties of Haemopoietic Stem Cells?
- Self renewal
- High proliferative potential
- Differentiation potential for all lineages of cells in blood stream
- Long term activity throughout individuals lifespan
- Experimental proof from stem cells transplanted from 1 mouse to another over several generations- stem cells keep working.
What are the origins of Haemopoiesis?
- Fertilised ovum= replicates= morula embeds in wall= trophoblast; areas where hemopoieses can start.
- Yolk sac
What is the timescale of Haemopioesis in the embryo?
Haemopioesis strats in Aorto-Gonado- Mesonephros
• Day 27: Haemopioesis starts at in aorta gonad mesonephros region.
• Day 35: Expands rapidly
• Day 40: then disappear at day 40- ‘disappearance’ correlates with haematopoietic stem cells to foetal liver which becomes subsequent type of haemopioesis
• Area in base of aorta- is the AGM where primitive stem cell they then perculate through aorta wall to the liver.
What is the function of Blood Cells?
Blood Cell Function • Oxygen transport • Coagulation (haemostasis) • Immune response to infection • Immune response to abnormal cells (screens out senescent & malignant cells)
What are erythrocytes? What happens if they are reduced? What happens when they are increased?
RED BLOOD CELLS •Bi-concave discs, 7.5 µM diameter •Lifespan in blood: 120 days (3mnths) •Contain Hb •333,200 x 106 red cells •Reduced RBCs= ANAEMIA •Raised RBCs= POLYCYTHEMIA (Relative polycythaemia- when plasma vol reduced so looks like RBCs high)
What are some common leukocytes? What is the function of leukocytes?
WHITE BLOOD CELLS • Functions: immunity & host defence • Granulocytes- have cytoplasmic granules; o Neutrophils o Eosinophils o Basophils • Monocytes • Lymphocytes
What are neutrophils?
- Phagocytes
- Polylobed nuclei (n.o. of lobes can indicate anaemia)
- Most common EBC in adult blood (~ 10x109 per litre)
What is neutrophilia?
Increased numbers of Neutrophils= neutrophilia e.g. bacterial infec/ inflamm
What is neutropenia?
decreased numbers of Neutrophils = neutropenia e.g. side effect of drug (steroids act on neutrophils on endothelial wall = cause neutrophils to leave wall & float out into blood (demargination))
What is eosinophilia ?
Increased numbers of Eosinophils = eosinophilia e.g. parasitic infec, allergies (e.g. asthmatic)
What is basophilia ?
Increased numbers of basophils: e.g. chronic myeloid leukaemia (member of myloprolif neoplasms)
What are monocytes?
- Phagocytic &; antigen presenting cells
* Migrate to tissues- then i.d. as ‘macrophages’ or ‘histiocytes’ e.g. kupffer cells (liver), Langerhans cells (skin).
What is monocytosis ?
•Increased numbers of monocytes= MONOCYTOSIS e.g. tuberculosis
What is lymphocytosis?
•Increased numbers of Lymphocytes: lymphocytosis e.g.
oAtypical lymphocytes of glandular fever (infectious mononucleosis)
oChronic lymphocytic leukaemia
What is lymphopenia?
•Decreased numbers of Lymphocytes: - lymphopenia e.g.
o Post bone marrow transplant (lymphocyte cells one of last to regenerate after this
What are Natural Killers cells?
Lymphocytes
• Innate immune system
• Larger granular lymphocytes
• Recognise ‘non-self’ (cells, viruses)- go round body looking for strange/ infected cells
What are T-lymphocytes?
Lymphocytes • Adaptive immune system (specific response to attack) • Multiple sub-types e.g. CD4 antigen • Rearrange T-cell antigen recep • Cell-mediated immunity • Target- specific cytotoxicity • Interact with B cells &; macrophages • Regulate immune response
What are B-lymphocytes?
Lymphocytes
• Adaptive immune system
• Rearrange immunoglobulin genes to enable antigen specific antibody production (germline configuration edited)
• Humoral immunity- immunoglobulins
• Increased numbers of plasma cells (make antibodies)- PLASMOCYTOSIS (can be benign/ malignant) e.g. infec, myeloma
What is a Full Blood Count (FBC)?
• Hb conc
• Red cell parameters;
o MCV (mean cell vol- size of individual RBC)
o MCH (mean cell Hb- how much colour in RBC)
• White cell count (WCC)
• Platelet count
Interpreting Full Blood Count
• Be alert to technical probs e.g. thrombocytopenia (may be real/ artefact)
• If in doubt if its an actual prob- repeat taking blood test
• Abnormal results; flagged by lab, may trigger additional tests e.g. blood film
• Serious urgent abnormalities- lab staff will alert on-call docs e.g. new leukaemia
What is a Coagulation Screen?
• Tests measure time taken for clot to form when plasma is mixed with specified reagents
• Can analyse diff parts of coag cascade;
o Prothrombin time (repeat this when patient on warfarin)
o Activated Partial Thromboplastin Time (unrationed heparin)
o Thrombin time (overall view of coag system in patient)
How do you take a blood sample?
• Accurate full blood count (FBC) & correct blood film interpretation;
o Appropriate sample from patient
o Collected into EDTA (anticoagulant) anticoagulated blood;
- Mixed well (shake gently)
- [K2EDTA] = 1.5 - 2.2 mg ml-1
- Blood should be filled to the line on tube
o Samples to lab promptly- as EDTA artefact can affect results.
Where is bone marrow obtained from?
- Local anaesthetic
- Liquid marrow aspirated from post. Iliac crest of pelvis
use tool called trephine to make a hole
What is a reference range?
Establishing a Reference Range
• Define reference pop- should be relevant to test pop
• Consider if diff reference ranges needed for; adults vs children, men vs women etc.
• Determine expected range of interindividual variation
Reference Range
• Set of values for a given test that incorporates 95% of normal pop
• 95% is arbitrary convention- determined by collecting data from vast n.o. of lab tests
• More people sample- less likely will be distorted
95% Results Fall Within Reference Range = normal
- Differentiates between healthy & ill.
- False Neg &; False Positive= need to figure out how good is test.
What is Sensitivity?
- Proportion of ABNORMAL results correctly classified
- Expresses ability to detect a true abnormality
- Sensitivity= TP/ (TP+FN)
TP-true positive
FN- false negative
What is Specificity?
- Proportion of NORMAL results correctly classified by test
- Expresses ability to exclude abnormal result in healthy person
- Specificity= TN/ (TN+FP)
- (True normal people divided by all the normal people)
TN-true negative
FP- false positive
What is the importance of looking at clinical data as well as Normal Ranges?
• Know your patient- give clinical details
• A FBC may fall outside ‘normal range’ BUT be appropriate for the given clinical situation e.g. abnormal lymphocyte count;
o Post-splenectomy (spleen out) mild lymphocytosis
o 3 months post-bone marrow transplant lymphopenia (so lymphocyte levels should be low at that point)
What is Microcytic Hypochromic Anaemia ?
- MCV <80 fl & MCH <27 pg
- Small RBCs made due to not enough Hb
- Iron deficiency
- Thalassaemia
- Anaemia of chronic disease (some)
- Lead poisoning
- Sideroblastic anaemia (some cases)
MICROCYTIC- small red cells, iron deficiency, Hb disorders (e.g. sickle cell, sometimes chronic disease)
• Anaemic patient in front of you- look at MCV, have their drug history- lead you down one of them
What is Normocytic Normochromic Anaemia ?
- MCV 80-95 fl (normal) & MCH ≥ 27 pg (normal)
- Failure to make enough RBCs (e.g. if get rheumatoid arthritis- turns vol of bone marrow down so less RBCs produced, but RBCs themselves are normal)
- Many haemolytic anaemias
- Anaemia of chronic disease (some cases)
- After acute blood loss
- Renal disease
- Mixed deficiencies
- Bone marrow failure (e.g. post-chemo, infiltration by carcinoma)
• NORMOCYTIC- normal red cells, anaemia of chronic disease/ inflammatory
What is Macrocytic Anaemia?
- MCV >95 fl
- Megaloblastic (defective DNA synthesis = abnormally large erythroblasts): vit B12 or folate deficiency
- Non-megaloblastic: alcohol, liver disease, myelodysplasia, aplastic anaemia etc
What is Iron Deficiency Anaemia ?
- Small pale red cells (low MCV &; MCH)
- Variable size & shape- long thin ‘pencil’ cells.
- Common in women esp. if have multiple preg, don’t eat meat & menstruating
What is Vit B12 Deficiency?
- Hyper segmented neutrophils &; oval mastocytes
- Big RBCs.
- Neutrophils hypersegmented
What is Sickle Cell Anaemia?
- Hypoxia = red cell distorted = sickle cells.
* Sickle cells crisis = bone pain (medical emergency!)
What cellular changes occur in Erythropoiesis ?
- Nucleus changes; first open, then starts to shut down becomes darker, then extruded from RBC precursor (nucleus seen last time in reticulocyte- sometimes seen in bloodstream).
- Megaloblastic change- nucleus stays open longer (?)
What are the causes of Macrocytic Anaemia ?
Cause of Macrocytic Anaemia • Megaloblastic anaemia (i.e. with megaloblastic erythropoiesis)- e.g.; o vit B12 o folic acid deficiency o myelodysplastic syndrome • Macrocytic anaemia with normoblastic erythropoiesis e.g. o liver disease o alcohol o hypothyroidism o myelodysplastic syndrome • ‘Stress’ haemopoiesis e.g. o Haemolytic anaemia o Recovery from blood loss (macrocytes reflect high reticulocyte count- reticulocytes make machine mistakenly thinks MCV high )
What is Myelodysplastic Syndrome?
• Myeloid lineage disorder; red cells, platelets, white cells- any of these can be reduced
• How you are affected depends on which cytopenia you have (which cell type is reduced)
• Typically disease of mid-life &; older people, occasionally in young people
• May be complication of chemotherapy/ radiotherapy treatment
• Manifestations vary;
o Chronic anaemia (survival several yrs)
o Aggressive disease terminating in acute myeloid leukaemia- abnormal chromosome mutations in cells in marrow, lost of circulating blasts (primitive cells).
• Prediction of outcome in individual patients; Revised International Prognostic Scoring System (IPSS-R) for Myelodysplastic Syndromes Risk Assessment Calculator
What are the characteristics of Myelodysplastic Syndrome?
- Mutated stem cell makes myeloid clone of abnormal cells- replaces normal haemopoiesis
- Hyperproliferative, lots of cells in marrow but die prematurely
- Abnormal cells; morphological abnormal maturation, often die before leaving bone marrow
- Cellular marrow (ususally) but peripheral blood cytopenias
What are the lab features of Myelodysplastic Syndrome?
• Typical blood results; o Anaemia o Neutropenia o Thrombocytopenia (single cytopenias or as a pancytopenia) • Symptoms o Anaemia- fatigue, dyspnoea o Neutropenia- infec • Thrombocytopenia- bruising & bleeding
Bone Marrow in Myelodysplasia (MDS) is Cellular - Irregular shaped nuclei &cytoplasm
What are the subtypes of Myelodysplastic Syndrome?
- MDS has several subtypes- may be recognised morphologically/ genetically (chromosomes)
- e.g. Refractory Anaemia, Refractory anaemia with excess blasts, MDS 5Q-syndrome
- MDS 5Q syndrome- chromosome 5 partail loss of q arm (long arm as Q longer than the letter P)
What is the therapy for Myelodysplastic Syndrome?
•SUPPORTIVE;
o Red cell infusions (iron chelation for iron overload)
o Erythropoietin hormone (try to increase amount of RBCs they make)
o Platelet transfusions
•ACTIVE THERAPY- DISEASE SPECIFIC
o Lenalidomide for 5q- syndrome
o Azacytidine (if not fit for more intensive treatment e.g. transplant)
o Trial of immunosuppression in minority of patients
o Chemotherapy & bone marrow transplant (younger fitter patients)
What is Leucodepletion?
•Leucodepletion- whole blood filtered before further processing to remove white cells (reduce complications from WBCs & reduce transmission of creutzfeld Jacob disease)
What happens to blood after donation?
- Blood given by donor
- Leucodepletion- whole blood filtered before further processing to remove white cells (reduce complications from WBCs & reduce transmission of creutzfeld Jacob disease)
- Blood then centrifuged and separated into 3 components; RBCs, platelets &plasma
- Plasma either frozen (2 ways) or fractionated to produce plasma derived components which contain specific products
What are the Indications of Transfusion of RBC?
• 1 unit RBC;
o Usual transmission time: 1.30- 3 hrs
o 4 hr limit from removal from cold storage to end of transfusion
o Use blood warmer for rapid transfusion
• Stored at 4 degrees celcius for up to 35 days collection
• (Refer to Trust Blood Transfusion Policy)
• Most of plasma usually removed- leave ‘concentrated red cells’ &; replaced by solution of electrolytes, glycose & adenine (solution keeps RBCs healthy during storage)
What are the 2 stages in Pre-Transfusion Lab Testing for RBCs ):
(don’t need to do them for platelets/ plasma)
- Tests on Patient- ‘Group &; Screen’
• Determination of ABO & Rh(D) group (of RBCs of patients)
• Patients plasma ‘screened’ for antibodies against other clinically signif blood group antigens (other than ABO group);
o If -ve- no further testing
o If +ve- identify antibody; test patients plasma against panel of RBCs containing all clinically signif blood groups antigens (use antiglobulin test) - Compatibility Testing- ‘Crossmatching’
• Donor red cells with same ABO & Rh group as patient selected from blood bank
• Avoid any other groups the patients has antibodies against (detected in screen- if patient has antibodies choose blood that’s negative for the antigens)
• Crossmatching- patients plasma mixed with aliquots of donor RBCs- see if reac occurs (agglutination or haemolysis);
o If no reac- RBC units compatible, no risk of acute haemolysis so can transfuse the unit
o If reac- RBC units incompatible, risk of acute haemolysis so don’t transfuse the unit
Why we transfuse patients?
- Prevent anaemia symptoms- TRUE
- Improve quality of life of anaemic patients- TRUE
- Prevent ischaemic damage of end organs in anaemic patients- TRUE
What is anaemia?
Anaemia- condition in which n.o. of RBCs/ their O2 carrying capacity insufficient to meet physiological needs (which vary be age, sex, altitude, smoking &preg status)
• Can’t measure amount of O2 carried, so measure Hb level to determine anaemia;
o Non preg women (15yrs &; above) <120g/L
o Men (15yrs & above) <130g/L
What is the Mechanism of Adaptation to Anaemia?
- Increased cardiac output
- Increased cardiac artery blood flow
- Increased oxygen extraction
- Increased RBC 2,3 DPG (disphosphoglycerate)
- Increase EPO produc
- Increase erythropoiesis
- AIM of these mechanisms: maintain tissue oxygenation (prevent tissue hypoxia)- not all patients display symptoms, only those that can’t adapt to anaemia.
- Acute anaemia- resp rate more markedly increases than in chronic anaemia.
- Chronic anaemia- O2 extraction increased due to rise in 2,3 DPG, kidneys respond to hypoxia by increasing erythropoietin produc which results in increased erythropoiesis.
What are the symptoms of anaemia?
SYMPTOMS OF ANAEMIA= TISSUE HYPOXIA!!!
• So only patients that fail to use adaptation mechanisms to adjust to anaemia develop symptoms (not everyone)
• Not all patients develop symptoms of anaemia at same level of Hb- diff factors that affect adaptation mechanisms so each individual adapts diff (so don’t have 1 Hb threshold that determines if anaemic)
How we determine transfusion threshold that will prevent hypoxia in any individual patient?
Parameters That Affect Adaptation Mechanisms to Anaemia
• Underlying conditions that affect cardiac output, arterial blood flow, O2 saturation of Hb impair adaptation mechanisms to anaemia
• E.g. underlying conditions that can impair these mechanisms; cardiovasc diseases, resp diseases, age
• Patients with acute anaemia don’t have time to develop all adaptaion mechanisms so develop symptoms earlier than patients with chronic anaemia.
What do you need to consider when making the Clinical Decision to Transfuse?
Consider;
• Expected benefits; treat/ prevent anaemia & avoid long term effect of ischeamia symptoms
• Harms; adverse events of transfusion
• Cost; days of hospitalisation, use precious resource.
• Availability of alternatives (if can treat them, will treat them)
• Patient’s wishes (e.g. may refuse to be transfused)
• Clinical evidence
Then determine transfusion threshold &a target
How do you Determine Ideal Hb Conc for Various Groups/ Individual Patients?
- Transfusion threshold (trigger)- Hb conc at or below which packed RBCs usually ordered for transfusion. Hb conc clinically decided based on holistic assessment of patient’s needs. (Lowest Hb conc not associated with anaemia symptoms)
- Transfusions thresholds differ in various subgroups of patients depending on balance between mechanisms of adaptation to anaemia & O2 requirements.
- Triggers of transfusion protect patients from unnecessary transfusions.
When is transfusion needed in Acute Anaemia ?
• Transfusion thresholds for patients with acute anaemia: Hb 70-80g/L EXCEPT symptomatic patients (transfused until symptoms relieved), patients with acute coronary syndrome, patients with major haemorrhage.
• Why we transfuse RBCs- to restore O2 carrying capacity
• Recommended not to transfuse patients with a treatable cause of anaemia e.g. iron deficiency.
• Triggers:
o ≤70 g/L for patients with mild symptoms of anaemia
o ≤80 g/L for patients with cardiovascular disease
• Alternatives to RBC transfusions:
o Correction of treatable causes of anaemia: iron deficiency, B12 & folate deficiency, erythropoietin treatment for patients with renal disease
o Correction of coagulopathy: discontinuation of antiplatelet agents, administration of anti-fibrinolytic agents
• If blood loss >40% of their total blood vol- need a transfusion
When is transfusion needed in Chronic Anaemia ?
• E.g. due to haematological syndromes.
• Consider setting individual thresholds & Hb conc targets for each patient who needs regular blood transfusions for chronic anaemia.
• Objectives for patients on regular transfusions due to myeloid failure syndromes;
o Relieve symptoms of anaemia
o Improvement of Quality of Life
o Prevention of ischemic organ damage
• Start with threshold Hb 80-100g/dl
• Tailor decision for each patient, consider;
o Co-morbidites that affect cardiac/ resp function,
o Risk for developing iron overload
o Adaptaion to anaemia (compensatory mechanisms developed).
• Patients on chronic transfusion programmes as patients with MDS that are NOT on chemo &; NOT thrombocytopenic usually transfused with target Hb 8-10g/dl.
• Objectives for patients on regular transfusions due to thalassaemia;
o Suppress endogenous erythropoiesis
• Threshold 90-95g/L, target 100-120g/L
• Consider iron overload!
Why Transfuse Patients with platelets?
Platelets • Stored at room temp (22°C) • Shelf life 5 days from collection • ‘Adult therapeutic dose’- platelets from 4 pooled donations (or equivalent n.o. from a single apheresis donation) • Usual transfusion time: 30min/unit.
Why Transfuse Patients?
• Treatment of bleeding due to severe thrombocytopenia (low platelets) or platelet dysfunction
• To prevent bleeding
• We transfuse;
o Massive haemorrhage (keep platelet count above 75 x 109/L)
o Bone marrow failure (platelet count <10 x 109/L or <20 x 109/L, e.g. sepsis)
o Prophylaxis for surgery (more major surgery 50 x 109/L, CNS or eye surgery 100 x 109/L)
• Contraindications;
o Heparin induced thrombocytopenia &thrombosis
o Thrombotic thrombocytopenic purpura
What is Fresh Frozen Plasma? What is it used for? What is it contraindicated in?
Fresh Frozen Plasma (1 unit of FFP)
• Stored at -30°C for up to 24 mnths
• Thawed immediately before use (takes 20-30 min)
• Usual dose 12-15mL/kg (4-6 units for average adult)
• Usual transfusion time: 30mins/unit
• Main indications;
o Coagulopathy with bleeding/ surgery
o Massive haemorrhage
o Thrombotic thrombocytopenic purpura
• Do NOT transfuse fresh frozen plasma with;
o Warfarin reversal
o Replacement of single factor deficiency e.g. haemophilia
What is Prothrombin Complex Concentrate?
• Product used to reverse warfarin is Prothrombin Complex Concentrate (PCC);
o Plasma- derived vit K dependant factors (II, VII, IX, X)
o For emergency reversal of life-threatening warfarin over-anticoagulation (do not use FFP)
o Issued by transfusion lab- supply in A&;E
Why are transfusions kept CMV negative?
CMV NEGATIVE • To keep at-risk patients CMV free (50% of us CMV –ve) • Children <1 yr • Intrauterine transfusions • Congenital immunodeficiency • And unless known to be CMV IgG +ve • Preg women having elective transfusion
Why are transfusions irradiated?
IRRADIATED
• To prevent transfusion-associated graft vs host disease (rare) in specific T-cell immunodeficiency cases
• Intrauterine transfusions
• Congenital immunodeficiency
• Hodgkin lymphoma
• Stem cell/ marrow transplant patients
• After purine analogue chemo (e.g. fludarabine)
What are Acute Transfusion Reactions ?
Acute Transfusion Reactions (present <24hrs of transfusion)
Immunological;
• Acute haemolytic transfusion reac due to ABO incompatibility
• Allergic/ anaphylactic reac
• Transfusion related acute lung injury (TRALI)
Non immunological;
• Bacterial contamination
• Transfusion associated circulatory overload (TACO)
• Febrile non-haemolytic transfusion reac
What are Delayed Transfusion Reactions ?
Delayed Transfusion Reactions (present >24hrs of transfusion)
Immunological;
• Transfusion-associated graft vs host disease (TA-G v HD)
• Post transfusion purpura
Non immunological
• Transfusion Transmitted Infection (TTI)- viral/prion
• Iron overload
What are the Infective Risks of Blood Transfusion?
Infective Risks of Blood Transfusion
• Risk of transmitting Hep B/C/HIV viruses are low
Prion disease
• E.g. Crutzfeld Jacob disease
• Steps to ↓ risk of potential transmission by blood/ blood components; leucodepletion, UK plasma not used for fractionation, imported FFP for patients born after 1996.
What is ABO Incompatibility?
Acute Haemolytic Reac-ABO Incompatibility
• Due to transfusion of RBCs that express ABO antigens to a recipient that has preformed antibodies against these antigens.
• Antibodies will bind on RBC surface &; be haemolysed intravascularly.
• Due to release of free Hb (toxic) in circ;
o Hb deposited in distal renal tubule= acute renal failure
o Coagulation stim = microvascular thrombosis
o Stim cytokine storm
o Scavenges NO = generalized vasoconstriction
- Severe reacs may occur within 15min of transfusion
- Milder reacs may occur later but usually before end of transfusion
- 20-30% fatal
Acute Haemolytic Reac- ABO Incompatibility SIGNS & SYMPTOMS • Fever &; chills • Back pain • Infusion pain • Hypotension/ shock • Hemoglobinuria (may be 1st sign in anaethsized patients) • Increased bleeding (DIC) • Chest pain • Sense of ‘impending death’
What are Delayed Haemolytic Reactions?
- Due to immune IgG antibodies against RBC antigens other than ABO
- The antibodies formed after transfusion
- Other groups on the surface of RBCs such as Rh, Duffy (Fy), Kell(Kk), Kidd (Jk) and La
Delayed Haemolytic Reac- CLINICAL FEATURES • Onset 3-14 days after RBC transfusion • Fatigue • Jaundice • And/or fever
Delayed Haemolytic Reac- Lab Findings
• Drop in Hb
• Increased LDH (sign of haemolysis)
• Increased indirect bilirubin (sign of haemolysis)
• DIRECT ANTIGLOBULIN TEST (Coomb’s Test) +VE
What is Coomb’s test?
The Anti-human Globulin (Coomb’s) Test
• (Test has diff names; Coomb’s test, Anti-human globulin test (AHG), Direct Anti-globulin test (DAG)).
• To detect incomplete (IgG) antibodies
• In 1945 Coombs raised rabbit antibodies to human IgG (anti-human globulin or AHG)
• These used to detect IgG antibodies on RBCs
• Normal RBCs don’t have anything on surface, but if covered with antibodies reagent will create bridges between RBCs & they will agglutinate.
• Key test in blood transfusion
What is TRALI?
Transfusion Related Acute Lung Injury (TRALI)
• Antibodies in transfusion
• Estimated rate of fatalities 5-10%
• Donor has antibodies to antigens on recipient’s leucocytes- bind &; activate them causing them to release intracellular substance = activate endothelium.
• Almost always complicates transfusion of plasma rich components (platelets, FFP)
What is the Criteria for TRALI Diagnosis ?
• Sudden onset of ‘Acute Lung Injury’ within 6 hrs of transfusion
• Acute lung injury;
o Hypoxemia
o New bilateral chest x-ray infiltrates (due to fluid capill leak)
o No evidence of vol overload (heart size normal
What is the treatment for TRALI?
TRALI- Treatment, Clinical Course
• Majority of patients recover within 72-96hrs
• Mild forms of TRALI may respond to supplemental oxygen therapy.
• Severe forms may require mechanical ventilation and ICU support.
• Lab investigations; donor tested for HLA &granulocyte antibodies, recipient tested for neutrophil antigens
• Diagnosis confirmation; donor has antibodies against antigens that are expressed on recipients granulocytes
What is Transfusion-Associated Circulatory Overload (TACO)?
TACO Signs &; Symptoms • Symptoms; o Sudden dyspnea o Orthopnoea o Tachycardia o Hypertension o Hypoxemia • Signs; o Raised BP o Elevated jugular venous pressure
TACO Risk Factors • Elderly patients • Small children • Patients with compromised L ventricular function • Increased vol of transfusion • Increased rate of transfusion
TACO significantly underreported & often unrecognised by clinicians.
What are allergic reactions in response to transfusions?
• Due to antibodies in recipient’s serum against transfused proteins
• Usually after transfusion of plasma or platelets
• URTICARIAL RASH +/- wheeze;
o Often not severe
o Hypersensitivity to a ‘random’ plasma protein
• ANAPHYLAXIS
o Usually in patients that are IgA deficient & have anti-IgA antibodies
o Severe life-threatening reac soon after transfusion started
o Wheeze/asthma, high pulse, low BP (shock)
o With laryngeal oedema/ facial oedema
• Lab investigations: IgA quantification, anti-IgA antibodies testing
What are Febrile Non-haemolytic Transfusion Reactions (FNHTR)?
• Pyrexial reac
• During or soon after transfusion;
o Fever- rise in temp >1°C +/- shakes/ rigors
o +/- high pulse
• Unpleasant but not life threatening
• FNHTR due to cytokines/ other biologically active molecules that accumulate during storage of blood components.
• Less since leucodepletion of blood & platelets
• Discontinue transfusion until exclude ‘wrong blood’ or bacterial infec
• Self-limited reac (stop when stop transfusion)
What is Haemostatic Plug Formation?
Haemostatic Plug Formation
• Primary haemostasis- platelet plug formation &; vascular constric
o 1st line of defence to stop bleeding
o Reac of vessel wall
o When vascular integrity of blood vessels breached= blood exposed to collagen (underlying vessel)= platelets are activated=form a plug at site of injury in vessel
• 2ndry event (in fluid phase of blood) - coagulation cascade (makes clot)
o Series of sequential enzymatic reacs form active proteins to enzymes form fibrin clot which stabilises platelet clot
• Both primary &; 2ndry processed work together to form a haemostatic plug- prevent further bleeding
What is Platelet Adhesion & Activation ?
Platelet Adhesion & Activation
• When normal platelets in blood- inert
• When exposed to collagen in vessel wall after injury, von willie brand factor (blood protein) adheres to surface of collagen &; also binds to recep on platelet- forms glue between platelet & vessel wall
• This interaction 1st step that happens in platelet activation
• Platelets activated in result of adherence to vessel wall=produce new receps on surface e.g. glycoprotein 2b3a recep undergoes conformational change into a active fibrinogen recep
• This allows fibrinogen to bind to platelets (fibrinogen in blood forms bridge between platelets- keeps platelets together)
• Alpha granules & dense granules in platelets- releases contents (adhesion proteins)when platelets activated= +ve feedback loop to activate other platelets
• Thromboxane synthesised inside platelets; is a procoagulant proplatelet activating material, activates further platelets
• Builds up so more platelets activated= plug formation
• Receps for other small molecules (e.g. ADP)- bind to other activators in circulation
What is Thromboxane synthesis?
Thromboxane synthesis
• Cyclooxygenase enzyme leads to thromboxane synthesis
• Aspirin (anti-thrombotic) inhibits cyclooxygenase inside platelets reducing thromboxane synthesis
• Clopidogrel (antiplatelet)- antagonist at platelet ADP recep
• Also large protein 23ba antagonists (fibrinogen recep)- stop platelets aggregating (in patients with acute coronary syndromes)
What is the clotting cascade? Intrinsic and extrinsic?
Clotting Cascade
• 3 parts to clotting cascade; extrinsic, intrinsic & common pathway (tests look at each pathway)
• Prothrombin time- looks at extrinsic pathway
• APTT- looks at intrinsic pathway
• Thrombin clotting time- looks at common pathway
Extrinsic Pathway (& common pathway)
• Most important initiator of coag cascade
• Extrinsic & common system (most common way fibrin made in vivo)
• Not many steps- fast way of making thrombin & fibrin
1. Initiated when vessel damaged- cells in vessel wall express tissue factor on surface of cell (so not circulating in blood)
2. Tissue factor activates factor 7 (7a)
3. 7a + tissue factor activate factor 10 (to factor 10a)
4. F10a + F5 (cofactor) on phospholipid surface (activated platelet) activates F2 (prothrombin) to thrombin
5. Thrombin cleaves fibrinogen= fibrin monomers= polymerise to form fibrin clot
Intrinsic Pathway
• Intrinsic (doesn’t need external protein from fluid in blood unlike extrinsic where need tissue factor).
• Intrinsic pathway has more steps than extrinsic- slower way of getting fibrin
1. F12 activated when blood come into contact with foreign surface
2. F12a activates F11 to F11a,
3. F11a activates F9 to 9a (need phospholipid surface)
4. F9a with cofactor F8 activates factor 10 (phospholipid surface provided by activated platelets)
5. (Then back to common pathway- thrombin & fibrin formation)
- Deficiency of factors 8 & 9 in intrinsic pathway= haemophilias
- Patients deficient in F12 no bleeding disorder
- Deficient in f11- variable bleeding; so top end of intrinsic pathway may not be as important as extrinsic (but deficiencies of F8 & 9 is important- haemophilais)
- We think in vivo, extrinsic pathway quickly activated when vasc damage, small amounts of thrombin generated which feedsback into intrinsic pathway & directly converts F11 into 11a (without need for 12b) & also activates F8.
- On phospholipid surface 7a can activate F9- so these intrinsic & extrinsic pathways not completely separate BUT lab tests (in unphysiological env) can sep into intrinsic, extrinsic & common pathways
- Cross between platelet activation & 2ndry part of haemostasis
What are Anticoagulant mechanisms ?
- Natural anticoagulant protions; protein C & S and anti-thrombin III, & fibronolytic system (when clot forms body will slowly dissolve it)
- Fibrinolytic system- series of steps inert proteins activated
- End product of finbronlytic cascade = plasmin (breaks down fibrin clots)
- Plasmin from plasminogen & TPA.
- Alpha 2 anti-plasmin & Pi1 (inhibitor of plasminogen activator TPA), present in plasma- stop fibrinolytic system being activated in fluid phase of blood &localise fibrinolysis to surface of clots (ensures plasmin doesn’t start digesting & dissolving coagulation factors).
What are the different types of Bleeding Disorders ?
- Congenital: usually single defect- 1 of factors/ platelet proteins will be deficient, or von willie brand factor deficient
- Acquired: often multiple defects, several parts of haemostatic system affected e.g. lots of factors
- Platelet/ bleeding vessel wall: mucosal & skin- skin bleeding, bruising, mucosal bleeding e.g. epistaxis
- Coag defect (deficiency in 1 or more of coag proteins or enzymes): deep muscular &; joint bleeds, bleeding following trauma, deep tissue bleeding into joints & muscles e.g. in haemophilias
What causes defects in primary haemostasis?
Platelet/ Vessel Wall Defects
• All give rise to prolonged bleeding time
• Reduced platelet n.o. (not got enough platelets)
• Abnormal platelet function (drugs/ aspirin- antiplateltes)- platelets not working
• Abnormalities of vessel wall e.g. inherited- ehlers dan los syndrome (cause weakness in vessel wall), acquired- vit C deficiency (need vit c to make good collagen- scurvy is an acquired bleeding disorder)
• Abnormal interaction between platelets &vessel wall (von willie brand disease- lack of protein that causes adhesion to vessel wall)
What is Petechiae ?
- Do not blanch with pressure (e.g. angiomas)
* Not palpable (e.g. vasculitis)
What is Epistaxis ?
Epistaxis
• Mucosal bleeding
• If single symptom with nothing else- due to local cause in nose
• Often part of symptoms patients with primary haemostasis (e.g. von wilie brand disease) will have
What is disease Von Willie brand disease?
Von Willie brand disease- (defective PRIMARY haemostasis)
• Most common heritable bleeding disorder
• Men &; women affected
• Mucocutaneous bleeding main symptom including menorrhagia
• Post operative &post partum bleeding
• Variable penetrance for mild types
• Diagnosis of mild vWD difficult due to confounding factors e.g. exercise, stress &; illness
• Deficiency of von willie brand protein (bridge between platelets &collagen in vessel wall)
• Parts of it’s struc binds to a glycoprotein on platelets surface
• Consists of lots of subunits- large multimeric struc (lower molecular weight multimers not as good of a glue as high molecular weight- act as a better glue)
• Is a heterogeneous disorder- has diff types;
o TYPE 1 common type is a mild reduc in amount of VWBF produced (reduced amount of the normal protein), may have slightly reduced level of factor 8
o VWBF levels go up and down during the day in everyone e.g. go up if you have infec or after the gym, stress etc- so choosing right time to test people for the disease important &; need to retake test (1 result not representative)
o TYPE 2 normal amounts of ABNORMAL von wilie brand protein (doesn’t have enough high molecular weight forms- dispoportinatley reduced), in lab see disproportion between VWB factor antigen levels & activity levels (how well its working)
o Type 1 &; 2 VWB disease- autosomal DOMINANT inheritance
o Type 1 has variable penetrance in families
o Blood group O- have slightly lower levels of VWBF (doesn’t mean they have the disease)
o TYPE 3/ SEVERE VWB disease- ABSENT VWBF, more severe; e.g. spontaneous bleeding events
o VWBF also carrier for Factor 8 in plasma, so type 3 VWB disease will also have low F8 level too (not because they can’t make F8 but because molecule carrying F8 not there)- so may have coag disorder clinical manifestations
o VWB disease most common reason why a female will have low factor 8 level.
• 1 in 2 chance of passing it onto child regardless of sex of child but variable penetrance (some people may not get manifestation if have mild form of the disease)
Treatment
• Most patients don’t need treatment in day to day life, need treatment when got symptoms/ being challenged e.g. at dentist, surgical operation, having a baby, heavy period
• Antifibrinolytics: tranexamic acid- (e.g. for simple dental work, heavy periods, stop a nose bleed)
• Desmopressin (DDAVP) given by subcutaneous inject (vasopressin hormone; sets tone of smooth muscle in blood vessels &; water balance (helps us to hang onto water)- helps to release VWBF stores into circ, temporarily raising VWBF in blood)- type 1 of this disease respond to this treatment
• Desmopressin temporary, if repeat it too many times then patient will stop responding to it (tachyphylaxis) (never give it more than 3 times), it also causes water retention so need to watch amount of water taking &; fluid restrict patient
• Factor concentrates containing vWD (plasma derived blood donations where plasma sep by being centrifuged)
• To get vaccinated against hep as with treatment would be exposed to pooled plasma (plasma form diff blood donations)- when treatment that exposes them to diff types of blood.
• COCP (combined oral contraceptive pill) for menorrhagia- causes women to have lighter periods
How do results of clotting tests help determine which factors are affected?
- These clotting screening tests not diagnostic- tell us something wrong but not what’s wrong
- But they can narrow down what specific factors want to assay.
- E.g. if get isolated prolongation of APTT want to isolate factors in intrinsic pathway (F 9, 8, 11 & 12)
- If isolated elongation of prothrombin time want to isolate F7
- If both abnormal- assay all of them (may be single defect in common pathway or could be more than 1 factor deficiency.
- F13 deficiency &; VWB disease (if don’t have F8 deficiency)- expect normal tests (deficiency screens won’t pick these up e.g. F13 comes after clot formed)
What are Heritable Coagulation Factor Deficiencies?
• F14- stabilises fibrin clot (comes after clot formation), covalently links fibrin monomers (stabilisin them)
• Deficiencies from factors all from single gene defects
• Most common disorders haemophilia A (F8 deficiency) &haemophilia B (F9 deficiency);
o Are x-linked
o Level of deficiency of these factors in blood correlate with clinical manifestations
F11 deficiency autosomal dominant or recessive
XII- Autosomal common
XI- Autosomal rare
VII- Auto recessive V.R
X,V,II,I, XIII- Auto recessive V.R
What is Haemophilia A & B ?
Haemophilia A & B • F8 &9 intrinsic pathway • F9- serene protease enzyme • F8- cofactor • APTT looks at intrinsic pathway so do APTT lab test- isolated prolongation of APTT in clotting screen picks up haemophilia
• Haemophilia A &; B CLINICALLY INDISTINGUISHABLE
• X linked recessive inheritance; females carriers- have about 50% of normal F8 would expect which is enough F8 to get by, don’t have bleeding disorder, some have less than 50% so have mild haemophilia (as 1 X chromosome normal & other carrying defective gene- non-random lyonisation is why some people have lower levels of F8 as normal X predominantly inactivated)
• Typically females carriers & males affected
• Haemophila A;
o Factor VIII deficiency
o 1 in 5,000 males
• Hemophilia B;
o Factor IX deficiency
o 1 in 30,000 males (more rare)
• Severity consistent between family members- if fam has history of severe haemophilia child will get severe, if mild history then child will get mild haemophilia.
• 30% haemophilia A are new mutations (no fam history)
What are the Degrees of Severity of Haemophilia ?
- Normal F 8 or 9 level= 50-150%
- Mild haemophilia F8 or 9 level= 6-50% (won’t spontaneously bleed, only bleed after trauma/ surgery)
- Moderate haemophilia F8 or 9 level= 1-5% (bleed following minor trauma, 1 or 2 spontaneous bleeds)
- Severe haemophilia F8 or 9 levels= <1%, common bleeds (muscle &; joint bleeds) e.g. often presents early in children e.g. when learning to walk
- General rule: less severe haemophilia gets- later in life will present &; be diagnosed haemophilia
What are the Types of Bleeds in Haemophilia?
- Spontaneous/ Post traumatic
- Joint bleeding (swollen, hot)= haemarthrosis
- Muscle haemorrhage- muscle fibres become damaged as a result of bleed
- Soft tissue (floor of mouth/ palate) e.g. haematomas under skin, retroperitoneal bleeding
- Life threatening bleeding (e.g. intercranial bleeding, bleeding around airway- can obstruct airway)
What is the treatment for Haemophilia?
- Replace deficient factor (missing clotting protein) using recombinant factor (not blood derived)- on demand or prophylaxis (prevention of bleeds)
- DDAVP (mild/ moderate Haemophilia A)
- Factor concentrates: Recombinant are products of choics
- Antifibrinolytic agents
- Vaccination against hep A &; B
- Supportive measures e.g. icing (ice pack), immobilisation, rest
Heamophilia A &; B Management
• Specialised centres; haemophilia centres &; comprehensive care centres, multidisciplinary approach, home treatment, patient education & social support, physio, orthopaedic advice &; treatment, treatment & diagnosis of liver disease, specialised management for HIV +ve patients, genetic counselling &parental diagnosis.
Ask haemophila patients who their haemophila centre is & talk to their centre.
What are the complications of treatment for Haemophilia?
Complications of Treatment of Haemophilia
• Transfusion transmitted infec; hep A, B, C etc, HIV, Parvovirus, vCJD
• Inhibitor development;
o Inhibitor- alloantibody against factor 8 or 9
o 25% in haemophilia A (more common in haemophilia A than B)
o Result in poor recovery and/or shortened ½ life of factor replacement therapy
o Poor clinical response to treatment- specialised management of bleeds
o Most occur early in course of condition (10 exposure days)
o Eradication of inhibitor (immune tolerance)
o Genetic predisposition
o Some transient (go away on their own), but some not transient- render treatment ineffective
o IF get inhibitor- more likely to bleed.
What suggests a vascular/ platelet defect?
Petechiae and superficial bruises
skin and mucous membranes
spontaneous
bleeding immediate prolonged and non-recurrent
What suggests a coagulation defect?
Deep spreading haematoma
Haemarthritis
Retroperitoneal bleeding
Bleeding prolonged and often recurrent
What are Acquired Bleeding Disorders ?
- Vit K deficiency
- Liver disease
- Massive transfusion syndrome
- Disseminated Intravascular Coagulation
- Iatrogenic
- Acquired inhibitors (antibodies that affect particular clotting factor/ mechanism)
How do you Assess Patients with Possible Bleeding Disorders?
- Clinical history important
- Date of onset, previous history of bleeding episodes &; clinical pattern
- Response to challenges e.g. surgery, dental extraction
- For young children; bleeding from umbilical stump, vaccinations, circumcision
- Requirement for medical/ surgical intervention
- Other systemic illness &; drug history
- Fam history
- Clinical examination: pattern of any bruising/ other evident haemorrhangic signs, digns of underlying disease, joints, muscles & skin.
How do you test for Possible Bleeding Disorders?
Tests
• Collect blood into tube that contains sodium citrate (removes sodium calcionate)- inhibits sample clotting in test tube
• Smaple in centrifuge in lab- all cellular components of blood drop to bottom, left with straw-coloured plasma at top.
• Do coag screening on plasma (with no platelets or RBCs in)
• Then do prothrombin time-……..
• Time how long it takes for prothrombin to form
• Recalcify & add substitiue factor(?)
• Prothrombin time
• APTT test;
o Add particular material
o Don’t add calcium
o Measure for certain length of time for clot to form- activation of factor 12 &; 11a.
• Thrombin clotting time (final step in common pathway);
o Add activated factor 2 (thrombin)- expect it to cleave fibrinogen….
o Don’t need to calcify
• Fibrinogen level
• Mixing test…
How to test whether its a Deficiency or Inhibitor?
- APTT prolonged
- Repeat with 50:50 mix patient to normal plasma
- Significant correction: deficiency
- No correction: inhibitor
What are common blockers of the Coagulation Cascade ?
• Gamma glutamyl carboxylase enzyme
• Warfarin- anticoagulant (can’t recycle vit k so eventually become vit K deficient)
• PIVKA- proteins induced by vit K absence
• Efficacy/ safety ratio for currently available therapies less than satisfactory due to their ill-defined, multitargeted activity
• Current available antithrombotic agents; heparins (UFH &Enoxaparin), vit K antagonists (warfarin) &; direct thrombin inhibitors (hirundins)
• Most used- heparin & vit k; have a range of actions on diff parts of the coag cascade (unpredictable clinical responses)
• Other limitations of antithrombotics-
o High incidence of serious adverse effects e.g. bleeding complications
o Monitor coag markers- burden of time & cost
o Narrow therapeutic margin
o Limited effectiveness in preventing VTE
• Factor Xa inhibitors- antithrombic agents designed to selectively target 1 core step in coag cascade leading to potent &; targeted effectiveness.
What happens as a result of Vit K deficiency ?
• Deficiencies of factors II, VII, IX, X
• Are fat soluble vits (stored in body longer than water soluble vits)
• Causes;
o Obstructive jaundice
o Prolonged nutritional deficiency
o Broad spectrum antibiotics
o Neonates (classical 1-7 days)- in UK given vit K to avoid haemorraghic disease of the newborn
What happens as a result of Liver Disease ?
- Cirrhotic coagulopathy major source of morbidity & mortality in patients with liver disease
- Increased risk of severe bleeding form invasive procedures/ surgery
- Conventional methods for treatment &prevention of bleeding are limited (may not increase deficient factors to hemostatic range, required vol for transfusion may be too large, theoretical risk of viral transmission)
- Thrombocytopenia, prolonged PT & hyperfibrinolysis- most freq haematological abnormalities in this pop
- Cirrhotic patients with prolonged PT have low levels of vit-k dependant coag factors- role in bleeding tendency
- Platelet dysfunction (plasma induced cleavage of surface glycoproteins)
- Reduced plasma conc of all coag factors (reduced synthesis) except FVIII
- Delayed fibrin monomer polymerisation due to altered fibrinogen glycosylisation (xs sialic acid)
- Excessive plasmin activity
What is a Massive Transfusion ? What Haemostatic Abnormalities are present?
Massive Transfusion
• Transfusion of vol equal to patients total blood vol in less than 24hrs or
• 50% blood vol lost within 3 hours
Haemostatic Abnormalities in Massive Transfusion
• Due to dilutional depletion of platelets &coagulation factors
• Due to DIC (due to tissue damage)- risk factors extensive trauma, head injury, prolonged hypotension
• Due to underlying disease e.g. liver or renal drug treatment or surgery
What are the ?Dilutional Effects on Haemostasis
- Citrate used as anticoagulant in RBCs
- Thrombocytopenia- at least 7-8 litres in adults usually transferred before probs likely
- Coag factor depletion- mainly factors V & VIII & fibrinogen
- DIC common
- Citrate toxicity uncommon- hypothermia & neonates- increases susceptibility
- Hypocalcaemia =- no clinically signif effect on coag
What is the Pathophysiology of DIC ?
- Occurs because of a trigger to activate coagulation cascade in vivo.
- Acute DIC
- Chronic DIC
- Activation of fibrinolytic system
- Generalised disruption in physiological balance of procoag &; anticoag mechanisms
- Consumption of clotting factors &; platelets
- Microvascular thrombosis (tissue ischaemia & organ damage)
- Fibrinolysis activation
- Microangiopathic haemolysis
What are the causes of Acute DIC?
- Sepsis
- Obstetric complications
- Trauma/ tissue necrosis
- Acute intravascular haemolysis e.g. ABO incompatible blood transfusion
- Fulminant liver disease
What are the causes of Chronic DIC?
- Malignancy
- End stage liver Disease
- Severe localised intravascular coagulation
- Obstetric- retained dead foetus
What are the lab tests for DIC?
- Rarely get DIC without thrombocytopenia
- Fibrinogen conc usually lower (may not be pathologically lowered)
- FBC &; blood film
- Coag screen; PT (70% prolonged), APTT (50% prolonged), TCT (usually prolonged), fibrinogen conc, FDP or D-dimer (elevated in 85%).
- Not 1 single diagnostic test- scoring systems sometimes used
