Special stains (esterase, MPO, Prussian blue, Kleihauer-Betke)

5 ASCP MLS practice questions on Special stains (esterase, MPO, Prussian blue, Kleihauer-Betke), part of Hematology. The first 5 are below; the first 5 with answers and explanations (tap Show answer after you have chosen), the rest in Learn with a free account.

1. Question 1 · Easy

   Ring sideroblasts on a Prussian blue-stained marrow are erythroid precursors with iron deposited in which structures?

   1. ALysosomes scattered in the cytoplasm
   2. BNuclear fragments at the cell edge
   3. CRibosomes clustered near the Golgi
   4. DMitochondria that encircle the nucleus

   Show answerHide answer

   Answer: D · Mitochondria that encircle the nucleus

   When heme synthesis is blocked, iron that enters the erythroblast's mitochondria cannot be inserted into protoporphyrin and builds up there. These mitochondria sit around the nucleus, so Prussian blue shows a ring of blue granules. A ring sideroblast has at least 5 granules covering at least one third of the nuclear circumference. They are typical of MDS with SF3B1 mutation and of inherited and acquired sideroblastic anemias.

   • A: A few scattered granules in lysosomes (siderosomes) describe normal sideroblasts.
   • B: Nuclear fragments are Howell-Jolly bodies, which contain DNA, not iron.
   • C: RNA on ribosomes gives basophilic stippling, which does not stain with Prussian blue.

2. Question 2 · Medium

   A Kleihauer-Betke slide from a pregnant woman, with acceptable controls, shows every red cell evenly stained a moderate pink. There are no ghost cells and no separate dark-staining population. The most likely explanation is:

   1. AHereditary persistence of fetal hemoglobin (HPFH)
   2. BA large fetomaternal hemorrhage at delivery
   3. CNormal adult cells with no fetal hemoglobin
   4. DDelta-beta thalassemia trait (heterocellular)

   Show answerHide answer

   Answer: A · Hereditary persistence of fetal hemoglobin (HPFH)

   In the classic deletional (pancellular) forms of HPFH, fetal hemoglobin is spread evenly through all red cells (a pancellular pattern), so every cell resists acid elution to the same degree and stains uniformly. A fetomaternal bleed gives a separate population of darkly stained fetal cells among pale maternal ghosts. Delta-beta thalassemia gives a heterocellular pattern, with cells of varying intensity. When HPFH makes the slide hard to read, flow cytometry, which measures the amount of Hb F per cell, is preferred to quantify a fetal bleed.

   • B: A fetal bleed shows a separate group of dark fetal cells among maternal ghosts.
   • C: Normal adult cells lose their hemoglobin in the acid buffer and appear as ghosts.
   • D: Delta-beta thalassemia spreads Hb F unevenly, so staining varies from cell to cell.

3. Question 3 · Medium

   Which esterase stains monocytic cells strongly and is inhibited by sodium fluoride?

   1. AAlpha-naphthyl butyrate esterase
   2. BLeukocyte alkaline phosphatase
   3. CTartrate-resistant acid phosphatase
   4. DNaphthol AS-D chloroacetate esterase

   Show answerHide answer

   Answer: A · Alpha-naphthyl butyrate esterase

   Alpha-naphthyl butyrate and alpha-naphthyl acetate are the nonspecific esterases, strongly positive in monoblasts and monocytes. Adding sodium fluoride abolishes the monocytic reaction, which confirms that the cells are monocytic. Naphthol AS-D chloroacetate is the specific esterase of the granulocytic line and is not fluoride sensitive. A double esterase stain shows both lines on one slide.

   • B: LAP is found in mature neutrophils and helps separate CML from a leukemoid reaction.
   • C: TRAP is the classic marker of hairy cell leukemia.
   • D: Chloroacetate (specific) esterase marks granulocytic cells and is not fluoride sensitive.

4. Question 4 · Hard

   A 62-year-old has 45% blasts and promonocytes in the marrow, with granulocytic and monocytic cells each above 20%. MPO is positive in 30% of blasts. A double esterase stain shows a chloroacetate-positive population and a butyrate-positive population, and fluoride abolishes the butyrate reaction. These findings best fit:

   1. AAcute myeloid leukemia with maturation
   2. BAcute myelomonocytic leukemia
   3. CAcute monocytic leukemia
   4. DAcute megakaryoblastic leukemia

   Show answerHide answer

   Answer: B · Acute myelomonocytic leukemia

   Acute myelomonocytic leukemia has both a granulocytic and a monocytic component, each at least 20% of marrow cells. The chloroacetate (specific) esterase marks the granulocytic cells, and the fluoride-sensitive butyrate (nonspecific) esterase marks the monocytic cells, so the double stain shows both lines. MPO is positive in the granulocytic blasts. In acute monocytic leukemia, at least 80% of the leukemic cells are monocytic, so the chloroacetate-positive population would be small.

   • A: AML with maturation lacks a large fluoride-sensitive monocytic population.
   • C: Acute monocytic leukemia has at least 80% monocytic cells and few granulocytic cells.
   • D: Megakaryoblasts are MPO negative and are identified by CD41 and CD61.

5. Question 5 · Easy

   Which cytochemical stain reacts with the primary granules of myeloblasts and is negative in lymphoblasts?

   1. AAlpha-naphthyl butyrate esterase
   2. BLeukocyte alkaline phosphatase
   3. CPeriodic acid-Schiff
   4. DMyeloperoxidase

   Show answerHide answer

   Answer: D · Myeloperoxidase

   Myeloperoxidase is stored in the primary (azurophilic) granules of granulocytic cells; it can be shown in myeloblasts, is strong from the promyelocyte stage on, and also stains Auer rods. Lymphoblasts lack it, so if at least 3% of the blasts are MPO positive, the blasts are classed as myeloid. Monoblasts are usually negative and monocytes stain only weakly. Sudan black B gives a similar pattern, and flow cytometry can detect MPO protein directly.

   • A: Butyrate esterase marks monocytic cells, not granulocytic blasts.
   • B: LAP is found in mature neutrophils, not in blasts.
   • C: PAS often shows coarse block positivity in lymphoblasts, not myeloblasts.