Overview
Leukemia is a disease of unknown etiology characterized by uncontrolled, abnormal and widespread proliferation of leukocytes and their precursors in bone marrow and blood. It is classified by clinical course (acute vs chronic) and cell lineage (myeloid vs lymphoid).
The FAB (French-American-British) classification uses blast morphology on Romanowsky stains, while the WHO classification adds immunophenotyping and cytogenetics. Accurate laboratory classification directly guides chemotherapy selection and prognosis.
Learning Objectives
After this lesson you will be able to…- Classify Acute Lymphoblastic Leukemia (ALL) by FAB subtype.
- Describe the FAB classification of Acute Myeloid Leukemia (AML).
- Explain the cytochemistry stains used in the diagnosis of leukemia.
- Explain the role of immunophenotyping in leukemia diagnosis.
- Describe Chronic Myeloid Leukemia, Chronic Lymphocytic Leukemia, myeloproliferative disorders and myelodysplastic syndromes.
Clinical Story
Why This MattersA 4-year-old boy presents with fever, pallor and easy bruising. His CBC shows a WBC count of 65 x10⁹/L with 80% blasts. The lab technologist performs cytochemistry — Myeloperoxidase and Sudan Black B are negative, while PAS shows block positivity. This distinguishes ALL from AML in minutes, letting the oncologist start the correct induction chemotherapy protocol without delay.
Core Concepts
Acute leukemia has sudden onset and rapid progression, characterized by blast cells in peripheral blood and bone marrow. Chronic leukemia has gradual onset and slow progression, characterized by more mature cells. The FAB classification (1990s) is based on blast morphology; the WHO classification (2002) adds immunophenotyping and cytogenetics.
Myeloperoxidase (MPO) and Sudan Black B (SBB) are positive in myeloid blasts, negative in ALL. PAS shows block positivity in ALL blasts. Acid phosphatase aids diagnosis of T-cell ALL and hairy cell leukemia (positive with and without tartaric acid inhibition in hairy cell leukemia). Esterase stains (specific and non-specific) differentiate granulocytic from monocytic lineage. Leukocyte Alkaline Phosphatase (LAP) is markedly decreased in CML.
Affects children 2–10 years commonly, with good prognosis in childhood. FAB subtypes: L1 (small uniform blasts, 14–16µm), L2 (larger, heterogeneous, 15–18µm, 1–2 nucleoli), L3 (18–20µm, vacuolated cytoplasm, Burkitt-type). MPO/SBB negative; PAS block-positive. Immunophenotyping: B cells are CD19+, CD10+; T cells are CD2+, CD5+, CD7+. FAB requires >30% marrow blasts; WHO requires >20%.
Affects young adults. FAB subtypes: M0 (undifferentiated, CD13+/CD33+/CD117+), M1 (minimal maturation, Auer rods), M2 (with maturation), M3 (promyelocytic, faggot cells, t(15;17)), M4 (myelomonocytic, inv16 variant with eosinophilia), M5 (monocytic, >80% monocytoid cells), M6 (erythroleukemia, >50% erythroid), M7 (megakaryoblastic, CD41+/CD61+). Auer rods are pathognomonic of AML when present.
CML: affects young adults; blood film resembles a marrow preparation with the full myeloid maturation series and basophilia; LAP is markedly decreased; Philadelphia chromosome t(9;22) is the hallmark cytogenetic finding. CLL: affects older age groups; 70–90% mature lymphocytes with smear cells on blood film; flow cytometry shows B-cell markers plus aberrant CD5 positivity.
Chronic myeloproliferative disorders include CML, polycythemia rubra vera, myelofibrosis, and essential thrombocythemia. Myelodysplastic syndromes (preleukemia) include refractory anemia, sideroblastic anemia, and refractory anemia with excess blasts (RAEB) — these often progress to AML.
Laboratory Principle
Cytochemical stains exploit lineage-specific enzymes or cytoplasmic products (peroxidase, lipids, glycogen, esterases) to distinguish myeloid from lymphoid blasts using color reactions visible under light microscopy. Immunophenotyping uses fluorescently labeled monoclonal antibodies against lineage-specific surface/cytoplasmic antigens, analyzed by flow cytometry, providing a far more precise lineage assignment than morphology alone.
Equipment Required
Reagents & Materials
| Reagent / Material | Concentration / Grade | Purpose | Storage |
|---|---|---|---|
| 3,3-diaminobenzidine (DAB) in phosphate buffer pH 7.3 | Substrate for MPO | Identify myeloid blasts | Prepare fresh, protect from light |
| Sudan Black B dye | Lipophilic stain | Stains granulocyte/monocyte granules | Room temperature |
| Periodic acid + Schiff reagent (leucobasic fuchsin) | PAS kit | Detect glycogen in ALL blasts | 2–8°C, protect from light |
| Naphthol AS-BI phosphate + hexazotised pararosanaline | Acid phosphatase kit | T-cell ALL / hairy cell leukemia diagnosis | 2–8°C |
Step-by-Step Procedure
Make thin, well-spread Romanowsky-stained smears from peripheral blood and bone marrow aspirate.
Examine blast size, nuclear-cytoplasmic ratio, nucleoli, chromatin pattern and cytoplasmic inclusions (Auer rods).
Run MPO, SBB, PAS, esterase and acid phosphatase stains as indicated to distinguish myeloid from lymphoid lineage.
Label cells with monoclonal antibody panels to confirm lineage (myeloid, B-cell, T-cell, megakaryocytic, erythroid).
Perform karyotyping, FISH or RT-PCR to detect diagnostic translocations (e.g. t(15;17), t(9;22)) and guide targeted therapy.
Flow Diagram
Quality Control
Use a known positive control (e.g. a pregnant female sample for LAP scoring) alongside test slides. Score neutrophils systematically (0 to 4+) for LAP using a standardized counting method.
Participate in hematopathology slide review programs and flow cytometry proficiency testing to validate blast classification consistency.
Reference Values
Normal Ranges⚠️ Reference ranges may vary between laboratories. Always apply your laboratory's established reference intervals.
Clinical Interpretation
| Finding | Possible Significance | Action / Follow-up |
|---|---|---|
| Blasts negative for MPO/SBB, PAS block positive | Acute Lymphoblastic Leukemia | Immunophenotype for B/T lineage; start ALL protocol |
| Blasts with Auer rods, MPO/SBB strongly positive | Acute Myeloid Leukemia | Subtype by FAB/WHO; test for t(15;17) if M3 suspected |
| Markedly decreased LAP score + t(9;22) | Chronic Myeloid Leukemia | Confirm Philadelphia chromosome; start tyrosine kinase inhibitor therapy |
Common Errors & How to Avoid Them
Cause: Poorly spread smears obscure fine cytoplasmic inclusions.
Prevention: Prepare thin, well-feathered smears and scan multiple fields under oil immersion.
Cause: Hypogranular variant of M3 lacks the classic heavy azurophilic granulation.
Prevention: Always correlate with cytogenetics — t(15;17) is diagnostic of M3 even when granulation is minimal; treat as a hematologic emergency due to DIC risk.
Cause: Some AML subtypes (M0, M7) look morphologically similar to ALL.
Prevention: Always confirm lineage with flow cytometry immunophenotyping before finalizing classification.
Laboratory Tips from the Bench
Auer rods, when present, are diagnostic of AML and rule out ALL — always scan carefully for these thin pink rod-like structures.
"Faggot cells" packed with Auer rods are characteristic of AML M3 (promyelocytic leukemia) — recognize this urgently due to DIC risk.
"MPO/SBB positive = Myeloid; PAS block positive = Lymphoid (ALL)" — a quick cytochemistry rule of thumb.
Important Notes
Promyelocytic leukemia (M3) is associated with life-threatening disseminated intravascular coagulation and requires urgent recognition and treatment with ATRA (all-trans retinoic acid).
In ALL, cytospin preparation of cerebrospinal fluid is used to detect small numbers of lymphoblasts with minimal cell distortion, important for staging and treatment planning.
Interactive Quiz
Test Your KnowledgeFlashcards
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Clinical Case Study
Apply Your KnowledgePresents with easy bruising, gum bleeding and severe fatigue for one week. On examination, mild splenomegaly and multiple ecchymoses are noted.
Hypergranular promyelocytes with numerous Auer rods (faggot cells) and evidence of DIC (deranged coagulation, low platelets) strongly suggest AML M3 — a hematologic emergency requiring immediate treatment.
- →AML M3 must be recognized rapidly due to associated DIC risk.
- →Confirm with cytogenetics for t(15;17) before starting ATRA therapy.
- →Coagulation profile monitoring is critical alongside CBC in suspected M3.
Frequently Asked Questions
Morphology alone can be misleading — some AML subtypes (M0, M7) closely resemble ALL blasts. Immunophenotyping provides definitive lineage assignment and detects minimal residual disease during follow-up.
The WHO lowered the diagnostic threshold from >30% to >20% marrow blasts, recognizing that patients with 20–30% blasts and typical cytogenetic abnormalities behave clinically like acute leukemia rather than MDS.
Yes — MDS is often called "preleukemia" because it frequently progresses to AML, particularly the refractory anemia with excess blasts (RAEB) and RAEB-T subtypes.
Quick Revision
10-Minute ReviewKey Takeaways
- Leukemia classification integrates morphology (FAB), immunophenotyping and cytogenetics (WHO).
- Cytochemistry stains distinguish myeloid from lymphoid blasts rapidly at the bedside.
- AML has 8 FAB subtypes (M0–M7), each with distinct morphology and markers.
- ALL has 3 FAB subtypes (L1–L3) and B/T immunophenotype categories.
- CML shows the Philadelphia chromosome and decreased LAP; CLL shows aberrant CD5 on B cells.
- Myeloproliferative and myelodysplastic disorders are related chronic marrow conditions with leukemic transformation potential.
Competency Checklist
Track Your MasteryReferences
- NIOS Hematology and Blood Bank Technique — Lesson 23: Leukemia.
- Bennett JM et al. Proposals for the classification of acute leukaemias (FAB co-operative group).
- WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues.