Haematology Flashcards
What happens to the FBC in pregnancy?
- Mild anaemia
- –Red cell mass rises (120 -130%)
- –Plasma volume rises (150%)
- Net dilution, hence why anaemia
- Macrocytosis
- –Normal
- –Folate or B12 deficiency
- Neutrophilia
- Thrombocytopenia
- –increased platelet size
- Platelet decline mainly happens towards the end of pregnancy, past the 28thwk, due to increased activation and clearance of platelets;
- leaves a proportion of women with a platelet count less than 150x10^9/L
- During normal pregnancy the normal platelet count falls
- Expansion of Red cell mass, so causes a mild anaemia due to an even higher increase in plasma volume – complete around 2nd trimester
- RBC become larger, which is normal
- Rise in neutrocyte count
- Decreased platelets, increase in platelet size as they are released at lower maturity
How much iron and folate is needed during pregnancy?
•Iron requirement
- •300mg for fetus
- •500mg for maternal increased red cell mass
- •RDA 30mg;
- • Increase in daily iron absorption:1-2mg to 6mg
•Folate requirements increase
- •Growth and cell division
- •Approx additional 200mcg/day required
•Iron deficiency: may cause IUGR, prematurity, postpartum haemorrhage
Iron best absorbed on empty stomach, with tea/coffe prevents absorption; legumes are good sources
Most iron is recycled, but no physiological excretion mechanisms; absorption increased due to hepcidin, which increases ferroportin
Iron/folate supplementation during pregnancy:
- WHO recommends 60mg Fe +400mcg folic acid daily during pregnancy
- Cochrane review
- –Fe/Folate supplements had no effect on measures of maternal or fetal outcome
- –Maternal Hb higher, Fe reserves higher, fetal ferritin higher
- RCOG guidelines:
- •Folic acid
- •Advise reduces risk of neural tube defects
- •Supplement before conception and for ≥ 12 weeks gestation (up until 2nd trimester)
- •Dose 400μg / day
- •Iron
- •No routine supplementation in UK
- •Folic acid
What is the cause of thrombocytopaenia in pregnancy?
- •Physiological:
- •‘gestational’/incidental thrombocytopenia
- •Pre-eclampsia
- •Immune thrombocytopenia (ITP) – can cause a lot of immune dysregulation, so lots of immune conditions
- •Microangiopathic syndromes – many more prevalent in pregnancy popn
- •All other causes: bone marrow failure, leukaemia, hypersplenism, DIC etc.
- The lower the platelets, the higher the chance of a physiological problem, with ITP being the greater proportion and then pre-eclampsia too in those with platelets less than 70x10^9/l
What is gestational thrombocytopaenia?
- Physiological decrease in platelet count ~ 10%
- >50x109/l sufficient for delivery (>70 for epidural, due to spinal haematoma which has severe side effects)
- Mechanism poorly defined
- Dilution + increased consumption
- Baby not affected
- Platelet count rises D2 – 5 post delivery
What is pre-eclampsia and it’s relation to thrombocytopaenia?
- •50% get thrombocytopenia
- •Proportionate to severity
- •Probably due to increased activation and consumption
- •Associated with coagulation activation
- •(incipient DIC – normal PT, APTT)
- •Usually remits following delivery
What is immune thrombocytopaenia in pregnancy?
- •5% of thrombocytopenia in pregnancy
- •TP may precede pregnancy
- •Early onset
- •Treatment options (for bleeding or delivery)
- •IV immunoglobulin
- •Steroids etc.
- •(Anti-D where Rh D +ve) – not in UK anymore
- •Baby may be affected – which is why it is important to distinguish between this and gestational thrombocytopaenia
- •Unpredictable (platelets <20 in 5%)
- •Check cord blood and then daily
- •May fall for 5 days after delivery
- •Bleeding in 25% of severely affected (IVIG if low)
- •Usually normal delivery
- •Only treat if count is <20
- •Need to reduce the risk of head trauma – not using voltuse and careful with forceps and not using invasive methods of examining the baby like fetal scalp electrodes
What are microangiopathic syndromes in pregnancy?
MAHA:
- –Deposition of platelets in small blood vessels
- –Thrombocytopenia
- –Fragmentation and destruction of rbc within vasculature
- –Schistocytes are main feature – mechanical shearing
- –Organ damage (kidney, CNS, placenta), as the schistocytes are deposited
- Film: fragments, low platelets and polychromasia
As you can see Pre-eclampsia, HELLP and the primary thrombotic microantiopathies (TTP and HUS) share a number of features such as thrombocytopenia and MAHA. However, by carefully noting specific clinical characteristics, may be able to differentiate.
Overlap between but management varies esp wrt platelet transfusions i.e not in TTP
Important point to note is that the defintitve therapy for preeclampsia and HELLP is delivery of the fetus, while delivery does not alter the course of thrombotic microangiopathies. However, these respond to plasma exchange (incl HUS cf non pregnancies)
Some: may or may not be associated coagulopathy
BUT: delivery doesn’t change course of TTP/HUS
Why is mortality during pregnancy from VTE/haemorrhage not decreasing?
2003 – 5: Many possible factors lie behind the lack of decline in the maternal mortality rate. They include
- rising numbers of older or obese mothers,
- women whose lifestyles put them at risk of poorer health
- growing proportion of women with medically complex pregnancies.
Because of the rising numbers of births to women born outside the UK, the rate may also be influenced by the increasing number of deaths of migrant women. These mothers often have more complicated pregnancies, more serious underlying medical conditions or may be in poorer general health. They can also experience difficulties in accessing maternity care.
The commonest cause of Direct deaths was again thromboembolism and as seen there has been a rise in amniotic fluid embolism, a rare and largely unavoidable condition. This may be due to better pathological identification leading to a defined and confirmed cause of death.
Pulmonary embolism still remains the leading Direct cause of maternal death in the United Kingdom with a mortality rate of 1.56 per 100,000 maternities.
Need to consider: With increasing rates of obesity, more and further air travel, a rise in the average age at childbearing and caesarean section rates of around 23%, it is pleasing that the number of maternal deaths from thromboembolism has hardly changed since 1985-87. This is almost certainly due to increasing vigilance among obstetricians and midwives and the careful application of thromboprophylaxis protocols. The fall in deaths from postpartum embolism after caesarean section shows the effectiveness of this strategy.
The same strategy has since be applied to prevent deaths in early pregnancy and postpartum deaths after vaginal delivery.
Confidential Report on Maternal Deaths
Has led to:
Improved assessment of risk
Public health education: identify women at risk because of their weight, family history or past history to seek advice before becoming pregnant. RCOG guidelines 2004
Increased recognition of symptoms in early pregnancy - chest pain / SOB / leg pain
Diagnosis - Increased awareness that diagnostic tests (VQ / CXR / Venogram/ CTPA) are safe
Treatment
- • Wider use of thromboprophylaxis – heparin, and won’t get osteopaenia
- • Therapy should be given pending the results of further testing
What are the coagulation changes in pregnancy?
- Factor VIII and vWF increase 3-5 fold
- Fibrinogen increases 2 fold
- Factor VII increases 0.5 fold
- (Factor X)
- HYPERCOAGULABLE!
- Protein S falls to half basal
- PAI-1 (endothelial cells) increase 5 fold
- PAI-2 produced by placenta
- HYPOFIBRINOLYTIC
Cause:
Rapid control of bleeding from placental site (700ml/min) at time of delivery – help to reduce risk of haemorrhage during delivery. Most causes of bleeding during delivery are due to lack of contraction of the uterus – uterine atony
Net effect is a procoagulant state:
- • Increased thrombin generation
- • Increased fibrin cleavage
- • Reduced fibrinolysis
- • Interact with other maternal factors
- •D-dimers are raised during pregnancy but can’t be used to determine clots
- Increased rate of thrombosis
Thromboembolic disease: deaths from pulmonary embolism in pregnancy are highest in the 1-13wks and 6 weeks postpartum (HIGHEST)
Deaths from PE are highest in postpartum - with RF: BMI as the highest, personal/family Hx VTE, air travel, hyperemesis gavidarum, OHSS, unrelated surgery
Incidence of thrombosis in pregnancy:
- 1 per 1000 <35 years
- 2 per 1000 >35 years
- Relative risk approx. x10
- 1/1000 for pregnancy and 0.5 in puerperium
- One third are post partum (only 6 weeks)
- Doppler and VQ are safe to perform in pregnancy
- D-dimer often elevated in pregnancy
- –Not useful for exclusion of thrombosis
Factors that increase the risk of thrombosis in pregnancy:
All
- Changes in blood coagulation
- Reduced venous return
- ~85% Left DVT
- Vessel wall
Variable
- Hyperemesis/dehydration
- Bed rest
- Obesity
- BMI>29 3x risk of PE
- Pre-eclampsia
- Operative delivery
- Previous thrombosis/thrombophilia
- Age
- Parity
- Multiple pregnancy
- Other medical problems:
- -HbSS, nephrotic syndrome
- IVF: ovarian hyperstimulation
How is thromboembolic disease prevented and treated during pregnancy?
- •Women with risk factors should receive prophylactic heparin +TED stockings
- •Either throughout pregnancy
- •Or in peri-post- partum period
- •Highest risk get adjusted dose LMWH heparin
- •Mobilise early
- •Maintain hydration
Management:
- •LMWH as for non-pregnant
- •Does not cross placenta
- •RCOG recommend once or twice daily
- •Do not convert to warfarin (crosses placenta)
- •After 1st trimester monitor anti Xa
- •4 hour post 0.5-1.0u/ml
- •Stop for labour or planned delivery, esp. for epidural
- •Epidural: wait 24 hours after treatment dose, 12 hours after prophylactic dose
Complications of pregnancy:
- •Hypothesis:
- •An increased tendency to thrombosis is associated with impaired placental circulation
- •Resulting in:
- •Fetal growth restriction (IUGR)
- •Recurrent miscarriage
- •Late fetal loss
- •Abruptio placentae
- •Severe PET
- •Biologically plausible but not proven
What is antiphospholipid syndrome and its complications in pregnancy?
Antiphospholipid Syndrome (APLS): Recurrent miscarriage + persistent Lupus anticoagulant (LA)/ anticardiolipin antibodies (ACL)
- Adverse pregnancy outcome: three or more consecutive miscarriages before 10 weeks of gestation
- One or more morphologically normal fetal losses after the 10th week of gestation
- One or more preterm births before the 34th week of gestation owing to placental disease.
- Venous or arterial clot presentation
Treatment: with aspirin and heparin to increase live birth rate to 71%
Give examples of thrombophilias whihc may or may not be associated with pregnancy complications:
- AT, PC,PS deficiency
- Factor V Leiden
- PTG20210A (high prothrombin)
- Hyperhomocysteinemia (HHC)
What is post partum haemorrhage?
Fatal bleeding in pregnancy
- MBRRACE 2009 -2011
- –14 deaths from haemorrhage
- •placenta praevia
- •Placenta accreta
- principal reason for hysterecetomy
- Use of Major Obstetric Haemorrhage protocols
- Determine placental site if previous C-Section
Non-fatal bleeding in pregnancy:
- Post Partum Haemorrhage (PPH) :
- > 500 mL blood loss
- 5% of pregnancies have blood loss >1 litre at delivery.
- Requiring transfusion post partum
- –1% after vaginal delivery
- –1-7% after C-Section
Mechanisms of PPH:
- major factors are
- –uterine atony
- –trauma
- haematological factors minor except
- – dilutional coagulopathy after resuscitation
- – DIC in abruption, amniotic fluid embolism etc.
What is DIC in pregnancy?
Coagulation changes in pregnancy predispose to DIC.
Decompensation precipitated by:
- –Amniotic fluid embolism
- –Abruptio placentae
- –Retained dead fetus
- –Preeclampsia (severe)
- –Sepsis
What is an amniotic fluid embolism?
- ‘the most catastrophic event in modern obstetrics’
- 1 in 20000-30000 births
- Sudden onset shivers, vomiting, shock, DIC
- 86% mortality
- –16 deaths in last triennium
- Presumed due to Tissue Factor in amniotic fluid entering maternal bloodstream
- Almost all >25 years
- Usually third trimester
- –Drugs used to induce labour e.g. misoprostol increase risk
What haemoglobinopathies do we screen for at birth?
- a° thalassaemia (Hb Bart’s, g4)
- Death in utero, hydrops fetalis
- b° thalassemia
- Transfusion dependent
- HbSS (sickle cell disease)
- Life expectancy 43 yrs
- Other compound HbS syndromes
- Symptomatic, stroke etc.
- Some compound thalassaemias
- Transfusion dependent, iron overload
- Prevalence area:
- High ≥ 1.5/10 000 : universal
- Low < 1.5/10 000 : selected
- Family Origin Questionnaire (FOQ)
- FBC: Red cell indices
- –MCH <27 possible thalassaemia trait
- –MCH <25 possible α thal trait
- •Alpha thal requires DNA analysis*
- HPLC
- –Identifies Hb variants eg: S, C, E
- –Quantifies Hb A2 (>3.5% → β thal)
- Aim to complete by 12/40 (incl partner testing where req’d)
Counselling:
- Important disorders are all recessive
- Therefore if mother is heterozygous partner should be tested.
- Combinations as important as homozygous states
- Options
- –Proceed
- –Prenatal diagnosis@
- •CVS sampling (10-12 weeks)
- •Amniocentesis (15-17 weeks), fetal blood sampling
- –Ultrasound screening for hydrops
What is the effect of sickle cell disease in pregnancy?
- •Hb SS (sickle cell anaemia),
- •HbS/clinically abnormal Hb e.g. HbC; βthal
- •~100 pregnancies/year in SCD females in UK
- •Vaso-occlusive crises become more frequent
- •Anaemia and existing chronic diseases exaggerated
- •Complications:
- •Fetal growth restriction,
- • Miscarriage, Preterm labour, ? Pre-eclampsia
- • Venous thrombosis
- •Management
- •Red cell transfusion (top up or exchange)
- • Prophylactic transfusion
- •reduces number of vaso-occlusive episodes
- •Not clear whether affects fetal or maternal outcome
- •Alloimmunisation -extended phenotype: Rh D c E, Kell
What is the difference between IDA and thalassaemia trait?
Hb: Normal or ¯, IDA; thal trait: Normal (rarely ¯)
MCH: Low (in proportion to Hb) IDA; thal trait Lower for same Hb
MCHC: Low IDA; thal trait: Relatively preserved
RDW: Increased IDA; thal trait: Normal
RBC: Low or normalIDA; thal Increased
Hb electrophoresis: Normal in IDA; Hb A2 ↑ in β-thal trait; Normal in α-thal trait
What are the haematological changes that can occur in systemic disease?
- •Soluble factors:
- •raised FVIII in Inflammation > Thrombosis risk.
- •Erythrocytes
- •Raised {altitude/hypoxia or Epo secreting tumour}
- •Reduced
- •BM infiltration or deficiency disease {Vit B12 or Fe}
- •Shortened survival {Haemolytic anaemia}
- •Platelets
- •Raised {Bleeding, Inflammation, splenectomy}
- •Reduced
- •BM infiltration or deficiency disease {Vit B12 }
- •Shortened survival {ITP, TTP}
- •Leucocytes
- •Raised {Infection, Inflammation, corticosteroids}
- •Reduced
- •BM infiltration or deficiency disease {Vit B12
What are the differentials of a pt with newly diagnosed lymphoma who develops jaundice, anaemia and raised LDH?
•Pre-hepatic jaundice
- •Lymphoma Stage 1 & auto immune haemolytic anaemia
•Hepatic
- •Lymphoma Stage 4 with BM and liver infiltration
•Post-hepatic
- •Lymphoma Stage 3 with pathological nodes compressing the bile duct & anaemia of inflammation
What is anaemia associated with malignancy or systemic disease?
•Anaemia may be the 1st presentation of occult malignancy
- •Fe deficiency
- •Leucoerythroblastic anaemia
- •Haemolytic anaemias
•Anaemia of Inflammation (chronic disease)
Iron deficiency:
- •Laboratory findings
- •Microcytic hypochromic anaemia
- •Reduced ferritin, transferrin saturation
- •Raised TIBC
- •Fe deficiency is bleeding until proven otherwise! (must find the cause)
- •Often menorrhagia in pre menopausal women
- •Blood loss in men and post menopausal women
- •Occult blood loss
- •GI cancers
- •Gastric
- •Colonic/rectal
- •Urinary tract cancers
- •Renal cell carcinoma (physicians tumour !)
- •Bladder cancer
What is leuco-erythroblastic anaemia?
•variable degree of anaemia, unremarkable MCV, look at the features of the cells
Morphological features in the blood film
- •Teardrop RBCs (+aniso and poikilocytosis)
- •Nucleated RBCs (normal in bone marrow, not in blood – in ADULTS)
- •Immature myeloid cells (normal in bone marrow, not in blood), so bigger, granules in the cytoplasm
Hints at bone marrow issue
Cancers form in organ, or metastasised into organ
Bone marrow + blood are the same dispersed organ
IDA in post menopausal woman = bowel cancer with blood loss
What are some common lab features of all haemolytic anaemias?
- Anaemia (though may be compensated)
- Reticulocytosis – healthy bone marrow compensating for short life of RBC
- Unconjugated bilirubin raised (pre-hepatic)
- LDH raised
- Haptoglobins reduced - the protein that in humans is encoded by the HP gene. In blood plasma,haptoglobin binds to free hemoglobin, compared to hemopexin that binds to free heme, released from erythrocytes with high affinity and thereby inhibits its oxidative activity
What are the 2 groups of haemolytic anaemias?
•Inherited: Defects of the red cell (covered in year 2)
- •Membrane: eg Hereditary Spherocytosis
- •Cytoplasm/enzymes: eg G6PD deficiency
- •Haemoglobin: Sickle cell disease (structural) Thalassaemia (quantitative)
• Acquired: Defects of the environment {systemic disease} in which the Red cell finds itself (exception, PNH an interesting but post-graduate disease)
- •Immune mediated
- Immune Haemolytic anemia
- •spherocytes
- •DAT +ve (Direct antiglobulin or Coombs) – determines between immune and non-immune disease
- Associated with systemic diseases involving Immune system
- •Malignancy : eg Lymphoma or CLL
- •Auto immune: eg SLE
- •Infection: eg mycoplasma
- •Idiopathic
- Immune Haemolytic anemia
- •Non-Immune mediated
- •Infection
- •Malaria – non-immune acquired HA
- •Micro-angiopathic Haemolytic anaemia (MAHA)
- •Underlying adenocarcinoma
- •Haemolytic uraemic syndrome
- RBC fragments and thrombocytopaenia on the film
- •Infection
What is the pathophysiology of microangiopathy?
Adenocarcinomas, low grade DIC/HUS
- Platelet activation
- Fibrin deposition and degradation
- Red cell fragmentation (microangiopathy)
- Bleeding (low platelets and coag factor deficiency)
Low grade activation – only should activate with wounds/tissue damage; can systemically activate the coag system, with platelets adhering to the vessels. With fibrin deposition and red cell fragmentation (caused by pushing through the microcirculation at high pressures)
What are the different WBC changes in systemic disease?
Causes of neutrophilia:
- •corticosteroids
- •underlying neoplasia - reactive
- •tissue inflammation (e.g.colitis, pancreatitis) - reactive
- •myeloproliferative/ leukaemic disorders – proliferative disorder
- •Pyogenic infection (pus forming bacteria)
Abnormal myeloid count:
- •Reactive/Infection : Neutrophilia + toxic granulation no immature cells
- •Malignant:
- • Neutrophilia plus basophilia & immature cells myelocytes. Suggest a myeloproliferative (CML)
- •Neutropenia plus Myeloblasts suggests acute leukaemia (AML)
Eosinophilia:
- Reactive eosinophilia
- •Parasitic infestation
- •allergic diseases e.g. asthma, rheumatoid, polyarteritis,pulmonary eosinophilia.
- •Underlying Neoplasms, esp. Hodgkin’s, T-cell NHL (reactive eosinophilia)
- •Drugs (reaction erythema multiforme)
- Chronic eosinophilic leukaemia
- •Eosinophils part of the “clone”
- •FIP1L1-PDGFRa Fusion gene
Monocytosis:
- Rare but seen in certain chronic infections and primary haematological disorders
- •TB, brucella, typhoid
- •Viral; CMV, varicella zoster
- •sarcoidosis
- •chronic myelomonocytic leukaemia (MDS)
Reactive lymphocytes:
- Raised {lymphocytosis}
- •EBV, CMV, Toxoplasma
- •infectious hepatitis, rubella, herpes infections
- •autoimmune disorders
- •Sarcoidosis
- Reduced {Lymphopenia}
- •Infection HIV
- •Auto immune disorders
- •Inherited immune deficiency syndromes
- •Drugs (chemotherapy)
Peripheral blood lymphocytosis:
Mature lymphocytes -> occurs with reactive/atypical lymphocytes (IM) and small lymphocytes and smear cells (CLL/NHL)
Immature lymphoid cells in Peripheral Blood -> lymphoblasts (ALL)
Clonality:
polyclonal has equal amounts of kappa and lambda chains or 60/40
Monoclonal has a majority of one type of chain, like 99:1
This is because each b cell should proliferate the same amount, a malignant proliferation will be the same B cell with the same either K or L chain which means 99% of the chains will be the same
What are the different methods of haemo-oncological diagnosis?
Morphology
- •architecture of tumour
- •cytology
- •cytochemistry
Cytogenetics
- •conventional karyotyping
- •fluorescent in-situ hybridisation
- •Interphase FISH
- •Metaphase FISH
- •As it is a genetic disease
Molecular genetics
- •mutation detection
- •direct sequencing
- •Pyrosequencing
- •PCR analysis
- •gene expression profiling
- •whole genome sequencing
Immunophenotype
- •flow cytometry
- •immunohistochemistry
Why are the diagnosis of leukaemia and lymphoma comples?
BASICS 1
Classify based on normal blood cell and their stage of maturity
- •Normal lympho-haemopoietic system is complex
- •Multiple lineages: myeloid, erythroid, T cells B cells
- •Multiple stages of differentiation eg myeloblasts>neutrophils or B lymphoblasts to Plasma cells
•Classify the malignant cells and aim to link to their normal cell counterpart
- •Primitive lymphoid blast cells expressing B cell marker > B cell Acute lymphoid leukaemia
- •Mature lymphoid cells expressing T cell antigens and involving skin> cutaneous T cell lymphoma
- •Mature erythrocytes with JAK2 mutation> polycythaemia vera
BASICS 2
Tissue biopsy to establish diagnosis
- 1.Morphology
- 1.malignant cells; large or small, mature or immature?
- 2.Lymph node diffuse invasion or forming follicles?
- 2.Immunophenotype (flow cytometry or Immunohistology)
- 1.myeloid or lymphoid? T or B lineage?
- 2.stage of maturation precursor or mature?
- 3.Cytogenetics (translocations or FISH studies)
- 1.confirm morphology eg Philadelphia Chromosome > CML.
- 2.Prognostic information eg 17p del in CLL
- 3.t(8;14) activates c-myc oncogene in Burkitt Lymphoma
- 4.Molecular genetics (PCR, pyro sequencing)
- 1.JAK2 mutation in suspected polycythemia vera
- 2.BCR ABL cDNA detection and quantification
BASICS 3:
In clinic the Histopathologist’s precise classification is used to:
- •Predict the likely clinical course eg
- •Burkitt Lymphoma highly aggressive needs urgent treatment
- •Chronic myeloid leukaemia has a chronic phase followed by a blast transformation
- •Polycythaemia vera is generally an indolent disorder
- •Chose appropriate treatment eg
- •An abl tyrosine kinase inhibitor for CML
- •Combination chemotherapy for acute leukaemias
- •Chemo+immunotherapy for lymphomas
- •Watch and wait for indolent lymphomas
BASICS 4
•Associated clinical problems generally relate to;
- •Lympho-haemopoietic failure (a dispersed organ!)
- •Bone marrow : anaemia, infection (neutrophils) bleeding (platelets)
- •Immune system: recurrent infection
- •Excess of malignant cells
- •Erythrocytes (polycythemia): impair blood flow >stroke or TIA(monocular)
- •Massively enlarged lymph nodes (lymphoma)> compress structures, bowel, vena cava, ureters, bronchus.
- •Impair organ function
- •CNS lymphoma
- •Skin lymphoma
- •Other problems
What are the different acquired somatic mutations which cause leukaemia and lymphoma?
Cellular proliferation - type 1:
- Mutations in Tyrosine Kinase genes cause excess proliferation (no effect on differentiation)
- •BCR ABL -> CML; JAK2 -> Myeloproliferative Disorder
Impair/block cellular differentiation type 2:
- Mutations in nuclear transcription factors may block differentiation. If present along with a proliferation mutation can cause acute leukaemia
- •PML RARA in acute promyelocytic leukaemia
Prolong cell survival - anti-apoptosis:
- Mutations in apoptosis genes may occur in lymphomas
- •BCL2 and Follicular lymphoma
What are the different morphologies of lymphomas?
B cell Acute lymphoblastic lymphoma
- TdT +ve
- CD19 +ve
- Surface Immunoglobulin –ve (hasn’t rearranged in the bone marrow)
Multiple myeloma
- TdT negative
- Surface Immunoglobulin +ve
- CD138 negative
What are the consequences of thromboembolism?
- Death
- Recurrence
- Thrombophlebitic syndrome (recurrent pain, swelling and ulcers – due to a previous clot damaging the vessels)
- Pulmonary hypertension
- Mortality 5%
- 20% in first 2 years and 4% pa thereafter
- Severe TPS in 23% at 2 years (11% with stockings)
- 4% at 2 year
Prevalent cause of morbidity and mortality, especially in hospital patients
Significant sequelae (death is rapid) but it is preventable (thromboprophylaxis)
May be an indicator of underlying disease (cancer)
Virchow’s triad = blood, vessel wall and blood flow
What are the blood factors involved in thrombosis?
Viscosity
- Haematocrit - polycythaemia
- Protein/paraprotein – malignant melanoma
Platelet count
Coagulation system
- Net excess of procoagulant activity
Procoagulant factors:
V, VIII, XI, IX, X, II, Fibrinogen, platelets
Fibrin formation is the final output
Anticoagulant factors:
TFPI, Protein C, Protein S, thrombomodulin, EPCR, antithrombin, fibrinolysis
What are the rates of thrombosis free survival in different deficiencies?
Antithrombin has the steepest decline in thrombosis free survival
Factor V leiden – not such a powerful effect
What are the vessel wall factors involved in clotting?
Vessel wall is usually antithrombotic:
- Expresses anticoagulant molecules
- Thrombomodulin
- Endothelial protein C receptor
- Tissue factor pathway inhibitor
- Heparans
- Does not express tissue factor
- Secretes antiplatelet factors
- Prostacyclin
- NO
Heparans help antithrombin work
Thrombomodulin helps activate thrombin
Usually trying to help stop clots form
Vessel wall injury/inflammation makes it prothrombotic:
Stimulus:
- Infection
- Malignancy -
- Cancer – prothrombotic state – more spread/aggressive = more clot risk
- Cancer causes inflammation and release of thrombotic factors
- Vasculitis
- Trauma
Effects:
- Anticoagulant molecules (eg TM) are down regulated, heparans are shed
- Adhesion molecules upregulated
- TF may be expressed
- Prostacyclin production decreased
- Hypoxia is also a cause
- Procoagulant state
How do we use anticoagulant therapy?
High dose – therapeutic
Low dose - prophylactic
•Immediate - increases anticoagulant activity
- •Heparin
- •Direct acting anti-Xa and anti-IIa (rivaroxaban, epixiban)
- •Anti-Xa
- •Rivaroxaban, apixaban, edoxaban
- •Anti-IIa -> Dabigatran
- •Properties
- •Oral administration
- •Fixed dose
- •Immediate acting –peak in approx. 3-4 hours (cf LMWH)
- •Also useful in long term
- •Short half-life
- •No monitoring
- •Anti-Xa
- •Unfractionated heparin -> iv infusion
- •Low molecular weight heparin (homogenous, reliable and predictable in behaviour) -> sub cut
- •Reliable pharmacokinetics so monitoring not usually required, but do need good renal function
- •Can use anti-Xa assay (because it blocks this factor) if needed eg:
- •Renal failure (CrCl<50)
- •Extremes of weight or risk
- •Unfractionated heparin (UFH)
- •Variable kinetics
- •Variable dose-response
- •Always monitor therapeutic levels with APTT or anti-Xa
- •Pentasaccharide (core of heparin molecules) -> sub cut
- •All act by potentiating antithrombin – inhibitor of enzymes hence immediate effect
- •Provide immediate effect (eg for treatment of thrombosis)
- •Long term disadvantage - injections, risk of osteoporosis
- •Variable renal dependence – according to size of molecule
•Delayed - reduces procoagulant activity
- •Vitamin K (2,7,9,10) antagonists
- •Warfarin
- •Given orally
- •Indirect effect by preventing recycling of Vit K BASIC EFFECT!
- •Therefore onset of action is delayed
- •Levels of procoagulant factors II, VII, IX & X fall because vit K is not present, so can’t make these factors
- •Levels of anticoagulant protein C and protein S also fall
- ALWAYS essential to monitor - measure effect = INR, derived from prothrombin time
- Difficult to monitor due to many interactions:
- •Dietary Vitamin K
- •Variable absorption
- •Interactions with other drugs
- •Protein binding, competition/induction of cytochromes
- •Teratogenic and crosses the placenta, so can’t be given during pregnancy
RISKS:
Anticoags - good at preventing thrombosis BUT increase risk of bleeding, so need to find the point at which the risk of thrombosis and bleeding are balanced
What is the lymphoreticular system?
- •Generative LR tissue
- –Bone marrow and thymus
- –Function - generation/maturation of lymphoid cells
- •Reactive LR tissue
- –Lymph nodes and spleen
- –Function - development of immune reaction
- •Acquired LR tissue
- –Extranodal lymphoid tissue
- •E.g. Skin, stomach, lung
- –Function - development of local immune reaction
- –Extranodal lymphoid tissue
Cells:
- •Lymphocytes
- •Classified according to function.
- –B lymphocytes
- •Express surface immunoglobulin
- •Antibody production
- –T lymphocytes
- •Express surface T cell receptor
- •Regulation of B cell and macrophage function
- •Cytotoxic function
- –B lymphocytes
- •Classified according to function.
- •Accessory cells
- –Antigen presenting cells
- –Macrophages
- –Connective tissue cells
LN, cortex -> T cell in thymus
B cell in BM -> prelymphoblast stage, where they then move into the circulation as naïve b cells and into LN; B cell follicles in cortex are called mantle cells, (mantle zones) where they then enter the GC and then become Marginal zone cells/plasma cells/memory b cells
B cell area:
- •Paracortical Tcell zone
- Lymphoid follicle
- •Mantle zone
- –Naïve unstimulated B cells
- •Germinal center
- –B cells
- –Antigen presenting cells
- –This is where B cells enlargen and which bind antigen epitopes are selected and activated
- •Mantle zone
T cell area:
Comprises
- •T cells
- •Antigen presenting cells
- •High endothelial vessels
- •This is where T cells which bind antigen epitopes are selected and activated
What are lymphomas?
•Definition
- –Neoplastic proliferation of lymphoid cells forming discrete tissue masses.
•Site
- –Arise in and involve lymphoid tissues (including acquired lymphoid tissue - extranodal lymphomas)
- •lymph nodes, bone marrow and/or blood (the lymphatic system)
- • lymphoid organs; spleen or the gut-associated lymphoid tissue
- •Skin (often T cell disease)
- •Rarely “anywhere” (CNS, occular, testes, breast, etc.)
•Classification
- –Two main clinically distinct groups:
- •Hodgkin lymphoma
- •Non-Hodgkin lymphoma
- –B cell type (most common) - low and high grade
- –T cell type (rarer)
- –Other (v rare)
- –Combination clinical, histological, immunohistochemical and molecular data resulting in well defined entities with distinct behaviour. This has treatment and prognostic implications
- Cf leukaemia – lymphoid proliferation presenting mainly with widespread involvement BM blood rather than tissue, some overlap
- Hodgkin’s vs non-Hodgkin’s – clinically distinct, different treatment and prognosis.
- REAL – Revised European American classification of Lymphoid Neoplasms
Basic principles:
- •B cell Non-Hodgkin lymphomas most common type (80-85 %)
- •Can arise at different stages of lymphocyte development and activation
- –Therefore in certain lymphomas the neoplastic lymphoid cell resembles a normal counterpoint both in morphology and in the pattern of CD markers expressed – follicular lymphoma is an example; NHL is widespread at presentation
- •Neoplastic lymphoid cells circulate in blood
- –Hence often disseminated at presentation (although this may be sub-microscopic). Exception is Hodgkin lymphoma and some very early NHL
- •Lymphoid neoplasms may disrupt normal immune system
- –Therefore patients may develop immunodeficiencies
- –Marrow effacement, which compromises the ability to fight infections
What is the pathogenesis of lymphoma?
- Neoplastic proliferation of lymphoid cells – clonal
- Mutation in genes to allow uncontrolled cell growth
- –Normal lymphocytes undergo controlled genomic “instability” of lymphoid cells - mistakes in this process produce neoplastic mutations
- –Inherited disorders – inherited disorder resulting in increased/abnormal genomic instability
- –Viral agents – EBV, HTLV-1
- –Environmental agents – mutagens, chronic immune stimulation (e.g H pylori)
- –Iatrogenic causes – radiotherapy, chemotherapy
•Immunosuppression predisposes to development of lymphoma
- –Infection
- –Loss of surveillance
How do we diagnose, stage and treat lymphoma?
HODGKINS LYMPHOMA:
Stage:
- •Staging: Following pathological diagnosis of a lymph node biopsy patients are ‘staged’ this has prognostic significance and also may determine the best approach for therapy.
- •FDG-PET/CT scan
- •Consider biopsy of other site if possibly infiltrated e.g. liver
- NB cHL spreads contiguously
- Diaphragm is the line for staging
Stage
- •I; one group of nodes
- •II; >1 group of nodes same side of the diaphragm
- •III; nodes above and below the diaphragm
- •IV; extra nodal spread
- •Suffix A if none of below, B if any of below
- •Fever
- •Unexplained Weight loss >10% in 6 months
- •Night sweats
- •Spleen is a giant LN for this classification
Treatment:
- •ABVD
- •Adriamycin
- •Bleomycin
- •Vinblastine
- •DTIC
- ABVD, is given at 4-weekly intervals.
- •ABVD is Effective treatment
- •Preserves fertility (unlike MOPP the original chemo)
- •Can cause (long term)
- •Pulmonary fibrosis
- •cardiomyopathy
- •EVERYONE NEEDS CHEMO!
- •ABVD 2-6 cycles (depending on stage)
- •PET CT
- •Interim: post x2 cycles, response assessment
- •End of Treatment: Guides need for radiotherapy
- •+/- Radiotherapy (optional)
- •PET CT
- •Relapse {salvage chemotherapy}
- •High dose chemotherapy + Autologous PB stem cell transplant as support
- •Relapse post salvage
- •Anti Cd30-MAME conjugate (brentuximab vedotin) – binds to reed-sternbeg cells, anti-cd30 which are internalised and then taken in and cause the cell to die (cytotoxic)
- •PD1 checkpoint inhibitor (nivolumab)
Radiotherapy:
- •Modern practice involved field only
- •Low/negligible risk of relapse within field – careful with neck and mediastinal, so will get rid of all cancers within a field
- •Risk of damage to normal tissue (collateral damage)
- •Ca breast (risk 1:4 after 25 years)
- •Leukaemia/mds (3%@10years)
- •Lung or skin cancer
- •Combined modality treatment greatest risk of 2o malignancy (two mechanisms of DNA damage)
•Outcome of therapy
- •Older patients generally do less well as do those with lymphocyte-depleted histology.
Prognosis:
- Cure rate ranges from 50-90%.
- •Over 80% of patients with stage I or II disease are cured
- •Only 50% of stage IV patients are cured
•What does cure mean ?
- • Overall 80% are long term survivors (can we improve)
- • 10% die from relapse of HL (first 10 years)
- •10% die from treatment complications (after 10 years)
- •“Curing” cHL in a 25-year old woman using chemo plus radiotherapy does not guarantee long term survival
- •Can cure them but some may still succumb to cancers due to rtx or chemo OR due to recurrence of HL
What are the diffeent types of lymphomas?
What is the long term prognosis following HL treament?
With HL you are more likely to die of relapse within 5yrs, but after 10yrs will die more likely from 2ry malignant condition or Cardiovasc events
- HL is a curable disease overall approx 80%
- Intensify therapy: more cures & more secondary cancers -> chemo + radiotherapy with 10% of therapy complication
- Reduce Therapy: less cures & less secondary cancer; chemo only with 10% die of cHL
What are marginal zone lymphomas?
- •Is a Marginal zone NHL involving extranodal lymphoid tissue (ie mucosa-associated lymphoid tissue MALT)
- •Comprise ~ 8% of all NHL
- •Chronic antigen stimulation
- •Sjogrens syndrome ; parotid lymphoma (MZL)
- •H.Pylori ; Gastric MALT lymphoma (MZL)
- •Hashimoto’s Thyroid; Thyroid (MZL)
- •Lachrymal gland (?Psittaci infection)
- •Median age at presentation 55-60y
- •Most commonly arise in stomach, usually present with dyspepsia or epigastric pain
- •Usual presentation is Stage I[E] (stage 1 so only 1 node, but extranodal symptoms)
- •‘B’-symptoms uncommon
Gastric MALT Stage I-II disease
- •Omep 20mg/Clarith 500mg/amox 1gm bd
- •Repeat breath test at 2 months
- •Repeat endoscopy every 6 months for 1st 2years then annually
- Results
- •Durable remission in 75% of patients
- •response may be delayed until 1yr
- •If fails eradication therapy then may require chemotherapy
What are the clinical issues of CLL and the treatment?
- •Suportive treatment
- •Vaccination
- •Anti-infective prophylaxis and treatment
- •Specific scenarios
- •Auto-immune cytopaenias
- •High grade (Richter) transformation
- •Leukaemia directed treatment
- •Tailored to patient
•Prophylaxis and treatment of infections
- – Account for 50% of all CLL related deaths
- – Most are bacterial, but fungal and viral are becoming increasingly prevalent
- – Prophylaxis
- •Aciclovir
- •PCP prophylaxis for those receiving fludarabine or alemtuzumab (Campath)
- •IVIG is recommended for those with hypogammaglobulinemia and recurrent bacterial infections
- •Immunisation against pneumococcus, and seasonal flu
- •Avoid live vaccines – herpes zoster
•Auto-immune phenomena
- • 1st Line Steroids. 2nd Line Rituximab
- •Irradiated Blood products if risk of TA GVHD
What is the treatment for leukaemia and its indications?
- • Incurable by chemotherapy
- •Watch and wait versus active treatment
- •Balance tx with comorbidities
- • Conventional not to treat Stage A
- •What are the indications for treatment?
- •If required tailor treatment (age/co-morbidities)
- • Aim of therapy obtain response/remission
- •Disease will relapse
- •2nd line therapy
- •Go-go (up to 70y) tolerate all tx; slow go (up to 80y) tolerate less heavy tx and no-go (>80y) don’t tolerate tx
- • Young patients may be cured by allogeneic SCT
Indications:
- Watch and wait unless
- •Progressive lymphocytosis
- –lymphocyte doubling time <6 months
- •Progressive marrow failure
- •Hb < 100, platelets <100, neutrophils <1
- •Massive or progressive lymphadenopathy/splenomegaly
- •Systemic symptoms (B symptoms)
- •Autoimmune cytopenias (treat with steroids)
- •Progressive lymphocytosis
1st line chemo - tp53 intact:
- •FCR
- •Fludarabine Cyclophosphamide Rituximab (anti CD20 moab)
- •Rituximab-Bendamustine
- •Obinutuzumab (anti CD20) +Chlorambucil (single agent chemo)
- •Supportive care only – blood transfusion and abx
High risk cases:
- •How to manage high risk cases
- •Patients with TP53/17p deleted CLL 1st Line
- •Refractory disease or early relapse (<24 months)
- •Won’t benefit from further chemo, so give new tx
- •New agents
- •Ibrutinib (Bruton Tyrosine Kinase Inhibitor)
- Take tablet 2x per day and with minimal side effects compared to chemo dose
- Eventually after many years the cancer can become mutant to the BTK inhibitor
- •Venetoclax (anti Bcl2 oral agent)
- promotes apoptosis of CLL cells; in high risk CLL p53 mutated 85% response
- Chimeric antigen receptor T cells
- Survive because it needs to constantly be stimulated via BCR otherwise undergoes apoptosis -> live on as memory cell; signalling pathway includes BTK (germ line mutations lead to b cell aplasia); Bcl 2 is proapoptotic
- •Ibrutinib (Bruton Tyrosine Kinase Inhibitor)
How are plasma cells made?
- Plasmablasts are still growing up – which need to undergo class switch recombination; they downregulate some tf which are needed for other b cells; and upreg the ones specific to plasma cells -> which drive the expansin of the secretory apparatus of the plasma cells -> which plasma cells make ab and secretory proteins need to be folded in a particular method in ER which needs complex quality control methods -> then passed to golgi with modificaltions and secreted -> plasma cells have highest secretory capacity
- Plasmablasts secrete 10,000 IG molecules per second
- Encountering antigen drives a virgin B cell to generate a low-affinity plasma cell or stimulates its migration to a germinal centre.
- In the germinal centre, affinity maturation occurs and is mediated through two processes: somatic hypermutation and antigen selection.
- Subsequently, class switch recombination occurs, leading to the development of immunoglobulin (Ig) isotypes.
- Once this process is complete, the plasmablast leaves the germinal centre and migrates to the bone marrow where it becomes a long-lived plasma cell that produces antibody.
- The machinery that is necessary to generate these physiological DNA rearrangements can malfunction, leading to mutations in crucial oncogenes and tumour suppressor genes, and malignant change.
- Key challenges for a plasma cell include switching off cellular characteristics that are no longer required, such as cell cycling, activating programmes that are essential for antibody production, and undergoing apoptosis if they do not find a receptive niche in the bone marrow.
- Failure to complete these programmes correctly could potentially leave active cellular processes, which may result in the features of myeloma.
- The key transcription factors underlying this coordinated differentiation process are also shown. BCL-6, B cell lymphoma 6; BLIMP1, B lymphocyte-induced maturation protein 1; CIITA, MHC class II transactivator; ID3, DNA-binding protein inhibitor ID3; PAX5, paired box gene 5; XBP1, X box-binding protein 1.
What is the pathogenesis of multiple myeloma?
Dna usually cut to realign sequences doesn’t happen properly – abnormal realignment, which then also has oncogenes which are under the control of IG genes regulatory element. This is a plasma cell so needs to transcribe lots of ig genes -> so upstream oncogene become upregulated which drives expansion of normal plasma cel and initial transformation process, which the pick up other mutations -> kras/nras -> leads to myeloma
Multiple myeloma has complex heterogeneous cytogenetic abnormalities.
MM is more genetically similar to a solid cancer rather than a blood cancer
Most non-hyperdiploid tumours have IgH translocations that involve several recurrent chromosomal loci, including 11q13 (cyclin D1), 6p21 (cyclin D3), 4p16 (FGFR3 and MMSET), 16q23 (MAF) and 20q11 (MAFB).
55–60% of patients have a hyperdiploid karyotype, which confers a better prognosis than those with non-hyperdiploid disease.
What is the clonal evolution of MM?
Clonal evolution – shows how Darwinian evolution and linear evolution is how clones develop in MM; not all MM cells are the same in one patient; after different treatemtns we get subclones from tx which appear and can’t be treated by those other tx
What is true or relative polycythaemia?
–Polycythaemia raised Hb concentration and Haematocrit %
- •Relative (or pseudopolycythaemia - lack of plasma) (non-malignant)
- •True (excess erythrocytes)
- –Secondary (non-malignant)
- –Primary (myeloproliferative neoplasm)
Red cell mass INCREASE + plasma volume NORMAL => True polycythaemia, either 2ry polycythaemia (elevated EPO) or 1ry polycythaemia (reduced EPO)
2ry =
Raised EPO:
Appropriate:
- high altitude
- hypoxic lung disease
- cyanotic heart disease
- high affinity hb
- We produce enough EPO to produce enough RBC -> at sea level; in altitude increase RBC production to increase O2 capacity so need increased EPO levels
- High affinity hb – bind to O2 too tightly and don’t release it at the muscles
Inappropriate:
- Renal disease (cysts, tumours, inflammation)
- Uterine myoma
- other tumours (liver, lung)
Red cell mass NORMAL + plasma volume DECREASE => relative (pseudo) polycythaemia, due to alcohol, obesity, diuretics
What are the phases of CML?
- Abl is for TK
- Bcr – break point cluster region -> lots of chromosomal break points cluster around this region
- Translocation of abl on to chromosome 22
- A fusion gene results on the derived c/some 22
- This leads to the synthesis of an abnormal protein with TK activity greater than the normal ABL protein
- Limit of detection according to the number of dividing cells obtained from the bone marrow, often 1 in 50 sensitivity
- Normal to have translocations -> but no stage at normal development of myelocytes does the DNA undergo recombination so this is unfortunate
- So now have a constituatively active TK due to the translocation
