The Quantitative detection
of different types of Hb variants through HPLC Technique: Report of 428 cases
in Indian population.
Abhinav Shrivastava*, S.K. Mohapatra,
Arpita Roy, S. N. Das and Gaurav Tyagi
Gayatri College of
Pharmacy, Sambalpur, Odisha.
ABSTRACT:
Identification of disorder
like thalassemia syndromes is immensely important epidemiologically and aid in prevention of more serious
hemoglobin disorders. High performance liquid Chromatography (HPLC) forms an
important tools and accurate and speedy diagnosis of various hemoglobin
disorders. The study was performed on BIO RAD Variant using beta thalassemia
short program. The blood samples are taken from various age groups. Abnormal
hemoglobin fraction on HPLC was seen in 69 cases of the 428 cases displayed.
Detection of this abnormal hemoglobin particularly keeping in mind in high
prevalence of Hb A2 will help in prevention of more serious hemoglobinopathies
including beta thalassemia major. HPLC form a rapid and accurate tool in early
detection and management of various hemoglobin disorders
KEYWORDS: HPLC, hemoglobin variant, thalassemia
INTRODUCTION:
Accurate and timely detection of various Hb variant
including beta thalassemia trait can prevent occurrence of more serious
disorders like thalassemia major in new born abnormalities of hemoglobin (Hb)
synthesis are among the most common inherited disorders of men can be
quantitative (thalassemia syndrome) or qualitative (variant HbS )1.
About 428 cases had came for diagnosis of abnormalities in hemoglobin have been studies from 1st
April 2010 to 15th April 2010 at S.R.L Diagnostic Lab Gurgaon in
Indian Population. Of these thalassemia syndrome particularly beta thalassemia
major and certain alpha thalassemia are serious and a major case of morbidity.
The
abnormal hemoglobin fraction according to the age group are 12 cases of age
between 0- 2 years, 8 cases of age between 2- 10 years, 6 cases of age between
11-20 years, 22 cases of age between 21-30 years, 19 cases of age between 31-50
years and 2 cases of above 50 years of individual were seen in the above cases.
Potential interaction between various Hb variant in heterozygous state may be
lead to serious homozygous Hb variant in the off springs. Double heterozygous
state between certain variants also leads to hematological defect. Cation
exchange HPLC offers a reliable tool for early, accurate detection thereby
aiding in prevention and management of various hemoglobinopathies.
MATERIAL AND
METHOD:
The
test was performed on and instrument manufactured by BIO RAD laboratories USA.
The instrument, known as BIO RAD “VARIANT” (beta thalassemia short program)
utilizes the principle of High performance liquid chromatography (HPLC). An Hb
A2 / F Calibrator and two levels of controls (BIORAD) were analyzed at the
beginning of each run. The total area
acceptable was between- one to three million. A total of 428 cases received
from 1st April 2010 to 15th April 2010 for Hb variant
analysis were studied. The geographical distribution of all the cases
predominantly includes part of Northern India (New Delhi, Haryana, Punjab,
Uttar Pradesh and Jammu and Kashmir).
Adult blood contain primarily Hemoglobin A (HbA), a
small percentage of Hemoglobin A2 (HbA2), and trace amount of Fetal hemoglobin
(HbF). Carriers of beta thalassemia have levels of hemoglobin A2 and F which
can greater than 3.5 % and 2 % of the total hemoglobin, respectively2 (Figure-1).
Determination of concurrently elevated levels of hemoglobin of A2 and F has
become the most practical means of diagnosis carriers of the beta thalassemia
gene3. Methods for the quantitation of hemoglobin A2 include
electrophoresis4 and anion-exchange column chromatography5.
Method for quantitation of hemoglobin F includes electrophoresis alkali
denaturation and radical immunodiffusion (RID).
High performance liquid chromatography (HPLC), which can
be relatively fast and reproducible method, has been used for the determination
of various hemoglobin6,7, including hemoglobin A28 and F.
The BIORAD VARIANT instrument is a fully automated HPLC system which can be
percentage for hemoglobin A2 and F and to provide qualitative determinations of
abnormal hemoglobin. The most commonly occurring hemoglobin variants include
hemoglobin D, S, C and E 9. Presumptive identification of these
hemoglobin variants is made using retention time windows, such as “D-window”,
“S-Window” and “C-Window”.
Specimen are subsequently inject into the analysis
stream at 6.5 minute intervals for a through put of 9 samples per hours. Two
duals- piston pumps and a pre-programmed gradient control the elution buffer
mixture passing through the analytical cartridge (column). The ionic strength
of elution buffer mixture is increased by raising the percent contribution of
elution buffer 2(sodium phosphate buffer, contain less than 0.05% sodium azide
as a preservative). As the ionic strength of mixture increases more strongly
retained hemoglobin elute from the analytical cartridge.
A dual-wavelength filter photometer (415and 690 nm)
monitors the hemoglobin elution from the column, detecting absorbance changes
at 415 nm. The 690 nm secondary filter corrects the base line for effects
caused by mixing buffer with different ionic strengths. Changes in absorbance
are monitored and displayed as a chromatogram of absorbance various time.
Analysis data from detector is processed by the built-in integrator (or by
Clinical Data Management [CDMTM] software for upgraded VARIANT
Systems) and printed in sample reports. Some examples are:-
Figure
1: (A) Chromatogram of Normal proportion of Hb variants in age of 1 year 6
month, and (B) Chromatogram of Abnormal proportion of HbA2 of age 31 year
respectively.
Figure
2: (C) Chromatogram of Abnormal proportion of HbA2 and HbF in age 10 month, and
(D) Chromatogram of Abnormal proportion of Hb iron (P3) of age 24 year
respectively.
Figure
3: (E) Chromatogram of Abnormal proportion of HbF and HbS of age 6 year, (F) Chromatogram of
Abnormal proportion of HbF and HbS of age 15 year respectively.
To aid in interpretation of results, window (range)
have been established for most frequently occurring hemoglobin base on their
characteristic retention time. Retention time is the elapsed time from the
injection of sample to the apex of a hemoglobin peak. Each hemoglobin variant has a characteristic
retention time. The total area of each analysis range from 1000, 000 to 3000,
000mvolts٠second. Result should not be reported if
the area is outside this range.
The retention of HbA2 of the calibrator is used to set
the appropriate operating temperature for the cartridge (column). The optimum
retention time is 3.65 minutes. The
temperature adjustment table (Table1) to determine the appropriate operating
temperature for the cartridge. For example, if the observe retention time of Hb
A2 in the calibrator is 3.73 minute; the necessary temperature adjustment is +30C.
By adding 30C to the standard temperature setting of 350C
the new adjusted temperature setting should be 380C.
Table 1 Retention Time and Temperature
Adjustment
|
RT of HbA2 in
Calibrator |
Temperature
Adjustment 0C |
|
3.47 |
-7 |
|
3.49 |
-6 |
|
3.52 |
-5 |
|
3.55 |
-4 |
|
3.57 |
-3 |
|
3.60 |
-2 |
|
3.62 |
-1 |
|
3.65 |
0 |
|
3.68 |
+1 |
|
3.70 |
+2 |
|
3.73 |
+3 |
|
3.75 |
+4 |
|
3.78 |
+5 |
|
3.81 |
+6 |
|
3.83 |
+7 |
Limitations:
The
VARIANT Beta thalassemia short program provides an area present determination
of hemoglobin A2 and F, as well as qualitative separation of normal and
commonly occurring abnormal hemoglobin. Other less frequently occurring variant
may also elute within the established analyte identification windows.
For the positive conformation of any particular
hemoglobin variant, globins chain analysis may be require.
·
Elevated
levels of HbA2 may be masked by concurrent iron deficiency anemia.
·
Minor
component of Hb S and other hemoglobin variants eluting after HbA2 may co-elute
with HbA2. This may result in a falsely elevated area percent value for HbA2.
·
Hemoglobin
D and E have been observed to co-elute with Hb A2. Specimen determine to have
Hb A2 level greater than 10% should be tested fro the possible presence of
hemoglobin variant interference.
·
Diabetic
specimen typically exhibit an elevated P2 Peak and additional peak identified
as “unknown”, may also found between the normal position of HbF and P2. If the
area of unknown peak is larger than the area of HbF, the additional peak may be
mistakenly identified as HbF. This
should be suspected if there is and additional peak in the HbF window.
RESULT AND
CONCLUSION:
Our
studied included predominantly population of North India. The total of 428
cases were studied of these 359 cases blood samples displayed normal range of
different Hb variant and 45 sample displayed abnormal range of HbA2, 23 samples
displayed abnormal range of HbF, 3 samples displayed abnormal range of HBS (S
Window), 1 samples displayed abnormal range of HbD (D-Window) and 3 samples
displayed abnormal range of Hb iron (P3) in remaining case.
Cation
exchange HPLC is emerging as one of the best methods for screening and
detection of various hemoglobinopathies with rapid reproducible and precise
result. Conditions with borderline HbA2 need careful interpretation. Iron
deficiency may lead a low HbA2 and hence may mask a thalassemia trait whereas
B12 / Folate deficiency may lead to slightly raised HbA2 leading to a fast
diagnosis of trait. Careful evaluation of indices with iron profile will
usually help in such cases. Thalassemia major and inter media constituted
approximately 0.6% of cases. Children with thalassemia tends present within two
years of life and are dependent on regular blood transfusion. Clinical feature
includes anemia, microcytosis, and hypochromia with target cell.
To conclude, HPLC forms a rapid, accurate and
reproducible tool for early detection and management of hemoglobinopathies and
variants. This is especially important in view of high incident of beta
thalassemia trait in the Indian subcontinent. Early detection of trait will
prevent occurrence of thalassemia major in off spring. Detection of other
variant becomes important due to complex interaction in cases with double
heterozygous and homozygous state, which may lead to severe hematological and
abnormalities.
ACKNOWLEDGEMENTS:
SRL Ltd. Gurgaon for providing the laboratory
facilities and for carrying out quantitative analysis.
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Received on 01.10.2010
Accepted on 04.11.2010
© A&V Publication all right reserved
Research J. Pharmacology and
Pharmacodynamics. 2(6): Nov. –Dec. 2010, 397-400