Cytochemistry

• It found in the primary granules

  of cells in myeloid lineage (neutrophil,

  eosinophil, & monocyte; phagocytes)

  except lymphocyte & erythroid cell; role is

  to kill in phagocytosis

  Peroxidase
• Myeloperoxidase is to differentiate this two:

  • Acute myelogenous/monocytic leukemia (AML)
  • Acute Lymphocytic leukemia (ALL)
• Principle of Myeloperoxidase

  Peroxidase in azurophilic granules + 3-amino-9-ethylcarbazole (H2O2)
• Positive result in Myeloperoxidase

  Insoluble Reddish-brown precipitate
• Cells that are positive in Myeloperoxidase

  Neutrophil

  Eosinophil

  Monocytes

  * Except blast forms
• Reaction is frequently parallel with SBB

  reaction

  Myeloperoxidase
• Principle in Specific Esterase


  Esterase + Napthol AS-D chloroacetate

  =red granulation

  (combines with fast red violet LB)

  Hydrolysis
• Principle in Non-specific Esterase

  Esterase + Alpha-naphthyl acetate, butyrate = dark brown granulation

  (combines with fast blue BB) Hydrolysis
• Upon addition of this substance it inhibits monocytic esterase only, that results in no color of cell.


  Sodium Fluoride (NaFl)
• Positive cell in combination of Esterase and Napthol AS-D chloroacetate.


  Granulocytic Cell (Red granulation)
• Positive cell in combination of esterase and Alpha-naphthyl acetate, butyrate.

  Monocytic cells (Dark brown granulation)
• Low Substrate Specificity (Non-Specific

  Esterase)

  Acetate, butyrate
• High Substrate Specificity (Specific

  Esterase)

  Chloro-acetate
• Cause False-Negative that inhibited Esterase Activity.

  • Mercury
  • Acid solution
  • Heat
  • Iodine
• Iso-enzyme numbers in Specific Esterase & seen on this cell.

  1, 2, 7, 8, 9; seen in Mast Cells &

  neutrophil
• Iso-enzyme numbers in non-specific esterase & seen in what cells.

  3, 4, 5, 6; seen in Monocytes,

  megakaryocyte, plasma cells
• Acid Phosphatase Principle

  All normal hematopoietic cells (ACP) + L- tartrate + Naphthol AS-BI Phosphoric Acid = No Color
• Present of Hairy Cell (tartrate resistant) with L-tartrate and Naphtol AS-BI phosphoric acid, results to

  Granular Red Precipitate
• Acid Phosphatase positive in what type of cell

  Hairy Cell Leukemia
• Isoenzymes of Acid Phosphatase:

  2,4: Neutrophil, monocyte

  3: lymphocyte, platelet

  5: Hairy Cell (HCL)
• Inhibit most acid phosphatase except Isoenzyme 5 present in Hairy cell leukemia, which is resistant to this.

  L-tartrate
• Positive in TRAP(Tartrate Resistant Acid Phosphatase) stain. Acid phosphatase reaction after incubation with tartaric acid.

  Hairy Cell Leukemia
• The only cell involve in Alkaline Phosphatase / Leukocyte Alkaline Phosphate (LAP)

  Neutrophil
• How to get LAP score

  Use for LAP score:

  100 neutrophils are counted and scored.

  Number of neutrophils rated in each

  category (grading of 0-4)multiply by the grading and all scores are added
• Fast Blue RR Normal Range Lap score

  32-182
• Fast violet B Normal Range Lap score

  12-180
• What is the principle of Alkaline Phosphatase

  Blood smears are fixed in citrate buffered acetone. When places in the incubation solution containing naphthol AS-MX phosphate and fast blue RR, the alkaline phosphatase present in the WBC liberates naphthol AS-MX, which then couple with fast blue RR to form an insoluble compound.
• Sudan Black B Principle

  Stains various lipids (sterols, phospholipids, and neutral fats) = Dark Purple – Black granulation
• Positive cells in Sudan Black B

  • Neutrophil
  • Basophil
  • Eosinophil
  • Monocyte
• Sudan Black B used to distinguish:

  Acute myelogenous

  and monocytic leukemia from

  lymphocytic leukemia
• A Fat-Soluble dye that stains phospholipids, neutral fats and sterols.

  Sudan Black B
• Principle of Periodic Acid Schiff (PAS)

  Glycogen 𝑃𝑒𝑟𝑖𝑜𝑑𝑖𝑐 𝐴𝑐𝑖𝑑 → Di-aldehyde + Schiff Reagent = Bright reddish (magenta) pink color

  (Oxidation)
• It is oxidized to aldehyde by Periodic Acid, Schiff reagent reacts with aldehyde to form Insoluble red colored precipitate


  Glycogen/Carbohydrate
• Positive in PAS

  Neutrophil except blast forms

  RBC in erythroleukemia (FAB

  M6)

  Eosinophil

  Basophil

  Monocyte

  Lymphocyte

  Thrombocyte

  Megakaryocyte

  Leukemic lymphoblasts (ALL –

  L1 – L2)

  Gauchers cell
• Negative in PAS

  • RBC
  • Myeloblast
  • Monoblast
• It is the oxidizing agent in PAS, that converts Glycogen to Aldehyde.

  Periodic Acid
• What are the two components of Schiff reagent that serve as stain.

  Pararosaniline and Sodium Metabisulfte
• Stain that binds with acid mucopolysaccharides in blood cells

  Toluidine Blue
• It is used for recognition of Mast Cell and Basophils

  Toluidine blue
• These are found in the cytoplasm of developing cells in BM in the form of Ferric (Fe+3)


  Siderotic granules