Flow cytometry immunophenotyping (leukemia, lymphoma, subsets, PNH)

13 ASCP MLS practice questions on Flow cytometry immunophenotyping (leukemia, lymphoma, subsets, PNH), part of Hematology. The first 13 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 · Hard

   A 69-year-old man has a WBC of 168 × 10^3/µL (168 × 10^9/L), splenomegaly, a pleural effusion and a facial rash. Most blood cells are medium-sized lymphocytes with a single visible nucleolus and small cytoplasmic blebs. Flow cytometry: CD2+, CD3+, CD5+, CD7 bright, CD4+, CD8−, CD25−, CD26+, CD1a−, TdT−, CD34−; B-cell markers are negative. HTLV-1 serology is negative, and he has no erythroderma. FISH shows a rearrangement involving TCL1A on chromosome 14. What is the most likely diagnosis?

   1. AT-cell prolymphocytic leukemia
   2. BSézary syndrome
   3. CAdult T-cell leukemia/lymphoma
   4. DT-lymphoblastic leukemia

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   Answer: A · T-cell prolymphocytic leukemia

   TdT, CD1a and CD34 are negative, so these are mature T cells, not lymphoblasts. Bright CD7, retained CD26 and absent CD25 separate this disease from Sézary syndrome, in which CD7 and CD26 are usually lost and erythroderma is typical, and from adult T-cell leukemia/lymphoma, which is strongly CD25 positive and HTLV-1 positive. A very high count, splenomegaly, effusions and skin involvement by prolymphocytes are typical of T-cell prolymphocytic leukemia. Rearrangement of TCL1A, usually inv(14)(q11q32) or t(14;14), is its hallmark. The disease is aggressive, and the cells strongly express CD52, a target for antibody therapy.

   • B: Sézary cells usually lose CD7 and CD26, and the patient has no erythroderma.
   • C: Adult T-cell leukemia/lymphoma shows strong CD25 and is HTLV-1 positive.
   • D: T-lymphoblasts express TdT and often CD1a or CD34.

2. Question 2 · Medium

   The dot plot shows FLAER against CD24 on gated neutrophils. What does the plot show?

   

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   1. AA PNH clone in about 76% of neutrophils
   2. BNormal neutrophils with no PNH clone
   3. CMonocytes contaminating the neutrophil gate
   4. DA PNH clone in about 24% of neutrophils

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   Answer: D · A PNH clone in about 24% of neutrophils

   FLAER binds the GPI anchor itself, and CD24 is a GPI-linked protein on neutrophils, so normal neutrophils are positive for both (upper right, 75.9%). Cells that lack the GPI anchor are negative for both (lower left, 23.6%), a PNH clone of about 24% of the neutrophils. Granulocytes are the preferred cells for sizing the clone because transfusion and hemolysis shrink the red cell clone but not the white cell clone. Monocytes are FLAER-positive and lack CD24, so they would fall in the lower right.

   • A: 75.9% is the normal, GPI-positive population.
   • B: The 23.6% double-negative population is a PNH clone.
   • C: Monocytes are FLAER-positive and would fall in the lower right quadrant.

3. Question 3 · Medium

   Refer to the CD45 versus side scatter dot plot of a bone marrow aspirate. Which numbered population contains the monocytes?

   

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   1. APopulation 4
   2. BPopulation 1
   3. CPopulation 2
   4. DPopulation 3

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   Answer: C · Population 2

   CD45 brightness increases as leukocytes mature, and side scatter reflects granularity. Monocytes are bright for CD45 with intermediate side scatter (population 2), between lymphocytes and granulocytes. Lymphocytes are the brightest CD45 cells with the lowest side scatter (population 3), granulocytes have moderate CD45 and high side scatter (population 1), and blasts are CD45-dim with low side scatter (population 4). This CD45 gating is the usual first step in finding an abnormal blast population.

   • A: Population 4 is CD45-dim with low side scatter: blasts.
   • B: Population 1 has moderate CD45 and high side scatter: granulocytes.
   • D: Population 3 has the brightest CD45 and the lowest side scatter: lymphocytes.

4. Question 4 · Hard

   A 66-year-old man has a WBC of 31.0 × 10^3/µL (31.0 × 10^9/L), splenomegaly and enlarged lymph nodes. His B cells are CD20 bright, FMC7-positive and CD10-negative, with bright lambda-restricted surface immunoglobulin. The CD5 versus CD23 plot is shown. The most likely diagnosis is:

   

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   1. AFollicular lymphoma
   2. BChronic lymphocytic leukemia
   3. CMantle cell lymphoma
   4. DLymphoplasmacytic lymphoma

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   Answer: C · Mantle cell lymphoma

   The plot puts 88% of the B cells in the lower right quadrant: CD5-positive and CD23-negative. CLL is also CD5-positive, but it is CD23-positive with dim CD20, dim surface immunoglobulin and usually no FMC7, so the bright CD20 and sIg and the FMC7 here point away from CLL. A clonal CD5+, CD23− B-cell population with these features is typical of mantle cell lymphoma, confirmed by cyclin D1 overexpression or t(11;14). Follicular lymphoma is CD10-positive and CD5-negative, and lymphoplasmacytic lymphoma is usually CD5-negative, with plasmacytoid cells and an IgM paraprotein.

   • A: Follicular lymphoma is CD10-positive and CD5-negative.
   • B: CLL cells are CD23-positive, with dim CD20 and dim surface immunoglobulin.
   • D: Lymphoplasmacytic lymphoma is usually CD5-negative and makes an IgM paraprotein.

5. Question 5 · Hard

   A 34-year-old with HIV has WBC 4.0 × 10^3/µL (4.0 × 10^9/L) with 25% lymphocytes. CD4-positive T cells are 20% of lymphocytes. What is the absolute CD4 count?

   1. A20 cells/µL
   2. B800 cells/µL
   3. C1,000 cells/µL
   4. D200 cells/µL

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   Answer: D · 200 cells/µL

   Absolute lymphocytes = 4,000/µL × 0.25 = 1,000/µL. Absolute CD4 = 1,000/µL × 0.20 = 200 cells/µL (0.2 × 10^9/L). Multiplying the WBC directly by the CD4 percentage skips the lymphocyte step and gives 800. This dual-platform calculation carries the errors of both the CBC and the flow result, so many labs count directly with beads (single-platform method).

   • A: 20 is a tenfold decimal error in converting 10^3/µL to cells/µL.
   • B: 800 applies the CD4 percentage to the whole WBC count.
   • C: 1,000 is the absolute lymphocyte count, before the CD4 step.

6. Question 6 · Medium

   Flow cytometry of a lymph node shows CD19-positive B cells with a kappa to lambda ratio of 15:1. This most strongly suggests:

   1. AA clonal B-cell lymphoma
   2. BA clonal T-cell lymphoma
   3. CNormal B-cell precursors (hematogones)
   4. DReactive polyclonal B-cell hyperplasia

   Answer and explanation in LearnFree account

7. Question 7 · Easy

   Which CD antigen is present on all leukocytes and is plotted against side scatter to gate cell populations?

   1. ACD38
   2. BCD34
   3. CCD45
   4. DCD71

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8. Question 8 · Medium

   Which lymphocyte subset pattern is typical of untreated, advanced HIV infection?

   1. ALow CD4 count; CD4:CD8 ratio well below 1
   2. BNormal CD4 count; low CD8 count
   3. CLow CD8 count; CD4:CD8 ratio above 3
   4. DHigh CD4 count; CD4:CD8 ratio above 3

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9. Question 9 · Easy

   In PNH testing, the fluorescent reagent FLAER binds directly to:

   1. AFetal hemoglobin in the cell
   2. BCD59 on the cell surface
   3. CComplement bound to red cells
   4. DThe GPI anchor itself

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10. Question 10 · Medium

    In PNH red cell testing with anti-CD59, which cells are classed as type III?

    1. ACells with CD59 but no glycophorin A
    2. BCells with partial loss of CD59
    3. CCells that completely lack CD59
    4. DCells with normal CD59 expression

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11. Question 11 · Hard

    A 40-year-old with dark morning urine, a raised LD and a negative DAT received 2 units of red cells last week. Flow cytometry shows 38% FLAER-negative, CD24-negative granulocytes but only 9% type III red cells. The lower red cell figure is best explained by:

    1. APNH red cells lysed and diluted by donor cells
    2. BNormal CD24 loss on immature granulocytes
    3. CHigher sensitivity of the red cell CD59 assay
    4. DDonor red cells carrying no GPI-linked proteins

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12. Question 12 · Medium

    Where do myeloblasts typically fall on a CD45 versus side scatter plot?

    1. ABright CD45 and low side scatter
    2. BBright CD45 and moderate side scatter
    3. CDim CD45 and low side scatter
    4. DModerate CD45 and high side scatter

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13. Question 13 · Hard

    Blasts express CD34, CD19, CD10 and TdT. What is the most likely diagnosis?

    1. AChronic lymphocytic leukemia
    2. BB-lymphoblastic leukemia
    3. CAcute myeloid leukemia
    4. DT-lymphoblastic leukemia

    Answer and explanation in LearnFree account