HPLC.pptx

HPLC
Principle, Uses, Interpretation,
Results
MODERATOR: PRESENTED BY:
Dr. Maneesh Sulya Dr. Parul Murab

HPLC?
HIGH PERFORMANCE: Originally referred as high pressure liquid
chromatography since high pressure is applied using a pump
system.
Due to its high efficiency and performance, it is now referred as high
performance.
LIQUID CHROMATOGRAPHY: A physical method of separation,
identification and quantification of components in which
components to be separated are distributed between a liquid
mobile phase and a solid stationary phase in a definite direction.

INTRODUCTION: CHROMATOGRAPHY
 Chromatography is a physical method of separation between:
STATIONARY PHASE MOBILE PHASE
The substance on which adsorption Solvent which carries the analyte.
of analyte takes place.
Can be solid, gel, liquid or Can be liquid or gas.
combination.
 TECHNIQUE IS DIVIDED ON BASIS OF:
Chromatographic bed: column or planar
Physical state of mobile phase: gas or liquid
Separation mechanism: adsorption, ion exchange , ion pair , affinity, gel permeation
Elution techniques: Isocratic , gradient
Scale of operation: analytical(no recovery) , preparative

SYSTEM TYPE?
 LIQUID CHROMATOGRAPHY: Liquid mobile phase
 COLUMN CHROMATOGRAPHY: Adsorption of substance on stationary phase.
-Separation of adsorbed substance using mobile phase.
-recovery of individual components by continuous flow of
mobile phase.
 HIGH PRESSURE CHROMATOGRAPHY-15-75kg/cm²
 ION EXCHANGE( cation) OR ADSORPTION MECHANISM or both.
 GRADIENT ELUTION TECHNIQUE
 REVERSED PHASE Chromatography-non polar stationary phase and polar mobile phase.

PRINCIPLE
Mixture of molecules
(normal and variant
haemoglobin) with a net
positive charge is separated
into its components by their
adsorption onto a negatively
charged stationary
phase(resin) in
chromatographic
column/cartridge.

Cations in the mobile phase(buffer A
and B) competes with adsorbed
proteins(haemoglobin molecules) for
anionic binding sites.
Buffer A: weak affinity for anionic
binding sites.
Buffer B: strong affinity for anionic
binding sites.

The adsorbed positively charged
haemoglobin molecules are eluted from
column into liquid phase at a rate
related to their affinity for stationary
phase.
It is then detected optically, identifying
the haemoglobin variants by their
retention times and quantified by
computing the area under
corresponding peak in elution profile.
Flow rate: 1.5mL/min
Pressure shouldn’t fluctuate >5%

WHY USE HPLC?
 Simultaneous analysis
 High resolution
 High sensitivity
 Good repeatability
 Easy to fractionate and detect
 Quantification is possible
 Less labour intensive
 Small sample is adequate
Though it is easy to detect and quantify haemoglobin variants , HPLC is a
screening tool and the definite confirmation is possible with DNA
analysis.

APPLICATION FIELDS
 Haemoglobin profiling in suspected haemoglobinopathies:
-Diagnosis of β Thalassemia Trait
-Diagnosis of rare Haemoglobins
-Diagnosis of Hereditary Persistance of Fetal Haemoglobin
-Antenatal screening for Thalassemia and Sickle Cell Disease
-Pre-operative screening of HbS cases
 HbA1C detection
 Pharmaceutical application
 Detect food adulterants
 To differentiate organic chemicals
 To differentiate proteins on basis of their structure

PREREQUISITES:
 AGE (>1yrs)
 HAEMOGLOBINOPATHIES ARE INHERITED DISORDERS.
Ethnic background
Family history
 TRANSFUSION HISTORY (deferred if transfused within 3 months)
 DRUG HISTORY (hydroxyurea, azacytidine, cytarabine)
 Complete blood counts
 PREGNANCY- may elevate HbF

SAMPLE PREPARATION
 After addition of diluents , calibration check and control check,
The whole blood EDTA sample is prediluted with 5µL sample in 1.5ml wash
solution in aliquots.
 These aliquots arranged in a rack of 10 with 2 controls(low and high) are
then inserted to be run.
#Dilution should be according to haematocrit of the sample.
High Hct- 1:400 dilution
Low Hct- 1:100 dilution

CHROMATOGRAM
The receiver receives a voltage signal and plots it versus time. The area counts of the peak
for each component are calculated as a percentage of total area counts.
RT- retention time

CHROMATOGRAM
HbA2
PEAK
P3 VALUE-
Sample
quality
AREA
Within 1-
4 million
BASELINE
• At 0.02OD
• Curved,convex
• Properly
constructed
• <8%- acceptable
• 8-14% -
contamination
• >14% -HbJ variant
• should be present
• Symmetrical , sharp
• bell shaped
• TAC Less-increase sample ratio
• TAC high-decrease the sample ratio

EXCEPTION:
 Hb Lepore:
Baseline- inverted v shaped
RT- 3.5mins
coelutes with HbA2 and usually <20%
Elevation of HbF in range 3-7%

 HbD IRAN-
Baseline- inverted v shaped
split peak
Coelutes with HbA2 ranging from 40-
48%
Split peak- unknown peak before HbA2
and coelution with HbA2

RETENTION TIME?
 RETENTION TIME is the time in minutes from the
sample injection to the maximum point of the elution
peak of normal Haemoglobin and variant
Haemoglobins.
 It depends upon the type of Haemoglobin molecules.

BIO-RAD D10 values
PEAK RETENTION
TIME(MINS)
WINDOW(MINS) ACCEPTANCE
CRITERIA(%)
A1a 0.21 0.16-0.26 <2.5
A1b 0.29 0.23-0.35 <2.5
F 0.485 0.41-0.56
LA1c/CHb1 0.715 0.56-0.87 <4.0
LA1c/CHb2 0.735 0.60-0.87 <3.5
A1c 0.83 0.66-1.00 3.7-18.4
P3 1.43 1.23-1.63 <8.0
Ao 1.70 1.55-1.85
A2 3.01 2.59-3.43
S 4.16 4.02-4.30 >10
C 4.80 4.70-4.90

ORDER OF ELUTION
(with respect to retention time)
HbA2 HbD HbS HbQ HbC
D window/Unknown S window Unknown C window
@ 3.01mins @3.8mins @4.16mins @ 4.4mins @4.8mins

Alpha Thalassemia
HbH and Hb Barts are seen as preintegration sharp peaks @RT 0.60-0.70mins

ALPHA GENE VARIANTS
 Caused by Point Mutations leading to replacement of single
amino acid.
 HbJ Meerut - Haematological parameters are normal-mildly
decreased.
- sharp peak in P3 window with RT 1.6-1.85 mins.
- HbJ >14% is significant.
 HbQ India - Unknown window at RT 4.70-4.90min with a sharp
peak.
- Anaemia is absent but can be present along
with iron/folic acid/Vit B12 deficiency.

 Hb Godavari at RT 4.90-5.30 mins
 HbI Philadelphia at RT 1.28-1.30mins
 Hb Fontainbleau at RT 1.28-1.30mins at descending arm of
HbA
 Being single gene mutation, these conditions are clinically
silent and hence difficult to diagnose.
 Presentation: Anaemia
Familial cyanosis(alters oxygen affinity)
Familial Polycythemia
Microcytosis on PS

HbF(α2γ2)
 Results below range to be reported as <0.8%*.
 If HbF >16.5% - May elute in LA1c/CHb or A1c window and no HbF is reported.
 If HbF is absent – check for A1c/LA1c/CHb peaks.
If <3.5% - HbF absent
If HbF 5-15% with normal blood counts – may suggest HbF Trait

HPFH Δβ Thalassemia
HbF 15-45% Towards Lower value(~20%)
Normal indices Microcytosis
Positive modified Betke’s test Variable
Increased HbF can be seen in:
• Inherited red cell aplasia
• Diamond Blackfan Syndrome
• Fanconi’s Anaemia
• Erythropoietic stress(Haemolysis , bleeding)

HbA2(α2δ2)
LOWER HIGH
Iron Deficiency Anaemia Megaloblastic Anaemia
Anaemia of Chronic Disease Sickle Cell Anaemia(4-4.5%) with associated α
Thalassemia
HbH Disease HIV +ve person on ART
Sideroblastic Anaemia Hyperthyroidism
Δβ Thalassemia Thalassemia Trait

Beta Thalassemia Major(BIORAD variant)
• HbF levels are higher in
absence of β chain than
reduced synthesis.
• It ranges from 30-95%.

Beta Thalassemia Intermedia
• HbF is variable depending
upon whether the patient is
dependent on transfusion or
not.
• Marked increase of HbF(10-
35%) with concomitant
variable reduction in HbA
seen.
• HbA2 may be reduced/
normal/ elevated.

Beta Thalassemia Trait
• HbA2 = 3.9-8% is diagnostic.
• HbA2 3.3-3.7% -assess Iron status
As IDA decreases HbA2 levels.
Also B12 / Folic acid deficiency
increases HbA2 levels.
• HbF is normal/mildly increased
to 1-4%
• HbA2 = 7-9% usually found in
gujratis and sindhis.

BETA GENE VARIANTS
 HbE – Elutes with HbA2
 HbD – Unknown peak at 3.8 +/- 0.1 mins
 HbS – S window after HbA2
 HbC – C window
 22-50% - heterozygous condition
 70-90% - homozygous condition
 Compound conditions

HbE
(substitution of glutamic acid by lysine at β26 position)
HbE Trait
HbE coelutes with HbA2 (RT 3.69-3.79mins)
HbA2 <= 9% AND HbE >15%
Cases are clinically normal.
HbF usually normal.
HbE Disease
Major peak at A2 (85-95%).
RT similar as HbA2 BUT HbA2 <9% which helps to
differentiate A2 from HbE.
Cases have mild anaemia and splenomegaly.
HbF mildly increased/normal.

HbD
(substitution of glutamic acid by glutamine at β121 position)
Prevalent in Punjab and
Gujarat.
Cases are normal.
Detected when family
studies are carried out.
HbA2 is usually <2%
HbD TRAIT

HbD Disease
Presentation-
mild anaemia
with/without
splenomegaly.
D window 87.9%
at RT 4.20mins.

HbS
(substitution of glutamic acid by valine at β6 position)
HbS shows D window at
RT 4.30-4.50 mins.
HbS homozygous-
HbS = 70-90% total Hb
HbF = 10-30%
HbAo almost nil.

HbC
(substitution of glutamic acid by lysine at β6 position)
 HbC Trait cases are normal.
 HbC Disease cases have mild anaemia.
Cases on HPLC shows-
C Window ~75-90% at RT 4.90 -
5.30mins
HbC Disease

HbS - β Thalassemia
Clinical manifestation are variable
and severity depends on β
Thalassemia gene.
Red cell indices are decreased with
increased RBC count.
HPLC PATTERN-
HbS >50%
HbA 5-10%
HbF and HbA2 elevated.

HbE – β Thalassemia
Mutation in β chain and unstable nature
of HbE ~ SEVERITY
Variable presentation with decreased red
cell survival time.
HPLC PATTERN-
HbF -15-50% and HbE -50-80% in
untransfused cases.
HbA varies according to severity and may
be upto 25%.

HbD β-Thalassemia
Aymptomatic
Mild anaemia
Similar to β Thalassemia Trait
HPLC PATTERN-
HbD-70-90% as unknown peak
HbF- 3-20%
HbAo reduced.
HbA2 elevated

HbS-HbE
Asymptomatic
Mild anaemia
Paediatric patients have a milder
presentation than adults.
HPLC PATTERN-
HbS- 30-50%
HbE- 30-50%
HbA <10%
Mild elevation of HbF

HbS-HbD
Clinical presentation is of Sickle Cell
Anaemia.
Hb varies in the range of 6-10 g/dl.
HPLC PATTERN-
Unknown peak at RT 3.8mins <50%
HbS at S window <50%
HbF 10-20%
HbA2 -1.5-2.6%

Hereditary Persistence of Fetal
Haemoglobin(HPFH)
• Increased HbF in adults without
haematological abnormalities.
• Homozygous condition - total absence
of HbA with HbF >90%
• Heterozygous condition –
• HbF –10-30% with normal indices.

BILIRUBIN Preintegration peak
RT - < 0.63mins

COELUTION: DRAWBACK
 HbE and Hb Lepore may overlap HbA2.
 HbD IRAN may overlap HbA2.
 HbA2 falsely elevated in HbS upto 4-4.5%
 Increased HbF may elute in HbA1c peak.
 Increased HbA1c may elevate HbF.
 Increased Bilirubin in plasma gives preintegration peaks in
same areas as HbF and HbH.

Parameters Observed value Reference Value (Adults)
Haemoglobin 11.8gm/dl 12-16 g/dl
PCV 33.9% M:40-50 ; F:36-46%
RBC count 5.56 M/cumm M:4.5-5.5 M/cumm ;F:3.8-4.8 M/cumm
MCV 60.9 fL 76-96 fL
MCH 21.2pg 27.5-31.5pg
MCHC 34.8g/dl 30-35 g/dl
RDW-CV 17.3 % 11.6-12.2 %
RDW-SD 36.2 fL 40.5-52.5 fL
RETIC COUNT: 4.5-5% Adults:0.5-2.5% ; Neonates:4-6%
PS : RBC series shows mild anisopoikilocytosis with Microcytic Hypochromic RBC’s.
Few elliptocytes seen.
Haemoglobin Chromatography
HbAo 82.6 % 95-98 %
HbA2 2.0 % 2.2-3.8 %
HbF 0.8% 00- 01 %
HbS 00% 00%
Impression: Hb HPLC report is suggestive of Normal Hb chromatography.
Disclamer: HPLC is not a screening test for α- Thalassemia.

PCV 38.6% M:40-50% ; F:36-46%
RBC count 6.55M/cumm M:4.5-5.5 M/cumm ;F:3.8-4.8M/cumm
MCV 59.0 fL 76-96 fL
MCH 20.3pg 27.5-31.5pg
RDW-CV 17.7 % 11.6-12.2 %
RDW-SD 38.0 fL 40.5-52.5 fL
RETIC COUNT: 3-4% Adults:0.5-2.5% ; Neonates:4-6%
PS : RBC series shows mild to moderate anisopoikilocytosis with microcytic
hypochromic RBC’s. Few elliptocytes, schistocytes and target cells seen.
HbAo 79.7% 95-98 %
HbA2 5.6% 2.2-3.8 %
HbF 0.9% 00- 01 %
HbS 00% 00%
Impression: Blood picture and increased HbA2 is suggestive of
β THALASSEMIA TRAIT.
Advice: Hb HPLC of siblings and spouse.
-DNA molecular study for confirmation.

PCV 42.5% M:40-50% ; F:36-46%
RBC count 5.20M/cumm M:4.5-5.5 M/cumm ;F:3.8-4.8M/cumm
MCV 81.7 fL 76-96 fL
MCH 30.4pg 27.5-31.5pg
RDW-CV 12.6 % 11.6-12.2 %
RDW-SD 42.0 fL 40.5-52.5 fL
RETIC COUNT: 01% Adults:0.5-2.5% ; Neonates:4-6%
SICKLING TEST: POSITIVE
PS : RBC series shows normocytic normochromic RBC’s. Occasional elliptocytes
and target cells seen.
HbAo 48.3 % 95-98 %
HbA2 2.5% 2.2-3.8 %
HbF 0.8% 00- 01 %
HbS 38.9%(@ RT 4.15mins) 00%
Impression: Hb HPLC shows increased HbS, normal HbF and normal HbA2.
Blood picture and increased HbS is suggestive of SICKLE CELL TRAIT.
Advice: Hb HPLC of parents, siblings and in future spouse.

PCV 21.6% M:40-50% ; F:36-46%
RBC count 62.29M/cumm M:4.5-5.5 M/cumm ;F:3.8-4.8 M/cumm
MCV 94.4fL 76-96 fL
MCH 28.4pg 27.5-31.5pg
RDW-CV 17.4% 11.6-12.2 %
RDW-SD 62.1 fL 40.5-52.5 fL
RETIC COUNT: 02% Adults:0.5-2.5% ; Neonates:4-6%
PS : RBC series : Decreased in total number with moderate degree of
anisopoikilocytosis showing normocytic normochromic and normocytic
hypochromic RBCs. Polychromatic cells, target cells, schistocytes, elliptocytes and
sickle shaped RBCs seen along with few microspherocytes.
Test value Reference value
HbF 14.8% 00- 01 %
HbAo 2.9% 95-98 %
HbA2 1.2% 2.2-3.8 %
HbS 71.2% 00%
Impression: Blood picture, HPLC and positive sickling test are suggestive of
Sickle Cell disease.
Advice: Hb HPLC of siblings and spouse.
Disclamer: HPLC is not a screening test for Sickle cell anaemia.

TO SUMMARIZE:
RBC series on PS and Reticulocyte count to be
documented.
SPECIAL TESTS CHECKLIST- if not done , they are to be performed
simultaneously.
(sickling test, Modified Betke’s Test, Reticulocyte stain)
ASK FOR AGE,FAMILY HISTORY,TRANSFUSION
HISTORY,DRUG HISTORY,ETHNICITY
EDTA SAMPLE FOR HPLC+2 PS SLIDES

If all parameters are within normal range, proceed to read
the graphs.
Check for control readings- baseline , P3 value , HbA2
peak , Total area
Take 5µL Whole blood sample + 1.5ml wash solution in
aliquots and keep it in rack to be run.
Sample to be refrigerated until run in the HPLC system.

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