WBC Lecture 3: Myeloproliferative Disorders, FCM

Myeloproliferative neoplasms

Clonal hematopoietic disorders caused by gene mutations in HSCs; increase in proliferation of and accumulation of mature erythrocytes, granulocytes, and platelets

10:1

M:E ratio in CML

300×10⁹/L

Approximately 15% of CML patients exhibit hyperleukocytosis (total WBC counts greater than (——)), risk of leukostasis

Decreased

LAP score in CML

• Gaucher-like (sea blue)

• Green-gray crystals

Describe macrophage characteristics that can be observed in CML (2)

• Granulopoiesis: ↑

• Erythropoiesis: ↓

• Megakaryopoiesis: ↑/N

• Reticulin: ↑

BM characteristics in CML (granulopoiesis, erythropoiesis, megakaryopoiesis, reticulin)

Small

Describe megakaryocyte characteristics that may be observed in CML

• Hepatomegaly

• Splenomegaly

Describe extramedullary tissue characteristics that can be observed in CML (2)

• Hyperuricemia

• Uricosuria

• Secondary gout

• Uric acid stones

CML symptoms (4)

• Cytogenetic analysis: t(9;22)

• qRT-PCR: BCR-ABL 1 transcript

• FISH: BCR-ABL 1 fusion gene

Genetic analysis for CML (3)

• PBS incubated with ALP substrate → precipitate on bands and seg neuts

  • Napthol-phosphate substrate and diazo dye at alkaline pH

• 100 neuts and bands counted → score (color intensity) multiplied by # cells

• Normal range: 15-170

How to get a LAP score (3)

• Poor response to treatment

• More: chromosomal changes, anemia, basophils, abnormal PLT, micromegakaryocytes, megakaryocytic fragments, circulating blasts (10-19%)

• Less: Mature neuts, platelets

• Transformation to AML/ALL

CML blast crisis characteristics (treatment, increased values (7), decreased values (2), transformation)

• Signal transducers and activators of transcription (STAT)

• EPO

• Polycythemia vera

Normal JAK2 is related to (——) proteins which are activated following the binding of cytokines (——), leading to cell proliferation; in (——) mutated JAK2 activates the JAK/STAT signaling pathway independent of cytokines

• Major

  • Elevated Hb and HCT

  • BM hypercellularity with trilineage growth (panmyelosis)

  • JAK2 mutation

• Minor:

  • Low serum EPO

WHO criteria for PV diagnosis (4)

Normal or increased

Polycythemia vera LAP score

• Splenomegaly

• Hepatomegaly

• Generalized vascular engorgement

• Inc risk: hemorrhage, tissue infarction, thrombosis

Polycythemia vera symptoms (6)

• Toxic vacuolization: 2-4+

• Toxic granulation: 2-4+

• Dohle bodies; Frequent

• Eosinophilia: 0

• Basophilia: 0

• LAP: High

• Ph xm: (-)

Leukemoid reaction characteristics (toxic vacuolization, toxic granulation, dohle bodies, eosinophilia, basophilia, LAP, Ph xm)

• Toxic vacuolization: 0-1+

• Toxic granulation: 0-1+

• Dohle bodies; Rare

• Eosinophilia: 1-3+

• Basophilia: 1-3+

• LAP: Low/0

• Ph xm: (+) >90%

CML characteristics (toxic vacuolization, toxic granulation, dohle bodies, eosinophilia, basophilia, LAP, Ph xm)

• Red cell mass always increased

• EPO decreased

• O2 sat normal

• Leukocytosis and thrombocytosis

• BM myelopoiesis

• Splenomegaly

Characteristics of PV that differentiates it from hypoxia (RBC mass, EPO, O2 sat, elevated cells, BM, EM tissue)

• Red cell mass can be increased

• Only erythroid hyperplasia in BM

• EPO increased

• O2 sat decreased

Hypoxia characteritics that differentiates it from PV (RBC mass, BM, EPO, O2 sat)

• 600-2000×10⁹/L

• Large masses of PLT aggregates

• Abnormal morphology (giant/bizarre/MK fragments)

  • (generally normal, but can vary)

PLT characteristics in essential thrombocythemia (3)

• Elevated WBCs

• Neutrophilia with slight left shift

WBC characteristics in essential thrombocythemia (2)

• RBCs normal or slightly decreased

• nRBCs rare

RBC characteristics in Essential thrombocythemia (2)

• Hemorrhage (bleeding tendencies from abnormal PLTs)

• Thrombosis (PLTs > 2000×10⁹/L)

Complications associated with polycythemia vera (2)

Primary myelofibrosis

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

• PDGF-β

• Fibrosis

• Extramedullary hematopoiesis

In primary myelofibrosis, fibroblastic growth factors such as (——) are released; normal bone marrow tissues are eventually replaced by (——) and lead to dry tap in biopsies; over time, this leads to progressive bone marrow failure, eventually leading to (——)

• PV

• ET

• PMF

MPNs associated with JAK 2 mutation (3)

• Immature granulocytes

• nRBCs

• Dacrocytes (+ other bizarre shapes)

• Giant PLTs

Primary myelofibrosis characteristics in a PBS (4)

• WBC: ↑

• RBC: N/↓

• PLT: N/↑

• Molecular abnormalities: Ph t(9;22), BCR-ABL 1

CML characteristics (WBC, RBC, PLT, molecular abnormalities)

• WBC: N/↑

• RBC: ↑

• PLT: N/↑

• Molecular abnormalities: JAK2 V617F (or other JAK2)

Polycythemia vera characteristics (WBC, RBC, PLT, molecular abnormalities)

• WBC: N/↓

• RBC: N/↓

• PLT: ↑

• Molecular abnormalities: JAK2 or other mutation

Essential thrombocythemia characteristics (WBC, RBC, PLT, molecular abnormalities)

• WBC: N/↑/↓

• RBC: N/↓

• PLT: N/↑/↓

• Molecular abnormalities: JAK 2 or other mutation

Primary myelofibrosis (WBC, RBC, PLT, Molecular abnormalities)

• BM aspirate → heparin

• BM → fine needle biopsy

• PB → EDTA

• Lymphoid tissue

• Body cavity fluid/solid tissue w/ hematologic malignancy

Specimens most commonly analyzed for flow cytometry in hematology (5)

• A: Erythroid and PLT

• B: Blast

• C: Neutrophils

• D: Monocytes

• E: Lymphocytes

Identify the flow cytometry populations of a BM sample

• SSC = complexity

• FSC = size

In flow cytometry, side scatter is proportional to (——), while forward scatter is proportional to (——)

• SCID

• HIV

What can flow cytometry test for with immune deficiencies

CD34

What can flow cytometry test for with stem cell enumeration

• CD 55

• CD 59

What can flow cytometry test for with PNH

RNA binding dyes (toluene orange)

What can flow cytometry test for with reticulocytes

Ab against HbF, Rh D, or both, or CA

What can flow cytometry test for with FMH (4)

DNA content

What can flow cytometry test for with cell cycle analysis

• HLA

• Flow cross match

  • (Donor lymph + pt serum → incubate AHG F(ab)/FITC)

What can flow cytometry test for with transplantation (2)

FMH volume = (% fetal cells) (1800) (1.22)

How to calculate fetal maternal hemorrhage volume from FC analysis

• WB/BM stored long with anti-coagulants

• WB/BM aspirate delivered not fresh or inappropriate anticoagulant

• Tissue sample not in appropriate media

• Harsh vortex on cell pellets

• Aggressive mechanical disruption of tissue

• Cell pellets kept dry for a long time

• High concentration cell suspension

Reasons for unacceptable specimens due to compromised cell viability (WB/BM 2, tissue 2, cell pellets 2, cell suspension)