1. MT 417 – Clinical Hematology II Manual/Special Tests Unit
Acute Leukemia Stain Panel Exercise
AL Stain Panel Questions KEY

2. Stain Panels used to aid the diagnosis of acute leukemia may include the Peroxidase, Sudan Black B, Specific Esterase, Nonspecific Esterase and PAS stains.
The Peroxidase stain procedure is representative of the typical steps in other cytochemical staining procedures.

3. 1. The peroxidase stain, in conjunction with other cytochemical stains, is most useful for identifying subtypes of:
a. Acute myeloid leukemia
a. Acute myeloid leukemia
b. Acute lymphoid leukemia
c. Chronic leukemias
d. Non-Hodgkin’s lymphoma
Stains are valuable for identifying types of myeloid leukemia but have less diagnostic value for lymphoid leukemias with the advent of immunophenotyping. Cell marker results are essential to the diagnosis of lymphoid disorders. These stains, which are done to identify the blast cell lineage in acute leukemias, are not required for chronic leukemias.

4. 2. What is the usual patient specimen?
Patient bone marrow smears are used most often. Fingerstick, EDTA or heparinized blood smears are acceptable IF circulating blast cells are present.
3. What should you consider when selecting a control?
The control used is blood from a normal individual that has a high normal WBC count with plenty of neutrophils and some lymphocytes & monocytes (a higher number of monocytes is needed for an esterase control). The control must exhibit the appropriate staining reactions before interpretation of the patient staining patterns by the pathologist can be done.

5. 4. List in correct order the following Peroxidase stain procedure steps:5
Validate staining on control slide
Incubate slides in staining substrate solution
Fix slides
Verify acceptable patient specimen
Select appropriate control as dictated by staining procedure and prepare smears 4 3 1 2

6. 5. What cells stain peroxidase positive?
Peroxidase positive cells include all stages of granulocytes (neutrophils) and monocytes. For this method, positive cells will show blue granulation. Lymphocytes and erythrocytes should stain peroxidase negative.
5. What other cytochemical stain do you expect to show the same staining pattern?
The SBB (coarse, black granules) parallels the staining pattern of the peroxidase stain.

7. 6. Is it important to memorize the “color” of cytochemical stains?
No, the stain color will vary depending on the substrate used. It is important to know the staining color of positive cells for your particular procedure so that you can validate that the stain “worked”.

8. 7. An acute leukemia panel typically includes all of the following EXCEPT:
a. Peroxidase and SBB
b. Specific and nonspecific esterases
c. PAS and LAP
c. PAS and LAP
The PAS stain is not routinely included in an acute leukemia stain panel but is done if acute Erythroleukemia is suspected. PAS reactions in cases of ALL are variable and often negative making the immunophenotype of greater value. The LAP stains seg & band neutrophils so is not useful for acute leukemia.

9. 8. Positive peroxidase and SBB staining indicates blasts of myeloid rather than lymphoid origin. Esterase stains:
a. Help differentiate myeloid leukemia subtypes….. AML, AMML, and AMonoL.
b. The specific esterase is strongly positive in granulocytes and uses the substrate naphthol AS-D Chloroacetate.
c. The nonspecific esterase is strongly positive in monocytes and substrates used are alpha-naphthyl Acetate or Butyrate.
d. Specific and nonspecific esterase staining is performed on the same slide in the dual/combined esterase.
e. All of the above
e. All of the above

10. 9. In the diagnosis of acute leukemia, interpretation of a patient’s staining pattern results must be correlated with the:
a. immunophenotype
b. morphologic features of blood and bone marrow cells
c. genotype
d. All of the above
d. All of the above
Staining results frequently do not fit the ‘expected’ pattern and the malignant cell lineage cannot be clearly defined. Diagnosis is never based on a single diagnostic tool. All results…cytochemical stains, cell markers, cell morphology, and cyto genetics…must be correlated.

11. You are now ready to interpret the validity of
an acute leukemia stain panel on the case slides provided and report your results on the yellow worksheet found in the Special Tests Unit of the Hemo II manual.
The blood & bone marrow of case patient SM is below.
SM Blood
SM Marrow