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Hemoglobin electrophoresis, HPLC and sickling tests

18 DOH Abu Dhabi (HAAD) lab licensing practice questions on Hemoglobin electrophoresis, HPLC and sickling tests, part of Hematology. The first 18 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

    Newborn screening in a UAE hospital tests a heel-prick sample from a term baby boy at 48 hours by HPLC; he has never been transfused. The result is Hb F 80%, Hb S 13% and Hb A 7%, with no other variant and no Hb Bart's. The mother's premarital screening showed Hb A 60% and Hb S 37%, with a normal MCV; the father has not been tested. The laboratory reports each newborn pattern by listing the hemoglobins present in decreasing amount. The most likely interpretation is:

    1. AFSA pattern: probable Hb S/beta-plus thalassemia
    2. BFAS pattern: trait with alpha-thalassemia
    3. CFAS pattern: probable sickle cell trait
    4. DFS pattern: probable sickle cell anemia
    Show answer

    Answer: A · FSA pattern: probable Hb S/beta-plus thalassemia

    At birth Hb F dominates, and the adult hemoglobins are listed after it in decreasing amount. Here Hb S (13%) exceeds Hb A (7%), an FSA pattern: the baby makes some beta-A chains but fewer than beta-S chains, which fits Hb S/beta-plus thalassemia, a form of sickle cell disease. A baby with sickle cell trait makes more Hb A than Hb S (FAS), and an FS pattern with no Hb A fits Hb SS or Hb S/beta-zero thalassemia. Coexisting alpha-thalassemia lowers the Hb S share and shows Hb Bart's at birth. The result is confirmed on a new sample with parental or DNA studies (the father may carry beta-thalassemia), and the baby is referred for sickle cell care.

    • B: Alpha-thalassemia lowers the Hb S share and shows Hb Bart's at birth.
    • C: In sickle cell trait Hb A exceeds Hb S; here Hb S is larger.
    • D: An FS pattern has no Hb A; this baby has 7% Hb A.

  2. Question 2 · Hard

    At premarital screening, a 27-year-old man has a normal CBC and a negative sickle solubility test, and his variant runs with Hb A on acid agarose. His HPLC chromatogram is shown. His fiancée has sickle cell trait. What is the risk for each pregnancy?

    Figure: MedExams
    HPLC chromatogram with peaks at 1.08, 1.31, 1.70, 2.46, 3.60 and 4.12 minutes. Peak table: F 0.6%, P2 3.4%, P3 3.5%, A0 51.0%, A2 2.6%, D-window 38.9%.
    1. A1 in 2: compound Hb S/D-Punjab disease
    2. BNo risk: Hb D-Punjab is harmless
    3. C1 in 4: sickle cell anemia (Hb SS)
    4. D1 in 4: compound Hb S/D-Punjab sickling disease
    Show answer

    Answer: D · 1 in 4: compound Hb S/D-Punjab sickling disease

    A 38.9% peak at 4.12 minutes falls in the D window; with a negative solubility test and migration with Hb A on acid agarose, this is Hb D-Punjab (D-Los Angeles) trait. Hb D-Punjab is clinically silent alone, but it takes part in Hb S polymerization, so Hb S/D-Punjab is a sickling disease similar to sickle cell anemia. Each partner passes on the variant allele with a chance of 1 in 2, so the chance that a child inherits both is 1/2 × 1/2 = 1 in 4. Hb SS is impossible here because only the fiancée carries Hb S.

    • A: Both partners must pass on their variant, which gives 1/2 × 1/2, not 1/2.
    • B: Hb D-Punjab co-polymerizes with Hb S, so the compound state sickles.
    • C: Hb SS needs Hb S from both parents, and he carries Hb D, not Hb S.

  3. Question 3 · Medium

    A healthy 23-year-old woman with MCV 86 fL and a normal ferritin has the HPLC chromatogram shown (Hb A2 reference 2.0-3.3%). How is her Hb A2 result best interpreted?

    Figure: MedExams
    HPLC chromatogram with peaks at 1.10, 1.32, 1.72, 2.48, 3.62 and 4.45 minutes. Peak table: F 0.8%, P2 3.5%, P3 3.6%, A0 50.6%, A2 4.1%, S-window 37.4%.
    1. ATrue rise: coexisting alpha-thalassemia trait
    2. BTrue rise: coexisting beta-thalassemia trait
    3. CTrue rise: iron deficiency raises Hb A2
    4. DOverestimated: Hb S adducts elute in the Hb A2 window
    Show answer

    Answer: D · Overestimated: Hb S adducts elute in the Hb A2 window

    The chromatogram shows Hb A0 50.6% and a 37.4% peak in the S window: Hb A greater than Hb S, the pattern of sickle cell trait. On cation-exchange HPLC, modified (adducted) forms of Hb S elute in the A2 window, so Hb A2 reads falsely high, often 3.5-4.5%, in people with Hb S. Beta-thalassemia on the other chromosome would lower Hb A and give Hb S above Hb A, with microcytosis, and her MCV is normal. Hb A2 in a sample containing Hb S should not be used to diagnose beta-thalassemia trait without a method that separates it.

    • A: Alpha-thalassemia lowers Hb A2 and the Hb S percentage rather than raising them.
    • B: Beta-thalassemia with Hb S would make Hb S exceed Hb A and lower the MCV.
    • C: Iron deficiency lowers Hb A2; it does not raise it.

  4. Question 4 · Medium

    On alkaline electrophoresis, a sample shows Hb A and a variant band of about 30% at the C position. The same sample on acid citrate agar is shown. The variant is most likely:

    Figure: MedExams
    Acid citrate agar gel with the application point in the middle. AFSC control: C and S bands toward the cathode, the A band just anodal to the origin and the F band further toward the anode. Patient: one heavy band at the A position and no other band.
    1. AHb S
    2. BHb E
    3. CHb C
    4. DHb D-Punjab
    Show answer

    Answer: B · Hb E

    At alkaline pH, Hb C, Hb E, Hb O-Arab and Hb A2 run together, so a band at the C position needs acid electrophoresis to name it. On citrate agar at pH 6.2 the patient shows a single band at the A position, so the variant now runs with Hb A. Hb E behaves this way, while Hb C would stay at the C position on acid agar too. A level of 25-30% is typical of Hb E trait, which is common in people from South and Southeast Asia.

    • A: Hb S would form a band at the S position on citrate agar, and it runs at the S position on alkaline gel.
    • C: Hb C would form its own band at the C position on citrate agar.
    • D: Hb D-Punjab runs with Hb A on acid agar, but at the S position, not the C position, on alkaline gel.

  5. Question 5 · Easy

    Hemolysates from a normal adult and a patient are run beside an AFSC control on cellulose acetate at pH 8.6, as shown. Which hemoglobin is absent from the patient's lane?

    Figure: MedExams
    Alkaline cellulose acetate gel, three lanes. AFSC control: bands at the C, S, F and A positions. Normal adult: a heavy band at the A position and a faint band at the C position. Patient: a heavy band at the S position, a lighter band at the F position and a faint band at the C position.
    1. AHb A2
    2. BHb A
    3. CHb S
    4. DHb F
    Show answer

    Answer: B · Hb A

    The patient's lane has a heavy band at the S position, a lighter band at the F position and a faint band at the C position, which in this setting is Hb A2. Nothing lines up with the A control band, the main band of the normal adult. Hb S with no Hb A fits sickle cell anemia or Hb S/beta-zero thalassemia. Alkaline electrophoresis cannot separate Hb S from Hb D or G, so the variant is confirmed by acid agar or HPLC and a solubility test.

    • A: A faint band sits at the C/A2 position, as in the normal adult.
    • C: A heavy band sits at the S position.
    • D: A band sits at the F position.

  6. Question 6 · Hard

    A healthy 35-year-old has Hb 148 g/L (14.8 g/dL), MCV 89 fL and MCH 30 pg. HPLC: Hb A 72%, Hb F 26%, Hb A2 2.0%. Acid elution shows Hb F spread evenly through all red cells. The most likely diagnosis is:

    1. AHereditary persistence of Hb F
    2. BBeta-thalassemia trait
    3. CDelta-beta-thalassemia trait
    4. DHb Lepore trait

    Answer and explanation in LearnFree account

  7. Question 7 · Easy

    Which hemoglobin is measured by its resistance to denaturation in strong alkali?

    1. AHb H
    2. BHb S
    3. CHb A2
    4. DHb F

    Answer and explanation in LearnFree account

  8. Question 8 · Hard

    A 24-year-old woman at premarital screening has Hb 96 g/L (9.6 g/dL), MCV 63 fL, ferritin 4 µg/L (reference 15-150 µg/L) and Hb A2 3.3% by HPLC (cut-off for beta-thalassemia trait 3.5%). Which action is most appropriate?

    1. ARepeat HPLC on a fresh sample tomorrow
    2. BReport beta-thalassemia trait as excluded
    3. CTreat the iron deficiency, then repeat Hb A2
    4. DReport beta-thalassemia trait from the MCV

    Answer and explanation in LearnFree account

  9. Question 9 · Medium

    A sickle solubility test can be falsely positive (turbid) in a sample with:

    1. AAge under 6 months
    2. BHemoglobin of 60 g/L
    3. CRecent red cell transfusion
    4. DMarked hyperlipidemia

    Answer and explanation in LearnFree account

  10. Question 10 · Medium

    An adult with Hb SS, never transfused, starts hydroxyurea. Which HPLC change over the following months best shows a response to treatment?

    1. AA rise in the Hb F percentage
    2. BA rise in the Hb A2 percentage
    3. CThe appearance of an Hb A peak
    4. DA shift in the Hb S retention time

    Answer and explanation in LearnFree account

  11. Question 11 · Hard

    A 30-year-old man at premarital screening has Hb 138 g/L (13.8 g/dL), MCV 71 fL, MCH 23 pg and a normal ferritin. The sickle solubility test is positive. HPLC: Hb A 68%, Hb S 28%, Hb A2 3.1%, Hb F 0.6%. The most likely interpretation is:

    1. ASickle cell trait with alpha-thalassemia
    2. BSickle/beta+-thalassemia
    3. CSickle cell trait alone
    4. DSickle cell trait with iron deficiency

    Answer and explanation in LearnFree account

  12. Question 12 · Easy

    In the tube solubility test for Hb S, which reagent component removes oxygen from the hemoglobin?

    1. ASaponin
    2. BPotassium ferricyanide
    3. CSodium dithionite
    4. DPhosphate buffer

    Answer and explanation in LearnFree account

  13. Question 13 · Medium

    Why can Hb A2 not be measured on alkaline cellulose acetate electrophoresis in a carrier of Hb C?

    1. AHb C precipitates at the point of application
    2. BHb C is destroyed in the alkaline buffer
    3. CHb C suppresses delta-globin synthesis
    4. DHb C and Hb A2 migrate to the same position

    Answer and explanation in LearnFree account

  14. Question 14 · Medium

    A laboratory must measure Hb A2 in people who carry Hb E. Why is capillary electrophoresis preferred over cation-exchange HPLC for this?

    1. AIt separates Hb F from Hb A
    2. BIt detects alpha-globin deletions
    3. CIt needs no calibrators or controls
    4. DIt separates Hb E from Hb A2

    Answer and explanation in LearnFree account

  15. Question 15 · Hard

    A 7-year-old new to a hospital in Al Ain received a red cell transfusion elsewhere 3 weeks ago. The sickle solubility test is positive. HPLC: Hb A 55%, Hb S 41%, Hb A2 2.8%, Hb F 1.2%. What is the best course?

    1. AReport sickle/beta+-thalassemia, as Hb A is present
    2. BReport sickle cell trait, as Hb A exceeds Hb S
    3. CRepeat the HPLC in one week and report that result
    4. DDefer the diagnosis and confirm it by DNA testing

    Answer and explanation in LearnFree account

  16. Question 16 · Medium

    An HPLC on a 3-month-old reports Hb A2 of 1.1%. Why can this result not exclude carrier status for beta-thalassemia?

    1. AHb A2 co-elutes with Hb F on HPLC
    2. BHb A2 has not yet risen to its adult level
    3. CHb A2 is masked by maternal hemoglobin
    4. DHb A2 is destroyed by the high Hb F

    Answer and explanation in LearnFree account

  17. Question 17 · Hard

    A 25-year-old with known sickle cell disease, not recently transfused, has Hb 50 g/L (5.0 g/dL) during an aplastic crisis, and the film shows sickle cells. A tube solubility test using the standard blood volume stays clear, and both controls perform correctly. What best explains the result?

    1. AHb F above 20% from hydroxyurea
    2. BReagent dithionite that has lost activity
    3. CMarked hyperlipidemia in the sample
    4. DToo little hemoglobin in the test tube

    Answer and explanation in LearnFree account

  18. Question 18 · Easy

    In cation-exchange HPLC for hemoglobin variants, each hemoglobin is identified mainly by its:

    1. ARetention time
    2. BAbsorbance wavelength
    3. CMolecular mass
    4. DMigration distance

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