Haemoglobinopathy answers

Answers to Chromatograms1:

1. There are clearly two main peaks here; one at 4.41min and the usual expected HbA. The ratios do not fit with HbAS as HbA would be about 60% and HbS about 40%. This will be a transfused HbSS. In a SS patient one would not expect HbA2 at all as they are unable to produce normal alpha2-delta2 globins - so this is actually denatured HbS.

2. This is a straightforward HbAC - the ratio of 60:40 is expected with a beta chain variant (which is what HbC is) - as variant haemoglobins are not as stable as HbA and there will be a low level of haemolysis. The two small peaks correspond to glycated and denatured haemoglobin respectively.

3. This is a normal HPLC plot (refer back to the learning section on HPLC for a review of a normal chromatogram). This chromatogram comes directly after the previous one in this run - if there is presence of HbC on one chromatogram it may contaminate the following two chromatograms - and sometimes the peak may be even bigger - so it is important to check the previous chromatogram. HbC is one of the last to elute off the column and can remain for this reason on the following plot(s).

4. Again the elevated HbA2 is denatured HbS in this case. There is an elevated HbF here; the possibilities are that this patient has:

HbSS and on hydroxycarbamide (which is what this patient had);

HbS/Bthal sickle cell disease (remember a beta thalassaemia gene can result in an increased amount of HbF);

HbSS with HPFH (relatively rarer)

Of interest also is the v small peak you note in the HbA0 region. This is not HbA but likely glycated HbS - the reason the relative peak is lower compared with HbA1c is because HbS is more vulnerable to haemolysis and so there will be a correspondingly lower level of glycated HbS.

5. Differentials might include HbEF (unlikely with the relative proportions); HbEE with HPFH (v rare). Most likely is this patient has HbE/Bthal (elevated HbF is a giveaway, as should the blood film and FBC if available. This cannot be a beta thalassaemia trait because HbA0 is non existent and the HbA2 is too high (>60%).

6. Three peaks are present - at HbA, HbS and HbC. Possibilities are that this could be a transfused HbSS with HbAC blood (but the relative proportions of S and C are roughly 50:50) - so this is most likely to be a transfused HbSC.

7. With the right FBC indices, an isolated elevated HbA2 to about 5% is diagnostic of a beta thalassaemia trait. There are no realistic differentials in this case (too low for HbE trait; and slightly too high for a patient to be on highly active anti-retroviral therapy) - one might expect this to be about 3-4% as it only increased HbA2 by 10% overall.

8. Again as before with question 4 - possibilities are HbSS on hydroxycarbamide; HbS/Bthal or HBSS with HPFH.

9. This patient has an elevated HbA2 at 3.9% and so is above the 3.5% threshold required for diagnosis of beta-thalassaemia trait. Note also the elevated HbA1c and so it is likely that he/she is also diabetic.

10. This is HbAE - and although the normal expectation is that HbA = 60% and HbE = 40%; we see that the relative amount of E only reaches 25%. A rough level of between 15-25% for a variant haemoglobin suggests that the patient has a coinherited alpha chain variant (alpha chain variants will also decrease the amount HbE as the patient will be less able to produce HbE, which contains alpha chains.

11. This is a classic chromatogram of HbH disease. The very first peak is bilirubin, and the initial Hbs that elute give a classic saddle pattern.

12. HbAC

13. Transfused HbSS - or a patient who is HbAA and has had a transfusion with blood from a sickle cell trait donor (much less likely).

Answers to Chromatograms2:

Note these have no lab numbers but will be displayed in order. You may have found these easier having done the ones above but it is also establishing familiarity with the gels and their running positions (make sure you have memorised the common variants!).

14. HbSC - elevated A2 because of denatured S. Small peak of denatured C next to the C peak

15. HbAD - The HPLC shows a peak that doesn't coincide with the S-window; in addition there is a band running on the alkaline gel in the SDG position - most likely to be a D band. Differentials include HbAG - as the peak could be a HbG.

16. HbA with HPFH. Not a beta thal trait as HbA2 is normal. Alternatively this could be a neonate of less than six months who is transitioning to adult from fetal haemoglobin.

17. HbAC. Glycated and denatured peaks for the C band are also visible at 4.54 and 4.90 mins respectively.

18. HbSC

19. HbAC

20. HbAC

21. HbE/Bthal. Band which runs with C could also be E or O-Arab.

Answer to Chromatograms3:

22. The clue should be in the oxygen saturations of the patient possibly indicating a methaemoglobinaemia. These plots may present with either something which elutes off very quickly from the machine (in which case there will be a rapid peak on the left of the machine). Be aware that certain chemicals, drugs and other enzymopathies can also cause a methaemoglobinaemia and can only be detected by blood gas analysers and NOT on the HPLC. In this case, the methaemoglobinaemia can be traced to a Hb M-Iwate, which is the peak eluting off at the S window. The shape of the peak is wrong for this to be a transfused patient S, and the clinical history would not fit with this picture. In addition, there is a notable P3 peak.