Flow cytometry and lymphocyte subsets

6 DOH Abu Dhabi (HAAD) lab licensing practice questions on Flow cytometry and lymphocyte subsets, part of Immunology and Serology. The first 6 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 · Medium

   A man with HIV infection has a lymphocyte count of 1.50 × 10^9/L. The plot shows CD4 and CD8 staining within his lymphocyte gate. His absolute CD4 count is:

   

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   1. A2.1 × 10^9/L (2,100 cells/µL)
   2. B0.21 × 10^9/L (210 cells/µL)
   3. C0.78 × 10^9/L (780 cells/µL)
   4. D0.14 × 10^9/L (140 cells/µL)

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   Answer: B · 0.21 × 10^9/L (210 cells/µL)

   CD4-positive, CD8-negative cells in the upper left quadrant are 14% of the lymphocytes. In this dual-platform method, absolute CD4 = lymphocyte count × CD4 fraction = 1.50 × 0.14 = 0.21 × 10^9/L, or 210 cells/µL. The CD4:CD8 ratio, 14 ÷ 52 = 0.27, is inverted, as is usual in HIV infection. Single-platform counting with beads is preferred where possible, because it avoids errors carried over from a separate analyzer count.

   • A: 2.1 × 10^9/L has a tenfold decimal error.
   • C: 0.78 × 10^9/L uses the CD8 quadrant (52%).
   • D: 0.14 × 10^9/L treats the percentage as the count, leaving out the lymphocyte count.

2. Question 2 · Medium

   Why are helper T cells counted as CD3-positive, CD4-positive cells rather than as all CD4-positive cells?

   1. AMonocytes also carry CD4
   2. BActivated T cells lose their CD4
   3. CNeutrophils carry high levels of CD4
   4. DB cells also carry CD4

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   Answer: A · Monocytes also carry CD4

   Monocytes express CD4 at a lower density than helper T cells, so counting every CD4-positive event would include them and overestimate the helper T-cell count. Gating on CD3, usually within the bright CD45, low side scatter lymphocyte region, restricts the count to T cells. Most CD4 panels therefore combine CD45, CD3, CD4 and CD8. On the plot, monocytes appear as CD4-dim, CD3-negative events.

   • B: Activation does not remove CD4 from helper T cells.
   • C: Neutrophils are not a source of CD4 events in these counts.
   • D: B cells express CD19 and CD20, not CD4.

3. Question 3 · Medium

   Why is the CD4 percentage often used rather than the absolute CD4 count to assess HIV in children under 5 years?

   1. AChildren have no CD8-positive T cells
   2. BAbsolute counts are high and vary with age
   3. CCD4% cannot be measured in adults
   4. DAbsolute counts need a bone marrow sample

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   Answer: B · Absolute counts are high and vary with age

   Young children normally have much higher lymphocyte and CD4 counts than adults, and these fall with age, so absolute counts need age-specific cut-offs. The CD4 percentage varies less with age and is less affected by changes in the total white count, so it is preferred in children under 5. From about 5 years of age, adult-type absolute counts are used.

   • A: Children do have CD8 cells; this is not the reason.
   • C: CD4% is measured at every age; adults are usually followed by absolute count.
   • D: CD4 counts are done on peripheral blood.

4. Question 4 · Easy

   Blood in EDTA for a CD4 count should be kept until testing at:

   1. AFrozen (-20 °C)
   2. BRefrigerated (2-8 °C)
   3. CRoom temperature (18-25 °C)
   4. DBody temperature (37 °C)

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   Answer: C · Room temperature (18-25 °C)

   Whole blood for lymphocyte immunophenotyping is held at room temperature and tested within the time the method allows, usually 24 to 48 hours. Refrigeration can damage leukocytes and change their scatter and antigen staining, and freezing destroys them. Warming to body temperature speeds cell death. Clotted, hemolyzed or over-age samples should be rejected.

   • A: Freezing lyses the cells.
   • B: Refrigeration can harm leukocytes and alter scatter and staining.
   • D: Warming to 37 °C speeds cell death.

5. Question 5 · Hard

   A man with HIV on stable ART has two dual-platform CD4 results 3 months apart. Visit 1: WBC 6.4 x10^9/L, lymphocytes 30%, CD4 25% of lymphocytes. Visit 2, during an acute viral illness: WBC 3.2 x10^9/L, lymphocytes 30%, CD4 25%. The best interpretation is:

   1. A480 cells/µL at both visits, as CD4% is unchanged
   2. B480 to 240 cells/µL, from the low WBC; repeat after recovery
   3. C1,600 to 800 cells/µL, from the low WBC
   4. D480 to 240 cells/µL, a sign that ART is failing

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   Answer: B · 480 to 240 cells/µL, from the low WBC; repeat after recovery

   Absolute CD4 = WBC x lymphocyte fraction x CD4 fraction: 6.4 x 0.30 x 0.25 = 0.48 x10^9/L (480 cells/µL) at visit 1, and 3.2 x 0.30 x 0.25 = 0.24 x10^9/L (240 cells/µL) at visit 2. The CD4 percentage did not change, so the fall comes from the short-lived leukopenia of the viral illness rather than from HIV destroying CD4 cells. The count should be repeated after recovery, and the viral load is the main test for treatment failure. Leaving out the lymphocyte percentage gives the falsely high 1,600 and 800.

   • A: The absolute count does fall, because it depends on the WBC.
   • C: 1,600 and 800 leave out the lymphocyte percentage.
   • D: The stable CD4% and the illness point to leukopenia; viral load judges ART failure.

6. Question 6 · Easy

   How does a single-platform method obtain the absolute CD4 count?

   1. ACounting against a known number of beads
   2. BCounting stained cells in a hemocytometer
   3. CMultiplying the CD4% by a lymphocyte count
   4. DEstimating cell numbers from forward scatter

   Answer and explanation in LearnFree account