Deck 5: Laboratory Analysis of Hemoglobin Variants

Question 1

Suppose HPLC results suggest Hb S trait.Why should this be confirmed with the sickle solubility test?

A)Retention characteristics of hemoglobins vary significantly between chromatographic runs, requiring this confirmation step.
B)With chromatography, there is always the possibility of unknown hemoglobins co-eluting with the suspected hemoglobin.
C)The use of chromatographic windows has been associated with a high degree of imprecision.
D)The sickle solubility test is the only technique available to quantify Hb S.

Answer 1

With chromatography, there is always the possibility of unknown hemoglobins co-eluting with the suspected hemoglobin.

Question 2

Hb F is the major hemoglobin fraction present in neonates and is found elevated in various hematologic conditions.Which of the following problems may be encountered in the HPLC analysis of Hb F when it is greatly elevated?

A)The measured retention time may fall outside the Hb F window, causing the peak to be labeled as "unknown."
B)Hb F will be measured as part of the background and produce no discernable chromatographic peak
C)Hb F will crystallize and block flow through the chromatographic column.
D)Hb F will break into fragments, producing multiple peaks for detection.

Answer 2

The measured retention time may fall outside the Hb F window, causing the peak to be labeled as "unknown."

Question 3

Many HPLC systems utilize "windows" to aid in the interpretation of hemoglobin chromatographic analyses.Which of the following best describes chromatographic windows?

A)time intervals associated with changes in mobile phase ionic strength during gradient analysis
B)time intervals associated with detector measurement at two different wavelengths
C)time intervals during which specific hemoglobin fractions are expected to elute
D)time interval between the analysis of two different samples during which the chromatograph re-equilibrates

Answer 3

time intervals during which specific hemoglobin fractions are expected to elute

Question 4

In the chromatographic analysis of variant hemoglobins, it is the retention time that determines the _________ and the absorbance that determines the _________.

A)percent concentration, hemoglobin identity
B)need for further testing, hemoglobin purity
C)hemoglobin purity, need for further testing
D)hemoglobin identity, percent concentration

Question 5

Your lab uses the BioRad HPLC system for hemoglobin analysis.As you set up for the analysis, you power up the instrument to allow it to reach operational temperature and prepare the mobile phase buffer.When the instrument and mobile phase are ready, you begin pumping the mobile phase through the system to allow the instrument to equilibrate.You next inject your calibrator.The resulting chromatogram shows nothing but a flat baseline.You inject a larger volume of calibrator and get the same result.Which of the following would explain these results?

A)The column heater is not set too high.
B)The detector is set at the wrong wavelength.
C)Chromatographic windows are not assigned properly.
D)The mobile phase was not prepared to the correct ionic strength.

Question 6

Given that Hb Barts elutes first and Hb C elutes last using cation exchange HPLC, which of the following statements most accurately describes the charge of these hemoglobins under chromatographic conditions?

A)Hb C is more positively charged than Hb Barts.
B)Hb Barts is more positively charged than Hb C.
C)Hb C has no positive charge, whereas Hb Barts has a positive charge,
D)Hb C is more negatively charged than Hb Barts

Question 7

Quantification of Hb A2 following separation by cation-exchange HPLC produced a result of 15% in an Hb AA patient.Which of the following describes a concern the technologist should have regarding this result?

A)There may be other hemoglobin variants co-eluting with Hb A2, causing the Hb A2 result to be falsely elevated.
B)The low Hb A2 result may be falsely decreased because of an integration error caused by a rising baseline.
C)Quantification of Hb A2 by HPLC is no longer recognized by CAP because of significant intralaboratory variation in Hb A2 measurements.
D)Glycation of Hb A2 may cause its concentration to vary significantly, based on the extent to which Hb A2 is glycated.

Question 8

Standard hemoglobin electrophoresis on cellulose acetate at pH 8.6 is utilized in some laboratories for the evaluation of hemoglobins.In many cases, this is followed by hemoglobin electrophoresis on the same patient sample, using citrate agar at pH 6.2.What is the purpose of this second electrophoretic analysis?

A)to confirm the results of the electrophoresis in the cellulose acetate (alkaline) system
B)to aid in the identification of hemoglobins that co-migrate in the cellulose acetate (alkaline) system
C)to replace the need for chromatographic analysis
D)to give the technologist confidence that the initial results are correct, since the second technique will produce identical results as the first technique

Question 9

Your laboratory provides preliminary identification and confirmation of hemoglobin fractions.Suppose your laboratory initially identified a hemoglobin fraction by cation-exchange HPLC.Which of the following techniques should be selected to confirm the presence of the hemoglobin fraction?

A)cation-exchange HPLC on a different HPLC instrument
B)anion-exchange HPLC
C)column chromatography
D)isoelectric focusing

Question 10

Which of the following is considered the most powerful analytical tool in the laboratory analysis of hemoglobin variants?

A)electrophoresis
B)high-pressure liquid chromatography
C)radial immunodiffusion
D)densitometry

Question 11

Normal hemoglobin fractions seen in HPLC include all but which of the following?

A)Hb A
B)Hb A2
C)Hb C
D)Hb F

Question 12

Isoelectric focusing (IEF) may be utilized to investigate unresolved HPLC hemoglobin results.What advantage of this technique makes it useful in this situation?

A)IEF is a technique that is readily adaptable to automation, resulting in a quick and simple method to resolve questionable HPLC hemoglobin results.
B)IEF is a technique that does not depend on the charge of the hemoglobin fractions, providing a method that complements HPLC analysis of hemoglobin fractions.
C)IEF results in the formation of discrete sharp bands, allowing clear separation of the various hemoglobins present in the patient sample.
D)IEF provides an enhanced method of detection that may allow the technologist to see fractions not detected by HPLC.

Question 13

The Singer 1-minute denaturation test and the Betke 2-minute alkali denaturation test is useful for measuring Hb ________ and is based on this hemoglobin's ________.

A)A, resistance to alkaline solutions
B)A, denaturation by alkaline solutions
C)F, resistance to alkaline solutions
D)F, denaturation by alkaline solutions

Question 14

Which of the following variations between hemoglobins allows for their separation by electrophoresis and chromatography?

A)variations in the amino acid composition of hemoglobins
B)variations in the heme composition of hemoglobins
C)variations in the amount of oxygen carried by hemoglobins
D)variations in the concentration of hemoglobins in the sample under analysis

Question 15

Hb F may be acetylated in vivo.What is the effect of acetylated Hb F on the measurement of total Hb F (acetylated and non-acetylated) by HPLC?

A)The peak produced by Hb F will be much larger in the presence of the acetylated form because of the increased absorptivity of the acetyl group, resulting in an overestimation of total Hb F levels.
B)The acetylated form of Hb F cannot be detected by HPLC systems, causing the level of total Hb F to be underestimated.
C)The acetylated form of Hb F will have a different retention time from the non-acetylated form, resulting in the need to sum both forms to determine the total Hb F level.
D)The presence of the acetylated form of Hb F will produce baseline drift, causing the analysis of total Hb F to be unreliable.

Question 16

Bilirubin is a common interference encountered in the HPLC analysis of hemoglobins, because patients with high levels of bilirubin will show a sharp peak in the same general area as:

A)Hb Barts
B)Hb D-Punjab
C)Hb G-Philadelphia
D)Hb F

Question 17

Which of the following steps are performed automatically by HPLC systems used in hemoglobin analysis?

A)dilution of the blood sample
B)hemolysis of the blood sample
C)injection of the blood sample in the HPLC system
D)all of the above are performed by automated HPLC systems

Question 18

Consider the separation of Hb 1, Hb 2, and Hb 3 by cation-exchange HPLC.The initial mobile phase pH causes Hb 1 to have the greatest positive charge, Hb 2 to have minimal positive charge, and Hb 3 to be neutral.Predict the order of elution from the stationary phase as the chromatographic run continues from the initial mobile phase to mobile phases with increasing concentrations of cations.(first to elute to last to elute)

A)Hb 1, Hb 2, Hb 3
B)Hb 1, Hb 3, Hb 2
C)Hb 2, Hb 1, Hb 3
D)Hb 3, Hb 2, Hb 1

Question 19

Hb A2 is often falsely elevated in the presence of __________ and falsely reduced in the presence of ________.

A)Hb S, Hb D-Punjab
B)Hb F, Hb D-Punjab
C)Hb S, Hb C
D)Hb F, Hb C

Question 20

Perhaps the most significant disadvantage of HPLC analysis of hemoglobins is:

A)the technique is only qualitative and not quantitative
B)the technique is associated with a high degree of imprecision
C)the minor differences between the various hemoglobin fractions cause hemoglobins to elute in very narrow, overlapping windows
D)post-translational modification of hemoglobins produces fractions that often elute with the same retention time as other hemoglobin fractions