Haematology Flashcards
1
Q
Examples of primary haematological disorders
A
Primary disorders usually arise from DNA mutation(s)
inherited (germline gene mutated):
FIX: deficiency > haemophilia B, Excess > FIX Padua (gene therapy use)
Erythrocytes: Deficiency> B globin gene mutation > Hb S, Excess > High affinity Hb mutation >Erythrocytosis
acquired (somatic gene mutated) BM rapid turnover system! (more common in blood cancers)
Erythrocytes: Excess> JAK2 > Polycythaemia vera, Deficiency> PIG A >PNH paroxysmal nocturnal haemoglobinuria
Soluble factors No acquired DNA mutations because not rapidly dividing cells (hepatocytes/endothelial cells)
2
Q
Examples of secondary haematological disorders
A
Secondary disorders are changes in haematological parameters in response to a non-haematological disease or scenario. eg
Factor VIII:
Excess > inflammatory response/pregnancy
Deficiency>2ndry to anti-FVIII auto antibodies (acquired haemophilia A)
3
Q
Systemic (non-haematological) conditions causing haematological abnormalities
A
Chronic Inflammation:
raised FVIII levels> increased Thrombosis risk.
Erythrocyte (Hb) count
Raised {altitude/hypoxia or Epo secreting tumour}
Reduced - BM infiltration or deficiency {Vit B12, or Fe} disease, Shortened survival {Haemolytic anaemia}
Platelet count
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 }
4
Q
Anaemia; malignancy or systemic disease
A
may be first sign of systemic disease or occult malignancy eg.
Folate deficiency and Howell Jolly bodies > Coeliac
Fe deficiency > bowel or gastric cancer, peptic ulcer, IBD, renal cell carcinoma, blood cancer (urinary tract ones more rare)
Leucoerythroblastic anaemia > primary infiltration in bone marrow; blood cancer leukaemia/lymphoma/myeloma), metastatic breast/lung/prostrate cancer, could also be miliary TB or severe fungal infection, if massive splenomegaly -> myelofibrosis
Haemolytic anaemia > acquired immune and non-immune
Anaemia of Inflammation (chronic disease)
5
Q
Leuco-erythroblastic anaemia
A
= red cell and white cell precursor anaemia
peripheral bloodfilm features: teardrop RBCs, nucleated RBCs, immature myeloid cells (myelocytes) - these features are present normally but in bone marrow not peripheral blood
6
Q
Haemolytic anaemia lab results
A
anaemia
reticulocytosis (may cause modest elevation of MCV [upper limit of normal])
bilirubinaemia (unconjugated/pre-hepatic cause)
raised LDH
reduced haptoglobins (binding protein for Hb)
7
Q
Immune haemolytic anaemia (acquired)
A
spherocytes
positive direct antiglobulin test
associated with systemic diseases:
immunological disorders (lymphoma, chronic lymphocytic leukaemia)
infection (mycoplasma)
idiopathic
8
Q
Non-immune haemolytic anaemia (acquired)
A
DAT negative
infection of erythrocytes (malaria)
Micro-angiopathic haemolytic anaemia (MAHA) = acquired associated with systemic disease -> underlying adenocarcinoma or haemolytic uraemic syndrome
9
Q
MAHA
A
bloodfilm features: RBC fragments, thrombocytopenia
Adenocarcinomas, low grade DIC
Platelet activation
Fibrin deposition and degradation
Red cell fragmentation (microangiopathy)
Bleeding (low platelets and coag factor consumption)
10
Q
Types of white blood cells
A
Bone marrow:
blasts (myeloid and lymphoid) (usually <5% of BM cells if excess consider leukaemia)
promyelocytes
myelocytes
Peripheral blood:
Phagocytes
Granulocytes: Neutrophils, Eosinophils, basophils
Monocytes
immunocytes
T lymphocytes
B lymphocytes
NK cells
11
Q
high WCC with blasts in peripheral blood
A
acute myeloid leukaemia
12
Q
high WCC with myelocytes in peripheral blood
A
chronic myeloid leukaemia
13
Q
Reactive neutrophilia causes
A
pyogenic infection
corticosteroids
underlying neoplasia
tissue inflammation (eg. colitis, pancreatitis, myocarditis, MI)
neutrophils + toxic granulation, no immature cells
14
Q
Malignant neutrophil abnormalities
A
Neutrophilia plus basophilia & myelocytes. Suggestive of chronic myeloid leukaemia (CML)
Neutropenia plus myeloblasts suggests acute myeloid leukaemia (AML)
15
Q
Reactive Eosinophilia
A
Parasitic infestation
Allergic diseases e.g. asthma, rheumatoid, polyarteritis, pulmonary eosinophilia.
Neoplasms, esp. Hodgkin’s, T-cell NHL (reactive eosinophilia)
Drugs (reaction erythema multiforme)
16
Q
Chronic eosinophilic leukaemia
A
Eosinophils part of the “clone”
FIP1L1-PDGFRa Fusion gene
17
Q
Monocytosis
A
Rare but seen in certain chronic infections and primary haematological disorders:
TB, brucella, typhoid
Viral; CMV, varicella zoster
sarcoidosis
chronic myelomonocytic leukaemia (MDS)
18
Q
Raised lymphocyte count secondary/reactive causes
A
EBV, CMV, Toxoplasma (Infectious mononucleosis IM)
infectious hepatitis, rubella, herpes infections
autoimmune disorders
Sarcoidosis
19
Q
Reduced lymphocyte count secondary/reactive causes
A
Infection: HIV
Auto immune disorders
Inherited immune deficiency syndromes
Drugs (chemotherapy)
20
Q
Lymphocytosis in peripheral blood morphology
A
Mature lymphocytes (PB):
- reactive/atypical lymphocytes (IM)
- small lymphocytes and smear cells (CLL/NHL)
Immature Lymphoid cells in PB:
Lymphoblasts; Acute Lymphoblastic Leukaemia (ALL)
21
Q
Determining clonality in b-cell lymphocytosis using light chain restriction
A
polyclonal
Kappa &
Lambda -> 60:40 -> reactive
Monoclonal
kappa only or
lambda only -> 99:1 -> malignant
22
Q
Haemato-oncology diagnosis
A
Morphology: architecture of tumour, cytology, cytochemsitry
Immunophenotype: flow cytometry, immunohistochemsitry
cytogenetics: conventional karyotyiping, fluorescent in-situ hybridisation (interphase or metaphase FISH)
molecular genetics: mutation detection (direct and pyrosequencing), PCR analysis, gene expression profiling, whole genome sequencing
Morphology
malignant cells; large or small, mature or immature?
Lymph node architecture (diffuse invasion or forming follicles?)
Immunophenotype (flow cytometry or Immunohistology)
myeloid or lymphoid?
T or B lineage?
stage of maturation (precursor or mature? )
Cytogenetics (translocations: 1) fusion gene or 2)deregulated oncogene )
confirm morphological diagnosis eg
Philadelphia Chromosome > CML.
t(8;14) activates c-myc oncogene in Burkitt Lymphoma
Prognostic information eg 17p del in CLL
Molecular genetics (PCR, pyro-sequencing)
JAK2 mutation in suspected polycythemia vera
BCR ABL cDNA detection and quantification
23
Q
Common blood cancer presentations and problems
A
Lympho-haemopoietic failure (a dispersed organ!):
Bone marrow (myeloid): anaemia, bacterial infection (neutrophils) bleeding (platelets)
Immune system (lymphoid): recurrent viral or fungal infection
Excess of malignant cells:
Erythrocytes or leucocytes: impair blood flow >stroke or TIA
Massively enlarged lymph nodes (lymphoma)> compress hollow tubes: bowel, vena cava, ureters, bronchus.
Infiltrate and impair other organ function:
CNS lymphoma
Skin lymphoma
Kidney failure (light chain deposition from myeloma)
Miscellaneous problems:
hyepercalcaemia
hypermetabolism
24
Q
Lymphomas
A
Definition:
The term ‘lymphoma’ means a neoplastic (malignant) tumour of lymphoid cells.
Lymphomas usually found in:
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” (breast kidney){*Immune privilege sites CNS, occular, testes}
Incidence:
There are approximately 200 new cases per year for every million of the population (around 10,000 new cases a year in the UK).
Non-Hodgkin’s Lymphomas 80%
Hodgkin Lymphoma 20%
25
Types of lymphoid malignancies
Precursor malignancies:
B or T cell lineage
Mature B cell malignancies:
Non Hodgkin Lymphoma (NHL)
Or
Hodgkin Lymphoma
Mature T cell malignancies:
T cell or NK cell Non Hodgkin
Lymphoma (NHL)
26
Mechanisms of risk factors for NHL subtypes
Constant antigenic stimulation:
Bacteria infection (chronic)
Auto immune disorders
Viral Infection
(direct viral integration of lymphocytes)
Loss of T cell function and EBV infection plus EBV infections of B cells:
Loss of T cells (HIV infection untreated )
Iatrogenic immunosuppression
27
Chronic antigen stimulation and NHL
(Bacterial or auto immune antigenic drive)
B cell Non Hodgkin Lymphoma Marginal zone sub type (MZL):
- H.Pylori : Gastric MALT (mucosa associated lymphoid tissue) (MZL of stomach)
- Sjogren syndrome : MZL of
- Hashimoto’s : MZL of thyroid
Enteropathy associated T-Cell Non Hodgkin lymphoma (EATL):
- Coeliac disease/Gluten: small intestine EATL
28
Direct viral integration and NHL
HTLV1 retrovirus infects T cells by vertical transmission
Caribbean, Japan (and world wide) endemic infection
Risk of Adult T cell leukaemia lymphoma (ATLL) is 2.5% at 70 years
ATLL is a subtype of T cell Non Hodgkin Lymphoma
29
Loss of T cell function and EBV-driven NHL
EBV infection:
- EBV infects B lymphocytes, healthy carrier state post glandular fever.
- EBV driven proliferation of B cells is associated with surface expression of EBV antigens. Proliferating B cells targeted and killed by EBV specific cytotoxic T cell response
Loss of T cell function:
- HIV (in uncontrolled infection there is x60 increased incidence of B NHL )
- Iatrogenic (transplant immunosuppression)
PTLD (post transplant lymphoproliferative disorder)
Loss of cytotoxic T cell function can cause failure to eliminate EBV driven proliferation of B cells
30
The lymphoreticular system
3 types of tissue
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
31
Cells of the lymphoreticular system
2 categories:
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
Accessory cells:
Antigen presenting cells
Macrophages
Connective tissue cells
32
B cell area in lymphoid tissue
Paracortical Tcell zone
Lymphoid follicle:
Mantle zone- Naïve unstimulated B cells
Germinal center - B cells and Antigen presenting cells
This is where B cells which bind antigen epitopes are selected and activated
33
T cell area in lymphoid tissue
Comprises:
T cells
Antigen presenting cells
High endothelial vessels
This is where T cells which bind antigen epitopes are selected and activated
34
Lymphoma pathogenesis
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
35
WHO lymphoma classification
HODGKIN LYMPHOMA
Classical
Lymphocyte predominant
NON-HODGKIN LYMPHOMA
B cell:
- Precursor B cell neoplasms
- Peripheral B cell neoplasms (Low and High grade)
T cell:
- Precursor T cell neoplasms
- Peripheral T cell neoplasms
36
Key cell surface markers for T and B cells
T = CD3, CD5
B = CD20
these normal cell markers may be decreased in lymphoma
abnormal markers may be expressed in lymphoma eg. cyclin D1 (not present normally)
37
Common B cell NHLs
Low grade: (small lymphocytes)
- Follicular lymphoma
- Small lymphocytic lymphoma/chronic lymphocytic leukaemia
- Marginal zone lymphoma
High grade: (big lymphocytes)
- Diffuse large B cell lymphoma
- Burkitt’s lymphoma
Aggressive:
- Mantle cell lymphoma
38
Follicular lymphoma
Clinical:
- Lymphadenopathy MA/elderly
Histopathology:
- Follicular pattern
- Germinal centre cell origin CD10, bcl-6+
Molecular:
- 14;18 translocation involving bcl-2 gene
Indolent but can transform to high grade lymphoma (incurable, median survival 12-15 years, may require 2-3 cycles of chemotherapy during this time)
39
SLL/CLL
Clinical:
- MA/elderly; nodes (SLL) or blood (CLL)
Histopathology:
- Small lymphocytes, Naïve or post-germinal centre memory B cell
- CD5, CD23 + (CLL)
Molecular:
- Multiple genetic abnormalities
Indolent, but can transform to high grade lymphoma (Richter transformation)
40
Marginal zone/ MALT lymphoma
Arise mainly at extranodal sites (many sites, e.g. gut, lung, spleen)
Thought to arise in response to chronic antigen stimulation (e.g. by Helicobacter in stomach)
Post germinal centre memory B cell
Indolent but can transform to high grade lymphoma
Can treat low grade disease with non-chemotheraputic modalities - i.e. remove antigen
- E.g Helicobacter eradication
41
Mantle cell lymphoma
Clinical:
- MA male predominence
- Lymph nodes, GI tract
- Disseminated disease at presentation
Histopathology:
- Located in mantle zone
- Pre-germinal centre cell
- Aberrant CD5, cyclin D1 expression
Molecular:
- 11;14 translocation
- Cyclin D1 over expression
Median SR 3-5 yrs
42
Burkitt's lymphoma
Clinical:
- Jaw or abdominal mass children/young adults (endemic, sporadic, immunodeficiency)
- EBV associated
Histopathology:
- Germinal center cell origin
- “starry-sky” appearance
- very high Ki-67 index
Molecular:
- C-myc translocation (8:14, 2:8, 8;22)
Aggressive disease (fastest proliferating malignancy)
NB: Tumour lysis syndrome can occur during treatment but even before treatment
43
Diffuse large B cell lymphoma
Clinical:
- MA/elderly
- Lymphadenopathy
Histopathology:
- Germinal center (CD10) or post-germinal center B cell
- Sheets of large lymphoid cells
- Germinal center phenotype = good prognosis
- p53 positive, high proliferation fraction = poor prognosis
44
Peripheral T cell lymphomas
MA/elderly
Lymphadenopathy and extranodal sites
Large T lymphocytes
Often with associated reactive cell population, esp eosinophils
Aggressive
45
Special forms of T cell lymphomas
Adult T cell leukaemia/lymphoma:
- Caribbean and Japan
- Associated with HTLV-1 infection
- arises from CD4 positive T cells (also express CD25)
- acute and chronic presentations
- aggressive esp in acute form
Enteropathy associated T cell lymphoma:
- Some patients with long standing coeliac disease
- arises from intra-epithelial CD8 positive T cells
- T cells form mass in bowel
- very aggressive
- treatment: gluten avoidance
Cutaneous T cell lymphomas:
E.g. mycosis fungoides
- begins as long-standing patches/plaques on skin in sun-protected areas and progresses to nodules and tumours
- arises from CD4 positive T cells
Anaplastic large cell lymphoma
- CD3 and CD2 cell markers but may have null cell marker phenotype
46
Anaplastic large cell lymphoma
Clinical:
- Children/young adults
- Lymphadenopathy
Histopathology:
- Large “epithelioid” lymphocytes
- T cell or null phenotype
Molecular:
- 2;5 translocation
- Alk-1 protein expression
Aggressive
Alk-1 positive better prognosis
47
Hodgkin's lymphoma (HL)
More often localised to a single nodal site
Spreads contiguously to adjacent lymph nodes
Classical:
(Several subtypes)
- Nodular sclerosing
- Mixed cellularity
- Lymphocyte rich and lymphocyte depleted
Lymphocyte predominant:
- Some relationship to non-Hodgkin’s lymphoma
48
Classical HL
Clinical:
- Young and MA
- Often involves just single lymph node group (painless lymphadenopathy)
- may cause obstructive symptoms
- constitutional symptoms (B symptoms/FLAWS, rare: pruritus and alcohol-induced painful lymphadenopathy)
Thought to be germinal center/post germinal center B cell origin
EBV associated
CD30 +, CD15+, CD20-
Histopathology:
- Sclerosis, mixed cell population in which scattered Reed-Sternberg and Hodgkin cells with eosinophils
Moderately aggressive
49
Nodular lymphocyte predominant HL
Clinical: Isolated lymphadenopathy
Germinal centre B cell (positive for some germinal centre B cell markers)
No association with EBV
CD20+, CD30-, CD15-
Histopathology: B cell rich nodules with scattered L&H cells
Indolent
Can transform to high grade B cell lymphoma
50
Lymphoma presentations
Painless progressive lymphadenopathy
Palpable node
Extrinsic compression of any “tube”
Eg Ureter, Bile duct, large blood vessel, bowel, trachea, oesophagus
Infiltrate/impair an organ system
E.g. skin rash, ocular&CNS, liver failure
Recurrent infections
Constitutional symptoms (FLAWS)
Coincidental e.g. FBC, Imaging
51
HL classification
Classical HL:
Nodular sclerosing 80% Good prognosis (causes the peak incidence in young women)
Mixed cellularity 17% Good prognosis
Lymphocyte rich (rare) Good prognosis
Lymphocyte depleted (rare) Poor Prognosis
Nodular Lymphocyte predominant HL 5% (disorder of the elderly multiple recurrences)
52
HL staging
(using lymph node biopsy and PET)
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
53
cHL nodular sclerosing subtype
Young women(>men) 20-29 years
Neck nodes and mediastinal mass(may be massive and compress SVC or trachea)
May have B symptoms
Needs a Tissue diagnosis
54
Combination chemotherapy for cHL
ABVD:
Adriamycin
Bleomycin
Vinblastine
DTIC
given at 4-weekly intervals.
ABVD is Effective treatment
Preserves fertility (unlike MOPP the original chemo)
Can cause (long term): Pulmonary fibrosis, cardiomyopathy
Chemotherapy (essential for cure)
ABVD 2-6 cycles (depends:stage&interim response)
PET CT
Interim: After x2 cycles, response assessment
End of Treatment: Guides need for additional radiotherapy
+/- Radiotherapy (see next slide)
Relapse {salvage chemotherapy}
High dose chemotherapy + Autologous PB stem cell transplant as support
55
Radiotherapy for HL
Modern practice involved field only
Adv: Low/negligible risk of relapse within field
Disadv:
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 (chemo + radio) greatest risk of 2o malignancy
56
Diffuse large B cell NHL Treatment
Treated by x 6-8 cycles of R-CHOP (Rituximab-CHOP)
combination chemotherapy using a mixture of drugs usually including an anthracycline (e.g. doxorubicin).
Combination drug regimens e.g. CHOP
Cyclophosphamide 750 mg/m2 i.v. D1
Adriamycin 50 mg/m2 i.v. D1
Vincristine 1.4 mg/m2 i.v. D1
Prednisolone 40 mg/m2 p.o. D1‑D5
R is Immunotherapy using the anti CD20 monoclonal antibody Rituximab
Aim of therapy is curative (overall approx 50%)
Relapse:
High dose chemotherapy and Auto stem cell transplant
CAR-T cell therapy (Chimeric antigen receptor)
57
Follicular lymphoma treatment
At presentation Watch and wait only treat “if clinically indicated”:
Nodal extrinsic compression: bowel, Bile duct, ureter, vena cava,
Massive painful nodes, recurrent infections
Treatment:
combination Immuno-chemotherapy R-COP or R-CHOP
Treatment is not curative, may require 2nd or 3rd line
Median survival 13 years (wide range)
58
Chronic lymphocytic leukaemia
Proliferation of mature B-lymphocytes
Commonest leukaemia in the western world
Caucasian
UK incidence 4.2/100,000/year
Age at presentation median 72 (10% aged <55yrs)
Relatives x7 increased incidence
(cancer primarily of bone marrow and blood, whereas lymphoma typically present with lymphadenopathy initially)
59
CLL lab findings
Lymphocytosis between 5 and 300 x 109/l
Smear cells
Normocytic normochromic anaemia
Thrombocytopenia
Bone marrow lymphocytic replacement of normal marrow elements
B lymphocytes that are CD5 and CD19 positive (usually don't express CD5 as this is T cell marker) - will also have other normal b cell markers
60
CLL prognosis
Highly variable natural history:
Initially 5-10 years good health until progression to a 2-3 year terminal phase
Rapid progression to death within 2-3 years
In a disorder of elderly:
1/3 never progress
1/3 Progress, respond to CLL Rx (death from unrelated disorder)
1/3 Progress, require multiple lines of Rx, refractory disease, death from CLL
Cell based prognostic factors:
IgHV mutation status
CLL FISH cytogenetic panel
TP53 mutation status (Chromosome 17p del and/or TP53 point mutation)
Clinical staging systems:
Binet or Rai (clinical staging)
CLL IPI score
61
CLL clinical issues
Increased risk of infection
bone marrow failure
lymphadenopathy, splenomegaly, lymphocytosis
transform to high grade lymphoma
autoimmune complications (eg. immune haemolytic anaemia)
62
CLL supportive care
Sino-pulmonary infections:
Early Rx with antibiotics
Pneumocystis prophylaxis (may also require zoster ppx)
Recurrent infection + IgG < 5g/l > IVIG replacement therapy
Vaccinations:
Pneumococcal
Covid19
Seasonal flu
Avoid live vaccines
63
CLL indications to treat
Watch and wait unless:
Progressive lymphocytosis
>50% increase over 2 months
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 immunosuppression not chemotherapy)
CLL therapy:
Combination Immuno-chemotherapy (being superseded by targeted Rx)
Targeted Therapy (BTK inhibitor eg ibrutinib, BCL2 inhibitor [allowing apoptosis] eg. venetoclax)
Cellular therapy only for relapsed high risk cases (Allogeneic SCT, CAR-T therapy)
NB: risk of tumour lysis syndrome on initiating venetoclax treatment
64
Raised Hb causes
Polycythaemia = raised Hb concentration and Haematocrit %
- Relative (lack of plasma) (non-malignant)
- True (excess erythrocytes)
- Secondary (non-malignant)
- Primary (myeloproliferative neoplasm)
Myeloproliferative neoplasms (MPDs):
- Ph (Philadelphia Chromosome)negative
- Polycythaemia vera (PV)
- Essential Thrombocythaemia (ET)
- Primary Myelofibrosis (PMF)
- Ph positive:
- Chronic myeloid leukaemia (CML)
65
Causes of secondary polycythaemia (non-malignant)
(raised EPO)
Appropriate:
High altitude
Hypoxic lung disease
Cyanotic heart disease
High affinity haemoglobin
Inappropriate:
Renal disease (cysts, tumours inflammation)
uterine myoma
other tumours (liver, lung)
treatment often not indicated
66
Haematological malignancies
Myeloid:
Acute myeloid leukaemia (blasts >20%
Myelodysplasia (blasts 5-19%)
Myeloproliferative disorders
-Essential thrombocythaemia (megakaryocyte)
-Polycythemia vera (erythroid)
-Primary myeofibrosis
Chronic myeloid leukaemia
Lymphoid:
Precursor cell malignancy
-Acute lymphoblastic leukaemia (B & T)
Mature cell malignancy
-Chronic Lymphocytic leukaemia
-Multiple myeloma
-Lymphoma (Hodgkin & Non Hodgkin)
67
What processes are disrupted by mutation?
Impair/block cell differentiation (type 2)
Cellular proliferation (type 1)
Prolong cell survival (anti-apoptosis)
Mutation mechanisms:
DNA point mutations
Chromosomal translocations
- Creation of novel Fusion gene
- Disruption of proto-oncogene
68
Tyrosine kinase signalling in haemotopoeisis
Normal Tyrosine kinases:
Transmit cell growth signals from surface receptors to nucleus
Activated by transferring phosphate groups to self and downstream proteins
Normally held tightly in inactive state
Promote cell growth do not block maturation
Activating Tyrosine kinase mutations:
expansion increase in mature/end cells
Red cells; polycythaemia
Platelets; essential thrombocythaemia
Granulocytes; chronic myeloid leukaemia
69
Diagnosing philadelphia negative myeloproliferative disorders (MPD)
Based on combination of :
Clinical features
- Symptoms
- splenomegaly
FBC +/- Bone marrow biopsy
Erythropoietin level (epo)
Mutation testing (eg. JAK2)
Phenotype linked to acquired mutation
70
Polycythaemia Vera
Incidental diagnosis routine FBC (median Hb 184g/l, Hct 0.55)
Symptoms of increased hyper viscosity:
Headaches, light-headedness, stroke
Visual disturbances
Fatigue, dyspnoea
Increased histamine release:
- Aquagenic pruritus
Peptic ulceration
Test for JAK2 V617F mutation
Treatment:
Aim to reduce HCT : target HCT <45%
- Venesection
- Cytoreductive therapy hydroxycarbamide
Aim to reduce risks of thrombosis
- Control HCT
- Aspirin
- Keep platelets below 400x109/l (see treatment of ET)
71
Essential Thrombocythaemia
Chronic MPN mainly involving megakaryocytic lineage
Incidental finding on FBC (50% cases)
Thrombosis: arterial or venous
CVA, gangrene, TIA
DVT or PE
Bleeding: mucous membrane and cutaneous
Headaches, dizziness visual disturbances
Splenomegaly (modest)
Treatment:
Aspirin: to prevent thrombosis
Hydroxycarbamide: antimetabolite. Suppression of other cells as well.
Anagrelide: specific inhibition of platelet formation, side effects include palpitations and flushing
Prognosis:
Normal life span may not be changed in many patients.
Leukaemic transformation in about 5% after >10 years
Myelofibrosis also uncommon, unless there is fibrosis at the beginning
72
Primary Myelofibrosis
A clonal myeloproliferative disease associated with reactive bone marrow fibrosis
Extramedullary haematopoieisis
Primary presentation:
Incidence 0.5-1.5 /100000
Males=females
7th decade. Less common in younger patients
Other MPDs (ET & PV) may transform to PMF
Incidental diagnosis in 30%
Presentations related to:
Cytopenias: anaemia or thrombocytopenia
Thrombocytosis
Splenomegaly: may be massive
Budd-Chiari syndrome
Hepatomegaly
Hypermetabolic state:
Weight loss
Fatigue and dyspnoea
Night sweats
Hyperuricaemia
73
Primary myelofibrosis haematological findings
Blood film:
Leucoerythroblastic picture
Tear drop poikilocytes
Giant platelets
Circulating megakaryocytes
Liver and spleen
Extramedullary haemopoiesis in spleen and liver
DNA : JAK2 or CALR mutation
Bone marrow:
‘Dry tap’
Trephine:
Increased reticulin or collagen fibrosis
Prominent megakaryocyte hyperplasia and clustering with abnormalities
New bone formation
74
Primary myelofibrosis prognosis
Median 3-5 years but very variable
Bad prognostic signs:
- Severe anaemia <100g/L
- Thrombocytopenia <100x109/l
- Massive splenomegaly
Prognostic scoring system (DIPPS)
Score 0 -- median survival 15years
Score 4-6– median survival 1.3 years
75
Primary myelofibrosis treatment
Supportive: RBC and platelet transfusion often ineffective because of splenomegaly
Cytoreductive therapy: hydroxycarbamide (for thrombocytosis, may worsen anaemia)
Ruxolotinib: JAK2 inhibitor (high prognostic score cases)
Allogeneic SCT (potentially curative reserved for high risk eligible cases)
Splenectomy for symptomatic relief: hazardous and often followed by worsening of condition
76
Chronic myeloid leukaemia (CML)
Incidence 1-2/100,000
M:F 1.4:1
40-60 years @ presentation
Radiation exposure risk factor (Hiroshima & Nagasaki)
History:
Lethargy/ hypermetabolism/ thrombotic event : monocular blindness CVA, bruising bleeding
Exam:
Massive splenomegaly +/- hepatomegaly
FBC:
Hb and platelets well preserved or raised
Massive leucocytosis 50-200x109/L
Blood film :
Neutrophils and myelocytes (not blasts if chronic phase)
Basophilia
Treatment:
Imatinib (Abl tyrosine kinase inhibitor)
77
CML lab features
Leucocytosis between 50 – 500x109/l
Mature myeloid cells
Bi phasic peak Neutrophils and myelocytes
Basophils
No excess (<5%) myeloblasts
Platelet count raised/upper normal
(contrast acute leuk)
78
Philadelphia chromosome
Translocation of part of the long arm (q) of chromosome 22 to chromosome 9
And reciprocal translocation of part of chromosome 9, which includes the ABL oncogene to a specific breakpoint cluster region (BCR) of chromosome 22
A fusion gene results on the derived chromosome 22
This leads to the synthesis of an abnormal protein with TK activity greater than the normal ABL protein
Detection:
Conventional Karyotyping
FISH metaphase or interphase karyotyping
RT-PCR amplification and detection
79
Diagnosing and monitoring CML
FBC and measure leucocyte count
Cytogenetics and detection of Philadelphia chromosome
RT-PCR of BCR-ABL fusion transcript which can be quantified by RQ-PCR to determine response to therapy
Haematological response
Complete Haematological Response WBC<10x109/l
Cytogenetic response (on 20 metaphases)
Partial 1-35% Philadelphia positive
Complete 0% Ph positive
Molecular ( reduction in % BCR-ABL transcripts)
BCR-ABL transcripts reduce 100% > 10% > 1% > 0.1%
Major Molecular response (MMR) <0.1% (3 log reduction)
80
CML clinical course
Commence on oral TKI 1st generation
Monitor response FBC, Cytogenetics, RQ-PCR
CCyR at 12mo 97% FFP at 6 years (Fail to achieve CCyr 80%)
Average 95% 5 year survival
Annual mortality 2%
Reasons for not working:
Failure to achieve CCyR
Non compliance
Side effects; fluid retention pleural effusions
Loss of MMR
Acquiring abl point mutations leading to resistance
Evolution to blast crisis
81
Treatment algorithm CML
Chronic phase Tyrosine kinase Inhibitor (TKI)
Imatinib (1Gen,) Dasatanib Nilotonib (2G) Bosutinib (3G)
Failure (1) > switch to 2Gen or 3G TKI
No complete cytogenetic response @ 1year
Respond but acquire resistance
Failure (2) > consider allogeneic SCT
Inadequate response or intolerant of 2G TKIs
Progression to accelerated or blast phase
82
Myelodysplastic syndromes (MDS)
Biologically heterogeneous group of acquired haemopoietic stem cell disorders (~ 4 per 100,000 persons)
Characterised by:
The development of a clone of marrow stem cells with abnormal maturation resulting in -
functionally defective blood cells AND
a numerical reduction
This results in:
i. Cytopenia(s).
ii. Qualitative (i.e. functional) abnormalities of erythroid, myeloid and megakaryocyte maturation.
iii. Increased risk of transformation to leukaemia
Typically a disorder of the elderly.
Symptoms/signs are those of general marrow failure
Develops over weeks & months
83
MDS bone marrow and blood film features
Pelger-Huet anomaly (bilobed neutrophils)
Dysganulopoieses of neutrophils
Dyserythropoiesis of red cells (ringed sideroblasts on iron [prussian blue] staining)
Dysplastic megakaryocytes – e.g. micro-megakaryocytes
Increased proportion of blast cells in marrow (normal < 5%)
84
Diagnosis of MDS
requires:
1. cytopenia of at least on blood cell line
2. <20% blasts in blood and bone marrow
3. Either:
- characteristic cytogenetic or molecular findings without evidence of an alternate cause of these findings
OR
- morphological dysplasia in >/ 10% of nucleated cells of at least one cell lineage
85
WHO classification of MDS
Number of dysplastic lineages
Percentage of blasts in bone marrow and peripheral blood
Cytogenetic findings
Percentage of ringed sideroblasts
Number of cytopenias (based on criteria from the International Prognostic Scoring System - IPSS)
Hb < 100 g/L
Platelets < 100 x 10^9/L
Neutrophils < 1.8 x10^9/L
Monocytes < 1.0 x 10^9/L (if > 1.0 x 10^9/L then diagnosis is CMML)
86
Gene mutations in MDS
Driver mutations in MDS - carry prognostic significance:
TP53, EZH2, ETV6, RUNX1, ASXL1
Others: SF3B1, TET2, DNMT3A
Majority of common mutations are found more frequently in high risk MDS than in low risk MDS
87
MDS evolution
1. Deterioration of blood counts
* Worsening consequences of marrow failure
2. Development of acute myeloid leukaemia
– Develops in 5-50%< 1 year (depends on subtype)
– Some cases of MDS are much slower to evolve
– AML from MDS has an extremely poor prognosis and is usually not curable
3. As a rule of thumb
* 1/3 die from infection
* 1/3 die from bleeding
* 1/3 die from acute leukaemia
88
MDS treatment
At present, the only two treatments that can prolong survival are:
1. allogeneic stem cell transplantation (SCT)
2. intensive chemotherapy
but only a minority of MDS patients can really benefit from them
Majority:
1. Supportive care:
Blood product support
Antimicrobial therapy
Growth factors (Epo, G-CSF, TPO-Receptor Agonist)
2. Biological Modifiers:
Immunosuppressive therapy (Azacytidine )
Hypomethylating agents (Decitabine)
Lenalidomide (for del(5q) variant)
89
Bone marrow failure
1. PRIMARY (primarily in children)
Congenital: Fanconi’s anaemia (multipotent stem cell)
Diamond-Blackfan anaemia (red cell progenitors)
Kostmann’s syndrome (neutrophil progenitors)
Acquired: Idiopathic aplastic anaemia (multipotent stem cell)
2. SECONDARY (much more common)
Marrow infiltration:
Haematological (leukaemia, lymphoma, myelofibrosis)
Non-haematological (Solid tumours)
Radiation
Drugs
Chemicals (benzene)
Autoimmune
Infection (Parvovirus, Viral hepatitis)
90
Drugs that cause bone marrow failure
1. PREDICTABLE (dose-dependent, common):
Cytotoxic drugs
2. IDIOSYNCRATIC (NOT dose-dependent, rare):
Phenylbutazone
Gold salts
3. ANTIBIOTICS:
Chloramphenicol
Sulphonamide
4. DIURETICS:
Thiazides
5. ANTITHYROID DRUGS:
Carbimazole
91
Aplastic anaemia epidemiology
* 2-5 cases/million/yr (world-wide)
* All age groups can be affected
* Peak incidence:
i. 15 to 24 yrs
ii. over 60 yrs
92
Aplastic anaemia classification
IDIOPATHIC: Vast majority (70-80%)
INHERITED:
Dyskeratosis congenita (DC)
Fanconi anaemia (FA)
Shwachman-Diamond syndrome
SECONDARY:
Radiation: Predictable
Drugs: Predictable: Cytotoxic agents
Idiosyncratic: Chloramphenicol, NSAIDS
Viruses (Idiosyncratic): Hepatitis viruses
Immune: SLE
MISCELLANEOUS:
PNH (Paroxysmal Nocturnal Haemoglobinuria)
Thymoma
93
Idiopathic AA pathophysiology
Failure of BM to produce blood cells
* “Stem cell” problem (CD34, LTC-IC) [Long-Term Culture-Initiating Cells]
* Immune attack:
Humoral or cellular (T cell) attack against multipotent haematopoietic stem cell.
94
AA clinical presentation
Triad:
1. Anaemia Fatigue, breathlessness
2. Leucopenia Infections
3. Platelets Easy bruising/bleeding
95
AA diagnosis
1. Blood Cytopenia
2. Marrow Hypocellular
Classification:
Severe (SAA)
Non-severe (NSAA)
96
AA differential diagnoses
Hypoplastic MDS / Acute Myeloid Leukaemia
Hypocellular Acute Lymphoblastic Leukaemia
Hairy Cell Leukaemia
Mycobacterial (usually atypical) infection
Anorexia Nervosa
Idiopathic Thrombocytopenic Purpura
97
Severe AA
Camitta criteria:
2 out of 3 peripheral blood features
1. Reticulocytes < 1% (<20 x 109/L)
2. Neutrophils < 0.5 x 109/L
3. Platelets < 20 x 109/L
Bone marrow <25% cellularity
98
Bone marrow failure treatment
1. Seek and remove a cause (detailed drug & occupational exposure history).
2. Supportive: Blood/platelet transfusions (leucodepleted, CMV neg, irradiated)
Antibiotics
Iron Chelation Therapy
3. Immunosuppressive therapy (anti-thymocyte globulin, steroids, eltrombopag, cyclosporine A)
4. Drugs to promote marrow recovery
Oxymethalone, TPO receptor agonists (eltrombopag), ??G-CSF (prob not).
5. Stem cell transplantation (allogeneic)
6. Other treatments in refractory cases – e.g. alemtuzumab (anti-CD52, high dose cyclophosphamide)
99
AA treatment
SUPPORTIVE
Blood products:
Leucodepleted
(CMV negative)
(Irradiated)
Antimicrobials
Iron Chelation Therapy
(when ferritin > 1000 µg/L)
SPECIFIC TREATMENT – Idiopathic AA
Based on:
Severity of illness
Age of patient
Potential sibling donor
A. Immunosuppressive therapy – older patient
Anti-Lymphocyte Globulin (ALG)
Ciclosporin
Eltrombopag
B. Androgens – oxymethalone
C. Stem cell transplantation
Younger patient with donor (80% cure)
VUD/MUD for > 40 yrs (50% survival)
100
Late complications of immunosuppressive therapy for AA
1. Relapse of AA (35% over 15 yrs)
2. Clonal haematological disorders
Myelodysplasia
Leukaemia
~ 20% risk over 10 yrs
PNH (paroxysmal nocturnal haemoglobinuria)
May be a transient phenomenon
3. Solid tumours ~ 3% risk
101
Fanconi anaemia (FA)
The most common form of inherited aplastic anaemia.
Autosomal recessive or X-linked inheritance
Heterozygote frequency may be 1:300
Multiple mutated genes are responsible.
When these genes become mutated, this results in:
- Abnormalities in DNA repair
- Chromosomal fragility (breakage in the presence of in-vitro mitomycin or diepoxybutane)
102
Congenital abnormalities in FA
Congenital malformations may occur in 60-70% of children with FA:
Short Stature
Hypopigmented spots and café-au-lait spots
Abnormality of thumbs
Microcephaly or hydrocephaly
Hyogonadism
Developmental delay
No abnormalities 30%
103
FA complications
Aplastic anaemia
leukaemia
liver disease
myelodysplasia
cancer (epithelial)
104
Dyskeratosis congenita (DC)
An inherited disorder characterised by:
Marrow failure
Cancer predisposition
Somatic abnormalities
Patients may present with the Classical Triad of
Skin pigmentation
Nail dystrophy
Leukoplakia
105
DC genetic basis - Telomere shortening
3 patterns of inheritance
Abnormal telomeric structure & function is implicated.
Telomeres are:
found at the end of chromosomes
act to prevent chromosomal fusion or rearrangements during chromosomal replication
protect the genes at the end of the chromosome from degradation.
Telomere length is reduced in marrow failure diseases (especially short in patients with DC).
Maintenance of telomere length is required for the indefinite proliferation of human cells.
106
DC inheritance
X-linked recessive trait — the most common inherited pattern (mutated DKC1 gene - defective telomerase function).
Autosomal dominant trait — (mutated TERC gene - encodes the RNA component of telomerase).
Autosomal recessive trait — The gene for this form of DC has not yet been identified
107
How to choose a donor for allogeneic stem cell transplants
Well matched for tissue type - also known as HLA type
Ideally a sibling (one in four chance of matching with each sib)
If not, a volunteer unrelated donor or minimally mismatched family member
More recently, increased use of haploidentical family member – almost every patient has a donor
108
HLA system
A person’s tissue type comprises a set of distinct proteins called Human Leukocyte Antigens (HLA), found on the surface of most nucleated cells.
HLA molecules, or proteins, relevant in transplantation –
HLA-A, -B, -C, (class I), present peptide to CD8+ (cytotoxic T-cells)
HLA-DP,-DQ and -DR (class II), present peptide to CD4+ (helper T-cells)
HLA encoded by the Major Histocompatibility Complex, MHC, on chromosome 6.
Function – present foreign peptides to T cells
Routinely, HLA-A, -B and DR are typed for compatibility purposes.
109
Autologous transplantation
Give GCSF, collect stem cells and freeze, then thaw and reinfuse when pt is in remission after giving high dose chemotherapy
suitable for:
Acute leukaemia
Solid tumours
Autoimmune disease
Myeloma
Lymphoma
Chronic lymphocytic leukaemia
110
Allogeneic transplantation
high dose chemotherapy/radiotherapy is given and then bone marrow or peripheral blood cells from a donor are infused once patient is in remission
suitable for:
Acute leukaemia
Chronic leukaemia
Myeloma
Lymphoma
BM failure
Congenital immune deficiencies
111
Principles of transplantation
Identify disease unlikely to respond to standard treatment
Treat patient to remission
Identify a donor and collect stem cells
Give patient myeloablative therapy
Infuse stem cells
Continue immunosuppression & support patient through period of cytopenia
while collecting stem cells: have to make sure bone marrow sample is not contaminated by peripheral blood
112
Where do haematopoietic stem cells come from?
bone marrow
peripheral blood
umbilical cord (usually for children)
113
Complications of stem cell transplant (SCT)
Graft failure
Infections
Graft-versus-host disease (GVHD): allografting only
Relapse
114
Graft vs Host disease (GvHD)
An immune response when donor cells recognise the patient as ‘foreign’
Acute GvHD affects skin, gastrointestinal tract and liver
Chronic GvHD affects skin, mucosal membranes, lungs, liver, eyes, joints
pathophysiology: cytokine storm after chemotherapy drives GvHD
Grade 1-4 according to organ staging
Grade 4 = fatal
115
Risk factors for acute GvHD
Degree of HLA disparity
Recipient age
Conditioning regimen
R/D gender combination
Stem cell source (peripheral blood has more cells)
Disease phase
Viral infections (eg. CMV)
116
Treatment for acute GvHD
Corticosteroids
Calcineurin inhibitors: cyclosporin A, tacrolimus, sirolimus
Mycophenylate mofetil
Monoclonal antibodies (more aggressive)
Photopheresis
Total lymphoid irradiation
Mesenchymal stromal cells (more aggressive)
117
Acute GvHD prevention
Methotrexate
Corticosteroids
Calcineurin inhibitors: cyclosporin A, tacrolimus, sirolimus
CsA plus MTX
T-cell depletion (no longer used)
Post-transplant cyclophosphamide (remove active circulating T cells and prevent immune reaction)
118
Chronic GvHD
Immune dysregulation
Immune deficiency,
Impaired end-organ function
Decreased survival.
Diagnosis within 6 months of transplant, lasts 2-5 years
85% of survivors can discontinue treatment at that time
5-year survival is 70–80%, in persons with low risk cGVHD and those responding to corticosteroids.
Five-year survival is 30–40% for those with high-risk disease +/- failure of steroids
119
Risk factors for chronic GvHD
affects 50% of people who survive >1 year after transplant
Prior acute GvHD
Increased degree of HLA disparity
Male recipient: female donor
Stem cell source (PB>BM>UCB)
T-cell replete
Older donor age
Use of DLI
120
Main source of infection in neutropenic patients
Gram-positive from vascular access
Gram-negative from GI tract
121
Bacterial infections in neutropenic patients
In neutropenic patients, the causative organism is identified in approximately one third of patients
The most frequently isolated organisms are gram positive eg, staph epidermidis
Most deaths from sepsis are due to gram negative organisms eg e.coli, pseudomonas aeruginosa
Reduced incidence of infection using isolation measures and broad spectrum oral antibiotics
122
Managing neutropenic sepsis
Emergency situation
Defined as temperature >38 sustained for one hour, or single fever >39, in a patient with neutrophils <1.0 x 109/L
Assess patient: temperature, pulse, oxygen saturation and blood pressure. History and examination for evidence of source
Blood cultures, MSU, CXR
Initiate empirical broad spectrum antibiotics and supportive care
123
Fungal acquisition in neutropenic patients
Yeasts from translocation from the intestinal mucosa, or indwelling catheters
Moulds: inhalation, chronic sinusitis, skin, mucosa
NB: aspergillus is big killer
124
CMV
Member of herpes virus family: primary infection usually as a child, remains latent
Can be reactivated if immunosuppressed - can be problematic
Reactivation does not always result in infection - intervention at point where viraemia is low prevents infection
90% of London population has been exposed to CMV
125
Manifestations of CMV disease
Pneumonitis
Retinitis
Gastritis – colitis
Encephalitis
126
Preventing CMV disease
Twice weekly quantitative monitoring of peripheral blood viraemia to day 100
Thresholds for treatment together with evidence of increasing viral load
Ganciclovir/valganciclovir, lentamavir: oral and IV preparations.
Minimum of 2/52 treatment with clear evidence of reduction in viral load
127
Other viral complications of SCT
EBV: acute infection, PTLD (post-transplant lymphoproliferative disease)
Respiratory viruses: influenza, parainfluenza, respiratory syncytial virus, rhino, metapneumovirus, COVID-19
PAPOVA viruses: BK and haemorrhagic cystitis
Adenovirus
128
What affects the outcome of SCT?
Age
Disease phase
Gender of recipient and donor (if they don't match)
time to transplant from diagnosis
donor (sibling or unrelated)
129
What is acute leukaemia?
neoplastic condition characterised by:
- rapid onset
- early death if untreated
- immature cells (blast cells)
- bone marrow failure:
- anaemia, fatigue, pallor, SOB
- neutropenia, infections
- thrombocytopenia, bleeding
130
Acute myeloid leukaemia (AML)
dysfunction at the multipotent myeloid stem cell/progenitor cell, or pluripotent haematopoietic stem cell level leading to lack of maturation and build-up of immature cells
Increases with age
Prognosis worse with increasing age
40% of adults cured
Risk factors/causes:
Familial or constitutional predisposition
Irradiation
Anticancer drugs (esp platinum based drugs: inhibit DNA synthesis)
Cigarette smoking
Unknown
Multiple genetic hits
at least 2 interacting molecular defects
synergise to give leukaemic phenotype
131
Chromosomal abnormalities in AML
Translocation:
chromosomal translocation t(15;17) associated with acute promyelocytic leukaemia which has a 90% mortality rate in the first 24 hours.
Duplication:
Common in AML
Disease hotspots
+8
+21 gives predisposition
Possible dosage affect
extra copies of proto-oncogenes
Loss or deletion:
Common in AML
Disease hotspots
deletions and loss of 5/5q & 7/7q
Possible loss of tumour suppressor genes (p53)
Alternative explanation ‒ one copy of an allele may be insufficient for normal haemopoiesis
Possible loss of DNA repair systems
132
Abnormalities in leukaemogenesis (AML)
Type 1 abnormalities:
promote proliferation & survival
Type 2 abnormalities:
block differentiation (which would normally be followed by apoptosis)
133
Acute promyelocytic leukaemia with translocation t(15;17)
A very special type of acute leukaemia
The molecular mechanism is understood
Molecular treatment can be applied
The great majority of patients can now be cured
An excess of abnormal promyelocytes (inhibit vascular adhesion molecules which stimulate coagulation pathways)
Disseminated intravascular coagulation (DIC)
Two morphological variants but the same disease
bloods: no platelets, wcc may be normal
blood film: promyelocytes with auer rods (faggot cells)
134
AML vs ALL (how to differentiate)
Cytological features:
AML granular
AML has auer rods
Cytochemistry:
AML: myeloperoxidase, sudan black, NSA (all positive in AML, negative in ALL but not commonly used anymore)
Immunophenotyping
(myeloid cells have more side scatter and are usually bigger)
specific markers
135
Clinical features of AML
Bone marrow failure:
- Anaemia
- Neutropenia
- Thrombocytopenia
Local infiltration:
- Splenomegaly
- Hepatomegaly
- Gum infiltration (if monocytic)
- Lymphadenopathy (only occasionally)
- Skin, CNS or other sites
Hyperviscosity if WBC is very high, retinal haemorrhages, retinal exudates
136
AML blood films
Usually diagnostic: circulating blasts
Auer rods (proves myeloid)
ALL versus AML (if no granules or Auer rods how do you tell?)
“Aleukaemic” leukaemia --> if no leukaemia cells in blood then do bone marrow aspiration
137
AML Treatment
Supportive care:
- Red cells
- Platelets
- Fresh frozen plasma/ cryoprecipitate if - DIC
- Antibiotics
- Long line
- Allopurinol, fluid and electrolyte balance
Chemotherapy
Molecularly targeted therapy
Bone marrow transplantation
138
AML Chemotherapy
Damages DNA
Normal stem cells
- often quiescent
- checkpoints allow repair of DNA damage
Leukaemia cells
- continuously dividing
- lack of cell cycle checkpoint control
Combination chemotherapy
different mechanisms of action
synergy
non-overlapping toxicity
Mainly cell cycle specific drugs
4‒5 courses
remission induction × 2
consolidation × 2‒3
About 6 months of therapy
Consider transplantation if poor prognosis
139
Acute lymphoblastic leukaemia (ALL)
Peak incidence in childhood
Most common childhood malignancy
85% of children cured
Prognosis worse with increasing age
140
ALL Clinical features
Bone marrow failure — effects of
- Anaemia
- Neutropenia
- Thrombocytopenia
Local infiltration
- Lymphadenopathy (± thymic enlargement)
- Splenomegaly
- Hepatomegaly
- Testes, CNS, kidneys or other sites
Bone (causing pain)
141
Leukaemogenic mechanisms ALL
Proto-oncogene dysregulation
chromosomal translocation
Fusion genes
Wrong gene promoter
Dysregulation by proximity to T-cell receptor (TCR) or immunoglobulin heavy chain loci
Unknown – hyperdiploidy
142
ALL diagnosis
Clinical suspicion
Blood count and film
Bone marrow aspirate
Immunophenotyping
Cytogenetic/molecular genetic analysis
Blood group, LFTs, creatinine, electrolytes, calcium, phosphate, uric acid, coagulation screen
Why does immunophenotype matter?
AML and ALL are treated very differently
T-lineage (15%) and B-lineage (85%) ALL may be treated differently
Why does cytogenetic/molecular genetic category matter?
Ph-positive need imatinib
Treatment must be tailored to the prognosis
143
ALL Treatment
Specific therapy
systemic chemotherapy
CNS-directed therapy
Supportive care
blood products
antibiotics
general medical care
(Supportive care:
Central venous catheter
Red blood cell and platelet transfusions
Broad spectrum antibiotics for fever
Prophylaxis for Pneumocystis jirovecii infection
Hyperuricaemia: hydration, urine alkalinization and allopurinol or rasburicase
Hyperphosphataemia; aluminum hydroxide, calcium
Hyperkalemia: fluids, diuretics
Extreme leukocytosis (WBC > 200 × 109/l): leukapheresis
Sometimes haemodialysis)
Children: 5-year disease-free survival 80%
Adults: 5-year disease free survival 30‒40%
144
Haematology in pregnancy
Blood count changes
– Thrombocytopenia in pregnancy
Coagulation changes
– Thromboembolic disease
– Complications of pregnancy
– DIC syndromes
Haemoglobinopathy
– Haemoglobinopathy screening
– Sickle cell disease and pregnancy
Immune disorders
145
FBC in pregnancy
Mild anaemia
– Red cell mass rises (120 -130%)
– Plasma volume rises (150%)
Macrocytosis
– Normal
– Folate or B12 deficiency
Neutrophilia
Thrombocytopenia
– increased platelet size
146
Blood demands in 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
147
Iron and folate supplements in pregnancy
Folic acid
– Advise reduces risk of neural tube defects
– Supplement before conception and for ≥ 12
weeks gestation
– Dose 400μg / day
n Iron
– No routine supplementation in UK
148
Anaemia in pregnancy
Definition
– Hb < 110 g/l 1st trimester
– Hb < 105 g/l 2nd and 3rd trimester
– Hb < 100 g/l postpartum
Diagnosis of Iron def anaemia
↓ Hb, ↓MCV , ↓ MCH,
MCHC, film
- Known haemoglobinopathy
Check serum ferritin Treat if ferritin < 30 μg/l
- No
haemoglopinopathy/unknown
Trial of oral iron, +/- haemoglobinopathy screen
149
Causes of thrombocytopenia in pregnancy
Physiological:
– ‘gestational’/incidental thrombocytopenia
Pre-eclampsia
Immune thrombocytopenia (ITP)
Microangiopathic syndromes
All other causes: bone marrow failure, leukaemia,
hypersplenism, DIC etc.
150
Gestational thrombocytopenia
Physiological decrease in platelet count ~ 10%
>50x109/l sufficient for delivery (>70 for epidural)
Mechanism poorly defined
– Dilution + increased consumption
Baby not affected
Platelet count rises D2 – 5 post delivery
151
Pre-eclampsia and thrombocytopenia
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
152
ITP in pregnancy
5% of thrombocytopenia in pregnancy
– TP may precede pregnancy
– Early onset
Treatment options (for bleeding or delivery)
– IV immunoglobulin
– Steroids etc.
Baby may be affected
– 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
153
Microangiopathic syndromes in pregnancy
MAHA:
- Deposition of platelets
in small blood vessels
– Thrombocytopenia
– Fragmentation and
destruction of rbc
within vasculature
– Organ damage (kidney,
CNS, placenta)
154
Coagulation changes in pregnancy
Factor VIII and vWF increase 3-5 fold
Fibrinogen increases 2 fold
Factor VII increases 0.5 fold
(Factor X)
Protein S falls to half basal
PAI-1 increase 5 fold
PAI-2 produced by placenta
HYPERCOAGULABLE
HYPOFIBRINOLYTIC
Rapid control of bleeding from placental site (700ml/min)
at time of delivery
Increased thrombin generation
* Increased fibrin cleavage
* Reduced fibrinolysis
* Interact with other maternal factors
Net effects: a procoagulant state
Increased rate of thrombosis
155
VTE risk throughout pregnancy
highest risk at 0-6 weeks post delivery
156
VTE risk factors 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
157
Prevention of VTE in 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
158
VTE treatment in pregnancy
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
159
Chondrodysplasia punctata
Abnormal cartilage
and bone formation
Early fusion of
epiphyses
Nasal hypoplasia
Short stature
Asplenia
Deafness
Seizures
caused by warfarin use in first trimester (teratogenic)
160
Antiphospholipid syndrome in pregnancy
(acquired thrombophilia with pregnancy complications)
Antiphospholipid Syndrome (APLS): Recurrent
miscarriage + persistent Lupus anticoagulant (LA)
and/or antiphospholipid antibodies
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.
161
Fatal bleeding in pregnancy
due to placenta praevia and placenta accreta
= principal reason for hysterectomy
Use of Major Obstetric Haemorrhage protocols Determine placental site if previous C-Section
162
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
163
Causes of postpartum haemorrhage (4Ts)
Tone – Uterine Atony
Trauma – Laceration/Uterine rupture
Tissue – Retained placenta
Thrombin – Coagulopathy
164
Mechanisms of postpartum haemorrhage
major factors are
– uterine atony
– trauma
haematological factors minor except
– dilutional coagulopathy after resuscitation
– DIC in abruption, amniotic fluid embolism etc.
165
Pregnancy and DIC
Coagulation changes in pregnancy
predispose to DIC.
Decompensation precipitated by:
– Amniotic fluid embolism
– Abruptio placentae
– Retained dead fetus
– Preeclampsia (severe)
– Sepsis
166
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
167
Haemoglobinopathy screening in pregnancy aims
To avoid birth of children with:
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
168
IDA vs thalassaemia trait
IDA:
Hb: normal or low
MCH: low (proportional to Hb)
MCHC: low
RDW: increased
RBC: low or normal
Hb electrophoresis: normal
Thalassaemia trait:
Hb: normal, rarely low
MCH: lower for same Hb
MCHC: relatively normal
RDW: normal
RBC: increased
Hb electrophoresis: Hb A2 raised in beta-thal, normal in alpha-thal
169
Other important haem disorders in pregnancy
Haemolytic disease of the newborn (HDN)
Neonatal alloimmune thrombocytopenia
(NAIT)
Maternal immune responses against fetal antigens
requiring monitoring and intervention during
pregnancy.
170
Multiple myeloma
Malignancy of bone marrow plasma cells, the terminally
differentiated and immunoglobulin (Ig) secreting B cells
Myeloma plasma cells:
* home and infiltrate the bone marrow
* may form bone expansile or soft tissue tumours: plasmacytomas
* produce a serum monoclonal IgG or IgA: paraprotein or M-spike
* produce excess of monoclonal (κ or λ) serum free light chains
* Bence Jones protein: urine monoclonal free light chains
171
Multiple myeloma epidemiology
* Median age 67 years
* Incidence increases with age
* Only 1% of patients are younger than 40 years
* Men > women
* Black > Caucasian and Asians
* >17,600 people with myeloma live today in the UK
* Prevalence of myeloma in the community is increasing (ageing population)
(second most common haem malignancy, 19th in all cancers)
172
MM aetiology
Aetiology is unknown …
Risk factors
* Obesity increases the risk for myeloma
* Age
* Genetics
* Incidence in black population
* Sporadic cases of familiar myeloma
… but, myeloma is always preceded by a premalignant condition:
Monoclonal Gammopathy of Uncertain Significance (MGUS)
173
Monoclonal Gammopathy of Uncertain significance (MGUS)
the most common (known) premalignant condition
* incidence increases with age
* up to 1% - 3.5% in elderly population
* average risk for progression : 1% annually
* IgG or IgA MGUS -> myeloma
* IgM -> lymphoma
MGUS:
higher incidence of osteoporosis, thrombosis and
bacterial infection compared to general population
Diagnostic criteria for MGUS (WHO)
* Serum M-protein <30g/L
* Bone marrow clonal plasma cells <10%
* No lytic bone lesions
* No myeloma-related organ or tissue impairment
* No evidence of other B-cell proliferative disorder
174
Mayo criteria
used for MGUS risk stratification
3 parameters:
* Non-IgG M-spike
* M-spike >15g/L
* Abnormal serum free light chain (FLC) ratio
175
Smouldering myeloma
Both criteria must be met:
- serum monoclonal protein (IgG or IgA) >/ 30g/L or urinary monoclonal protein >/500mg per 24h and/or clonal bone marrow plasma cells 10-60%
- absence of myeloma defining events or amyloidosis
2019 IMWG Updated Risk Stratification model
* Bone marrow myeloma cells ≥20%
* M-spike ≥20g/L
* Serum FLC ratio ≥20
Low, intermediate risk: observation
High risk (≥2 factors): ? treatment
176
Pathogenesis of MM
Primary events
* Hyperdiploidy (60%)
q additional odd number Chr
* IGH rearrangements (Chr 14q32)
q t(11;14) IGH/CCND1
q t(4;14) IGH/FGFR3
q t(14;16) IGH/MAF
Common secondary events
* KRAS, NRAS
* t(8;14) IGH/MYC
* 1q gain / 1p del
* del 17p (TP53)
* 13- / del 13q
- bone destruction
- anaemia
- angiogenesis
- immunosuppression and infections
(myeloma cells interact with bone marrow microenvironment)
177
MM diagnostic criteria
≥10% plasma cells in bone marrow or plasmacytoma + ≥1 CRAB or MDE
CRAB
C: Hypercalcaemia
calcium >2.75mmol/L
R: Renal disease
creatinine >177μmol/L or eGFR <40ml/min
A: Anaemia
Hb <100g/L or drop by 20g/L
B: Bone disease
One or more bone lytic lesions in imaging
MDE (myeloma defining events):
* Bone marrow plasma cells ≥60%
* Involved : uninvolved FLC ratio >100
* > 1 focal lesion in MRI (>5mm)
178
Bone disease in MM
80% present with bone disease
* Proximal skeleton
* Back (spine), chest wall and pelvic pain
* Osteolytic lesions, never osteoblastic
* Osteopenia
* Pathological fractures
* Hypercalcaemia
Imaging:
- whole body CT scan low-dose
- CT / FDG-PET scan
- whole body diffusion-weighted MRI (bone marrow cellularity and active vs treated disease)
179
Bone disease emergencies in myeloma
Cord compression
* Diagnosis & treatment within 24hrs
* MRI scan
* Ig and FLC studies +/- biopsy
* Dexamethasone
* Radiotherapy
* Neurosurgery: rarely required
* Stabilise unstable spine
* MDT meeting
Hypercalcaemia
* Presents with drowsiness, constipation, fatigue, muscle weakness, AKI
* Fluids, steroids, zoledronic acid
180
MM kidney disease
Definition:
– Serum creatinine >177μmol/L (>2mg/dL ) or eGFR <40ml/min (CDK-EPI)
– Acute kidney injury and result of myeloma
* 20-50% acute kidney injury at diagnosis
* 2-4% of newly diagnosed patients will require dialysis
* 25% develop renal insufficiency at relapse
Cause of myeloma kidney disease:
- Cast nephropathy is caused by high serum free light
chains (FLC) levels and Bence Jone proteinuria
- Hypercalcaemia, loop diuretics, infection,
dehydration, nephrotoxics
181
Infections in MM
Complex humoral and cellular
immunodeficiency
* Immunoparesis: low serum normal Igs
* Myeloid, T cells and NK cells impairment
* Chemotherapy impairs immune response
* Myeloma immune evasion
182
MM diagnostic workup
Immunoglobulin studies
* Serum protein electrophoresis
* Serum free light chain levels
* 24h Bence Jones protein
Bone marrow aspirate
and biopsy
* IHC for CD138
FISH analysis
* Should include at least
high risk abnormalities (eg. t(4;14) FGFR3/IGH and del17p)
Flow cytometry immunophenotyping
* Diagnosis
* MRD
183
MM and AL amyloidosis
* MGUS or myeloma in the background
* Misfolded free light chains aggregate into amyloid
fibrils in target organs
* The amyloidogenic potential of light chains is more
important than their amount
* Amyloid fibrils stain with Congo Red, are solid, non-branching and randomly arranged with a diameter of
7 – 12 nm
* Lambda light chain is involved in 60%
- IGLV6-57 in kidney
- IGLV1-44 in cardiac
184
AL amyloidosis
Common target organs: kidney, heart, liver, neuropathy
Clinical presentation:
* Nephrotic syndrome (70%)
– Proteinuria (not BJP!), peripheral oedema
* Unexplained heart failure à determinant of prognosis – Raised NT-proBNP
– Abnormal echocardiography and cardiac MRI
* Sensory neuropathy
* Abnormal liver function tests
* Macroglossia
185
Monoclonal gammopathy of renal significance (MGRS)
Definition
“…MGRS applies specifically to any B-cell clonal lymphoproliferation where there are:
1. one or more kidney lesions caused by mechanisms related to the produced
monoclonal immunoglobulin (Ig) and
2. the underlying B cell clone does not cause tumor complications or meet current
hematological criteria for immediate specific therapy”
MGRS pathology
* Rare disease, several subtypes
* Demonstration of the involved
monoclonal Ig or light chain is
possible in most cases
* Work up similar to myeloma
* Many patients will require
myeloma-type treatment aiming
to renal survival
186
MM therapy
Melphalan
* Nitrogen mustard derivate, in use since the 1960’s
* Backbone of myeloma therapy until late 1990’s
* High-dose melphalan 200mg/m2 still in use in Autologous SCT
Cyclophosphamide
* Widely used in combination with steroids and/or other drugs
* Immunomodulation and microenvironment
Dexamethasone and Prednisolone
* Induce apoptosis in myeloma cells
* Strong synergy, part of almost all combination regimens
187
Immunomodulatory drugs for MM
- Cereblon E3 ligase modulators (CELMoDs)
- Proteasome inhibitors
- therapeutic monoclonal antibodies
188
Proteasome inhibitors in MM
Bortezomib – 2003
* Currently approved for first line or relapse
* iv or s/c use
* Neuropathy is main toxicity
Carfilzomib - 2012
* More potent than
* Approved in relapse
* iv only
* thrombocytopenia, cardiotoxicity
Ixazomib - 2015
* Approved in relapse, in combination
* Oral drug
* Favourable toxicity profile
(important in removal of misfolded proteins)
189
Therapeutic monoclonal Abs in MM
Daratumumab is the first therapeutic moAb approved for multiple myeloma (2015)
* CD38 is strongly expressed in
normal and malignant plasma cells
* Not a lineage specific marker
Daratumumab monotherapy
in relapsed/refractory myeloma
36% of patients with multiple previous
treatments and refractory disease to
standard myeloma therapies responded to
daratumumab
190
Novel immunotherapy in MM
Belantamab mafodotin: anti-BCMA toxin conjugate (keratopathy is common toxicity)
Bispecific T cell engagers (BiTE) [targets: CD3 and BCMA]
anti-BCMA CAR T-cells
191
Treatment algorithm in new diagnosis MM
Transplant-eligible patients
Fit and typically <65 years old
Induction:
anti-CD38, PI + IMiD + Dex
(D-VTD, D-VRD)
x4-6
Autologous Stem Cell
Transplantation
Consolidation x2
(D-VTD, D-VRD)
Maintenance until PD
Low dose Lenalidomide
Transplant-ineligible patients
Frail and usually >65 years old
Daratumumad - Lenalidomide + Dex
Or
Bortezomib – Cyclophosphamide- Dex
Or
Daratumumab-Bortezomib –
Cyclophosphamide- Prednisolone
192
Indications for emergency blood films
An unexpected life-threatening condition
Acute anaemia
ITP and TTP (thrombotic thrombocytopenic purpura)
Acute leukaemia
Burkitt lymphoma
Acute kidney injury
Infection
193
Unexpected life-threatening condition
low platelets; consider travel history, blood film may show malaria
Babesiosis: Babesia microti, looks a bit like malaria but there are extracellular parasites as well as ring forms (fever in immunosuppressed individual, endemic to USA)
HIV-positive people may have atypical infections eg disseminated histoplasmosis (looks like bubbles in cells)
194
Acute anaemia blood films
Megaloblastic anaemia: hypochromic red blood cells (central pallor), hypersegmented neutrophils
Paroxysmal cold haemoglobinuria: agglutination, spherocytes, Erythrophagocytosis, Atypical lymphocytes
Hereditary spherocytosis with parvovirus B19 infection: spherocytes, lack of reticulocytosis --> red cell aplasia (PMH of neonatal jaundice?, presents with rash, fever, pallor)
Haemolysis: Polychromasia, Spherocytes, Erythroblasts, Normal platelet count
195
Haematuria vs haemoglobinuria
haematuria: when you centrifuge the urine, there will be red blood cells at the bottom and urine at the top
if not: haemoglobinuria
196
Causes of haemolytic anaemias
Acute intravascular haemolysis resulting from anti-A in the immunoglobulin preparation: direct antiglobulin test positive, Anti-A eluted from the red cells
G6PD deficiency is cause
197
Anaemic neonates
Haemolytic disease of the newborn
G6PD deficiency (breast feeding may be relevant – has mother eaten fava beans or is she taking a relevant drug?)
Maternal parvovirus infection (virus has crossed the placenta)
Fetomaternal haemorrhage
198
Thrombocytopenic blood films
with petechiae: meningococcal sepsis (on blood film)
Thrombotic thrombocytopenic purpura: lack of platelets and can see red cell fragments
Acute (promyelocytic) leukaemia: leukaemic cells are unusual as they are primitive cells but cytoplasm is full of granules
(can result in death from DIC)
Severe thrombocytopenia in pregnancy: Haemolysis Elevated Liver enzymes Low Platelets ‒ HELLP syndrome
An emergency because of the need to deliver the baby
A caesarean section was done with a successful outcome
Malaria: travel history is important
199
Haemolytic causes of acute renal insufficiency
Multiple myeloma
Haemolytic uraemic syndrome
Thrombotic thrombocytopenic purpura
Tumour lysis syndrome in high grade haematological neoplasms
AML or lymphoma with hyperuricaemia
(blood film will help diagnosis)
200
Infective causes of anaemia
(diagnosis by blood films)
Malaria
Giemsa
Capnocytophaga canimorsis
Babesia (maltese cross formation on film)
Borrelia (Africa history, like a squiggle on film)
201
Normal red cell life span
120 days
(haemolysis = shortened red cell survival) in sickle cell can be as short as 5-7 days
202
Haemolysis classification
Intravascular - within circulation
Extravascular - removal/destruction by reticuloendothelial system
Acquired or genetic
203
Extravascular haemolytic anaemias
autoimmune
alloimmune
hereditary spherocytosis
204
Intravascular haemolytic anaemias
malaria
G6PD deficiency
mismatched blood transfusion
cold antibody haemolytic syndromes
drugs
MAHA (HUS, TTP)
paroxysmal nocturnal haemoglobinuria
205
Hereditary haemolytic anaemias (inherited)
Disorders of membrane:
cytoskeletal proteins
cation permeability
Disorders of red cell metabolism
Disorders of haemoglobin:
thalassaemia
sickle cell
unstable Hb variants
206
Consequences of haemolysis
anaemia
erythroid hyperplasia with increased rate of red cell production and circulating reticulocytes
increased folate demand
susceptibility to effect of parvovirus B19 (infect erythroid cells and arrest maturation)
propensity to gallstones (increased bilirubin due to haem catabolism)
increased risk of iron overload and osteoporosis
207
Clinical features of haemolytic disorders
pallor
jaundice
splenomegaly
pigmenturia
family history
208
Lab findings of haemolytic disorders
anaemia
increased reticulocytes
polychromasia (take up both eosinophilic and basophilic dye [blueish] --> reticulocytes)
hyperbilirubinaemia
increased LDH
reduced/absent haptoglobins
haemoglobinuria
haemosiderinuria (iron excreted - prussian blue or perl's stain)
209
Defects in red cell membrane (disorders)
Hereditary spherocytosis (vertical interaction)
Band 3
Protein 4.2
ankyrin
beta spectrin
Hereditary elliptocytosis (horizontal interaction)
alpha spectrin
beta spectrin
protein 4.1
210
Hereditary spherocytosis
genetic defect of red cell cytoskeleton
family history in 75% (typically AD)
in vitro red cells show increased sensitivity to lysis in hypotonic saline (osmotic fragility test)
reduced binding of dye eosin-5-maleimide
Blood film: lack of central pallor, smaller, more densely stained
FBC: increased MCV
211
Hereditary elliptocytosis
blood film: elliptical or ovalocytic shapes of red cells, no polychromasia, FBC is normal
212
Hereditary pyropoikilocytosis
Blood film: fragmented red cells, variation in shapes, budding of membrane
(homozygous form of hereditary elliptocytosis --> may lead to extreme haemolysis requiring blood transfusion)
213
G6PD deficiency
(glucose-6-phosphate dehydrogenase)
prevalent in areas of malaria endemicity
X-linked (clinical effects seen predominantly in hemizygous males and homozygous females)
G6PD enzyme catalyses first step in pentose phosphate pathway -> generates NADPH required to maintain intracellular glutathione
214
Clinical effects of G6PD deficiency
neonatal jaundice
acute haemolysis (triggered by oxidants [drugs/fava beans]/infection)
chronic haemolytic anaemia (rare)
215
Lab findings in G6PD deficiency resulting in acute haemolysis
haemoglobinuria
blood film: contracted cells, nucleated red cells, bite cells (cytoplasm removed), hemi- ghosts (haemoglobin removed to one side)
methylviolet stain: heinz bodies
asymptomatic state: unremarkable
216
Drugs that can trigger haemolysis in G6PD deficiency
anti-malarials (primaquine)
antibiotics: sulphonamides, cirpoflox, nitrofurantoin
Other: dapsone, vitamin K
fava beans
moth balls
217
Pyruvate kinase deficiency
(defect in glycolytic pathway)
blood film: akinocytes (short projections from red cells), spherocytes
218
Pyrimidine 5-nucleotidase deficiency
defect in nucleotide metabolism pathway
(pyrimidine is toxic to red cells and is broken down by pyrimidine 5-nucleotidase)
results in prominent basophilic stippling on blood film
219
First line investigations for haemolytic anaemias (to identify cause)
direct antiglobulin test (autoimmune)
urinary haemosiderin/haemoglobin (intravascular)
osmotic fragility / dye-binding test(membrane defect)
G6PD +/- PK activity
haemoglobin separation A and F (haemoglobin disorders)
heinz body stain (oxidative haemolysis)
Ham's test/flow cytometry of GPI-linked proteins (paroxysmal nocturnal haemoglobinuria)
thick and thin blood films (malaria)
220
Management of haemolytic anaemias
folic acid supplements
avoidance of precipitating factors (eg. oxidants in G6PD def)
red cell transfusion/exchange
immunisation against blood-borne viruses (eg. hep a and b)
monitor for chronic complications (cholelithiasis, iron overload, osteoporosis) - cholecystectomy for symptomatic gallstones
splenectomy if indicated
221
Indications for splenectomy
significant benefit in:
PK deficiency
hereditary spherocytosis
severe elliptocytosis/pyropoikilocytosis
thalassaemia
immune haemolytic anaemia
but pt at risk of overwhelming sepsis (capsulated bacteria; pneumococcus, meningococcus etc) --> penicillin prophylaxis
222
Criteria for splenectomy
(with conditions indicated)
transfusion dependence and iron chelation therapy
growth delay
physical limitation (usually if Hb < 8g/dL)
hypersplenism
Age: 3-10 years to maximise prepubertal growth
(before 3; spleen plays large role in immunity)
223
Haemoglobin Hammersmith
rare and severe unstable haemoglobin variant
severe electrophoretically silent heinz body haemolytic anaemia
mutation disrupts haem contact
reduced oxygen affinity
