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Year : 2014  |  Volume : 5  |  Issue : 1  |  Page : 19-22

Transfusion associated unknown peak on HPLC chromatogram: A diagnostic dilemma

1 Department of Pathology, Lady Hardinge Medical College and Kalawati Saran Children's Hospital, New Delhi, India
2 Department of Pediatrics, Lady Hardinge Medical College and Kalawati Saran Children's Hospital, New Delhi, India

Date of Web Publication2-May-2014

Correspondence Address:
Sunita Sharma
Department of Pathology, Lady Hardinge Medical College, New Delhi - 110 001
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1658-5127.131821

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A pre-transfusion blood sample with a well-spread and well-stained peripheral smear is indispensible for diagnosis of hemolytic anemia. However, apparent hemoglobinopathies have been reported in previous literature. They are known to cause delay in diagnosis and treatment. We report a case of newly diagnosed β-thalassemia major infant, in which an abnormal peak was seen at retention time 4.71 minutes on Hemoglobin-High Performance Liquid Chromatography (Hb-HPLC). This peak was associated with history of recent blood transfusion and was confirmed by family study and with repeated values of HPLC.

Keywords: Hemoglobinopathy, high performance liquid chromatography, transfusion, β-thalassemia major

How to cite this article:
Chauhan R, Sharma S, Rai P, Chandra J. Transfusion associated unknown peak on HPLC chromatogram: A diagnostic dilemma. J Appl Hematol 2014;5:19-22

How to cite this URL:
Chauhan R, Sharma S, Rai P, Chandra J. Transfusion associated unknown peak on HPLC chromatogram: A diagnostic dilemma. J Appl Hematol [serial online] 2014 [cited 2020 Apr 1];5:19-22. Available from: http://www.jahjournal.org/text.asp?2014/5/1/19/131821

  Introduction Top

Hemoglobin-High Performance Liquid Chromatography (Hb-HPLC) is routinely performed to diagnose various hemoglobinopathies. Scientific literature describes a few unusual peaks in HPLC chromatograms that led to diagnostic dilemma and delay in treatment. Recent blood transfusion from donors who are the carriers of hemoglobinopathy can lead to spurious peaks in Hb-HPLC chromatograms.

  Case Report Top

A 7-month-male, from Western Uttar Pradesh, India was brought to the outpatient department with complaints of increasing pallor and distension of abdomen. Clinical examination revealed pallor and hepatosplenomegaly.

Laboratory investigations including complete blood counts (CBC) and peripheral blood film examination were done. CBC was obtained using Sysmex KX-21 autoanalyzer. HPLC was performed on 2 mL ethylenediaminetetraacetic acid (EDTA) blood sample using Bio-Rad VARIANT™ II instrument with β-thalassemia short program.

Hemogram showed hemoglobin 79 gm/L (126 ± 15 g/L), total leukocyte count 18.3 × 10 9 /L (12 ± 6 × 10 9 /L) and platelet count 130 × 10 9 /L (200-550 × 10 9 /L). Red cell count was 2.5 × 10 12 /L (4.7 ± 0.6 × 10 12 /L) and Red Cell Distibution Width (CV) was 44.7% (12.8 ± 1.2%). Mean cell volume, mean cell hemoglobin and mean cell hemoglobin concentration were 65.9 fL (76 ± 8 fL), 21.4 pg (27 ± 3 pg) and 325 g/L (330 ± 30 g/L), respectively. Corrected reticulocyte count was 4.8%. Peripheral blood film showed leukoerythroblastic picture. Twenty nucleated red cells per 100 WBCs were present. Red cells were microcytic hypochromic with marked anisopoikilocytosis and presence of elliptocytes, target cells, tear-drop cells and polychromatophils. Few normocytic normochromic red cells were also seen. A possibility of β- thalassemia major was suggested and Hb-HPLC was advised.

Hb-HPLC chromatogram revealed raised Hb F (47.6%), with Hb A 0 (35.9%), Hb A 2 (3.5%) and a peak of 9.1% at retention time (RT) of 4.71 minutes [Figure 1].
Figure 1: Chromatogram of the patient

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In view of chromatogram findings, possibility of interaction of heterozygous Hb Q India and β- thalassemia major was considered. However, value of Hb A 0 was high. Therefore, prior history of blood transfusion was taken. The patient had received one unit of packed cells from a private hospital a week before presentation. This raised a possibility of transfusion associated peak at 4.71 minutes in otherwise β- thalassemia major patient. Family study was performed. Both mother and father showed evidence of heterozygous state for β-thalassemia on Hb-HPLC. [[Figure 2] a and b] However, there was no abnormal peak at RT 4.71 minutes in both the parents. The donor segment could not be tested due to history of blood transfusion from a private center. The patient was followed up at thalassemia day care center and another sample was taken 4 weeks later. There was disappearance of the transfusion-associated peak at RT 4.71 minutes. Thus, peak at 4.71 minutes was attributed to prior blood transfusion and a final diagnosis of β-thalassemia major with transfusion-associated peak at RT 4.71 minutes was made.
Figure 2: (a) Chromatogram of mother (b) Chromatogram of father

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  Discussion Top

Transfusion-associated hemoglobinopathies are uncommon cause of misdiagnosis in the interpretation of Hb-HPLC chromatograms. This can lead to repeated testing and delay in diagnosis and treatment. [1] In some cases, transfusion-associated abnormal hemoglobins have led to disastrous consequences, for example, significant morbidity in an infant due to transfusion of blood from sickle cell trait donor has been reported. [2] Therefore, careful interpretation and follow-up is necessary to make a correct diagnosis.

Gupta et al., have described 3 cases of β-thalassemia major with transfusion-associated peaks. [1] The percentage of these hemoglobins ranged from 9.9 to 18.5 in the 'S' window. Kzarski et al., defined transfusion-induced hemoglobinopathy as the transfusion of a unit of non-AA blood that could lead to the consideration of an alternate hemoglobin variant in the recipient. They detected 52 occurrences of transfusion induced hemoglobinopathies in 32 recepients including Hb C, Hb S, Hb O Arab. [3] The initial percentage of abnormal hemoglobins in the recipients ranged from 0.8 to 14% in the study. Other studies describe the occurrence of Hb S, Hb C, Hb O Arab, Hb D and of β-thalassemia trait associated with blood transfusion. [3],[4],[5],[6],[7]

Hb Q India, a rare structural alpha chain variant, has a retention time of 4.7 ± 0.01 minutes. [8] Hb Q India heterozygotes have a value of 20% and are clinically asymptomatic. However, interaction of Hb Q India with β-thalassemia leads to reduction in its level to 7-9% due to post-translational control mechanism. [9] Thus, a hemoglobin peak of 9.1% observed at RT of 4.71 minutes in the present case could be an interaction of Hb Q India with β-thalassemia or transfusion-associated peak from asymptomatic Hb Q India heterozygous donor. Absence of this peak in both the parents and its disappearance from latter sample confirmed the diagnosis of transfusion-associated hemoglobinopathy. Joutovsky et al. have described Hb Hasharon at the RT 4.70-4.90 minutes. [10] However, Hb Hasharon is found in Jewish families of Ashkenazi origin in Israel and thus was excluded. [11]

Abnormal peaks on Hb-HPLC chromatograms can be detected by (1) sudden appearance or disappearance of abnormal peak and (2) low percentage of abnormal hemoglobins. [3] Family studies of parents and siblings should be done to rule out such incidental hemoglobinopathies.

This case highlights the importance of careful interpretation of Hb-HPLC data. A proper workup by taking history of recent blood transfusion, family study and follow-up should be done to rule out transfusion-associated hemoglobinopathy. Testing donor segments can be done to confirm the occurrence of hemoglobinopathy in the donor. [3] Some authors have suggested screening of blood donors for hemoglobinopathies. [2],[4] Also, this case emphasizes the importance of pre-transfusion sampling for Hb-HPLC to overcome such diagnostic challenges. If possible, testing for Hb-HPLC should be delayed in case of already transfused subjects. Family study can be performed meanwhile to assist in reaching a diagnosis.

  References Top

1.Gupta SK, Sharma M, Tyagi S, Pati HP. Transfusion induced hemoglobinopathy in patients of beta-thalassemia major. Indian J Pathol Microbiol 2011;54:609-11.  Back to cited text no. 1
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2.Novak RW, Brown RE. Multiple renal and splenic infarctions in a neonate following transfusion with sickle trait blood. Clin Pediatr (Phila) 1982;21:239-41.  Back to cited text no. 2
3.Kozarski TB, Howanitz PJ, Howanitz JH, Lilic N, Chauhan YS. Blood transfusions leading to apparent hemoglobin C, S, and O Arab hemoglobinopathies. Arch Pathol Lab Med 2006;130:1830-3.  Back to cited text no. 3
4.Lippi G, Mercandante M, Alberta C, Franchini M. An unusual case of a spurious, transfusion-acquired haemoglobin S. Blood Transfus 2010;8:199-202.  Back to cited text no. 4
5.Suarez AA, Polski JM, Grossann BJ, Johnston MF. Blood transfusion-acquired hemoglobin C. Arch Pathol Lab Med 1999;123:642-3.  Back to cited text no. 5
6.Jain S, Dass J, Pati HP. Transfusion associated peak in Hb-HPLC chromatogram- a case report. Mediterr J Hematol Infect Dis 2012;4:e2012006.  Back to cited text no. 6
7.Wong P, Tapprom A, Jermnim N, Charoenporn, Kanthiyawong S. A blood transfusion leading to misdiagnosis of beta-thalassaemia carrier status. Blood Transfus 2010;8:69-70.  Back to cited text no. 7
8.Rao S, Kar R, Gupta SK, Chopra A, Saxena R. Spectrum of haemoglobinopathies diagnosed by cation exchange-HPLC and modulating effects of nutritional deficiency anaemias from north India. Indian J Med Res 2010;132:513-9.  Back to cited text no. 8
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9.Phanasgaonkar S, Colah R, Ghosh K, Mohanty D, Gupte S. Hb Q (India) and its interaction with beta-thalassaemia: A study of 64 cases from India. Br J Biomed Sci 2007;64:160-3.  Back to cited text no. 9
10.Joutovsky A, Hadzi-Nesic J, Nardi MA. HPLC retention time as a diagnostic tool for hemoglobin variants and hemoglobinopathies: A study of 60000 samples in a clinical diagnostic laboratory. Clin Chem 2004;50:1736-47.  Back to cited text no. 10
11.Alberti R, Mariuzzi GM, Marinucci M, Bruni E, Tenteri L. Haemoglobin Hasharon in a north Italian community. J Med Genet 1975;12:294-6.  Back to cited text no. 11


  [Figure 1], [Figure 2]


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