CORRESPONDENCE 817

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DOI: http://dx.doi.org/10.1016/j.pathol.2017.07.012
A simple means to differentiate
labile HbA1c from haemoglobin
variant on Bio-Rad Variant II Turbo
2.0 cation-exchange HPLC method
Sir,
Accurate measurement of glycated haemoglobin A1c
(HbA1c) is important for optimal management of diabetes.
Cation-exchange high performance liquid chromatography
(CA-HPLC) methods can be interfered with by haemoglobin
(Hb) variants,1,2 which are prevalent in many parts of the
world, including South-East Asia (prevalence: Singapore
3.5%, Thailand up to 30%).3,4 The Variant II Turbo 2.0
analyser (HbA1c program, Bio-Rad Laboratories, USA) is a
CA-HPLC method. Certain Hb variants (e.g., HbJ) can elute
in the labile HbA1c window that precedes HbA1c, and
interfere with its measurement.
Labile HbA1c is a reversible intermediary formed acutely
between glucose and Hb, and can rise considerably after
ingestion of carbohydrate.5 The glucose attached to labile
HbA1c can spontaneously dissociate over time when the
ambient glucose is low. It is important to differentiate labile
HbA1c formed after a meal from co-eluting Hb variant for
proper interpretation of the chromatograms. We explored
the use of a decrease in native labile HbA1c after diluting
whole blood with instrument diluent and incubating in 37 C
water bath as a simple means to differentiate it from co-
eluting Hb variants, which cannot be reduced in this
manner.
Residual K2EDTA samples collected from subjects
without Hb variants for routine HbA1c testing (n = 121) were
centrifuged at 3000 g for 5 min. Following this, 3 mL of the
packed red blood cells were mixed with 1.5 mL (i.e., 1:500
dilution ratio) of the Variant II Turbo diluent to standardise
the total area count to 1.5e2.5 million (within the manufac-
turer’s recommendation of 1.5e3.5 million total area count).
Next, the samples were incubated in a water bath at 37 C for
4 h before re-analysis on the Variant II Turbo 2.0 analyser.
Since the concentration of labile HbA1c is dependent on
HbA1c,6 the degree of reduction in labile HbA1c after in-
cubation was expressed as labile HbA1c to HbA1c ratio. The
reference interval for the post-incubation labile HbA1c to
HbA1c ratio from subjects without Hb variants was calcu-
lated. The reference interval was then applied to samples with
known Hb variant after identical incubation. The results of
the experiments are summarised in Table 1.
Of note, urea can dissociate in vivo to form isocyanic acid
and react with Hb to form carbamylated Hb. It is elevated in
patients with chronic renal disease. In the Variant II Turbo 2.0
analyser, carbamylated Hb co-elutes with labile HbA1c and
may confound the above interpretation. In the presence of a
history of chronic renal disease or significantly elevated
serum urea concentration, an elevated labile HbA1c may be
clarified by Hb phenotyping, if clinically indicated.
Incubating EDTA whole blood samples in diluent at 37 C
for 4 h reduces the labile HbA1c fraction. The use of the
labile HbA1c:HbA1c ratio can effectively differentiate Hb
variants eluting in the labile HbA1c window, because the
apparent elevated labile HbA1c due to the presence of an Hb
variant does not decrease after incubation, as shown by
subjects with HbJ.
It is important to identify the presence of Hb variant on
chromatogram and suggest confirmatory testing where
clinically indicated, since it can affect the relationship with
the glycaemic status of the patient.7 However, the investi-
gation strategy adopted by a laboratory (e.g., at what con-
centration of labile HbA1c should one begin to investigate
for the presence of Hb variant) should be individualised,
taking into account the prevalence of Hb variant in the
population served by the laboratory. More than 600 Hb
variants have been reported thus far. It would be prudent to
verify the performance of the laboratory manipulation
described above on Hb variants that are prevalent in the
local population, particularly if they differ from the five Hb
variants described in this study. When in doubt, laboratory
practitioners can use this simple manipulation and the labile

Table 1 Summary of the Variant II Turbo results before and after incubation at 37 C for haemoglobin (Hb) variants

Haemoglobin (Hb) subtype n Pre-incubation Post- incubation

Labile HbA1c Labile HbA1c: Labile HbA1c HbA1c Labile HbA1c:
HbA1c HbA1c ratio (% remaining) HbA1c ratio

Normal Hba 121 1.6e4.8 4.9e14.2 0.18e0.55 1.1e3.1 (51e90%) 4.8e14.3 0.13e0.41
Heterozygous HbE 12 1.1e2.1 5.5e9.8 0.12e0.37 0.9e1.4 (61e91%) 5.4e9.8 0.11e0.25
Heterozygous HbS 11 1.0e2.3 5.0e9.7 0.20e0.27 0.8e1.6 (59e85%) 5.1e10.0 0.14e0.21
Heterozygous HbD 12 1.0e2.7 5.0e10.2 0.17e0.28 0.9e1.4 (52e90%) 4.8e10.4 0.12e0.21
Heterozygous HbQ-Thailand 7 1.2e2.2 5.6e9.1 0.21e0.30 0.9e1.6 (64e79%) 5.6e9.2 0.14e0.22
Heterozygous HbJ 12 3.5e7.9 4.6e8.0 0.74e1.03 3.5e7.3 (92e100%) 4.5e8.3 0.76e1.02

a
The values shown for normal haemoglobin are the 95% reference intervals while those shown for the other haemoglobin subtypes are the minimum and
maximum values.
818 CORRESPONDENCE Pathology (2017), 49(7), December

HbA1c:HbA1c ratio to clarify suspicious peak eluting at 1. Loh TP, Cheng WL, Kao SL, Thai AC, Sethi SK. Effects of haemoglobin
E traits on HbA1c measurement by two cation-exchange HPLC and two
labile HbA1c window. immunoturbidimetric methods. Pathology 2014; 46: 265e6.
2. Cheng WL, Neo SF, Chew S, Sethi SK, Loh TP. HbG-Honolulu in-
Conflicts of interest and sources of funding: The authors terferes with some cation-exchange HPLC HbA1c assays. Clin Chem Lab
Med 2016; 54: e77e9.
state that there are no conflicts of interest to disclose. 3. Saw S, Loh TP, Yin C, Sethi SK. Identification of hemoglobin variants in
samples received for glycated hemoglobin testing. Clin Chim Acta 2013;
Wan Ling Cheng 415: 173e5.
4. Viprakasit V, Lee-Lee C, Chong QT, Lin KH, Khuhapinant A. Iron
Siew Fong Neo chelation therapy in the management of thalassemia: the Asian perspec-
Suru Chew tives. Int J Hematol 2009; 90: 435e45.
Sunil Kumar Sethi 5. Higgins PJ, Bunn HF. Kinetic analysis of the nonenzymatic glycosylation
Tze Ping Loh of hemoglobin. J Biol Chem 1981; 256: 5204e8.
6. Loh TP, Peng WK, Chen L, Sethi SK. Application of smoothed contin-
uous labile haemoglobin A1c reference intervals for identification of
Department of Laboratory Medicine, National University potentially spurious HbA1c results. J Clin Pathol 2014; 67: 712e6.
Hospital, Singapore 7. Loh TP, Sethi SK, Wong MS, Tai ES, Kao SL. Relationship between
measured average glucose by continuous glucose monitor and HbA1c
measured by three different routine laboratory methods. Clin Biochem
Contact Tze Ping Loh. 2015; 48: 514e8.
E-mail: tploh@hotmail.com
DOI: http://dx.doi.org/10.1016/j.pathol.2017.08.014