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These pathways list the tests that will be done routinely on samples based on the diagnostic information provided and the HODS morphology screening of peripheral blood (PB) and bone marrow (BM).
If comprehensive testing to provide prognostic information is not required for your patient (for example, due to advanced age or poor performance status), please state this in the clinical details you provide on the request form and the standard diagnostic pathways will be adjusted accordingly.
Clinical trial patients
If your patient is being monitored on a clinical trial, then follow-up samples may not be needed as they will be performed as part of the trial by external laboratories.
Additional tests
If you require any additional tests to be performed by HODS, that are not included in the standard pathways, these can be requested in the clinical details or by direct discussion with a member of the HODS team.
AML, MDS, MDS/MPN at diagnosis
| AML | MDS, MDS/MPN aplastic anaemia other bone marrow failure disorders | |
|---|---|---|
| Morphology | Blast count. Grade any underlying dysplasia. Maturation/differentiation, if AML. Iron stain (presence or absence of ringed-sideroblasts). | Blast count; monocyte count. % of dysplasia per cell lineage. Iron stain. |
| Flow cytometry | Acute leukaemia panel. Establish leukaemia-associated immunophenotype (LAIP). | If increased blasts on morphology. Markers of aberrant myeloid maturation. |
| FISH | May 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) • 17p FISH if any other abnormalities | Can be requested if rapid TAT of cytogenetic changes required |
| Karyotype | Recurrent translocations and other abnormalities. | For IPSS. May use SNP array. |
| Molecular genetics | FLT-3 ITD and allelic ratio – rapid TAT FLT-3 TKD – rapid TAT NPM1 and quantitation if positive CEBPA 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 | Consider testing as per AML depending on blast count and clinical factors. |
| BM histology | May 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 |
AML, MDS, MDS/MPN at follow-up
| AML | MDS, MDS/MPN aplastic anaemia other bone marrow failure disorders | |
|---|---|---|
| Morphology | Blood cell counts Blast count | Blood cell counts Blast count |
| Flow cytometry | If LAIP established at diagnosis | |
| FISH | To determine cytogenetic remission, if known cytogenetic marker | If marker to determine cytogenetic remission (patient treated with curative intent). |
| Molecular genetics | If 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 |
| BM histology | Blast “equivalent(s)” % May not be required in non-transplanted patient. |
AML, MDS, MDS/MPN at relapse
| AML | MDS, MDS/MPN aplastic anaemia other bone marrow failure disorders | |
|---|---|---|
| Morphology | Blast count Dysplasia | Blast count % of dysplasia per cell lineage Iron stain (presence or absence of ringed-sideroblasts) |
| Flow cytometry | Acute leukaemia panel | If blasts > 20% |
| FISH | if cytogenetic marker present at diagnosis | May be appropriate |
| Karyotype | (Additional) abnormalities | May be appropriate |
| Molecular genetics | As for diagnosis (excluding CEBPA) | May be appropriate |
| BM histology | CD34 / CD117 Dysplasia | CD34 / CD117 Dysplasia, fibrosis, cellularity |
MPN at diagnosis
| MPN | CML | |
|---|---|---|
| Morphology | Blast count / megakaryocytes Assessment of dysplasia Iron stain | Blast count / megakaryocytes Basophilia |
| Flow cytometry | If blasts > 5% | If blasts > 5% |
| FISH | Eosinophilia: • 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 |
| Karyotype | Karyotyping may be considered in appropriate patients and is available upon request | t(9;22) Additional abnormalities |
| Molecular genetics | JAK2 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 |
| BM histology | Cellularity Megakaryocyte morphology Fibrosis CD34 / CD117 | Cellularity Megakaryocyte morphology Fibrosis CD34 / CD117 (Trephine biopsy may not be necessary if good aspirate obtained) |
MPN at follow-up
| MPN | CML | |
|---|---|---|
| Morphology | blood cell counts | blood cell counts basophilia / megakaryocytes |
| Flow cytometry | ||
| FISH | BCR-ABL1 | |
| Karyotype | t(9;22),additional abnormalities | |
| Molecular genetics | BCR-ABL1 E13A2/E14A2 transcript quantification. For rare / unusual BCR-ABL1 fusion transcript analysis, samples are sent to another GLH as per NHS England. | |
| BM histology | Cellularity Megakaryocyte morphology Fibrosis CD34 / CD117 |
MPN at transformation
| MPN | CML | |
|---|---|---|
| Morphology | blast count dysplasia iron stain (presence or absence of ringed sideroblasts) | blast count basophilia |
| Flow cytometry | blast phenotype | blast phenotype |
| FISH | if appropriate | |
| Karyotype | if appropriate | t(9;22),(additional) abnormalities |
| Molecular genetics | myeloid NGS if disease transformation in transplant candidate | BCR/ABL TKD mutation analysis |
| BM histology | fibrosis CD34 / CD117 | fibrosis CD34 / CD117 |
ALL at diagnosis
| ALL | |
|---|---|
| Morphology | Blast count |
| Flow cytometry | Acute leukaemia panel. Establish Leukaemia-Associated Immunophenotype (LAIP). |
| FISH | B-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) |
| Karyotype | SNP array |
| Molecular genetics | Confirm FISH findings. IgH/TCR to be sent to appropriate laboratories to determine MRD marker depending on patient age. |
| BM histology | Not required if diagnosis confirmed by flow in the PB or aspirate. If ambiguous lineage leukaemia in the PB or aspirate, trephine is required. |
ALL at follow-up
| ALL | |
|---|---|
| Morphology | Blast count |
| Flow cytometry | At every time point independent of presence of LAIP |
| FISH | To confirm cytogenetic remissions where appropriate |
| Karyotype | Not required |
| Molecular genetics | IgH/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 |
| BM histology | Not required routinely at follow up |
Mature LPD at diagnosis
| LPD | MM, MGUS | |
|---|---|---|
| Morphology | Lymphocyte and differential count. Lymphocyte morphology. Iron Stain if applicable. | Plasma cell and differential count. Iron Stain if applicable. |
| Flow cytometry | LPD 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. |
| FISH | May be performed for relevant abnormalities in CLL, MCL, PLL, FL, Burkitt lymphoma | Deletion 1q21, additional 1p32, TP53 deletion, IGH-FGFR3, IGH-MAF, IGH-CCND1, IGH-MAFB |
| Molecular genetics | In 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: • hotspot panel in pre-treatment samples without evidence of 17p deletion by FISH • IGVH mutation status (on request) | |
| BM histology | Assessment 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. |
Mature LPD at follow-up
| LPD | MM, MGUS | |
|---|---|---|
| Morphology | Lymphocyte count Lymphocyte morphology | Plasma cell count |
| Flow cytometry | Specific 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. |
| BM histology | Assess extent of residual lymphoma involvement | Assess extent of residual plasma cell involvement. |
Mature LPD at relapse
| LPD | MM, MGUS | |
|---|---|---|
| Morphology | Lymphocyte count Lymphocyte morphology | Plasma cell count |
| Flow cytometry | If significant change in morphology | Normally not indicated in cases of clear relapse of myeloma |
| FISH | CLL FISH may be indicated | May be required to detect therapeutic target, e.g. t(11;14) |
| Molecular genetics | CLL Hotspot panel may be indicated | |
| BM histology | Assess extent of lymphoma involvement. Assess for evidence of transformation. | Assess extent of plasma cell involvement. |