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HAEMOGLOBINOPATHY CASES on HPLC. Dr. Archana Vazifdar Head Pathologist Hindlabs , HLL Lifecare Ltd. Detector. Direction of flow. HPLC Automated system precalibrated column and gradient. Hemoglobin is eluted in a stepped manner by Buffers of Increasing Ionic strength .

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haemoglobinopathy cases on hplc

HAEMOGLOBINOPATHY CASESon HPLC

Dr. ArchanaVazifdar

Head Pathologist

Hindlabs, HLL Lifecare Ltd.

slide3

Detector

Direction of flow

HPLC

Automated system precalibrated column and gradient

slide5

Output

Time

CHROMATOGRAMS

Peak

Area

RT (min)

Total Area Count: 1-3 million

HbF: 1-2% RT: 1.03-1.13

HbA2: 1.75-3.25% (2-3.6%) RT: 3.63-3.64

slide6

P2- Glycemic status, upto 6% acceptable

P3- 6% acceptable, 6-15% deterioration

15-25% HbJ

slide12

Mother of 8 mnth old child

Heterozygous Beta thalassemia

hba2 normal
HbA2- Normal
  • RBC indices- Normal
    • Silent β thalassaemia
  • RBC indices s/o thal
    • Co existing IDA
    • Co inheritance of α thalassaemia
    • δβ thalassaemia

HbA2 LOW in α thalassaemia

slide18

All criteria fit provided there is NO history of recent blood transfusion

  • ALWAYS CORRELATE with clinical history with CBC & peripheral blood picture
  • Degenerated sample
slide19

Case 1:

2 yr/M, anemia, hepatosplenomegaly

Hb 7

MCV 84

MCH 28

MCHC 32.4

RDW 20.2

slide20

Elevated HbF:

  • HPFH (HbF 5-30%, CBC N, asymp)
  • Heterozygous δβthal
  • (HbF 3-20%, asymp)
  • Homozygous beta thal
  • Pregnancy
slide22

Case 2:

28/F, Severe anemia

slide23

Borderline HbA2 levels:

  • Silent carrier of thalassemia
  • βthal with superadded IDA
  • Macrocytosis
  • αβthalassemia
slide25

P3:

6% acceptable,

6-12% deterioration

15-25% HbJ

slide26

Hb: 6.5

MCV: 63

MCH: 23

MCHC: 26

RDW: 19.6

slide28

Tests may not be accurate if…

  • Patient had a blood transfusion within the past four months.
  • Patient has polycythemia (increased red blood cell production) or underlying anemia
  • If the patient is on certain medications
  • Aged/ degenerated sample