Cytochemistry and Immunochemistry
  • Stains used in the differentiation and classification of acute leukaemias (AL) according to the FAB system.
  • Myeloperoxidase (MPO) is found in azurophilic granules ie. in the immature granulocytic series (>3% of blasts should be positive for the myeloid group, and <3% for the lymphoid group)
  • These do not cause alteration of the cellular morphology
  • These criteria refer to the immature cells only
  • Muramidase is released by monocytes
  • TdT catalyses the addition of dinucleoside triphosphate onto the free end of ssDNA without a template
    • Not in normal, mature lymphocytes but is present in 65% of the total thymic population of lymphocytes
    • 1-3% of all normal BM cells are POS
    • T-ALL are TdT POS but B-ALL are NEG

Leukaemia Assay
Myeloperoxidase or Sudan Black B Esterases PAS
Periodic acid-Schiff		 Muramidase
"Lysozyme"		 TdT
Terminal deoxynucleotidyl transferase
Specific
a-Naphthol AS-D chloroacetate esterase		 Non-Specific
a-Naphthyl acetate esterase & a-naphthyl butyrate esterase
AML-M1 + ++ - - + - - - or +
AML-M2 + +++ ++ - + - +/- -
AML-M3 +++ +++ - + - ++ -
AML-M4 ++ +++ + +++ + +++ + - ++ -
AML-M5 + - +++ + - +++ -
AML-M6 - - - +++ - -
AML-M7 - - ++ - ++ ++ - -
ALL - - - (+ for CLL & T-ALL) +++ - +++
Normal Neutrophils +++ +++ - +++ - -
Normal Monocytes + - +++ +++ +++ -
Normal Lymphocytes - - - +/- - -
Platelets     + (a-Naphthyl a-e eg. Megakaryocytes), - (a-Naphthyl b-e eg. M7) +++    

Myeloperoxidase (MPO)

    • A lysosomal enzyme localised to the azurophilic granules of neutrophils and monocytes. Can be demonstrated in eosinophils and basophils.
    • Completed in minutes
    • Fixation in glutaraldehyde in PBS, rinsed and immersed substrate in Tris-HCl + H2O2, rinsed again and placed in enhancing solution. To clarify detail, the cells are counter-stained with Giemsa
    • Substrates include:
        .o-toluidine
        .3,3'-diaminobenzidine (DAB)
        .2,7-fluorenediamine (FDA)
        .3-amino-9-ethylcarbazole
    • Only really of value if POS, immature cells. NEG can result in M0, M7 (monoblasts) and ALL (lymphocytes and lymphoblasts)
    • Developing granulocytes are always POS. Strongly in promyelocytes and myelocytes but may be NEG in early myeloblasts. Almost all mature neutrophils give POS reactions as do eosinophils, basophils, promonocytes and monocytes.
    • Main function of this test is in the distinction between AML and ALL

Sudan Black B

     

    Parallels MPO, staining the lipid membrane around the granules, but it takes much longer.

Chloro-acetate esterase (CAE)

    • A specific esterase (capable of liberating naphthol from a naphthol AS-D chloroacetate substrate) present in granulocytes and mast cells distinct from that in monocytes and megakaryocytes
    • Auer rods are POS
    • Optimal pH of 7.0-7.6
    • Not inhibited by sodium fluoride (NaF)
    • Enzyme activity can be seen in paraffin embedded sections
    • Can use fast garnet, fast red violet or fast blue as chromogens in the reaction

Non-specific esterases

    • POS for the monocytic series

     

    a-Naphthyl Acetate Esterase (ANAE)

      • Sensitive but not specific (picks up T-lymphocytes (punctate staining pattern) as well as monocytes (diffuse staining pattern))
      • The T lymphocytic pattern is resistant to NaF while staining in monocytes is NaF sensitive and favoured at an alkaline pH
      • Method involves formaldehyde-PBS fixation. After drying, incubate for 45min in the presence of substrate and chromogen (hexazonium salt) and counterstained with methyl-green.
      • Helps diagnosis in AML of M5; in ALL of T-ALL and in CLL T-CLL (POS) fro B-CLL (NEG)
      • Erythroblasts are usually ANAE NEG, however they may react in megaloblastic anaemia and erythroleukaemia

     

    a-Naphthyl Butyrate Esterase (ANBE)

      • Similar results to above
       

      Specific but not as sensitive

Periodic Acid Schiff's (PAS) Stain

    • looking for abnormal amounts of glycogen, glycoproteins, mucoproteins and high molecular weight carbohydrates
    • Schiff's reagent reacts with aldehydes formed by periodic acid breaking down (oxidising) the C-C bond in CHOH-CHOH groups
    • Most cells POS but intensity and pattern varies
  • Another way of typing the variants is called immunophenotyping
  • Monoclonal antibodies toward cell-type restricted antigens are used in this highly specific method

Antigens Cluster of Differentiation (CD) Number FAB Nomenclature
M1 M2 M3 M4 M5 M6 M7
HLA-DR   + + - + +/- +/- +/-
MY9, LF3 CD33 + + + + +/- +/- +/-
MCS-2, MY7 CD13 + + + + + -  
Mo1, OKM1 CD11b - + +/- + + -  
LeuM1 CD15 - + +/- + + +/-  
Mo2, UCHM1 CD14 - - - + + -  
Glycophorin A   - - - - - + -
Glycoprotein IIb/IIIa CDw41 - - - - - - +
Glycoprotein Ib CDw42 - - - - - - +
von Willebrand factor (vWF) - - - - - - - +
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