Cytochemistry for Leukemias

leukocyte alkaline phosphatase (LAP)

to: differentiate cells of CML from leukemoid rxn

LAP activity is present in varying degrees in secondary granules (after myelocyte stage) of maturing granulocytes (segs & bands)

LAP values

normal: 13-113

leukemoid LAP is high bc more mature granulocytes to fight infn

CML LAP is lower bc premature cells

myeloperoxidase (MPO) P/P

to differentiate acute myeloid leukemia from acute lymphocytic leukemia

MPO = enzyme in primary granules (azurophilic, appear at promyelocyte stage) of granulocytes, beginning at late myeloblast, promyelocyte stage and throughout myeloid maturation

low activity in monocyte

rbcs or lymphocytes = NEG MPO

MPO pos values

super MPO pos = promyelocytes & Auer rods

most leukemic myeloblasts are usually MPO pos in AML

• MPO activity is limited in early myeloblast

neg for lymphs

Auer rods

abnormal fusion of primary granules in neoplastic myeloid blasts

possible to see AML-M1-3, super rare in M4, M5

m/c in M3 (APL)

Sudan Black B P/P

to distinguish AML from lymphocytic leukemia

dye stain phospholipid & other intracellular lipids found in primary & secondary granules of neutrophils, eosinophils, and in smaller qty in monocytes → more sensitive for early myeloid cells compared to MPO

• Auer rods are SBB+

• monocytic cells are neg (or W), lymphoblast are neg

SBB vs MPO: why is SBB better?

SBB is more sensitive to early myeloblasts

when fresh specimen isn’t available for MPO stain & when blast has MPO defic (in MDS)

specific esterase (chloracetate esterase)- CAE P/P

diff myelocytic & monocyclic leukemia

lysosomal enzymes, specific for granulocytic cells

CAE interpretations

POS = myeloid cells, Auer rods

neg-W = monocytes

neg = lymphocytes

can be used to ID granulocytes in formalin fixed tissues

nonspecific esterase (NSE) p/p

diff myelocytic & monocyclic leukemia

lysosomal enzymes specific for monocytes lineage

substrates: a-Napthyl acetate & a-napthyl butyrate

NSE interpretation

NSE exhibits strong activity in monocytic cells but v weak in granulocytes, megakaryocytes & lymphocytes

POS = bright red-brick color on monocytic cells

NSE w/fluoride inhibition

if fluoride added, only monocyte NSE will be inhibited

dual esterase stains

useful in ID monoblast & promonoblast populations in acute monoblastic leukemia & acute myelomonocytic leukemia

periodic acid Schiff (PAS) reaction

dx of erythroleukemia, erythroblasts ie subtype of AML-M6

AND dx of some acute lymphocytic leukemia

the stain indicates intracellular glycogen & related mucopolysaccharides

PAS interpretations

diffuse staining POS = erythroid popn in erythroblastic leukemias

and

block/coarse POS = lymphoblasts

neg = myeloid or monocytic blasts

tartrate resistant acid phosphatase (TRAP) p/p

dx of hairy cell leukemia (old gold standard)

acid phosphatase is in myelocytes, lymphocytes, monocytes, plasma cells, and plts

→ AP is inhibited in presence of L-tartrate → no color

→ AP in hairy cell will not be inhibited → POS stain

iron stain (Prussian Blue rxn) p/p

to detect Fe-contain granules (siderotic granules) found in CP of developing cells in BM/tissue

rxn occurs w treatment of sections in acid solutions of ferrocyanides (Perl’s rgt) → siderotic granules found in nRBCs and some retics

any ferric ion in tissue reacts w ferricyanide → Prussian blue → used to assess BM iron reserves → useful in evaluating pt w anemia, iron overload & myelodysplasia (MDS) + Pappenheimer bodies!

terminal deoxynucleotidyl transferase (TdT)

TdT = intra-nuclear enzyme in stem cell & immature lymphoid cells w/in BM

90% present in ALLs, but only 5-10% in AML

acute leukemia cytochemistry table

AML vs ALL cytochem tests