Hema 2 Lec Module 16: Cytochemistry

purplish to dark red granules

ACID PHOSPHATASE

result:

T-cell acute lymphoblastic leukemia

Provides a moderate activity for acid phosphatase, confined to the Golgi area of the cell.

Non-T-cell acute leukemia

negative/weak activity

Naphthol AS-BI

ACID PHOSPHATASE

substrate of choice:

TRUE

TRUE or FALSE

False negative results when reagents stored more than 2 weeks

Glycerin Jelly mounting media

What media is used when using fast Garnet GBC?

Routine mounting media

What media leech the reaction product within minutes?

0

ISOENZYMES

Gaucher cells

1 & 4

ISOENZYMES

Neutrophil, Monocytes

3a

ISOENZYMES

Lymphocytes, Platelets

3b

ISOENZYMES

Primitive cells and blasts

5

ISOENZYMES

Hairy cell

Tartrate-resistant acid phosphatase (TRAP)

Used to demonstrate/diagnose hairy cell leukemia.

ACP in isoenzyme band 5

The only isoenzyme resistant to L-(+) tartaric acid (TRAP).

TRUE

TRUE or FALSE

In tartrate-resistant acid phosphatase, hematopoietic cells showed a positive result.

pararosanilin and sodium metabisulfite

Schiff's reagent is a mixture of:

Leukocyte Alkaline Phosphate

Differentiate chronic granulocytic leukemia/chronic myelogenous leukemia from leukemoid reaction.

Citrate-formaldehyde acetone

Leukocyte Alkaline Phosphate

Fixative:

0

SCORE

No granules in the cytoplasm; No staining

1+

SCORE

Few small granules in the cytoplasm (could be counted); Faint and diffuse staining

2+

SCORE

Moderate staining granules in 50-80% of cytoplasm; Pale, with a moderate amount of blue staining

3+

SCORE

Strongly stained granules in 80-100% of cytoplasm and beginning to coalesce Strong blue precipitated staining

4+

SCORE

Strongly-stained granules packed into the cytoplasm (nucleus is the only visible component) Deep blue or brilliant staining with no visible cytoplasm

Periodic-Acid-Schiff (PAS)

Sudan Black B (SBB)

Prussian Blue Reaction

What are the 3 non-enzymatic techniques?

Sudan Black B

Demonstrates lipids (sterols, phospholipids, and neutral fats) present in the cells

Periodic-Acid-Schiff

Aid in the diagnosis of acute lymphocytic leukemia and FAB M6

bright reddish pink color

Aldehyde groups react with Schiff reagent producing a

Formalin or Ethanol

Periodic-Acid-Schiff

Fixative

FALSE

TRUE or FALSE

In periodic-acid-schiff, myeloid and monocytic cells showed a negative result.

Prussian Blue reaction

Indicated by a blue-green precipitate.

acute myeloid leukemia ; acute lymphocytic leukemia

Sudan Black B is useful in distinguishing __________ from ___________

Buffered formalin or acetone

Sudan Black B

Fixative:

Granulocytes, Monocytes-macrophages

Sudan Black B: Positive

Lymphoid cells

Sudan Black B: Negative

Perl's reagent (potassium ferrocyanide)

Prussian blue reaction is quantified by using _______.

thalassemia ; splenectomy

Increased numbers of siderocytes are seen in disorders such as _________ , or in patients after __________.

Immunocytochemistry

Is the use of a specific monoclonal or polyclonal antibodies against selected cell antigens.

M7

Factor VIII antibodies are used for accurate diagnosis of ________

Terminal deoxynucleotidyl transferase (TDT)

is a cell marker found on immature and neoplastic cells frequently seen in leukemic states.

Horseradish peroxidase

What is the plant enzyme of immunoperoxidase?

Peroxidase anti-peroxidase (PAP) method

What is the method used to detect enzyme immunochrmistry?

Lacks cross-reactivity with pseudoperoxidase of the RBCs.

What is the advantage of enzyme immunochemistry?

FALSE

TRUE or FALSE:

TDT is a non-template-directed DNA polymerase that catalyzes the reversible addition of deoxynucleotides to the 3' hydroxy groups on the end of the DNA.

Peroxidases

Used as a marker for primary neutrophilic granules.

Immunofluorescence or Immunoperoxidase

What are the methods used to detect TDT?

primitive lymphoid cells (lymphoblasts)

TDT is used to identify _________

Cytochemistry

A microscopic study identification of chemical constituents within an individual cell.

TRUE

Cytochemistry is used to identify malignant cell types, detect the presence of/lack of cellular constituents and cells exhibiting functional abnormalities.

A. TRUE

B. FALSE

Peroxidases

This catalyzes the oxidation of substance by hydrogen peroxide.

TRUE

Peroxidases (Myeloperoxidase / MPO) is routinely performed on all new acute leukemias

A. TRUE

B. FALSE

TRUE

Peroxidases (Myeloperoxidase / MPO) is not recommended for the demonstration of Auer rods.

A. TRUE

B. FALSE

FALSE

positive

Auer rods is peroxidase-negative.

A. TRUE

B. FALSE

2

Specimens for peroxidases should be stained within ___ weeks of collection.

FALSE

cannot withstand

Peroxidases can withstand high concentrations of methanol.

A. TRUE

B. FALSE

FALSE

does deteriorates

MPO enzyme does not deteriorates during storage.

A. TRUE

B. FALSE

Maturing neutrophil precursor

Pseudoperoxidase activity of RBCs

What are the positive controls for the enzymatic technique Peroxidases?

Buffy coat

What control is used for Cyanide- resistant peroxidase stain?

In Cyanide- resistant peroxidase stain, eosinophil what is the color for eosinophil which is strong positive?

Strongly positive (brown)

In the Cyanide-resistant peroxidase stain, eosinophil, what color indicates a strong positive result for eosinophils?

TRUE

The peroxidase present in the WBCs will catalyze the oxidation of 3-aminodiethylcarbozole by the hydrogen peroxide present in the stain forming an insoluble reddish-brown precipitate at the site of peroxidase activity

A. TRUE

B. FALSE

reddish deposits

What will be the color of the deposits when peroxidase activity is present?

immunocytochemical

Cytochemistry is divided into 3 groups:

- Enzymatic

- Non-enzymatic

- ________________________

Strongly positive (brown)

In the Cyanide-resistant peroxidase stain, eosinophil, what color indicates a strong positive result for eosinophils?

2 weeks

Specimens for the enzymatic technique Peroxidases should be stained within ___ weeks of collection.

Maturing neutrophil precursor

Pseudoperoxidase activity of RBCs

Positive controls used for the enzymatic technique - Peroxidases.

methanol

Peroxidases cannot withstand high concentrations of ___________

Esterases

This is a Lysosomal enzymes

Nine esterase bands

How many esterase bands should be demonstrated in gel electropheresis for enzymatic technique - Esterases?

Isoenzymes 1, 2, 7, 8, and 9

What are the isoenzymes found in neutrophils?

Isoenzymes 3, 4, 5, and 6

What are the isoenzymes found in monocyte?

TRUE

Monocytes and Histiocytes will show a very strong reactions in Alpha-Naphthyl Acetate Esterase (ANAE)

A. TRUE

B. FALSE

FALSE

Alpha-Naphthyl Butyrate Esterase (ANBE)

Naphthol AS-D Chloroacetate Esterase is use to demonstrate "nonspecific" esterases

A. TRUE

B. FALSE

TRUE

Alpha-Naphthyl Butyrate Esterase (ANBE) is used to distinguish monocytic leukemia from myeloid leukemias.

A. TRUE

B. FALSE

diazonium salt,

Naphthol compounds are coupled with ____________, forming highly colored deposits at the sites of enzyme activity.

Naphthol AS-D Chloroacetate Esterase

In this procedure, blood or bone marrow smears are incubated with naphthol AS-D chloroacetate in the presence of stable diazonium salt.

FALSE

POSITIVE- Cytoplasm of mast cell, neutrophils, neutrophil precursors, and monocytes

NEGATIVE- Eosinophils

Eosinophil will have a postive result for Naphthol AS-D Chloroacetate Esterase

A. TRUE

B. FALSE

RED

In alpha-naphthyl acetate esterase, the non-specific esterases found in the WBCs will hydrolyze the alpha-naphthyl acetate, then it will liberate the naphthyl compound which would then combine with the pararosanilin to produce what color of staining precipitate?

dark-red staining

Result for Monocyte, Macrophages, Megakaryocytes, and Platelets in alpha-naphthyl acetate esterase?

TRUE

Lymphocytes in alpha-naphthyl acetate esterase will result to dotlike stain

A. TRUE

B. FALSE

NEGATIVE

Myeloid and lymphoid cells will yield what result to α-Naphthyl butyrate esterase?

Buffered formalin acetone

Sodium nitrite

Hexazotized pararosanilin

Alpha-naphthyl acetate solution

Phosphate buffer

Mayer's Hematoxylin

Give one reagent for α-Naphthyl butyrate esterase

pH 7.6

ALPHA-NAPHTHYL ESTERASE

pH:

FALSE

negative

Positive - promyelocyte, myelocyte, metamyelocyte, bands and segmented neutrophils

In Naphthol AS-D Chloroacetate Esterase, myeloblasts, nonoblasts, promonocytes and monocytes will give a positive reaction

A. TRUE

B. FALSE

TRUE

In alpha-Naphthyl esterase, Monocytes will yield a negative reaction

A. TRUE

B. FALSE

Phosphatases

This hydrolyzes monophosphate esters at an acid or alkaline pH

Acid Phosphatase

What phosphatase is found in all hematopoietic cells and is in lysosomes?

Acid Phosphatase

Used in identification of T-acute lymphocytic

leukemia

TRUE

N,Ndimethylformaldehyde is stable at 4-10°C for 2 months

A. TRUE

B. FALSE

TRUE

The peripheral blood or bone marrow smears are fixed and incubated in a solution of naphthol AS-BI phosphoric acid and fast garnet GBC salt.

A. TRUE

B. FALSE

maroon

The naphthol AS-BI will then be released by enzymatic hydrolysis, and will be coupled with the fast garnet GBC salt at an acid pH forming an insoluble _________ dye deposit at the site of acid phosphatase activity