Intro to Leukemia + Cytochemical Stains

Question 1

What is leukemia?

Answer 1

Malignancy (cancer) of blood cells produced in the bone marrow, which do not function normally and over proliferate. Consequently, it becomes difficult for the body to fight infections, control bleeding, and transport oxygen. Patients have unexplained bruises and anemia

Question 2

What is the etiology of leukemia?

Answer 2

Mostly idiopathic (means we don’t know the cause), but contributing factors such as chemical toxins, radiation, microbes, heredity, and age are possible

Question 3

What is acute leukemia characterized by?

Answer 3

• Uncontrolled proliferation
• Arrested maturation
• Hiatus development (means that there’s accumulation of immature cells in blood and/or bone marrow)

Question 4

What is chronic leukemia characterized by?

Answer 4

• Uncontrolled proliferation
• Normal maturation

Question 5

Lab findings in acute leukemia?

Answer 5

• Mostly moderately increased WBC, sometimes decreased
• Build-up of immature WBCs
• Anemia (RBCs crowded out by malignant WBCS)
• Normal to decreased platelets (large, hypo granular, micromegakaryocytes)

Question 6

Lab findings in chronic leukemia?

Answer 6

• Increased to markedly increased WBCs of normal maturation
• Anemia
• Normal to increased platelets (large, hypo granular, micromegakaryocytes)

Question 7

Differential diagnoses options

Answer 7

• Reactive leukocytosis (viral lymphs)
• Cytopenias (aplastic anemia)
• Leukemoid reaction (severe infection, normal body response)

Question 8

What are the lab eval steps of leukemia?

Answer 8

1. CBC: numeric metrics, microscope morphology of abnormal cells
2. Follow-up tests: BM cytochemical stain, flow cytometry, molecular genetics, cytogenetics
3. Look for minimum residual disease where you look for slightest # of abnormal cells post-treat

Question 9

List the 4 common types of leukemia

Answer 9

1. Acute lymphoblastic leukemia (ALL)
2. Chronic lymphocytic leukemia (CLL)
3. Acute myeloid leukemia (AML)
4. Chronic myeloid leukemia (CML)

Question 10

Primary therapy for leukemia in 1900s?

Answer 10

Arsenic

Question 11

Current treatment of leukemia?

Answer 11

• Chemo
• Radiation therapy
• Immunotherapy (mAb, radio, vax, cytokines, donor lymph infusion)
• Targeted molecular therapy
• Transplant (BM/SC)

Question 12

Cytochemical stains used to be the only test available for which leukemia?

Answer 12

Acute
Now flow cytometry provides enough info to diagnose/manage disease

Question 13

List cytochemical stains

Answer 13

• Myeloperoxidase (MPO)
• Esterases (specific + non-specific + combined)
• Leukocyte alkaline phosphatase (LAP)
• Tartrate resistant acid phosphatase (TRAP)

Question 14

Why is TdT not a cytochemical stain?

Answer 14

It’s an immunofluorescent stain, so different principle

Question 15

Purpose of MPO?

Answer 15

Distinguish between myeloid and lymphoid leukemia (AML vs ALL)

Question 16

How do you interpret MPO results?

Answer 16

Only myeloblasts have primary granules with myeloperoxidase, not lymphoblasts. So, add H2O2 and see black/blue/brown ppt in myeloblasts.

Myelocytic and monocytic blasts are positive. But lymphs negative

Question 17

How do you interpret specific esterase (chloroacetate) stain?

Answer 17

It’s specific to granulocytic cells, so only myeloblasts positive (red stain). Monocytic/lymph/erythrocytic blasts all negative

Question 18

How do you distinguish between myelocytic and monocytic blasts using non-specific esterase (alpha-napthyl acetate)?

Answer 18

Incubate cells with NaF. NaF inhibits reaction in monocytic cells only, so appear negative. Myeloblasts still weakly positive.

Question 19

How do you interpret combined esterase stain?

Answer 19

• Red granules are myeloblasts
• Black/dark brown granules are monoblasts or promonocytes

Question 20

How do you do LAP method?

Answer 20

Count 100 PMNs and bands on a scale of 0-4+ based on amount of ppt in cell

Question 21

What leads to higher LAP reaction/score?

Answer 21

• Leukemoid reactions (bc LAP found in 2° granules of neutrophils)
• Some chronic myeloproliferative disorders (polycythemia vera, idiopathic myelofibrosis)

Question 22

How do you calculate total LAP score?

Answer 22

See pic

Question 23

Causes of decreased LAP?

Answer 23

• Malignant granulocytes (CML)
• Proximal nocturnal hemoglobinuria (PNH)
• Sideroblastic anemia
• Myeloproliferative disorders

Question 24

Causes of increased LAP?

Answer 24

• Left shift in leukemoid reaction
• CML of childhood Ph22 negative
• 3rd trimester of pregnancy
• Polycythemia vera

Question 25

How to interpret LAP score?-

Answer 25

• Normal: 15-70
• Leukemoid reaction: 150-365
• CML: 0-24 unless childhood Ph22 negative has high LAP

Question 26

What is the principle of TRAP?

Answer 26

• Acid phosphatase is present in almost all WBCs and it hydrolyzes the substrate napthol AS-BI phosphoric acid. The hydrolyzed substrate couples with a dye to form a red ppt
• All normal isoenzymes of acid phosphatase are inactivated by tartric acid and thus don’t stain

Question 27

TRAP method?

Answer 27

1. Incubate cells with tartric acid
2. Treat acid phosphatase in cells with napthol AS-BI phosphoric acid substrate
3. Normal cells don’t stain and malignant cells do stain

Question 28

Why does hairy enzyme leukemia isoenzyme #5 stain?

Answer 28

The malignancy renders it resistant to tartric acid

Question 29

How do you interpret Tdt?

Answer 29

It’s positive mainly in acute lymphocytic leukemias (L1 and L2). See bright fluorescence

Careful: Tdt has been observed in AML too!