JC42 (Medicine) - Leukaemia, Bone Marrow Transplant, Immunogenetics Flashcards
Leukocytosis
- 2 broad category of causes
- Normal Total WBC count in adult
- Typical adult reference range for differentials (/)
Leukocytosis: quantitative abnormality in WBC
Causes: can be classified into
→ Reactive as the result of complex interactions between cells, cytokines and peripheral, tissue and marrow WBC pool
→ Neoplastic as the result of acquired genetic changes leading to malignant transformation
Total WBC: 7.5 ± 3.5×109/L (4 – 11)
Neutrophil: 20 – 7.5
Lymphocytes: 1.5 – 4.0
Monocytes: 0.2 - 0.8
Eosinophil: 0.04 - 0.4
Basophil: 0.02 - 0.1
Outline questions to identify cause of lymphocytosis
- Fever and features of infection
- Bone pain: infection or leukemia
- S/S of inflammation, eg. rash, photosensitivity, joint pain
- S/S of malignancies, eg. hepatosplenomegaly, lymphadenopathy, palpable mass
- S/S of allergies, eg. rhinitis, asthma, rash, urticaria, angioedema, atopy esp for unexplained eosinophilia
- Symptoms of other lineages, eg. bleeding, anaemia
- TOCC esp travel for unexplained eosinophilia
- Past medical history
- Drug history
Describe the following PBS results + give D/dx
- Left shift
- Leucoerythroblastic picture
- High Blast count
- Auer rods, Faggot cells
- Atypical lymphocytosis
- Left shift: Immature granulocytes – Severe infection/ Sepsis or CML
- Leucoerythroblastic picture: Immature granulocytes + Nucleated RBC +/- Tear drop RBC – Marrow infiltration by Primary MF or metastatic CA
- High Blast count – >20% blast qualifies as acute leukaemia
- Auer road = myeloblastic cancer e.g. AML; Faggot cells (clustered auer rods) = APML
- Atypical lymphoctosis –* Infective mononucleosis*, viral or autoimmune diseases
Marrow exam
- Indication
- Site of sampling
- Tests
- Contraindication
Indication: Unexplained cytopenia/ leukocytosis
Site: PSIS, ASIS or Sternum
Samples:
Trephine biopsy for histological exam
IHC for immunophenotyping;
Aspirate for flow cytology and genetic studies
Contraindication: Severe bleeding disorders (e.g. hemophilia, DIC)
Blood cytochemistry test
- Function
- Technique
Detection of Myeloid lineage with dye: Myeloperoxidase, Sudan Black B stain
No good marker for lymphoid lineage
Blood immunophenotyping
- Function
- Methods
Function: Determine cell lineage by detection of antigens on cells-of-interest
Methods:
- **Immunohistochemistry (IHC) **on trephine biopsy: one marker only
- **Flow cytometry **on peripheral blood, marrow aspirate: co-expression of multi-markers and clonal populations of abnormal WBCs Markers
Myeloid lineage: myeloperoxidase, CD117, CD13, CD33
Lymphoid lineage:CD19, CD79a, CD22 (for B-ALL), CD3 (for T-ALL)
E.g. marker CD5 expression in CLL or Mantle cell lymphoma is rarely expressed in normal cells
Blood cytogenetic workup
Function
Methods
Function
- Diagnostic: eg. t(9;22) for CML, t(15;17) for APML
- Prognostic, eg. t(8;21) in AML carries good prognosis
- Selection of treatment, eg. allogeneic HSCT
Methods:
- Karyotyping: by use of Giemsa stain
- FISH: by use of fluorescence probes to detect specific DNA sequences
Blood Molecular genetic workup
Function
Methods
Function: Detect specific mutations in genes for diagnosis, prognostication, selection of Tx
Methods:* PCR-based,* next-generation sequencing
Leukopenia
Cut-off for Neutropenia and Lymphopenia
Severity cut-offs for Neutropenia
Neutropenia:** Absolute neutrophil count (ANC) <1.5**×109/L
Severity: 1-1.5 (mild), 0.5-1 (moderate), <0.5 (severe), <0.2 (agranulocytosis)
Lymphopenia: Absolute lymphocyte count (ALC) <1×109/L
Ddx Neutropenia + following PBS results
- Toxic granules (Dohle bodies)
- Bilobed/dysplastic neutrophils
- Hypersegmented neutrophils
- Blasts
- Leukoerythroblastic pattern
Toxic granules (Dohle bodies) → sepsis
Bilobed/dysplastic neutrophils → MDS
Hypersegmented neutrophils → megaloblastic anemia
Blasts → acute leukemia
Leukoerythroblastic pattern → marrow infiltration by primary MF or metastatic bone CA
Workup tests for Lymphopenia
Check HIV status
Lymphocyte function workup:
- IgGAM for Ig levels
- Lymphocye proliferation tests
- CXR for** thymus**
- CBC/PBS for lymphocyte sub-populations
Pancytopenia
Define cut-off for lineages
2 main pathological processes
Pancytopenia: ↓ in all peripheral blood lineages (Hb <12, ANC <1.8, PLT <150)
- Bone marrow failure: Bone Marrow infiltration/ replacement; Bone Marrow Failure
- Increase consumption: Peripheral consumption or Hypersplenism
Causes of Bone marrow failure
Congenital BM failure syndromes: Schwachman-Diamond syndrome, Fanconi anaemia, dyskeratosis congenita
Aplastic anaemia: idiopathic, secondary to drugs, A/I disorders, toxins…
Nutritional: alcoholism, megaloblastic anaemia
Infection-related marrow suppression: viral infection, eg. HIV, hepatitis, EBV
Haemophagocytic lymphohistiocytosis (HLH) due to genetic causes or secondary causes, eg. EBV infection, Kawasaki disease, macrophage-activation syndrome in A/I diseases
Myelodysplastic syndrome (classically pancytopenia in elderly)
Drug myelosuppression
Causes of peripheral consumption of blood cells
DIC: sepsis, APML, other disseminated malignancies
Thrombotic microangiopathy: TTP, HUS (usually just anaemia + thrombocytopenia)
Autoimmune-related: SLE
Paroxysmal nocturnal haemoglobinuria (acquired aplastic anaemia)
Causes of hypersplenism
Vascular from cirrhosis, pre-hepatic and post-hepatic congestion
Haematological malignancy, esp CML, MF
Hemoglobinopathies: β-thal intermedia/major, HbH disease
Autoimmune cytopaenia: ITP, AIHA
Infections: Malaria, EMV, CMV, HIV
Felty syndrome from RA
Leukemia”
- Cell of origin
- Classification/ types
- S/S
Leukemia: clonal malignant disorders of haematopoietic stem cell
Classification: acute (from immature cells) vs chronic (from mature cells)
□ Acute lymphoblastic leukaemia (ALL)
□ Acute myeloid leukaemia (AML)
□ Chronic lymphocytic leukaemia (CLL)
□ Chronic myeloid leukaemia (CML)
Clinical features:
□ Constitutional symptoms: Fever
□ Marrow suppression: anemia, thrombocytopenia S/S
□ **Organ enlargement **and tissue infiltration: Gum hypertrophy, Hepatosplenomegaly, Lymphadenopathy, Mediastinal mass, Testicular hypertrophy…
□ Asymptomatic unexplained increase in cell count
Approach to diagnose Leukemia”
5 steps to diagnosis of haematological malignancy (MCICM)
□ Morphology: peripheral blood smear (number, morphology, blasts), BM aspirate and trephine
□ Cytochemistry: MPO/Sudan black B for myeloid, no marker for lymphoid
□ Immunophenotyping: flow cytometry (peripheral blood, marrow aspirate), IHC (trephine only) for cell surface marker expression
□ Cytogenetics: karyotyping, FISH for chromosomal abnormalities
□ Molecular genetics: PCR, sequencing for genetic mutations
Acute leukaemia”
Clinical features
Diagnostic feature
Features of marrow failure
→ Anaemia: general fatigue, pallor, weakness
→ Leukopenia: frequent infections
→ Thrombocytopaenia: easy bruising, gum bleeding
- Severe bleeding is suggestive of APML → should look for lab evidence of DIC
Fever: usu due to infections (instead of tumour fever → more common in APML or ALL)
Organomegaly/ Cutaneous/gum involvement
Key diagnostic finding: >20% blasts in bone marrow or peripheral blood samples
First line investigations to diagnose Acute Leukemia”
□ CBC + differential WBC + manual count
□ PBS
□ Clotting profile, D-dimer, Fibrinogen (esp DIC in APL)
□ Biochemistry for TLS: RFT, Phosphate, Calcium, K, Urate, LDH
Bone marrow aspirate/biopsy - full workup MCICM
□ Morphology
□ Cytochemistry: MPO, Sudan Black B staining (suggestive of myeloid origin)
□ Immunophenotyping: distinguishing between cell lines
□ Cytogenetics (karyotyping, FISH)
□ Molecular genetics
Pre-treatment tests for acute leukaemia”
□ CXR - disease related complications, infections
□ LDH, urate, RFT, electrolytes: baseline for possible Tumor-lysis syndrome
□ Type and crossmatch for possible future transfusion
□ D-dimer, fibrinogen, Clotting profile if suspicious of APL or DIC
□ G6PD status: C/I many haematological drugs
→ Eg. septrin for PCP prophylaxis, rasburicase for TLS prophylaxis, co-trimoxazole
□ HBsAg, anti-HBc, anti-HBs ± HBV DNA, Anti-HCV: **Hepatitis B,C status **
□ Anti-HIV Ab: **HIV status **
□ HLA typing - candidates for HSCT
General management for acute leukaemia”
- Support
- Sepsis
- TLS
- Leukostasis
Support: nutrition, antiemetic, analgesia, reverse isolation, face mask, hand hygiene, low bacteria diet
→ RBC transfusion (anaemia)
→ PLT transfusion if PLT ≤10 or ≤20 if fever/bleeding
→ FFP if bleeding due to DIC
Sepsis/ neutropenic sepsis: workup source, resuscitate, empirical broad sepctrum antibiotics, antifungal prophylaxis
TLS: hydration + allopurinol or febuxostat or rasburicase
Leukostasis : Urgent leukopheresis
- APML: ATRA +/- As2O3
Phases of acute leukaemia treatment
-
Induction phase:High intensity chemotherapy, require intensive care for severe BM hypoplasia
2.Consolidation phase: multiple courses of chemo., require supportive care for BM hypoplasia
+ Maintenance phase: Low intensity chemo. for ALL ONLY
+ CNS prophylaxis against infiltration for ALL ONLY
HSCT for refractory/ poor prognosis
AML
Epidemiology
Risk factors
Pathogenesis (/)
Epidemiology: most common (80%) acute leukaemia in adults
Demographics: median age at dx ~65y, M>F = 5:3
Risk factors: characteristically a/w
□ Genetics: a/w** impaired DNA repair**, eg. trisomy 21, Fanconi anaemia, Bloom’s syndrome…
□ Environmental exposure a/w genetic mutation, eg. chemotherapy/RT-related, chemical exposure (eg. benzene)
□ Prior pre-leukemic conditions, eg. MDS, MPN
Pathogenesis: “two hit hypothesis”
□ Class I mutation: confer proliferative advantage (FLT3-ITD, c-Kit, Ras)
□ Class II mutation: impair haematopoietic differentiation (CEBPA)
6 subtypes of AML (/)
AML with recurrent genetic abnormalities (blast cells (even if <20%) with specific leukaemia-associated cytogenetic/molecular genetic abnormalities)
AML with myelodysplasia-related changes: features of dysplasia in at least 50% cells in ≥2 lineages
Therapy-related myeloid neoplasm (t-AML): AML mutation + previously treated by drugs (eg. etoposide, alkylating agent)
AML, not otherwise specified: absence of cytogenetic abnormalities
Myeloid sarcoma: solid tumor with myeloid blast cells, mostly extramedullary disease
Myeloid proliferation related to Down syndrome
Clinical presentation of AML
2 Complications
General fatigue
S/S of BM failure: profound anaemia, thrombocytopaenia, neutropenia
DIC in promyelocytic vatiant (APML)
Gum hypertrophy/infiltration + skin + CNS involvement in myelomonocytic and monocytic subtypes
Fever: infection
Complications:
Neutropenic fever: infection due to neutropenia
Leukostasis: due to very high blast count >300
»> respiratory symptoms (SOB, hypoxia), neurological (visual changes, headache, dizziness, tinnitus…)
AML
Typical results in CBC, PBS
CBC
NcNc anaemia and thrombocytopaenia
Leukocytosis or leukopenia
PBS
Circulating myeloblast ± Auer rods
AML”
Typical results in MCICM workup
Marrow
Hypercellular with replacement by myeloid blasts
Cytochemistry
Myeloperoxidase +ve (dark staining), Sudan Black B +ve
Immuno- phenotyping
Flow cytometry: expression of CD34, HLA-DR, CD117, CD13, CD33 (varies with AML subtypes)
Cytogenetics
- t(8;21)(q22;q22) + RUNX1-RUNX1T1
- inv(16)(p13.1q22) or t(16; 16)(p13.1;q22) + CBFB-MYH11
- t(15;17)(q24.1;q21.1) + PML-RARA (diagnostic of APL)
Molecular genetics
Eg. FLT3 mutation → predicts prognosis
Diagnostic criteria of AML (/)
1 of:
→ ≥20% blast in peripheral blood or BM aspirate
→ AML-defining genetic abnormalities
- t(8;21)(q22;q22) + RUNX1-RUNX1T1
- inv(16)(p13.1q22) or t(16; 16)(p13.1;q22) + CBFB-MYH11
- t(15;17)(q24.1;q21.1) + PML-RARA (diagnostic of APL)
→ Myeloid sarcoma
PLUS demonstration of myeloid lineage in leukaemic cells
D/dx AML
MDS, MDS/MPN: blast count may be marginal (i.e. approaching 20%)
ALL: some ALL cells may co-express myeloid markers or even be mixed phenotype acute leukaemia
Vitamin B12¸folate deficiency: pancytopenia, mimic erythroleukaemia
CML in blastic crisis
Investigations for APML”
Peripheral blood: leukopenia
Circulating malignant promyelocytes:
→ Morphology: violet granules ± bundles of Auer rods (‘faggot cells’)
→ Immunophenotype: MPO+, CD13+, CD33+
→ Genetics: t(15;17)(q24.1;q21.2);PML-RARA
CLL staging (/)
Donor selection criteria for HSCT”
Sources of donors
Human leukocyte antigen (HLA) matching: Determines risk of graft-versus-host disease in HSCT
- Syngeneic: from MZ twin → by definition 12/12 matched, no GVHD
- Matched sibling donor (allogeneic): 6/6 matched (HLA-A, -B, -DR)
- Matched unrelated donors: 8/8 matched (HLA-A, -B, -C, -DR)
- Mismatched related donors: <6/6 matched,
- Single antigen mismatched siblings (5/6) or Haploidentical donors (3/6)
unrelated donors from HK BM Donor Registry pool
Haploidentical donor - from ANY sibling, parent, child ± other relatives
ABO incompatibility doesn’t matter
Bone marrow harvest vs Peripheral blood progenitor cell for HSCT (/)
Advantages and Disadvantages
