WBC Lecture 3: Myeloproliferative disorders, FCM

1. What do you expect to see in the BM (bone marrow) investigation?
Findings expected:
- Hypercellular marrow: The bone marrow is packed with cells, particularly granulocytic precursors at all stages of maturation.
- Marked myeloid hyperplasia: Striking increase in myeloid-to-erythroid (M:E) ratio, often 10–50:1 (normal is ~2–4:1).
- Left shift: Abundant myeloblasts, promyelocytes, myelocytes, and metamyelocytes.
- Erythroid hypoplasia: Suppressed red cell precursors.
- Megakaryocytic hyperplasia: Often small, abnormal megakaryocytes.
- Basophilia and eosinophilia: Both seen in the bone marrow and peripheral blood.
- Chromosomal / genetic abnormality: Philadelphia chromosome t(9;22)(q34;q11), resulting in BCR-ABL fusion gene.

2. What are next steps and the expected results?
Next diagnostic steps:
1. Cytogenetic testing (karyotyping or FISH):
- Look for the Philadelphia chromosome t(9;22).
- Expected result: Positive in >95% of CML cases.
2. Molecular testing (RT-PCR or quantitative PCR):
- Detect and quantify the BCR-ABL fusion gene transcript.
- Expected result: Strong positive confirming diagnosis.
3. Additional supportive tests:
- Leukocyte alkaline phosphatase (LAP) score is very low in CML (as seen in this patient: 20, with reference 40–130).
- This helps distinguish CML from a leukemoid reaction (where LAP is high).

Expected interpretation:
- This 34-year-old woman’s presentation (fever, night sweats, weight loss, splenomegaly, marked leukocytosis with left shift, basophilia, eosinophilia, low LAP, and hyperuricemia) is highly consistent with Chronic Myeloid Leukemia (CML).
- Confirmatory cytogenetic and molecular tests would establish the diagnosis.
- Next steps in management would likely include targeted therapy (e.g., tyrosine kinase inhibitor such as imatinib).

Clonal hematopoietic disorders caused by gene mutations in HSCs, leading to increased proliferation of and accumulation of mature erythrocytes, granulocytes, and platelets.

Chronic myeloid leukemia (CML), Polycythemia vera (PV), Essential (primary) thrombocythemia (ET), Primary myelofibrosis (PMF).

PV, ET, and PMF (more frequent in PV).

Philadelphia (Ph) chromosome (t(9;22)) only in CML.

Approximately 15% of patients exhibit hyperleukocytosis

(total WBC counts greater than 300 × 109/L), risk of leukostasis

Hyperuricemia, uricosuria, risk of secondary gout, uric acid stones

Cytogenetic analysis for detection of t(9;22)

Detection of the BCR-ABL1 transcript using qRT-PCR

Detection of the BCR-ABL1 fusion gene using fluorescence in situ hybridization (FISH)

LAP score decreased in CML

PBS incubated with the substrate of ALP (naphthol-phosphate substrate and diazo dye at an alkaline pH)

A colored precipitate formed on the granules of neuts and bands

100 neutrophils and bands counted, and a score (from 0–4+) given depending on color intensity, score multiplied by the number of cells and added up

For example, 5 cells with 4+ staining, 5 cells with 3+, 25 cells with 2+, 45 cells with 1+, and 20 cells with 0 staining, will provide a LAP score of 130

Normal range: 15–170

Poor response to therapy and appearance of more chromosomal changes

More prominent anemia

Decrease in number of mature neuts and more basophils

Fewer platelets, more abnormal platelets, micromegakaryocytes, and megakaryocytic fragments

Increase of circulating blast count from 10% to 19% and transformation into either acute lymphoid or acute myeloid leukemia

Panmyelosis and JAK2 mutation

JAK2 is related to STAT (signal transducers and activators of transcription) protein which are activated following binding of cytokines (EPO here) leading to cell proliferation and differentiation

Mutated JAK2 causes constitutive activation of the JAK/STAT signaling pathway, (independent of EPO)

Three major: I–Elevated Hb and HCT, II–BM hypercellularity with trilineage growth (panmyelosis), III–Identification of JAK2 mutation

Low serum EPO levels

Pseudo-Gaucher cells are rare

Approximately 80% of patients manifest BM panmyelosis

100% of BM volume may exhibit hematopoietic cellularity

Splenomegaly, hepatomegaly, generalized vascular engorgement, and circulatory disturbances increase the risk of hemorrhage, tissue infarction, and thrombosis

Red cell mass always increased

EPO is decreased and arterial O₂ saturation is normal

Also has leukocytosis (>12) and increased PLTS (>400)

BM myelopoiesis and splenomegaly (>75%)

Red cell mass can be increased

In BM, only erythroid hyperplasia

EPO is increased and arterial O₂ saturation is decreased

Marked and persistent thrombocytosis; platelets range from 600–2000 × 10⁹/L (>450); large masses of platelet aggregates; abnormal platelet morphology including giant and bizarre forms and megakaryocyte fragments

WBCs ± elevated (can see neutrophilia with slight left shift); RBCs normal or slightly decreased (rare NRBC)

Often discovered in routine CBC

Patients can present with vascular occlusion or bleeding; symptoms include headache, dizziness, throbbing / burning in hands and feet, mucosal bleeding, seizures, and myocardial infarction

Most of the mutations described in PV also occur in ET but usually at a lower frequency

WBCs ± elevated – neutrophilia with slight left shift; RBCs are normal or slightly decreased, rare NRBC

Platelets greatly increased; generally ‘normal’ looking but can vary in size, shape, granulation; some giant platelets; and sometimes in clusters

Platelet function studies are abnormal and platelet aggregation is abnormal

When the PLT count is reduced to normal, the platelet function returns to normal

Complications include hemorrhage (bleeding tendencies – abnormal PLTs) or thrombosis (usually with PLTs > 2000 × 10⁹/L)

Abnormal clone of megakaryocytes stimulate the bone marrow fibroblasts to produce collagen

Fibroblastic growth factors released (PDGF-β); normal bone marrow tissue is gradually replaced with a fibrous scar-like material (fibrosis); dry tap BM during biopsy

Over time, this leads to progressive bone marrow failure

Results in extramedullary hematopoiesis

As with PV and ET, the JAK2 V617F mutation is involved and is found in 60% of PMF patients

Immature granulocytes and NRBCs, teardrops, giant platelets, and other bizarre RBC shapes

Increased platelets and a micromegakaryocyte

Tear drop poik is the result of narrowing and rigidity of splenic sinusoids

- Bone marrow (aspirate), in heparin
- Bone marrow FNB
- Peripheral blood, in EDTA
- Lymphoid tissues
- Body cavity fluids and solid tissues when they are suspected to harbor a hematologic malignancy

Blue: Neutrophils
Red: Red cells and PLTs
Black: Blasts
Aqua: Mature lymphocytes
Green: Monocytes

Figure A

3. C

1. A is larger than B and 3. B is smaller than A is correct

1. a

Immune deficiency (SCID, HIV, ……)

Stem cell enumeration (CD34)

PNH (CD55, CD59)

Reticulocytes (RNA binding dyes, e.g. TO)

FMH (Ab against HbF, RhD, or both, or CA)

Cell cycle analysis (DNA content)

Transplantation (HLA, flow cross match: donor lymphocytes + patient’s serum, then incubate with antihuman IgG F(ab)/FITC)

WB or BM samples stored for a long time with the anti-coagulants; WB or BM aspirates are NOT delivered to the lab while fresh or are collected in inappropriate anticoagulants

Tissue samples (BM FNB, LNB) are not transferred to the lab in appropriate media

Using harsh vortex on cell pellets and using aggressive mechanical disruption of tissues

Cell pellets are kept as dry for a long time or highly concentrated cell suspension is used

Keeping cells on ice can improve the viability as this slows down necrosis and the cell metabolism

Yes, FCM can be run on body fluids like CSF

Yes, frozen specimens can be used

The quality of the specimen will be influenced by transferring to the lab (time, condition), storage in the lab (time, condition), type of anti-coagulant, and staining procedure