Hindawi Publishing Corporation International Scholarly Research Notices Volume 2015, Article ID 498230, 1 page http://dx.doi.org/10.1155/2015/498230

Retraction Retracted: Detection of Abnormal Variants by HPLC Method: Common Problems with Suggested Solutions

International Scholarly Research Notices

Received 14 April 2015; Accepted 14 April 2015

Copyright © 2015 International Scholarly Research Notices. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Tepapertitled“DetectionofAbnormalHemoglobinVari- ants by HPLC Method: Common Problems with Suggested Solutions,” [1] published in International Scholarly Research Notices, has been retracted as it was submitted for publication without the knowledge and approval of one of the coauthors, Dr. Suman Lata Mendiratta. Te submitting author Dr. Dipti Kalita plagiarized data from the Talassemia Control Project headed by Dr. Suman Lata Mendiratta.

References

[1]L.Pant,D.Kalita,S.Singhetal.,“Detectionofabnormalhemo- variants by HPLC method: common problems with sug- gested solutions,” International Scholarly Research Notices,vol. 2014,ArticleID257805,10pages,2014. Hindawi Publishing Corporation International Scholarly Research Notices Volume 2014, Article ID 257805, 10 pages http://dx.doi.org/10.1155/2014/257805

Research Article Detection of Abnormal by HPLC Method: Common Problems with Suggested Solutions

Leela Pant,1 Dipti Kalita,1 Sompal Singh,1 Madhur Kudesia,1 Sumanlata Mendiratta,2 Meenakshi Mittal,2 and Alka Mathur3

1 Department of Pathology, Hindu Rao Hospital, Delhi 110007, India 2 Talassemia Control cell, Hindu Rao Hospital, Delhi, India 3 Department of Pediatrics, Hindu Rao Hospital, Delhi, India

Correspondence should be addressed to Dipti Kalita; [email protected]

Received 13 April 2014; Accepted 16 July 2014; Published 12 October 2014

Academic Editor: Josep Esteve-Romero

Copyright © 2014 Leela Pant et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Talassemia and thalassemic pose serious health problem leading to severe morbidity and mortality in Indian population. Plethora of hemoglobin variants is prevalent in multiethnic Indian population. Te aim of the present study was to analyze laboratory aspects, namely, hematological profle and HPLC fndings of the hemoglobin variants detected, and to discuss problems that we faced in diagnosis in a routine clinical laboratory. We screened a total of 4800 cases in a hospital based population of North India in a 2-years period of by automated HPLC method using the Variant Hemoglobin Testing System (Variant II Beta Talassemia Short Program, Bio-Rad Laboratories) under the experimental conditions specifed by the manufacturer. Whole blood in EDTA was used and red cell indices were determined using automated hematology analyzer. Wedetected 290 cases with abnormal variants in which beta thalassemia was the most common followed by . Here, we discuss the laboratory aspects of various hemoglobin disorders and diagnostic difculties in cases like borderline HbA2 values, presence of silent mutation, alpha thalassemia gene, and few rare variants which at times require correlation with genetic study. Special attention was given to HbA2 level even in presence of a structural variant to rule out coinheritance of beta thalassemia gene.

1. Introduction clinically signifcant variants. Te simplicity of the automated system with internal sample preparation, superior resolution, Talassemia is an autosomal recessive inherited group of rapid assay time, and accurate quantifcation of hemoglobin disorders of hemoglobin synthesis characterized by the fractions makes this an ideal methodology for the routine absence or reduction of one or more of the globin chains of clinical laboratory [2, 3]. hemoglobin. Te structural variants result from substitution With increasing global awareness and mass screening of one or more amino acids in the globin chains of the programs undertaken at various levels by health care sys- hemoglobin molecule [1]. Plethora of hemoglobin variants is tem, the responsibility for laboratory personnel has greatly prevalentinIndiaowingtoethnicdiversityofitspopulation enhanced in detection and prevention of this problem. with minimal to major clinical signifcance. Being recessively Awareness about the diagnostic problems as well as their inherited from the parents, the thalassemia and thalassemic solutions is very important so that one does not miss a single hemoglobinopathies pose serious health problem leading to case. severe morbidity and mortality in Indian population. Many studies have been published from India on tha- Detection of asymptomatic carriers by reliable laboratory lassemia and thalassemic hemoglobinopathies mostly putting methodsisthecornerstoneofpreventionofthisserious emphasis on epidemiology and screening [4–6]. health problem. Cation exchange high performance liquid Very few studies are available on the approach of diagno- chromatography (CE-HPLC) has become the preferred tech- sis and problems in routine diagnosis, especially in laborato- niquesuitableinIndianscenario,asitcandetectmostofthe ries with limited resources. 2 International Scholarly Research Notices

Te aim of the present study was to analyze laboratory Table 1: Distribution of hemoglobin variants. aspects,namely,hematologicalprofleandHPLCfndingsof thehemoglobinvariantsdetected,andtodiscussproblems Hemoglobin variant Number (%) that we faced in diagnosis in a routine clinical laboratory. Beta thalassemia trait (BTT) 216 (74.48) Beta thalassemia intermedia/major (BTI/M) 9 (3.10) 2. Material and Methods Hb E trait (HbAE) 28 (9.65) Hb E disease (HbEE) 2 (0.69) Tis was a prospective study carried out in the Department Hb E beta thalassemia (HbE-BT) 2 (0.69) of Pathology and Talassemia Control Cell, Hindu Rao Hb D trait (HbAD) 15 (5.17) Hospital,Delhi,for2years’period.Atotalof4800cases HbD-beta thalassemia (HbD-BT) 4 (1.38) were screened for presence of thalassemia or any structural HbS trait (HbAS) 9 (3.10) variant. Tese included all cases of microcytic hypochromic Delta beta thalassemia (deltaBTT) 3 (1.03) anaemia (MCV < 80 f, MCH < 27 pg, and RBC count Hb J Meerut 1 (0.34) > 5million/�l) not responding to conventional treatment, clinically suspected cases of , antenatal, Hb Hope 1 (0.34) and other cases coming to the department for thalassemia Total 290 screening. A 5 mL intravenous blood sample was collected in EDTA anticoagulant. Red cell indices were measured on an automated haematology analyzer (Sysmex KX 21). (4) Wash/diluent solution: deionized water. HbA2, HbF,and other haemoglobin variants were studied (5) Control: normal (HbF 1-2%, HbA2 1.8–3.2%) and by HPLC method used for chromatographic separation of abnormal (HbF 5–10%, HbA2 4–6%) controls. human hemoglobin [7–9]. We used the Variant Hemoglobin Testing System (Variant 2.3. Sample Collection and Preparation. Five milliliters II Beta Talassemia Short Program, Bio-Rad Laboratories (5 mL) of whole blood was collected in a vacuum collection Inc., Hercules, CA, USA) under the experimental conditions tube containing EDTA which can be stored at 2–8 degrees C specifed by the manufacturer [10]. for maximum 7 days if processing is delayed. No preparation wasrequiredunlessthesamplewasinatubeotherthanthe 2.1. Principle. TeVariantIIBetaTalassemiaShortPro- recommended tube or there was less than 500 �Lofsample gram utilizes principles of ion-exchange high-performance inthetube.Insuchcase,samplewasmanuallyprediluted. liquid chromatography (HPLC). Te samples are automati- Predilution was carried out by mixing 1.0 mL wash/diluents cally mixed and diluted on the Variant II Sampling Station with 5 �Lofwholebloodsample. (VSS) and injected to the analytical cartridge. Te Variant HbA2/F calibrators and normal and abnormal controls II Chromatographic Station (VCS) dual pumps deliver a were analyzed at the beginning of each run. programmed bufer gradient of increasing ionic strength to the cartridge, where HbA2/F are separated based on their 2.4. Interpretation of Reports. Reports and chromatograms ionic interaction with the cartridge material. Te separated generated were studied and interpreted by observing HbA2 HbA2/F then pass through the fow cell of the flter photome- and F concentration for beta thalassemia and retention ter where the changes in absorbance at 415 nm are measured. time and area percentage of other peaks and windows for An additional flter at 690 nm corrects the background structural variants. Each chromatogram shows peaks of Hb absorbance. Te Variant II CDM (CDM) Sofware performs A0, A2, and Hb F along with C window, D window, S window, reduction of raw data collected from each analysis. To aid in and two minor peaks, P2 and P3. Several hemoglobin variants the interpretation of results, windows have been established elutesamewindow;theywereprovisionallydiagnosedby for the most frequently occurring based on the retention time and area percentage keeping in mind the characteristic retention time. For each sample a sample report ethnicity of the patients. and a chromatogram are generated by CDM showing all Other relevant tests were done, for example, sickling test hemoglobin fractions eluted, their retention times, the area as supporting evidence of Hb S. Family study was carried of the peaks, and values of the fractions. out whenever possible and correlation with fndings of Hb electrophoresis result was done in few cases. 2.2. Reagents (1) Elution bufers (1,2): sodium phosphate bufer. 3. Results and Discussion (2) Whole blood primer: lyophilized human red blood 3.1. Results. Among 4800 cases screened, 290 (6.04%) cases cell hemolysate with gentamicin, tobramycin, and were detected with abnormal hemoglobin in this study. EDTA as preservative. Presumptive identifcation of hemoglobin variants was made (3) HbA2/F calibrator/diluent set: lyophilized human red primarily by retention time (RT) windows and area percent; blood cell hemolysate with gentamicin, tobramycin, however geographical factor, ethnicity, and clinical presen- andEDTAaspreservativeanalyticalcartridge.Dilu- tation were also taken into consideration. Distribution of ent contains deionized water. hemoglobin variants identifed is shown in Table 1.Teir International Scholarly Research Notices 3

Calibrated Retention Peak Peak name Calibrated Area (%) Retention Peak Peak name area (%) Area (%) time (min) area area (%) time (min) area Unknown — 0.0 0.96 1170 P1 — 0.1 0.74 1290 ∗ F 0.4 — 1.08 13120 F 29.1 — 1.13 326472 Unknown — 1.0 1.20 32945 P2 — 4.2 1.32 48837 P2 — 3.7 1.32 116596 P3 — 2.5 1.67 28868 Unknown — 0.6 1.48 17382 Unknown — 0.1 2.06 753 P3 — 4.1 1.72 129129 63.0 2.54 738847 A0 — ∗ A0 — 83.8 2.46 2647621 A2 2.2 — 3.58 28242 2 —∗ 3.68 A 5.6 — 201552 Total area: 1,173,30 3 159 5 ∗ F concentration = 0.4% Total area: , , F concentration =29.1 % 2 =5.6∗ ∗ A concentration % A2 concentration =2.2 % ∗ ∗ Values outside of expected ranges Values outside of expected ranges Analysis comments: Analysis comments: 45.0 45.0

37.5 37.5

30.0 30.0

22.5 22.5 (%) (%)

15.0 3.68 15.0 3.58 1.32 1.37 1.67 0.96 1.08 2.06 0.74 1.20 1.72 7.5 A2 7.5 1.48 2 2.54 A F 2.46 1.13 0.0 0.0 F

0 123456 0 123456 Time (min) Time (min) (a) (b)

Figure 1: (a) Chromatogram of beta thalassemia trait showing elevated HbA2 5.6% (RT 3.68 min) and HbF 0.4%. (b) Chromatogram showing elevated Hb F (29.1%) suggestive of HPFH.

Table 2: Mean values (mean ± SD) of red cell parameters and hemoglobin fractions in variants detected by HPLC.

Hb (g/dL) RBC ×106 cumm MCV (fL) MCH (pg) MCHC (g/dL) HbA (%) HbA2 (%) HbF (%) Other (%) Hb variant 10.0 ± 2.1 4.8 ± 1.02 70.7 ± 9.7 21.05 ± 3.2 29.7 ± 2.7 89.0 ± 2.5 5.2 ± 0.75 1.08 ± 0.7 BTT 4.78 ± 3.4 2.2 ± 1.3 73.2 ± 5.1 20.9 ± 2.1 28.5 ± 2.8 23.5 ± 22.6 4.06 ± 1.2 64 ± 28.6 BTM/I 10.7 ± 2.4 4.2 ± 1.05 81.8 ± 1.2 25.3 ± 3.4 31.5 ± 2.2 60.9 ± 6.6 26.6 ± 4.5 1.08 ± 1.2 HbAE 11.6 ± 1.0 5.9 ± 0.7 60.4 ± 0.7 19.8 ± 1.5 32.3 ± 2.1 3.8 ± 0.86 79.9 ± 2.5 3.92 ± 0.1 HbEE 8.5 ± 1.9 5.16 ± 1.5 63.4 ± 2.3 20.8 ± 1.3 30.3 ± 2.3 17.5 ± 9.65 61.6 ± 8.1 9.75 ± 4.7 HbE-BT 11.6 ± 2.0 4.3 ± 0.41 81.4 ± 7.3 26.2 ± 3.5 32.2 ± 2.8 52.4 ± 2.6 2.14 ± 0.5 1.02 ± 1.6 HbD36.7 ± 2 HbAD 9.65 ± 1.6 4.4 ± 0.97 67.9 ± 4.3 21.8 ± 1.8 32.1 ± 0.7 3.95 ± 2.3 4.67 ± 0.87 3.95 ± 2.3 HbD 79.7 ± 1.6 HbD-BT 9.6 ± 3.4 3.8 ± 1.4 79.6 ± 13.3 23.7 ± 5.16 30.4 ± 2.03 30.4 ± 2.03 3.25 ± 0.4 2.4 ± 2.9 HbS 29.7 ± 10.4 HbAS 11.7 ± 0.46 4.9 ± 0.49 76.3 ± 3 23.3 ± 1.5 31.4 ± 0.17 75.3 ± 0.19 2.43 ± 0.17 15.7 ± 2.6 deltaBTT 5.9 4.23 66.9 13.9 20.8 75.2 2 1.1 P3 20.0 Hb J Meerut 16.7 5.31 94.5 31.4 33.3 44.8 2.0 <1 P2 48.8 Hb Hope relevant RBC parameters and HPLC fndings are given in Majority were asymptomatic and detected during carrier Table 2. Chromatograms of some important variants are screening and family studies. shown in fgures (Figures 1, 2, 3, 4, 5,and6). Nine cases of beta thalassemia were detected which were Genotypes of some structural variants are shown in classifed as either major or intermedia depending upon Table 3. clinical severity. HbF was raised (20.7–97.1%), with variable As expected, beta thalassemia trait (BTT) was the most HbA2 (2.4–5.7%). Cases of thalassemia major presented common hemoglobin variant (74.48%) detected in our study within 1st year of life. Parental study was done to fnd out with elevated HbA2 level (>3.5%) and RT 3.63–3.69 min. carrier status and to confrm the diagnosis. 4 International Scholarly Research Notices

Calibrated Retention Peak Peak name area (%) Area (%) time (min) area 0.0 0.98 1013 Unknown — Peak name Calibrated Area (%) Retention Peak F 0.2 — 1.07 6584 area (%) time (min) area 1 — 0.1 0.73 866 Unknown — 0.7 1.25 19627 P ∗ 2 2.7 76646 F 30.1 — 1.12 470514 P — 1.35 2.7 0.7 1.52 18463 P3 — 1.83 42498 Unknown — 0.8 2.03 12667 Unknown — 2.0 1.74 55348 Unknown — 1.83 62326 Unknown — 4.0 2.25 62942 P3 — 2.2 0 7.0 2.48 109207 0 — 63.3 2.42 1778805 A — ∗ A A2 51.4 — 3.75 868101 A2 24.8∗ — 3.72 791409 1 566 79 Total area: 2,810,221 Total area: , , ∗ F concentration = 0.2% F concentration = 30.1 % ∗ ∗ A2 concentration =24.8 % A2 concentration =51.4 % ∗ ∗ Values outside of expected ranges Values outside of expected ranges Analysis comments: Analysis comments: 45.0 45.0

37.5 37.5 3.72

30.0 2 30.0 A 22.5 22.5 (%) (%) 15.0 15.0 2.48 1.35 1.74 1.63 2.25 1.52 1.25 1.07 1.83 2.03 0.98 0.73

7.5 1.12

7.5 3.75 2.43

0.0 F 0.0 F A 2

0 123456 0 123456 Time (min) Time (min) (a) (b)

Figure 2: (a) Chromatogram of HbE trait showing HbA2 24.8% (RT 3.72 min). (b) Chromatogram of E beta thalassemia showing elevated HbA2 51.4%, Hb F 30%.

Table 3: Genotypes of some common structural variants. with raised RBC counts and reduced MCV and MCH values. Parental study was carried out for confrmation. Abnormal variants Genotype Two cases of double heterozygous of HbE and beta Hb E beta26(B8)Glu → Lys, GAG → AAG thalassemia trait were found showing peak in HbA2 window Hb D Punjab beta121(GH4)Glu → Gln, GAA → CAA (53.5–69.7%) and raised HbF level (5–14.5%). Again, parental Hb S beta6(A3)Glu → Val, GAG → GTG and family study was carried out for confrmation. Hb D Iran beta22Glu → Gln, GAA → CAA NineteencasesshowedpeakintheDwindow(RT Hb Hope beta136(H14)Gly → Asp (GGT → GAT) 4.13–4.15 min), indicating presence of structural variant Hb OArab beta121(GH4)Glu → Lys, (GAA → AAA) hemoglobin D (Hb D) Punjab. Among these, 15 were HbD Hb C beta6(A3)Glu → Lys, GAG → AAG Punjab heterozygote showing Hb D level 31–40% and near Hb J Meerut alpha120(H3)Ala → Glu, GCG → GAG normal RBC parameters. Four cases had HbD level 77– 81.3%, with raised HbA2 levels (3.8–6%). Tese cases showed Hb Q India alpha64(E13)Asp → His, GAC → CAC reducedhemoglobin,MCV,andMCHlevels.Teywere provisionally diagnosed as double heterozygous of Hb D and beta thalassemia trait. Parental study and genetic study were done for confrmation. Tree cases showed raised HbF (13.7–19.7%), normal Nine cases had peak in S window (RT 4.27-4.28 min), HbA2 values (2.2-2.3%), and normal Hb level, raised RBC indicating presence of hemoglobin S (HbS). Eight of them count, and reduced MCV and MCH levels. Parental study was had HbS level 25–40%, HbF-0.2–7.4%. Mildly raised HbA2 done with a provisional diagnosis of delta beta thalassemia (3.3–3.7%) level was seen in 5 of them. Tere was one case trait and DNA study was advised. with marked reduction in Hb, MCV, and MCH levels and Hemoglobin E (HbE) was found to be the most common HbA2 10% and HbS 65.4%. Parental study was advised to structural variant with raised peak in A2 window (RT 3.76– confrm the double heterozygous status. Sickling test was 3.78 min). A total of 28 cases of Hb E trait were detected with foundtobepositiveinallthesecases. peak area ranging from 18.5 to 39% and mild elevation of HbF OnecaseshowedelevatedP3peak20%,withRT1.81min, level. Two cases of homozygous Hb E were detected showing suggesting Hb J Meerut with reduced MCV and MCH values. 77–83% HbE and 2–5% HbF. Hb level was mildly reduced Iron studies and family study were correlated. International Scholarly Research Notices 5

Calibrated Retention Peak Peak name area (%) Area (%) time (min) area ∗ 1.10 25943 Calibrated Retention Peak F 2.0 — Peak name area (%) Area (%) time (min) area 1.74 11676 ∗ Unknown — 0.8 F 10.8 — 1.12 99893 P3 — 3.4 1.88 48007 P2 — 3.2 1.35 29854 Unknown — 0.6 2.09 7847 P3 — 2.7 1.71 25154 A0 — 3.4 2.32 46975 A0 — 54.5 2.54 513793 A2 77.5∗ — 3.73 1255408 A2 3.0 — 3.63 30726 25.9 4.42 244100 Total area: 1,395,856 S window — ∗ F concentration = 2.0 % Total area: 943,519 ∗ ∗ F concentration = 10.8 % A2 concentration = 77.5 % A2 concentration =3.0% ∗ ∗ Values outside of expected ranges Values outside of expected ranges Analysis comments: Analysis comments: 45.0 45.0

37.5 37.5 4.42

30.0 30.0

22.5 22.5 1.12 (%) (%)

15.0 15.0 F 3.63 1.35 1.86 1.71 1.10 1.74 7.5 2.32 7.5 3.73 2 2.09 A F 2.54

0.0 2 0.0 A

0 123456 0 123456 Time (min) Time (min) (a) (b)

Figure 3: (a) Chromatogram of HbE homozygous showing HbA2 77.5%(RT 3.73 min). (b) Chromatogram of Hb S trait showing Hb S 25.9% (RT 4.42 min).

Onecaseshowedelevatedpeak(RT1.37min),48.8%with None of the above-mentioned methods can detect multiple normal hematological parameters. It was provisionally diag- hemoglobin fractions in a single step procedure. HPLC has nosed as Hb Hope afer further confrmation by hemoglobin many advantages over these methods and over the past electrophoresis. Family study and DNA analysis were advised. decades it has evolved as an excellent and powerful diagnostic tool for identifcation of most of the clinically signifcant 3.2. Discussion. India is an ethnically diverse country with Hb variants especially to beta thalassemia trait owing to marked regional variation. Tis diversity is refected in the its quantitative power and automation. HPLC is sensitive, presence of diferent hemoglobin variants in diferent ethnic specifc, reproducible, and less time consuming and requires groups. Due to migration, there is constant mixing of peoples less man power. Hence, it is ideal for a routine clinical from diferent regions. Many of these abnormal variants are laboratory with high work load. of little clinical signifcance in heterozygous state, but when Our study was carried out in a tertiary care hospital in combined with other variants they may give rise to severe Delhi, which represented not only north Indian population disease. Terefore there is always a need for a screening but a large migrant population of eastern and north east- method which can detect maximum variants. HPLC has ern parts of the country also. Apart from �-thalassaemia, the advantage of quantifying Hb F and Hb A2 along with common variants were encountered with various incidences: detecting other variants in a single screening test. HbE,HbDPunjab,andHbS—beingthemostcommonalong Besides HPLC, there are other analytical procedures used with a rare variant Hb Hope. for detection of thalassemia and hemoglobinopathies such Talassemia being the major concern in this study, as alkaline and acid electrophoresis, Hb A2 quantifcation quantitation of HbA2 and Hb F levels by HPLC was of prime by ion-exchange column chromatography, and Hb F quan- importance in our laboratory where facilities for genetic tifcation by alkali denaturation and radial immunodifusion studies are not available. However, parental study is also (9). Electrophoretic method does not separate all variants of great help in arriving at a conclusive diagnosis before from each other, and it is recommended by screening pro- referring patients for costly genetic studies. grams such that further tests should always be carried out HbA2 is constantly elevated in heterozygous beta tha- to confrm the presumed identity of an abnormal variant. lassemia carriers with values ranging from 3.5 to 7% with a 6 International Scholarly Research Notices

Calibrated Peak Peak name Retention area Calibrated Area (%) Retention Peak area (%) Area (%) time (min) Peak name area (%) time (min) area Unknown 0.0 0.96 720 0.1∗ 1.06 2002 — ∗ F — F 0.1 — 1.06 3545 Unknown — 0.5 1.21 6574 Unknown — 0.9 1.21 20718 P2 — 0.1 1.40 1014 P2 — 1.9 1.34 46113 P3 — 0.3 1.79 3598 Unknown — 0.4 1.52 9265 A0 — 4.2 2.13 60542 P3 — 2.1 1.72 51629 Unknown — 0.6 2.36 9102 1.98 2.52 Unknown — 0.5 12855 Unknown — 1.1 15738 Unknown — 2.0 2.13 47777 Unknown — 2.6 2.88 36721 A0 — 49.1 2.46 1194355 A2 2.4 — 3.67 38898 A2 2.3 — 3.69 60767 D window — 87.9 4.20 1263052 D window — 40.5 4.15 985084 Total area: 1,437,242 ∗ 2 432 82 = 0.1∗ F concentration = 0.1 % Total area: , , F concentration % 2 = 2.4% A2 concentration = 2.3% A concentration ∗ ∗ Values outside of expected ranges Values outside of expected ranges Analysis comments: Analysis comments: 45.0 45.0 37.5 37.5 30.0 30.0 22.5 22.5 (%) (%)

15.0 15.0 3.67 2.23 2.13 1.06 1.34 1.73 1.21 1.79 1.92 1.98 2.36 2.52 1.06 1.21 1.40 2.88 0.96

7.5 2.46 7.5 4.20 A 2 3.15 A 2 3.69 F F 0.0 0.0

0 1234 5 6 0 123456 Time (min) Time (min) (a) (b)

Figure 4: (a) Chromatogram of HbD Punjab trait showing HbD 40.5% (RT 4.15 min). (b) Chromatogram of Hb D Punjab homozygous showing Hb D 87.9%. mean of 5% [1]. During routine reporting, we faced certain was 2.0–3.3%. Few cases with macrocytic RBC indices had problems, regarding the cut of value of HbA2 for beta values as high as 3.4%. thalassemia trait. Diferent authors have established diferent Te variation of cut of value as well as borderline cut of values for HbA2 for diagnosis of beta thalassemia value by the same method leads to confusion resulting in trait, which ranges from 3.5 to 4%, although it has been underdiagnosis or overdiagnosis. In such situations, genetic recommended that each laboratory needs to establish their counseling with DNA analysis should be recommended. But own normal ranges [4, 11]. Rangan et al. used the term in a resource-limited country where population is huge but borderline to HbA2 levels 3.0-4.0% and found mutations in facilities of genetic or molecular tests are available only in a 32% people with HbA2 3.4–3.9% [12]. Similar fndings are few research laboratories, we may refer only antenatal cases described by Colah et al. [11]. In regions with high prevalence for genetic studies if one partner is a trait. For other cases, we of beta thalassemia researchers have taken variable values as can opt for parental study if possible or iron studies before referring them for costly genetic test. borderline, for example, 3.3–3.7% [13] and 3.1–3.9% [14]. On the other hand, there were few cases with symp- Iron defciency anaemia is a very common occurrence in tomatic refractory microcytic hypochromic anaemia with most of the screening populations, namely, school children borderline, normal, or reduced HbA2 levels. Tese cases and pregnant women in India. Tere are several studies should be investigated for presence of alpha thalassemia or its regarding the impact of iron defciency on HbA2 level and coinheritance with beta thalassemia gene. Alpha thalassemia controversy over its signifcance in screening of beta tha- is by far the commonest hemoglobinopathy in India, with lassemia trait [4, 15, 16]. Like Denic et al., we also suggest that highestprevalenceinPunjabipopulationinthenorthern concurrent iron defciency anaemia should be considered region [17]. Molecular genotyping of �-thalassemia helps in cases of borderline HbA2 with microcytic hypochromic to diagnose unexplained microcytosis and thus prevents anemia. On the other hand, we came across few cases with unnecessary iron supplementation [18]. 3.4–3.6% HbA2 with normal MCV and MCH values. It is Hb E, the most common structural variant in our study, is difcult to determine whether they are carriers of silent one of the world’s important mutations. Hb E trait, homozy- mutations or high normal HbA2 without genetic test. In our gous Hb E disease (Hb EE), and Hb E beta thalassemia are laboratory, range of HbA2 value with normal RBC parameter common in north eastern part of the country [19]. Hb E trait International Scholarly Research Notices 7

Peak name Calibrated Area (%) Retention Peak area (%) time (min) area Unknown — 0.2 0.91 6267 F 0.2 — 1.04 5136 — 0.6 1.21 17393 Peak name Calibrated Retention Peak Unknown area (%) Area (%) time (min) area 2 — 2.3 1.32 69227 ∗ P F 5.8 — 1.08 87528 P3 — 5.0 1.71 150318 P2 — 6.4 1.32 99803 Unknown — 0.9 2.14 28115 4.3 1.70 67364 2.44 P3 — A0 — 46.4 1397825 0 72.9 2.44 1145749 2 41.5∗ 3.60 1337204 A — ∗ A — A2 10.1 — 3.67 170271 Total area: 3,011,48 ∗ F concentration =5.8 % Total area: 1,570,71 F concentration = 0.2% 2 = 10.1∗ A concentration % 2 = 41.5∗ ∗ A concentration % Values outside of expected ranges ∗ Values outside of expected ranges Analysis comments: Analysis comments: 45.0 45.0

37.5 37.5

30.0 30.0

22.5 22.5 (%) (%) 3.67 15.0 15.0 1.06 1.71 1.32 A 2 1.32 F 2.14 1.70

7.5 7.5 1.04 1.21 0.91 2.44 2.44 0.0 0.0 F A 2 3.60

0 123456 0 123456 Time (min) Time (min) (a) (b)

Figure 5: (a) Chromatogram of showing HbA2 10.1% presumptive diagnosis of Hb Lepore. (b) Chromatogram of Hb D Iran showing HbA2 41%. and Hb EE are mild disorders. Detection of this variant is very It has been reported that coinheritance of beta tha- important because when combined with thalassemia or HbS, lassemia and HbD Punjab may result in symptomatic disease itgivesrisetomoderatetoseveredisease. and its detection is important in prenatal diagnosis. In cases, We observed that distinction between homozygous HbE where HPLC or Hb electrophoresis shows very high HbD disease and HbE beta thalassemia sometimes become dif- level and negligible HbA, one should look for raised HbA2 cult as both of these conditions show variably elevated HbE level and do parental study or DNA analysis for confrmation and Hb F with marked microcytic hypochromic picture. Hb of coinheritance. D Iran also comes as a diferential diagnosis. Compared to Hb A total of nine cases of Hb S were detected of which 5 cases Etrait,HbDIrantendstohavemoreareapercentageatthe showed elevated Hb A2 level (3.3−3.7%) with 25–40% HbS window of Hb A2 (usually more than 40%). Parental study, if and moderate to severe anaemia. Te mean values of HbA2 possible, may easily resolve the problem. Otherwise, genetic have been reported to be elevated in sickle cell syndrome in study should be done. previous reports [23, 24]. Te reason may be HbS adducts Hb D Punjab was the second most common structural (carbamylated and glycated) coeluting with HbA2. Parental variant in our study, mostly presented as asymptomatic het- studyshouldbedoneinsuspiciouscasesbeforereporting erozygous condition with normal hematological parameters. such case as sickle-beta thalassemia. Hb D Punjab occurs with greatest prevalence, that is, 2% We reported 3 cases of delta-beta thalassemia trait. among Sikhs in Punjab, and in Gujarat, reported prevalence Delta-beta thalassemia and hereditary persistence of fetal is 1% [20]. Hb D Punjab in the form of heterozygote hemoglobin (HPFH) constitute a heterogeneous group of Hb D trait, Hb S-D disease, and Hb D-thalassemia are disorders characterized by absent or reduced synthesis of commoner forms; however, homozygous form is very rare adult hemoglobin (Hb A) and increased synthesis of fetal [20, 21]. Few detailed studies are available on clinical, hema- hemoglobin (Hb F). Te distinction between HPFH and tological, and molecular analyses of Hb D Punjab in India delta-beta thalassemia is subtle and should be confrmed by [21, 22]. alpha-beta-globin chain synthesis ratio and DNA analysis 8 International Scholarly Research Notices

Calibrated Area (%) Retention Peak Peak name area (%) time (min) area 1 0.4 0.71 4107 Calibrated Retention Peak P — Peak name area (%) Area (%) time (min) area Unknown — 0.1 0.97 810 P1 — 0.9 0.74 26261 F 0.8 — 1.08 8958 F 0.2 — 1.10 6469 Unknown — 0.9 1.23 9703 P2 — 48.4 1.37 1387977 P2 — 2.1 1.34 23468 P3 — 2.0 1.73 58281 P3 — 25.5 1.69 290435 A0 — 45.0 2.50 1290273 A0 — 68.7 2.37 783063 2 3.0 96326 ∗ A — 3.69 A2 1.5 — 3.61 19024 Total area: 2,865,588 Total area: 1,139,569 F concentration = 0.2% A0 concentration =3.0% F concentration = 0.8% ∗ ∗ Values outside of expected ranges A0 concentration = 1.5 % ∗ Values outside of expected ranges Analysis comments: Analysis comments: 45.0 45.0

37.5 37.5 2.50 30.0 30.0

22.5 22.5 (%) (%) 15.0 15.0 3.61 3.69 1.34 1.09 0.71 0.07 1.23 7.5 1.10 7.5 0.74 1.73 A 2 2.37 A 2 1.69 F 1.37 F 0.0 0.0

0 123456 0 123456 Time (min) Time (min) (a) (b)

Figure 6: (a) Chromatogram of Hb Hope showing elevated P2 peak (48.4%). (b) Chromatogram of Hb J showing elevated P3 peak. since the distinction between these two conditions is not startofintegration[29]. We, however, did not fnd any such always possible from routine hematologic analyses. It is case. important to diferentiate between these two conditions Tis automated HPLC system is intended for separation especially in antenatal screening because HPFH is clinically and determination of area percentage for HbA2 and HbF and asymptomatic, but interaction of ��-thalassemia with �- for qualitative detection of abnormal hemoglobins. With the thalassemia can result in a severe disorder. integration of proper algorithms involving retention time, % Hb J Meerut was another rare variant detected in our Hb, and peak characteristics, a clinical laboratory is capable of study. Tis clinically asymptomatic variant has been reported identifying 75% of the common variants encountered without to interfere with HbA1c estimation [25]andtomaskbeta the need for confrmatory studies such as alkaline and acid thalassemia trait [26]. electrophoresis [9]. In the light of family study, ethnicity and Among the rarely encountered variants we detected one clinical data, we can detect most of the frequently occurring case of Hb Hope. In cation exchange HPLC method, it clinically signifcant variants by HPLC method alone. maybemistakenashighHbA1c,asitcomesinthesame HPLC has some limitations, including falsely decreased position as HbA1c. Hb Hope (beta 136(H14) Gly → Asp HbA2 levels in patients with the HbD Punjab trait, falsely (GGT → GAT)) is one of the unstable haemoglobin variants increased HbA2 levels in patients with HbS, and coelution of the beta-globin chain, which has been demonstrated in of various Hbs, including HbE, Hb Osu Christianborg, HbG people of various ethnic backgrounds. Few case reports of Coushatta, and Hb Lepore with HbA2 [30]. Interference Hb hope and its association with thalassemia and Hb E have from endogenous compounds or from drugs is not frequently been published from southeast Asia [27, 28]. Tis variant reported in literature. Howanitz et al. reported unknown is clinically silent and when associated with thalassemia tall peaks with elution times and shapes of hemoglobin may produce mild to moderate symptoms depending upon Barts on hemoglobin chromatograms that could not be genotype. confrmed by alkaline and acid gel electrophoresis mostly It has been reported that one can detect HbH form in patients with hemoglobin SS [31]. Te authors provided of alpha thalassemia on HPLC by visual analysis of the evidence that the peak is not hemoglobin Barts, but rather chromatogram plot as it produces a sharp peak before the bilirubin, and recommended exclusion of bilirubin before International Scholarly Research Notices 9 chromatographic results are interpreted as consistent with chromatography: its use in diagnosis and in the assessment of hemoglobin Barts. Determination of HbA2 and HbF is not cellular distribution of hemoglobin variants,” American Journal interfered by endogenous substances like triglyceride and of Hematology,vol.17,no.1,pp.39–53,1984. bilirubin levels up to 4600 mg/dL and 20 mg/dL, respectively [9] A. Joutovsky, J. Hadzi-Nesic, and M. A. Nardi, “HPLC reten- [10]. In diabetic patient labile HbA1c greater than 2.5% may tion time as a diagnostic tool for hemoglobin variants and adversely afect HbF quantitation [10]. In our study we did hemoglobinopathies: a study of 60000 samples in a clinical not experience such interference. diagnostic laboratory,” Clinical Chemistry, vol. 50, no. 10, pp. 1736–1747, 2004. [10] VARIANT II b thalassemia short program instruction manual. 4. Conclusion [11] R. B. Colah, R. Surve, P. Sawant et al., “HPLC studies in hemo- globinopathies,” IndianJournalofPediatrics,vol.74,no.7,pp. To conclude, RBC indices, HPLC fnding, and family study 657–662, 2007. are sufcient to detect and manage most of the hemoglobin variants prevalent in this country. However, one has to be [12] A. Rangan, P. Sharma, T. Dadu, R. Saxena, I. C. Verma, and M. Bhargava, “B-Talassemia mutations in subjects with borderline aware of the limitations and problems associated with the HbA 2 values: a pilot study in North India,” Clinical Chemistry diagnostic methods to avoid false negative diagnosis in day and Laboratory Medicine,vol.49,no.12,pp.2069–2072,2011. to day practice. Genetic studies are indicated to confrm bor- [13]A.Mosca,R.Paleari,R.Galanelloetal.,“Occurrenceof derline cases and to detect silent carriers of beta thalassemia, HbA2 borderlinephenotypes in areas with high prevalence of alpha thalassemia, and rare and novel variants in routine thalassemia,” in Proceedings of the 16th Meeting on EARCR, practice.TepresentstudyconductedusingHPLCrefects Oxford, UK, 2007. the magnitude of thalassemia and hemoglobinopathies in a [14] A. Giambona, C. Passarello, M. Vinciguerra et al., “Signifcance small hospital based population which may be in fact the tip of borderline values in an Italian population of an iceberg, but this type of study can defnitely help to with a high prevalence of �-thalassemia,” Haematologica,vol. increase awareness among both health care givers and general 93,no.9,pp.1380–1384,2008. population. [15]C.Passarello,A.Giambona,M.Cannata,M.Vinciguerra,D. Renda, and A. 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