Special Stains

Overview of Special Stains

Definition: Histochemical staining techniques used in hematology to identify specific cellular components or enzymatic activities that aid in the classification and diagnosis of hematologic disorders
Purpose:
- Differentiate Cell Lineages: Distinguish between myeloid, lymphoid, and erythroid cells
- Identify Abnormalities: Detect specific inclusions or enzymatic activities that are characteristic of certain diseases
- Classify Leukemias: Aid in the classification of acute leukemias
- Assess Iron Stores: Evaluate iron levels in the bone marrow and identify abnormal iron deposition
- Detect Fetal-Maternal Hemorrhage: Quantify fetal cells in the maternal circulation
Methods: Involve specific staining procedures performed on peripheral blood smears or bone marrow aspirate smears, followed by microscopic examination

Myeloperoxidase (MPO) Stain:

Principle: Detects the presence of myeloperoxidase, an enzyme found in the primary granules of myeloid cells (neutrophils, eosinophils, and monocytes)
Procedure:
1. Prepare a smear from bone marrow aspirate or peripheral blood
2. Fix the smear
3. Incubate the smear with a substrate (e.g., benzidine dihydrochloride or 3,3’-diaminobenzidine) and hydrogen peroxide (H2O2)
4. MPO in the cells catalyzes the oxidation of the substrate, producing a colored precipitate
5. Counterstain the smear (e.g., with hematoxylin)
6. Examine the smear under a microscope
Interpretation:
- Positive: Myeloblasts, promyelocytes, neutrophils, and eosinophils show granular staining pattern
- Negative: Lymphoblasts, megakaryocytes, and erythrocytes are negative
- Monocytes: Show weak, diffuse positivity
Clinical Significance:
- Differentiate AML from ALL: MPO is positive in myeloblasts and negative in lymphoblasts, helping distinguish between acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL)
- Subclassification of AML: Helps identify specific AML subtypes (e.g., AML with minimal differentiation may be MPO-negative)

Esterase Stains:

Principle: Detect the presence of esterase enzymes in myeloid and monocytic cells; two main types:
- Specific Esterase (Chloroacetate Esterase, CAE): Detects chloroacetate esterase, which is primarily found in granulocytes (neutrophils)
- Non-Specific Esterase (α-Naphthyl Butyrate Esterase, ANBE): Detects α-naphthyl butyrate esterase, which is found in monocytes and macrophages
Procedure:
1. Prepare a smear from bone marrow aspirate or peripheral blood
2. Fix the smear
3. Incubate the smear with the appropriate substrate (chloroacetate ester or α-naphthyl butyrate) and a diazonium salt
4. Esterase enzymes in the cells hydrolyze the substrate, and the released product reacts with the diazonium salt to form a colored precipitate
5. Counterstain the smear
6. Examine the smear under a microscope
Interpretation:
- Specific Esterase (CAE):
  - Positive: Neutrophils and their precursors show granular staining
  - Negative: Monocytes, lymphocytes, and erythrocytes are negative
- Non-Specific Esterase (ANBE):
  - Positive: Monocytes and macrophages show diffuse staining
  - Negative: Neutrophils and lymphocytes are negative
- Combined Esterase Stains:
  - Some procedures use both specific and non-specific esterase stains to better differentiate cell types
  - Inhibition of Non-Specific Esterase with Sodium Fluoride (NaF): Monocytic staining is inhibited by NaF, while granulocytic staining is not
Clinical Significance:
- Differentiate Myeloid and Monocytic Lineages: ANBE and CAE are used to identify leukemias of monocytic lineage (e.g., acute monoblastic leukemia) from myeloblastic lineage (e.g., AML with minimal differentiation)
- Classification of AML: Helps subclassify AML based on the degree of monocytic differentiation

Periodic Acid-Schiff (PAS) Stain:

Principle: Detects glycogen and other carbohydrate-containing molecules in cells
Procedure:
1. Prepare a smear from bone marrow aspirate or peripheral blood
2. Oxidize the smear with periodic acid
3. React the oxidized carbohydrates with Schiff reagent, forming a magenta-colored product
4. Counterstain the smear (e.g., with hematoxylin)
5. Examine the smear under a microscope
Interpretation:
- Diffuse Positivity: Seen in erythroblasts
- Block-Like Positivity: Seen in lymphoblasts
- Negative: Other cell types, staining is very light or not present
Clinical Significance:
- Diagnosis of Acute Lymphoblastic Leukemia (ALL): Lymphoblasts in ALL often show coarse, block-like PAS positivity
- Diagnosis of Erythroleukemia: Erythroid precursors show strong PAS positivity
- Identification of Certain Storage Diseases: Used to detect glycogen storage in certain lysosomal storage disorders

Prussian Blue Stain (Iron Stain):

Principle: Detects ferric iron (Fe3+) in tissues and cells Uses potassium ferrocyanide to react with ferric iron to produce insoluble blue complex
Procedure:
1. Prepare a smear from bone marrow aspirate
2. Fix the smear
3. Flood the smear with a solution of potassium ferrocyanide and hydrochloric acid (Perl’s Prussian blue reagent)
4. Counterstain the smear (e.g., with nuclear fast red)
5. Examine the smear under a microscope
Interpretation:
- Iron Granules:
  - Sideroblasts: Normal erythroblasts contain a few iron granules
  - Ringed Sideroblasts: Abnormal erythroblasts with iron-laden mitochondria encircling the nucleus (seen in sideroblastic anemias)
- Macrophages: Iron stores in macrophages can be assessed (increased in iron overload, decreased in iron deficiency)
Clinical Significance:
- Diagnosis of Sideroblastic Anemia: Identification of ringed sideroblasts in the bone marrow
- Assessment of Iron Stores: Evaluates iron levels in the bone marrow and helps differentiate between iron deficiency and anemia of chronic disease
- Diagnosis of Hemochromatosis: Detects iron overload in tissues

Sudan Black B (SBB) Stain:

Principle: Detects lipids (phospholipids, neutral fats, and sterols) in cells
Procedure:
1. Prepare a smear from bone marrow aspirate or peripheral blood
2. Fix the smear
3. Incubate the smear with Sudan Black B stain
4. Wash the smear
5. Counterstain the smear
6. Examine the smear under a microscope
Interpretation:
- Positive: Granulocytes, monocytes, and some blasts show black granular staining
- Negative: Lymphocytes and erythrocytes are negative
Clinical Significance:
- Differentiate AML from ALL: SBB is positive in myeloblasts and negative in lymphoblasts

Terminal Deoxynucleotidyl Transferase (TdT) Stain:

Principle: Detects the presence of terminal deoxynucleotidyl transferase (TdT), a DNA polymerase expressed in immature lymphocytes (T and B lymphoblasts)
Procedure:
1. Prepare a smear from bone marrow aspirate or peripheral blood
2. Fix the smear
3. Incubate the smear with an anti-TdT antibody
4. Add a secondary antibody conjugated to an enzyme (e.g., horseradish peroxidase)
5. Add a substrate that reacts with the enzyme to produce a colored product
6. Examine the smear under a microscope
Interpretation:
- Positive: Lymphoblasts show nuclear staining
- Negative: Mature lymphocytes, myeloid cells, and erythroid cells are negative
Clinical Significance:
- Differentiate AML from ALL: TdT is positive in most cases of T-cell ALL and some cases of B-cell ALL, but is usually negative in AML
- Diagnosis of Lymphoblastic Lymphoma: TdT is positive in lymphoblasts in lymphoblastic lymphoma

Tartrate-Resistant Acid Phosphatase (TRAP)

Principle: Detects the activity of the isoenzyme 5 of acid phosphatase, which is resistant to tartaric acid.
Procedure:
1. Prepare a blood smear from bone marrow aspirate
2. Incubate with naphthol AS-BI phosphate and fast red violet LB, with and without tartrate.
Interpretation:
- Positive: Hairy cell stains positive. All other cells are negative, including T lymphocytes, B lymphocytes, and monocytes.
Clinical Significance: Distinguish hairy cell leukemia from other B-cell neoplasms

Kleihauer-Betke (Acid Elution)

Principle: Detects fetal hemoglobin (HbF) in maternal blood
Procedure:
1. Prepare a blood smear from the maternal blood
2. Treat the smear with an acid solution, which elutes (removes) adult hemoglobin (HbA) from the red blood cells
3. Stain the smear with a counterstain (e.g., erythrosine)
4. Examine the smear under a microscope
Interpretation:
- Adult Red Blood Cells: Appear as “ghost cells” (pale or colorless) because the adult hemoglobin has been eluted
- Fetal Red Blood Cells: Contain HbF, which is resistant to acid elution, and stain pink
Quantitative Determination of Fetal Cells:
- Count the number of fetal cells per 100 adult cells under the microscope
- Calculate the percentage of fetal cells in the maternal circulation
Clinical Significance:
- Detection and Quantification of Fetal-Maternal Hemorrhage (FMH): FMH occurs when fetal red blood cells enter the maternal circulation
- Calculation of RhoGAM Dose: Used to determine the appropriate dose of RhoGAM (Rh immune globulin) to administer to Rh-negative mothers to prevent Rh sensitization
Calculation of RhoGAM Dose:
- Estimate the volume of fetal blood in the maternal circulation using the following formula: % Fetal Cells x Maternal Blood Volume = Volume of Fetal Blood (mL)
  - The maternal blood volume is estimated based on the mother’s weight and hematocrit
- One vial of RhoGAM (300 μg) covers 30 mL of fetal whole blood or 15 mL of fetal red blood cells
- Calculate the number of RhoGAM vials needed: Number of Vials = Volume of Fetal Blood (mL) / 30 mL
- Round up to the nearest whole number and add one additional vial for safety

Summary Table of Key Special Stains

Stain	Principle	Positive Reaction	Negative Reaction	Clinical Significance
Myeloperoxidase (MPO)	Detects myeloperoxidase enzyme in myeloid cells	Myeloblasts, promyelocytes, neutrophils, eosinophils	Lymphoblasts, megakaryocytes, erythrocytes	Differentiate AML from ALL; subclassify AML
Specific Esterase (CAE)	Detects chloroacetate esterase in granulocytes	Neutrophils and their precursors	Monocytes, lymphocytes, erythrocytes	Differentiation of AML subtypes
Non-Specific Esterase (ANBE)	Detects α-naphthyl butyrate esterase in monocytes and macrophages	Monocytes and macrophages (inhibited by NaF)	Neutrophils, lymphocytes, erythrocytes	Differentiation of AML subtypes
Periodic Acid-Schiff (PAS)	Detects glycogen and other carbohydrate-containing molecules	Diffuse positivity in erythroblasts; block-like positivity in lymphoblasts		Diagnosis of ALL and erythroleukemia; identify certain storage diseases
Prussian Blue	Detects ferric iron (Fe3+)	Iron granules in sideroblasts; iron stores in macrophages		Diagnosis of sideroblastic anemia; assessment of iron stores
Terminal Deoxynucleotidyl Transferase (TdT)	Detects TdT enzyme in immature lymphocytes	Lymphoblasts	Mature lymphocytes, myeloid cells, erythrocytes	Differentiate AML from ALL; diagnose lymphoblastic lymphoma
Kleihauer-Betke	Detects fetal hemoglobin (HbF) in maternal blood by eluting adult hemoglobin (HbA) in an acid solution	Fetal red blood cells stain pink	Adult red blood cells appear as “ghost cells”	Detection and quantification of fetal-maternal hemorrhage; calculation of RhoGAM dose
Tartrate-Resistant Acid Phosphatase (TRAP)	Detects the activity of the isoenzyme 5 of acid phosphatase, which is resistant to tartaric acid.	Hairy cell positive. All other cells are negative, including T lymphocytes, B lymphocytes, and monocytes	Useful in distinguishing hairy cell leukemia from other B-cell neoplasms

Key Terms:

Special Stains: Histochemical staining techniques used to identify specific cellular components or enzymatic activities
Myeloperoxidase (MPO): Enzyme found in the primary granules of myeloid cells
Esterase: Enzyme that hydrolyzes ester bonds
Specific Esterase (Chloroacetate Esterase, CAE): Esterase primarily found in granulocytes (neutrophils)
Non-Specific Esterase (α-Naphthyl Butyrate Esterase, ANBE): Esterase found in monocytes and macrophages
Periodic Acid-Schiff (PAS): Stain that detects glycogen and other carbohydrate-containing molecules
Prussian Blue Stain: Stain that detects ferric iron (Fe3+)
Ringed Sideroblasts: Erythroblasts with iron-laden mitochondria encircling the nucleus
Terminal Deoxynucleotidyl Transferase (TdT): DNA polymerase expressed in immature lymphocytes
Acute Myeloid Leukemia (AML): Aggressive leukemia of myeloid cells
Acute Lymphoblastic Leukemia (ALL): Aggressive leukemia of lymphoid cells
Lymphoblasts: Immature lymphocytes
Myeloblasts: Immature myeloid cells
Cytochemistry: Use of chemical reactions to identify cellular components
Fixation: Treatment of cells or tissues to preserve their structure and prevent degradation
Counterstain: A stain used to provide contrast to the primary stain and enhance visualization of cellular structures
Kleihauer-Betke: Detects fetal hemoglobin (HbF) in maternal blood
Auer Rods: Rod-shaped inclusions in myeloblasts, seen in AML
Basophilic Stippling: Presence of small, dark-blue granules scattered throughout the RBC cytoplasm
Heinz Bodies: Inclusions composed of denatured hemoglobin

Hemolysis

Other Studies