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Diagnostic Pathways

What and When in Haematopathology Diagnostics

These diagnostic pathways outline the routine tests performed by the Haematopathology and Oncology Diagnostic Service (HODS) and East Genomics Laboratory Hub (GLH). We base our testing on the diagnostic information you provide and our morphology screening of peripheral blood (PB) and bone marrow (BM) samples.

Key Points

  • Adjustments for Prognostic Testing: If comprehensive testing is not appropriate due to a patient's age or performance status, please indicate this in the clinical details. We will adjust the standard diagnostic pathways accordingly.

  • Clinical Trial Patients: For patients enrolled in clinical trials, follow-up samples may not be needed, as external laboratories involved in the trial will manage these.

  • Additional Tests: East GLH may offer tests beyond these standard pathways. You can request these in the clinical details or discuss them with a HODS team member.

Myeloid Neoplasms and Bone Marrow Failure Syndromes

Acute myeloid leukaemia (AML), Myelodysplastic Syndromes (MDS), Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPNs), Aplastic Anaemia, and other Bone Marrow Failure Disorders

Myeloproliferative Neoplasms (MPNs)

Lymphoid Neoplasms

Acute Lymphoblastic Leukaemia (ALL)

Mature Lymphoproliferative Disorders (LPDs) and Plasma Cell Disorders (Multiple Myeloma [MM], Monoclonal Gammopathy of Undetermined Significance [MGUS])

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

 
 
Investigation TypeAcute Myeloid Leukaemia (AML)MDS, MDS/MPNs,
Aplastic Anaemia, and other Bone Marrow Failure disorders
Morphology- Blast count
- Percentage of dysplasia per cell lineage, if AML
- Maturation/differentiation, if AML
- Grade any underlying dysplasia
- Iron stain to assess presence or absence of ring sideroblasts		- Blast count
- Monocyte count
- Percentage 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)
• chromosome 5 and 7 abnormalities
• t(15;17) (will also detect i17q)
• KMT2A (MLL) rearrangements
• 17p if any other abnormalities
NUP98 FISH is added if no other molecular MRD marker identified		Can be requested if rapid TAT of cytogenetic changes required, or only limited cytogenetic analysis needed.
Panel:
• -5/del5q
• -7/del7q
• Trisomy 8
• Del20q12
MECOM available as an additional probe
KaryotypeRecurrent translocations and other abnormalities.For IPSS. May use SNP array.
Molecular geneticsFLT3 ITD and allelic ratio – rapid TAT

FLT3 TKD – rapid TAT

NPM1 and quantitation if positive

Myeloid NGS

PML::RARA if t(15;17) detected by FISH

RUNX1::RUNX1T1 if t(8;21) detected by FISH

CBFB::MYH11 if inv(16) or t(16;16) detected by FISH

KMT2A translocation partner if 11q23 abnormality detected by FISH

If other fusion identified (e.g. by RNA fusion panel),samples sent to Viapath at Guy’s Hospital, London for baseline 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 or aparticulate aspirate, trephine is required.		Cellularity
Dysplasia
Fibrosis
CD34 / CD117
 

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

 
 
Investigation TypeAcute Myeloid Leukaemia (AML)MDS, MDS/MPNs,
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 Viapath at 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:
• Clinical worsening
• Morphological progression
• Cytogenetic progression
Bone marrow histologyBlast “equivalent(s)” %
May not be required in non-transplanted patient.		Dysplasia
Fibrosis
CD34 / CD117
 

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

 
 
Investigation TypeAcute Myeloid Leukaemia (AML)MDS, MDS/MPNs,
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

 
 
Investigation TypeMPNCML
MorphologyBlood cell countsBlood cell counts
Basophilia / megakaryocytes
Flow cytometry
FISHBCR::ABL1
Karyotypet(9;22),additional abnormalities
Molecular geneticsBCR::ABL1 E13A2/E14A2 transcript quantification.

For rare / unusual BCR::ABL1 fusion transcript analysis, samples are sent to another GLH as per NHS England.
Bone marrow histologyCellularity
Megakaryocyte morphology
Fibrosis
CD34 / CD117
 

Investigations for MPNs at transformation (acceleration/blast phase) 

 
 
Investigation TypeMPNCML
MorphologyBlast count
Dysplasia
Iron stain (presence or absence of ringed sideroblasts)		Blast count
Basophilia
Flow cytometryBlast phenotypeBlast phenotype
FISHIf appropriate
KaryotypeIf appropriatet(9;22),(additional) abnormalities
Molecular geneticsMyeloid NGS if disease transformation in transplant candidateBCR::ABL1 TKD mutation analysis
Bone marrow histologyfibrosis
CD34 / CD117		fibrosis
CD34 / CD117
 

Investigations for ALL at diagnosis

 
 
Investigation TypeALL
MorphologyBlast count
Flow cytometryAcute leukaemia panel.
Establish Leukaemia-Associated Immunophenotype (LAIP).
FISHB-ALL:
BCR::ABL1, ETV6::RUNX1, KMT2A
• If negative: PBX1::TCF3, HLF::TCF3
• 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 (performed externally) considered on individual case basis if case would otherwise would be B-ALL NOS.

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.
Iron Stain if applicable.		Plasma cell and differential count.
Iron Stain if applicable.
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, PLL, FL, Burkitt lymphomaDeletion 1q21, additional 1p32, TP53 deletion, IGH::FGFR3, IGH:MAF,IGH::CCND1, IGH::MAFB
Molecular geneticsIn appropriate cases;

Hairy cell leukaemia:
• BRAF V600E mutation analysis

Lymphoplasmacytic lymphoma:
• MYD88 L265P mutation analysis

Anaplastic large cell lymphoma:
• NPM::ALK fusion gene
• IGH::CCND1 fusion

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 (on request)
Bone marrow histologyAssessment of involvement and classification of disease.

FISH, 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 MRD.
Assessments can be performed upon request.
Bone marrow 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.