Indian J Hematol Blood Transfus

https://doi.org/10.1007/s12288-018-1004-3

CORRESPONDENCE

Elevated HbF Labelled as LA1C/cHb1 on BioRad D10 HPLC:

Missed Diagnosis of Homozygous Beta Thalassemia
Shruti Vaswani1 • Ranjeet Singh Mashon1 • Naveen Kakkar1

Received: 9 April 2018 / Accepted: 2 August 2018

Ó Indian Society of Hematology and Blood Transfusion 2018

Accurate and timely diagnosis, genetic counselling and elution region were misinterpreted as HbA0. Since the
prenatal diagnosis form the basis of control of thalassemias patient had been recently transfused, the diagnosis of
[1]. High performance liquid chromatography (HPLC) is a homozygous b thalassemia at our center was confirmed by
rapid, reproducible and accurate technique for the diag- DNA mutation study, which showed compound heterozy-
nosis of inherited hemoglobin disorders [2]. The Bio-Rad gosity for 619 bp del and CD8/9 (? G) mutation.
D10 hemoglobin testing system is an automated cation Case 2: A 2 years old girl presented with transfusion
exchange HPLC instrument for screening of inherited dependent severe anemia since 6 months of age and hep-
hemoglobin disorders. These are diagnosed by interpreta- atosplenomegaly. CBC showed microcytic hypochromic
tion of the chromatograms for percentages and retention anemia (Table 1). HPLC done outside showed HbA-
time [3]. Erroneous interpretation of the peaks may lead to 96.6%, HbA2-1.8% and no HbF. The clinician at our center
misdiagnosis with adverse clinical consequences. We pre- re-ordered HPLC as the clinical profile suggested beta
sent two patients in whom the diagnosis of b-thalassemia thalassemia major. HPLC showed raised HbF level, which
major was missed due to misinterpretation of HPLC however eluted in the adjacent peaks A1b (16.6%) and
chromatogram. LA1C/cHb1 (78.6%) with HbA2-2.7% and HbA0-1%.
Case 1: An 8 months old male child was referred with Outside HPLC chromatogram showed HbF elution in the
severe transfusion dependent anemia. Complete blood adjacent peaks-A1b (17.9%) and LA1C/cHb1 (73.6%),
count (CBC) showed microcytic hypochromic anemia which were misinterpreted to be HbA0 and the child was
(Table 1). HPLC done outside showed the child to be reported as normal (Fig. 1).
normal, mother as b thalassemia trait (HbA2-5.7%), while Accurate diagnosis of thalassemia is a key step in
the father’s report was not available. In view of clinical management and formulation of prevention strategies in
suspicion of thalassemia major, retesting was requested. the rest of the family members. HPLC plays a key role in
HPLC at our center showed HbF-23%, HbA2-4.2% and diagnosis of haemoglobin disorders. However, like any
HbA-55.9%. In view of raised HbF level and history of other technique, this system has its own limitations.
recent blood transfusion, thalassemia major was suspected. According to the manufacturer’s guidelines on Biorad D10
The outside HPLC report showed HbA1b (15.2%) and system, when HbF level is [ 16.5%, it can elute in the
LA1C/cHb1 (69.5%) peaks, HbA2-2.8% with no HbF in adjacent windows e.g., A1b and LA1C/cHb1 and no HbF
the designated region. HbA0 was reported as 95.1% with peak may be seen [4]. In our experience of 10 untransfused
no HbF (Fig. 1). The child was reported as normal on patients with beta thalassemia homozygosity with HbF
HPLC. The two prominent peaks adjacent to the HbF ranging from 58.9 to 98.7%, none of them showed raised
HbF peak but instead showed elution of HbF in the A1b
and LA1c/CHb1 peaks. The HbA1b in these patients ran-
& Naveen Kakkar ged from 12.3 to 20.6% while LA1c/CHb1 values ranged
[email protected]
from 46.6 to 80.3%. Elution of HbF was not seen in the
1
Department of Pathology, Christian Medical College and HbA1c region in any of these patients. Raised A1b ?
Hospital, Brown Road, Ludhiana, Punjab 141 008, India LA1c/CHb1 percentage is not seen in other hemoglobin

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 Indian J Hematol Blood Transfus

Table 1 Red cell indices in

Red cell haematological Case 1 Case 2
Case 1 and 2 (done outside and
at our center) Parameters Outside Our center Outside Our center

RBC (9 106/ll) 3.01 3.45 3.3 2.78

HGB (g/dl) 5.6 7.0 6.5 5.5
MCV (fl) 69.0 64.5 66.2 65.6
MCH (pg) 18.7 20.2 19.8 18.2
RDW-CV (%) 27.8 16.7 35.0 34.8

Fig. 1 HPLC chromatograms of cases of HPLC misinterpretation, A1b and LA1c/CHb1 peaks with no HbF in case 1 (outside report)
reported outside and at our center on Biorad D10 Hemoglobin testing and case 2 (in outside and our center’s report). #Test done on recently
system. a Normal chromatogram, b Case 1, c Case 2. Note the raised transfused blood sample

disorders. However HbA1a may be raised in the presence Helsinki declaration and its later amendments or comparable ethical
of fast moving hemoglogins (HbH and HB Barts). standards. No patient/subject identifying information has been dis-
closed in the manuscript. No patient/subject intervention was done
Accurate diagnosis requires that the cumulative per- and the subjects were not exposed to any risks during the study.
centages in the adjacent peaks (A1b and LA1C/cHb1) be
considered as HbF. However, in the patients reported here, Informed Consent Since the study involved a retrospective review
these instructions had not been followed leading to the HbF of cases from routine testing offered by the laboratory, separate
informed consent was not taken for the study. ‘‘For this type of study
sub-fractions being overlooked and misdiagnosis of beta formal consent is not required’’.
thalassemia major as normal. Both reports were from dif-
ferent outside laboratories. Such misdiagnosis can have
vital implications for the management and control of tha- References
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Chem 46:1284–1290
Compliance with Ethical Standards 3. Joutovsky A, Hadzi-Nesic J, Nardi MA (2004) HPLC retention
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Conflict of interest The authors declare that they have no conflict of binopathies: a study of 60000 samples in a clinical diagnostic
interest. laboratory. Clin Chem 50:1736–1747
4. Hemoglobin A1c/A2/F. Bio-Rad D10 dual program. UCSF Clin
Ethical Approval All procedures performed in studies involving Labs Chemistry. [Internet] www.labmed.ucsf.edu/proc-hgba1c_
human participants were in accordance with the ethical standards of a2_f_d10. Cited 6 Apr 2018
the institutional and/or national research committee and with the 1964

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