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Diagnostic Pathways

What and when in haematopathology diagnostics

  • Routine testing
    These pathways describe the routine tests performed by HODS and East GLH. Testing is based on the clinical information provided and HODS morphology screening of peripheral blood (PB) and bone marrow (BM) samples.

  • If prognostic testing is not appropriate
    If comprehensive prognostic testing is not appropriate because of age or performance status, please include this in the clinical details. The standard pathway will be adjusted accordingly.

  • Clinical trial patients
    For patients enrolled in clinical trials, follow-up samples may not be required if testing is being undertaken by external trial laboratories.

  • Additional tests
    Additional tests may also be available through East GLH. These can be requested in the clinical details or discussed with a member of the HODS team.

Myeloid Neoplasms and Bone Marrow Failure Syndromes

Acute Myeloid Leukaemia (AML) and Myelodysplastic Syndromes (MDS)

Myeloproliferative Neoplasms (MPN)

Lymphoid Neoplasms

Acute Lymphoblastic Leukaemia (ALL)

Mature Lymphoid Neoplasms

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Investigations for AML, MDS, and MDS/MPNs at diagnosis

 
 
Investigation TypeAcute Myeloid Leukaemia (AML)MDS, MDS/MPN,
Aplastic anaemia and other bone marrow failure disorders
MorphologyBlast count
% of dysplasia per cell-lineage, if AML
Maturation/differentiation, if AML
Grade any underlying dysplasia
Maturation/differentiation, if AML
Iron stain (presence or absence of ring sideroblasts)		Blast count; monocyte count
% of dysplasia per cell lineage
Iron stain
Flow cytometryAcute leukaemia panel
Establish Leukaemia Associated Immunophenotype (LAIP)		If increased blasts on morphology
Markers of aberrant myeloid maturation
FISHMay vary depending on clinical situation. If patients are potential candidates for remission induction chemotherapy:
AML FISH panel (all cases):
• t(8;21)
• inv(16)/t(16;16)
• chr 5 and 7 abnormalities
• t(15;17) (will also detect i17q)
• KMT2A (MLL) rearrangements
• 17p (TP53) deletion
• MECOM rearrangements
• NUP98 and NUP214 rearrangments if initial panel negative		Consider testing as per AML depending on blast count and clinical factors

MECOM available as an additional probe if SNP array rather than karyotype performed
KaryotypeRecurrent translocations and other abnormalitiesFor IPSS. May use SNP array.
Molecular geneticsFLT3 ITD and allelic ratio – rapid TAT
FLT3 TKD – rapid TAT
NPM1
IDH1 hotspot testing if aged > 65
Myeloid NGS panel
KMT2A translocation partner if 11q23 abnormality detected by on FISH
RNA fusion panel in selected patients in whom there is no disease-defining or risk stratifying genomic lesion after FISH, cytogenetics and NGS, or where FISH is suggestive of the possibility of a translocation.
If a fusion or NPM1 mutation identified, samples sent to Guy’s Hospital, London for baseline confirmation / identification of MRD marker		Consider testing as per AML depending on blast count and clinical factors.
Bone marrow histologyMay not be required if definite AML on PB and good aspirate sample.
If ambiguous lineage leukaemia, secondary AML, acute erythroid leukaemia, megakaryoblastic leukaemia or aparticulate aspirate, trephine is required.		Cellularity
Dysplasia
Fibrosis
CD34 / CD117
 

Note in APML, FISH and molecular confirmation by the MRD laboratory are required, but additional tests are not routinely added. 

 

Investigations for AML, MDS, and MDS/MPNs during follow-up

 
 
Investigation TypeAcute Myeloid Leukaemia (AML)MDS, MDS/MPN,
Aplastic anaemia and other bone marrow failure disorders
MorphologyBlood counts
Blast count		Blood counts
Blast count
Flow cytometryIf LAIP established at diagnosis
FISHIf marker to determine cytogenetic remissionIf marker to determine cytogenetic remission (patient treated with curative intent).
Molecular geneticsIf molecular marker of disease, MRD assessment will be performed as appropriate.

Tests not performed in-house are sent to Guy's and St Thomas’s laboratory, London.

Common tests are listed (rarer assays may be performed after discussion with Guy's MRD laboratory):
• NPM1
• PML::RARA
• RUNX1::RUNX1T1
• CBFB::MYH11
• KMT2A (MLL)–fusion transcripts
• NUP98::NSD1
• ETV::RUNX1		Consider if >6 months since last assessment, and presence of one or more of the following:
• Deteriorating blood counts
• Morphological progression
• Cytogenetic progression
Bone marrow histologyNot required unless concern about unexplained cytopenias or relapse
Routinely performed at day 100 post allograft
Blast “equivalent(s)” %		Dysplasia
Fibrosis
CD34 / CD117
 

Investigations for AML, MDS, and MDS/MPNs at suspected relapse

 
 
Investigation TypeAcute Myeloid Leukaemia (AML)MDS, MDS/MPN,
Aplastic anaemia and other bone marrow failure disorders
MorphologyBlast count
Dysplasia		Blast count
% of dysplasia per cell lineage
Iron stain (presence or absence of ringed-sideroblasts)
Flow cytometryAcute leukaemia panelIf blasts > 20%
FISHif marker at diagnosisMay be appropriate
Karyotype(Additional) abnormalitiesMay be appropriate
Molecular geneticsAs for diagnosisMay be appropriate
Bone marrow histologyCD34 / CD117
Dysplasia		CD34 / CD117
Dysplasia, fibrosis, cellularity
 

Investigations for MPNs at diagnosis

 
 
Investigation TypeMPNCML
MorphologyBlast count / megakaryocytes
Assessment of dysplasia
Iron stain		Blast count / megakaryocytes
Basophilia
Flow cytometryIf blasts > 5%If blasts > 5%
FISHEosinophilia:
• 4q12 FIP1L1::PDGFRA
• 5q33 PDGFRB break-apart
• 8p11 FGFR1 break-apart
• 9p24 JAK2 break-apart
• Consider ETV6 break-apart
• Consider 5q– in appropriate context		BCR::ABL1
KaryotypeKaryotyping may be considered in appropriate patients and is available upon requestt(9;22)
Additional abnormalities
Molecular geneticsJAK2 V617F mutation analysis.

If JAK2 V617F mutation not detected, then:
• JAK2 exon 12 mutation analysis (in suspected PV)
• CALR exon 9 and MPL exon 10 mutation analysis (in suspected ET or PMF)

KIT D816V mutation analysis (in suspected systemic mastocytosis).

BCR::ABL1 fusion gene to exclude CML (if thrombocytosis, otherwise molecularly negative).

Consider FIP1L1::PDGFRA fusion gene in appropriate context.

Myeloid NGS in myelofibrosis if potential transplant candidate or if full prognostic information required.		BCR::ABL1 fusion gene diagnostic qualitative PCR and quantitative PCR
Bone marrow histologyCellularity
Megakaryocyte morphology
Fibrosis
CD34 / CD117		Cellularity
Megakaryocyte morphology
Fibrosis
CD34 / CD117
(Trephine biopsy may not be necessary if good aspirate obtained)
 

Investigations for MPN at follow-up / suspected progression

 
 
Investigation TypeMPNCML
MorphologyBlood cell counts
Dysplasia
Perls' stain		Blood cell counts
Basophilia / megakaryocytes
Flow cytometryMay be relevant if increased blastsMay be relevant if increased blasts
FISHBCR::ABL1
KaryotypeKaryotype or SNP array may be relevant if progressive disease suspectedt(9;22),additional abnormalities
Molecular geneticsMyeloid NGS panel may be relevant if progressive disease suspectedBCR::ABL1 E13A2/E14A2 transcript quantification
Consider BCR::ABL1 TKD mutation if not in major molecular response
For rare/unusual BCR::ABL1 fusion transcript analysis, samples are sent to another GLH as per NHS England.
Bone marrow histology- Cellularity
- Megakaryocyte morphology
- Fibrosis
- CD34 / CD117		Fibrosis
CD34 / CD117
 

Investigations for ALL at diagnosis

 
 
Investigation TypeAcute Lymphoblastic Leukaemia (ALL)
MorphologyBlast count
Flow cytometryAcute leukaemia panel.
Establish Leukaemia-Associated Immunophenotype (LAIP).
FISHB-ALL:
BCR::ABL1, ETV6::RUNX1, KMT2A
• If negative, PBX1::TCF3, HLF::TCF3. IGH::MYC, then IGH::BCL2 and BCL6 breakapart if IGH::MYC present
• If negative: ABL class rearrangements and other probes

T-ALL:
MLL, BCR::ABL, p16, TRA/TRD, TRB, TRG, STIL, TLX3 (HOX11L2)
KaryotypeSNP array
Molecular geneticsConfirm FISH findings.
IgH/TCR to be sent to appropriate laboratories to determine MRD marker depending on patient age.
RNA fusion panel in all cases unless BCR::ABL1, ETV6::RUNX1, high hyperdiploidy or hypodiploidy.
NGS panel including TP53 if hypodiploid ALL
Bone marrow histologyNot required if diagnosis confirmed by flow in the PB or aspirate. If ambiguous lineage leukaemia in the PB or aspirate, trephine is required.
 

Investigations for ALL at follow-up

 
 
Investigation TypeALL
MorphologyBlast count
Flow cytometryAt every time point independent of presence of LAIP
FISHTo confirm cytogenetic remissions where appropriate
KaryotypeNot required
Molecular geneticsIgH/TCR to be sent to appropriate national MRD laboratories depending on patient age.

BCR::ABL1 MRD or KMT2A::x depending on diagnostic findings – in adult patients these are monitored routinely rather than IgH/TCR
Bone marrow histologyNot required routinely at follow up
 

Investigations for Mature LPD, MM, and MGUS at diagnosis

 
 
Investigation TypeLPDMM, MGUS
MorphologyLymphocyte and differential count.
Lymphocyte morphology.		Plasma cell and differential count.
Flow cytometryLPD Screen
Extended B and/or T panel as appropriate
Light chain expression (κ/λ) if B lineage neoplasm		Plasma cell phenotype including cytoplasmic light chain expression.
FISHMay be performed for relevant abnormalities in CLL, MCL, T-PLL, FL, high grade lymphoma, HSTCLDeletion 1q21, additional 1p32, TP53 deletion, IGH::FGFR3, IGH:MAF,IGH::CCND1, IGH::MAFB
Molecular geneticsIn appropriate cases;

Hairy cell leukaemia:
• BRAF V600E mutation analysis (if IHC negative)

Lymphoplasmacytic lymphoma:
• MYD88 L265P mutation analysis

T-cell LPD / lymphoma:
• TCR gene rearrangement (clonality)

B-cell LPD / lymphoma:
• IG gene rearrangement

Chronic lymphocytic leukaemia:
• TP53 sequencing by NGS (or broader NGS panel) in pre-treatment samples; IGVH mutation status in pre-treatment samples, if not previously tested

Mantle cell lymphoma:
• TP53 sequencing by NGS (or broader NGS panel)		TP53 on request
Bone marrow histologyAssessment of involvement and classification of disease.

FISH/clonality if required – may be possible depending on disease.		Extent of plasma cell involvement
Plasma cell phenotype if not already conclusively determined by flow cytometry
FISH, e.g. t(11;14),may be done if low level plasma cells in the liquid sample.
 

Investigations for Mature LPD, MM, and MGUS at follow-up

 
 
Investigation TypeLPDMM, MGUS
MorphologyLymphocyte count
Lymphocyte morphology		Plasma cell count
Flow cytometrySpecific B- or T-cell analysis to detect residual disease.
High resolution (0.01%) assays for CLL.		Normally not indicated at follow-up in myeloma.
Post-autograft.
Assessments can be performed upon request.
BM histologyAssess extent of residual lymphoma involvementAssess extent of residual plasma cell involvement.
 

Investigations for Mature LPD, MM, and MGUS at relapse

 
 
Investigation TypeLPDMM, MGUS
MorphologyLymphocyte count
Lymphocyte morphology		Plasma cell count
Flow cytometryIf significant change in morphology
Normally not indicated in cases of clear relapse of myeloma
FISHCLL FISH may be indicatedMay be required to detect therapeutic target, e.g. t(11;14)
Molecular geneticsCLL TP53 sequencing by NGS (or broader NGS panel) may be indicated
Bone marrow histologyAssess extent of lymphoma involvement.
Assess for evidence of transformation.		Assess extent of plasma cell involvement.