A term boy who has never been transfused has his state newborn screen on a dried blood spot collected at 30 hours of age. HPLC results, listed in order of amount: Hb F 79%, Hb S 14%, Hb A 7%. No Hb C, Hb E or Hb Bart's is detected. Healthy term newborns have mostly Hb F, with Hb A making up most of the rest. Both parents come from a region where sickle cell and beta-thalassemia genes are common, but neither has been tested. These results most likely indicate:

Sickle cell trait with alpha-thalassemia

Hemoglobin: qualitative (electrophoresis, HPLC, sickle solubility)

14 ASCP MLS practice questions on Hemoglobin: qualitative (electrophoresis, HPLC, sickle solubility), part of Hematology. The first 14 are below; the first 5 with answers and explanations (tap Show answer after you have chosen), the rest in Learn with a free account.

Question 1 · Hard
A term boy who has never been transfused has his state newborn screen on a dried blood spot collected at 30 hours of age. HPLC results, listed in order of amount: Hb F 79%, Hb S 14%, Hb A 7%. No Hb C, Hb E or Hb Bart's is detected. Healthy term newborns have mostly Hb F, with Hb A making up most of the rest. Both parents come from a region where sickle cell and beta-thalassemia genes are common, but neither has been tested. These results most likely indicate:
1. ASickle cell trait
2. BHb SS disease
3. CSickle cell trait with alpha-thalassemia
4. DHb S/beta-plus thalassemia
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Answer: D · Hb S/beta-plus thalassemia
In a newborn Hb F dominates, so the key finding is the order of Hb S and Hb A. In sickle cell trait the one normal beta gene makes more Hb A than Hb S, an FAS pattern. Here Hb S exceeds Hb A (FSA), so the gene opposite Hb S makes only a little normal beta chain, as in Hb S/beta-plus thalassemia. Hb SS disease would show no Hb A at all (FS), and alpha-thalassemia with trait would lower the Hb S share further and show Hb Bart's at birth. A transfusion can also give an FSA pattern, so the result is confirmed by repeat testing in infancy and by family studies.
- A: Trait gives more Hb A than Hb S (FAS); here Hb S exceeds Hb A.
- B: Hb SS disease produces no Hb A, giving an FS pattern.
- C: Alpha-thalassemia would keep Hb S below Hb A and show Hb Bart's at birth, and none is seen.
Question 2 · Hard
A 19-year-old with mild microcytic anemia has a positive sickle solubility test. Alkaline electrophoresis showed no Hb A, a heavy band at the S position and a lighter band at the C position. The citrate agar gel is shown. Which hemoglobins are present?
Figure: MedExams
1. AHb S and Hb D
2. BHb S and Hb C
3. CHb S and Hb E
4. DHb D and Hb C
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Answer: C · Hb S and Hb E
On citrate agar, Hb C runs furthest toward the cathode, Hb S less far, Hb A runs with Hb E, D and G, and Hb F moves furthest toward the anode. The patient's bands lie at the S and A positions, with nothing at the C position, so the band that ran at the C position on the alkaline gel is not Hb C. Hb E runs with Hb C on alkaline gels and with Hb A on acid gels, which fits both results. Hb SE disease is a mild sickling disorder with microcytosis, and the positive solubility test confirms the Hb S.
- A: Hb D runs with Hb S on alkaline gels, so it cannot explain the band at the C position.
- B: Hb C would give a band at the C position on citrate agar.
- D: Without Hb S the solubility test would be negative, and Hb C would show at the C position.
Question 3 · Medium
The HPLC chromatogram is from a healthy 30-year-old, with the analyzer's retention-time windows shaded. Which result does it show?
Figure: MedExams
1. AHb E trait
2. BHb C trait
3. CHb SC disease
4. DHb CC disease
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Answer: B · Hb C trait
The variant peak (35.8%) elutes at about 5.1 min, inside the C window, and Hb A0 (54.1%) is still the main hemoglobin. A heterozygote for Hb C has about 30-40% Hb C with the rest mostly Hb A, so this is Hb C trait, which causes no anemia. The A2 window holds a normal 3.1%, so the variant is not Hb E, which elutes there. A variant found by HPLC is confirmed by a second method, such as acid agar electrophoresis.
- A: Hb E elutes in the A2 window, which here holds a normal 3.1%.
- C: Hb SC disease shows a peak in the S window and no Hb A0.
- D: Hb CC disease has almost no Hb A0.
Question 4 · Easy
The alkaline electrophoresis (cellulose acetate, pH 8.6) shows an adult's hemolysate beside an AFSC control. The patient's pattern is most consistent with:
Figure: MedExams
1. ASickle cell anemia
2. BHb SC disease
3. CSickle cell trait
4. DHb C trait
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Answer: C · Sickle cell trait
At pH 8.6 hemoglobins are negatively charged and move toward the anode: Hb A runs furthest, then F, then S, with C (and A2, E and O) closest to the origin. The patient has a heavy band at the A position and a lighter band at the S position, the pattern of sickle cell trait, in which Hb S is about 35-40% of the total. The faint band at the C position is the normal Hb A2. Hb D and Hb G also run at the S position, so a positive solubility test or acid agar electrophoresis confirms that the band is Hb S.
- A: Sickle cell anemia has no Hb A band; Hb S predominates with some Hb F.
- B: Hb SC disease shows S and C bands of similar size and no Hb A.
- D: In Hb C trait the second band lies at the C position, not the S position.
Question 5 · Medium
The HPLC chromatogram is from an adult with microcytosis and normal iron studies. Taking an Hb A2 reference interval of 1.5-3.5%, the pattern is most consistent with:
Figure: MedExams
1. AHb Lepore trait
2. BAlpha-thalassemia trait
3. CHb E trait
4. DBeta-thalassemia trait
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Answer: D · Beta-thalassemia trait
The chromatogram shows no variant peak: Hb A0 dominates, Hb F is 1.8%, and the Hb A2 peak is 5.6%, above the reference interval. A raised Hb A2, often with a slightly raised Hb F, in a microcytic patient with normal iron is the hallmark of beta-thalassemia trait. Alpha-thalassemia trait gives a normal or low A2, while Hb E (about 25-30%) and Hb Lepore (about 10-15%) give much larger peaks in or near the A2 window. Iron deficiency lowers Hb A2, so it should be excluded before a borderline A2 is interpreted.
- A: Hb Lepore gives a peak of about 10-15% near the A2 window.
- B: Alpha-thalassemia trait has a normal or low Hb A2.
- C: Hb E elutes in the A2 window at about 25-30%, far above 5.6%.
Question 6 · Easy
On cellulose acetate electrophoresis at pH 8.4 to 8.6, which hemoglobin stays closest to the point of application?
1. AHb S
2. BHb A
3. CHb F
4. DHb C
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Question 7 · Medium
A 2-month-old with Hb SS confirmed by HPLC has a negative sickle solubility test. What is the most likely reason?
1. AEDTA is the wrong anticoagulant
2. BHyperlipidemia clouds the reagent
3. CHb SS reacts more weakly than trait
4. DHigh Hb F leaves too little Hb S
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Question 8 · Hard
Hemoglobin studies show: alkaline electrophoresis (pH 8.6), two bands of similar intensity at the Hb S and Hb C positions and no Hb A; acid citrate agar (pH 6.2), bands at the Hb S and Hb C positions; sickle solubility positive. These findings fit:
1. AHb S/O-Arab disease
2. BHb SE disease
3. CHb SC disease
4. DHb S/beta-zero thalassemia
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Question 9 · Hard
A 28-year-old has a normal CBC (MCV 88 fL). Sickle solubility: negative. Alkaline electrophoresis: Hb A and a second band of slightly lower intensity at the Hb S position. Acid citrate agar: a single band at the Hb A position. These results most likely indicate:
1. ASickle cell trait
2. BHb D or Hb G trait
3. CHb Lepore trait
4. DHb SD disease
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Question 10 · Hard
A 24-year-old who has never been transfused has Hb 10.8 g/dL (108 g/L), MCV 68 fL and a positive sickle solubility test. HPLC: Hb A 25%, Hb S 65%, Hb A2 5.5%, Hb F 4.5%. What is the most likely diagnosis?
1. ASickle cell anemia (Hb SS)
2. BHb S/beta-zero thalassemia
3. CHb S/beta-plus thalassemia
4. DSickle cell trait
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Question 11 · Medium
Hb C and Hb E migrate together on alkaline electrophoresis. Which method best separates them?
1. ACitrate agar at pH 6.0 to 6.2
2. BKleihauer-Betke acid elution
3. CAlkali denaturation test
4. DSickle solubility test
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Question 12 · Easy
A sickle solubility test turns turbid when Hb S is present because:
1. ASickled cells clump together in the tube
2. BDeoxygenated Hb S is insoluble in the buffer
3. CHb S resists denaturation by strong alkali
4. DHb S precipitates in isopropanol solution
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Question 13 · Hard
A 26-year-old blood donor, never transfused, has Hb 13.2 g/dL (132 g/L), MCV 71 fL, RBC 5.9 × 10^6/µL (5.9 × 10^12/L) and a normal ferritin. The sickle solubility test is positive. HPLC: Hb A 70%, Hb S 26%, Hb A2 3.0%, Hb F 1%. The most likely finding is:
1. AHb S/beta-plus thalassemia
2. BHb AS with alpha-thalassemia
3. CSickle cell trait alone
4. DHb AS with iron deficiency
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Question 14 · Medium
Which test confirms hemoglobin S and distinguishes sickle cell trait from sickle cell disease?
1. AThe sickle solubility (dithionite) test
2. BThe Kleihauer-Betke acid elution test
3. CHemoglobin electrophoresis or HPLC
4. DThe osmotic fragility test
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Hemoglobin: qualitative (electrophoresis, HPLC, sickle solubility)

A 19-year-old with mild microcytic anemia has a positive sickle solubility test. Alkaline electrophoresis showed no Hb A, a heavy band at the S position and a lighter band at the C position. The citrate agar gel is shown. Which hemoglobins are present?

The HPLC chromatogram is from a healthy 30-year-old, with the analyzer's retention-time windows shaded. Which result does it show?

The alkaline electrophoresis (cellulose acetate, pH 8.6) shows an adult's hemolysate beside an AFSC control. The patient's pattern is most consistent with:

The HPLC chromatogram is from an adult with microcytosis and normal iron studies. Taking an Hb A2 reference interval of 1.5-3.5%, the pattern is most consistent with:

On cellulose acetate electrophoresis at pH 8.4 to 8.6, which hemoglobin stays closest to the point of application?

A 2-month-old with Hb SS confirmed by HPLC has a negative sickle solubility test. What is the most likely reason?

Hemoglobin studies show: alkaline electrophoresis (pH 8.6), two bands of similar intensity at the Hb S and Hb C positions and no Hb A; acid citrate agar (pH 6.2), bands at the Hb S and Hb C positions; sickle solubility positive. These findings fit:

A 28-year-old has a normal CBC (MCV 88 fL). Sickle solubility: negative. Alkaline electrophoresis: Hb A and a second band of slightly lower intensity at the Hb S position. Acid citrate agar: a single band at the Hb A position. These results most likely indicate:

A 24-year-old who has never been transfused has Hb 10.8 g/dL (108 g/L), MCV 68 fL and a positive sickle solubility test. HPLC: Hb A 25%, Hb S 65%, Hb A2 5.5%, Hb F 4.5%. What is the most likely diagnosis?

Hb C and Hb E migrate together on alkaline electrophoresis. Which method best separates them?

A sickle solubility test turns turbid when Hb S is present because:

A 26-year-old blood donor, never transfused, has Hb 13.2 g/dL (132 g/L), MCV 71 fL, RBC 5.9 × 10^6/µL (5.9 × 10^12/L) and a normal ferritin. The sickle solubility test is positive. HPLC: Hb A 70%, Hb S 26%, Hb A2 3.0%, Hb F 1%. The most likely finding is:

Which test confirms hemoglobin S and distinguishes sickle cell trait from sickle cell disease?