High Performance Liquid chromatography INTERPRETATION.ppt
- 1. 1 - Flat baseline – The baseline should be properly constructed 2 - Peak profile & shape – Peaks should appear sharp and symmetrical 3 – Order of peaks – The peaks should follow the order F, P2, P3, Ao, A2 4 - Total peak area Should be 1 to 3 million 5 – Hb A2 and Hb F response- The new calibration factor should be – 0.7 to 1.3 6 - Hb A2 retention time – The retention time of Hb A2 for the calibrator should be 3.65 + 0.1 Checkpoints in the calibrator 1 4 3 2 6 5
- 2. Interpretation of chromatograms • Flat baseline • Total peak area • Hb A2 retention time • Peak profile & shape • Review the CBC data and Interpret result in conjunction with CBC • Consider ethnic origin • Related clinical information • Examine the relative percentages of the hemoglobin fractions found • Determine whether a variant is present • Consider the possibility of more than one hemoglobinopathy being present
- 3. Look for the following : F – Less than 2% P2 – changes with the glycemic status, upto 6 % acceptable P3 – upto 6 % acceptable , A0 – non glycated fraction of Adult hemoglobin A2 –normal range 2 to 4 %
- 5. Look for the following : Typical thalassemia carriers Hypochromic, Microcytic blood film Hb A2 > 4.0 % Hb F < 2.0% ( in some cases Hb F may also be elevated )
- 7. First evaluate age and transfusion history If transfusion is involved report with parental screening If transfusion interval is greater than 30- 60 days Look for the following : Variable degree of anemia Marked red cell changes Hb F elevated upto 90% Reduced Hb A Normal or elevated Hb A2
- 9. First evaluate age and transfusion history If transfusion is involved report with parental screening If no transfusion is involved Look for the following : Increased NRBC count Marked variation in shape and size Hb F elevated upto 90% Reduced Hb A Normal or elevated Hb A2
- 11. Hb (g/dl) MCV (fl) Hb F Hb E SEVERITY Normal 80 - 90 <1% 25-35% Asymptomatic Look for the following : Hb E elutes in the A2 window For a Hb E trait : Hb A2 will be between 25-35% Hb F will be normal
- 13. Hb (g/dl) MCV (fl) Hb F Hb E SEVERITY 10-12 65-75 >2% >60% Mild Look for the following : Hb E elutes in the A2 window For a Hb E homozygous: Hb A2 will be between >60% Hb F will be between 2-10% These values will be variable from patient to patient and will also vary if there is a history of blood transfusion
- 15. Hb (g/dl) MCV (fl) Hb F Hb E SEVERITY <10 <70 >10% >50% Severe Look for the following : Reduced indices Hb E elutes in the A2 window For a Hb E homozygous: Hb A2 will be between >50% Hb F will be between > 10% These values will be variable from patient to patient and will also vary if a history of blood transfusion is involved
- 17. Hb (g/dl) MCV (fl) Hb A2 Hb F Hb S SEVERITY Normal 80 - 90 < 4.0 % <1% 30- 40% Asymptomatic Look for the following : Normal indices Hb S elutes in the S window For a S trait : Hb A2 will be normal (however due to the elution of some glycated Sickle products the A2 may be elevated in some cases , do not consider it as a compound heterozygous case) Hb F will be normal Hb S will be between 30-40%
- 19. Hb (g/dl) MCV (fl) Hb A2 Hb F Hb S SEVERITY <12 70-75 < 5 % > 5% >50% Mild Hb S elutes in the S window Look for the following : For a Hb S homozygous: Hb A2 will be normal Hb F will be elevated Hb S will be > 50% These values will be variable from patient to patient and will vary if a history of blood transfusion is involved
- 21. Hb (g/dl) MCV (fl) Hb A2 Hb F Hb S SEVERITY <10 <70 > 5.0 % > 5% >50% Severe Hb S elutes in the S window If transfusion is involved report with parental screening Look for the following : Reduced indices For a Hb S thalassemia: Hb A2 will be elevated Hb F will be elevated Hb S will be > 50% These values will be variable from patient to patient and will vary if there is a history of blood transfusion
- 23. Hb (g/dl) MCV (fl) Hb A2 Hb F Hb D SEVERITY Normal 80 - 90 < 2% <1% 30-45% Asymptomatic Look for the following : Normal indices Hb D elutes in D window For Hb D trait : Hb A2 will be normal Hb F will be normal Hb D will be between 30-40%
- 25. Hb (g/dl) MCV (fl) Hb A2 Hb F Hb D SEVERITY <12 <75 <4% 3-6% >50% Variable between mild to severe Look for the following : Normal or reduced indices Hb D elutes in D window For a Hb D beta thalassemia : Hb A2 will be normal or elevated Hb F will be mildly elevated Hb D will be between >50%
- 27. Hb (g/dl) MCV (fl) Hb A2 Hb F Hb D Hb S SEVERITY </= 14 80-90 <4% <20% <50% >50% Variable between mild to severe Look for the following : Normal or reduced indices For a Hb S- Hb D disease : Hb A2 will be normal Hb F will be elevated Hb D will be < 50% Hb S will be < 50%
- 29. Hb (g/dl) MCV (fl) Hb A2 Hb F SEVERITY Normal Normal 40-48% <2% Asymptomatic Look for the following : Normal or reduced indices For a Hb D Iran trait : Hb A2 will be 40-48% Hb F will be normal
- 31. Hb (g/dl) MCV (fl) Hb A2 Hb F SEVERITY 12-14 80-90 10-18% <10% Asymptomatic Look for the following : Normal indices For Hb Lepore trait : Hb A2 will be 10-18% Hb F will be normal The retention time of Hb A2 will be earlier than normal - 3.45 to 3.6mins
- 33. Hb (g/dl) MCV (fl) Hb A2 Hb F Hb Q SEVERITY 12-14 80-90 <4% <2% 8-25% Asymptomatic Look for the following : Normal indices For Hb Q trait : Hb A2 will be normal Hb F will be normal Hb Q will elute at retention time 4.7 + 0.1 mins constituting 8-25%
- 35. Hb (g/dl) MCV (fl) Hb A2 Hb F SEVERITY 12-14 80-90 <4% <5-30 % Asymptomatic Look for the following : Normal indices For HPFH trait : Hb A2 will be normal Hb F will be 5-30%
- 36. Hb (g/dl) MCV (fl) Hb A2 Hb F SEVERITY <12 <75 <4% <3-20 % Asymptomatic Look for the following : Reduced indices For HPFH trait : Hb A2 will be normal Hb F will be 3-20%
- 38. Additional points • P3 – upto 6 % acceptable , – 6 to 12 % may indicate sample deterioration – 15 to 25 % indicate Hb J (refer to Dr Nardi’s algorithm) • Iron deficiency – Hb A2 found to be slightly lower • Megaloblastic anemia – Hb A2 found to be higher Unknown peak • May appear any where in the peak table • More than 1 unknown peak may be seen • Upto 6 % not significant • If, above 6% - look for the RT for Hb identification.