CLINICAL AND LABORATORY OBSERVATIONS

Is Hemoglobin D Trait Hematologically Silent: Comparison

With Healthy Controls and β-thalassemia Carriers
Deniz Aslan, MD

necessary consent from the institutional review board. Clinical

Summary: Hemoglobin D-Los Angeles, a recessively inherited details, laboratory parameters, and reasons for hematological
hemoglobin variant, has been introduced as hematologically silent assessment were obtained from case records. Diagnosis was
in the heterozygous state. However, as its compound heterozygosity established using high-performance liquid chromatography and
with other hemoglobinopathies may lead to a severe clinical phe-
confirmed by molecular analysis. Patient records were reviewed
notype, with hemoglobin S being the most serious, the detection of
carriers is important. To clarify the hematological picture, we for the presence of any accompanying iron deficiency (ID)
assessed erythrocyte parameters in D carriers and compared values (transferrin saturation <12%; serum ferritin <10 ng/mL),
in healthy controls and β-thalassemia carriers. Although values in D β-thalassemia minor (BTT) (normal body iron stores with Hb
carriers, in the absence of confounding factors, significantly differed A2 ≥ 3.5%), or any other variants (high-performance liquid
from thalassemia carriers (P < 0.05 for all), they were not similar to chromatography and genetic analysis), which could potentially
healthy controls. Microcytosis (absent in healthy controls) (mean affect erythrocyte parameters. Hb at presentation, hematocrit
corpuscular volume: 80.7 vs. 83.5 fL, P = 0.038) and erythrocytosis (HCT), mean corpuscular volume (MCV), mean corpuscular
(6 times more than in healthy controls) (red blood cell: 5.2 vs. hemoglobin (MCH), mean corpuscular hemoglobin concen-
4.7×1012/L, P = 0.002) were detected, making questionable the true
tration (MCHC), red blood cell (RBC) count, and red cell
silence of the D trait.
distribution width (RDW) were recorded. Results were com-
Key Words: hemoglobin D trait, erythrocyte parameters, micro- pared with healthy controls (n: 47, mean age: 12.4 y, range: 6 to
cytosis, erythrocytosis 54 y) and β-thalassemia carriers (n: 35, mean age: 16.3 y, range:
1 to 48 y). Statistical analyses were performed using SPSS, v.
(J Pediatr Hematol Oncol 2019;00:000–000) 21.0. Independent samples t test was used to compare 3 inde-
pendent groups for normally distributed variables. Arithmetic
mean and SD values were given as descriptive statistics for
quantitative data. A P-value <0.05 was considered to indicate a
A mong over 1000 hemoglobin (Hb) variants described to
date, hemoglobin D (Hb D) is the third most frequent
worldwide.1,2 It was initially named denoting the site of first
statistically significant difference.

discovery, hemoglobin D-Los Angeles (Hb D-LA).3 To date, 7

types of Hb D have been described; Hb D-LA is the one that can RESULTS
cause a serious Hb disorder. Although Hb D alone is a benign A total of 21 cases from 16 families were identified (10
condition, its coinheritance with other hemoglobinopathies may female individuals, 11 male individuals, mean age: 23.3 y,
have clinical significance4–9 and is important in populations with range: 3 to 45 y). Five cases were detected during premarital
high risk of hemoglobinopathies. Definitive diagnosis is estab- screening and 6 during a family study of the probands. The
lished by detecting the abnormal Hb molecule. However, as with remaining 10 cases were referred because of abnormal CBC
other hemoglobinopathies, complete blood count (CBC) is the results (Table 1).
first-line test for identifying Hb D. Unlike with thalassemia car- Molecular analysis revealed the underlying mutation as
riers, there is limited literature—in the form of infrequent case β-globin codon 121 GAA > CAA in all cases. Investigation
reports—with regard to the hematological profile of Hb D het- of body iron status in 19 cases showed ID in 3; none had a
erozygotes. To our knowledge, there are no reports concentrating second hemoglobinopathy.
on erythrocyte parameters or comparing results with controls. Hb, HCT, MCH, MCHC, and RDW values were
normal. Of 21 cases, microcytosis (MCV < 80 fL) was pres-
ent in 8 (38.1%) and erythrocytosis (RBC > 5×1012/L) in 15
PATIENTS AND METHODS (71.4%) (Table 1). In healthy controls, microcytosis was not
Medical records of Hb D subjects diagnosed in our present, and erythrocytosis was determined in only 12.8%.
Hematology Clinic between January 2009 and December Both abnormalities were present in the BTT group (100%
2018 were reviewed retrospectively, after obtaining the and 94.3%, respectively) (Supplementary Data, Supple-
mentary Digital Content 1, http://links.lww.com/JPHO/
Received for publication April 12, 2019; accepted September 16, 2019. A329).
From the Section of Hematology, Department of Pediatrics, Faculty of As expected, a significant difference in mean ± SD
Medicine, Gazi University, Ankara, Turkey. values was found between D carriers and BTT carriers
The authors declare no conflict of interest.
Reprints: Deniz Aslan, MD, Section of Hematology, Department of
(P < 0.05 for all) and between healthy controls and BTT
Pediatrics, Faculty of Medicine, Gazi University, Besevler, Ankara carriers (P < 0.05 for all) (Table 2). However, although
06500, Turkey (e-mails: [email protected]; [email protected]). mean ± SD values of Hb, HCT, MCV, MCH, MCHC, and
Supplemental Digital Content is available for this article. Direct URL RDW were within normal limits and similar between D
citations appear in the printed text and are provided in the HTML
and PDF versions of this article on the journal’s website, www.jpho-
carriers and healthy controls, mean MCV was found to be
online.com. significantly different from that of healthy controls (MCV:
Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved. 80.7 vs. 83.5 fL, P = 0.038). Mean RBC value was over

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 Aslan J Pediatr Hematol Oncol  Volume 00, Number 00, ’’ 2019

TABLE 1. Clinicolaboratory Findings of the Cases With Hb D-Los Angeles

Age Reason for Hb
(y)/ Hematological D-LA Hb HCT MCV MCH MCHC RBC RDW
Family Patient Sex (y) Assessment (%) (g/dL) (%) (fL) (pg) (g/dL) (×1012/L) (%) ID
I Case 1 13/M Microcytosis 38.7 12.6 37.5 77.2 26 33.6 4.9 14.9 +
(13.1)* (40.6)* (77.3)* (24.9)* (32.3)* (5.3)* (13.2)*
Case 2 39/M Family study 37.6 15.3 45.5 85.9 29 33.8 5.3 13.4 −
II Case 3 6/M Microcytosis 35.5 12.2 36.5 66.1 22.1 33.3 5.5 17.7 +
(13.2)* (39.1)* (74.6)* (26)* (34)* (5.7)* (15.7)*
Case 4 34/M Family study 42.5 16.5 48.4 83.4 28.4 34.1 5.8 14.1 −
III Case 5 3/M Microcytosis 34 13.9 38.9 77 27.4 35.6 5.1 14.2 −
Case 6 29/F Family study 36.8 12.8 36.9 85.1 29.5 34.7 4.3 14.4 −
IV Case 7 34/F Premarital screening 39.4 16.3 49.3 88.4 29.4 33.1 5.6 12.6 −
V Case 8 13/M Microcytosis 34.9 12.5 37.4 74.4 24.9 33.4 5 15.8 NA
VI Case 9 5/M Erythrocytosis 36 15.8 45.3 84 29.2 33.2 5.4 14.1 −
VII Case 10 3/F Microcytosis 40 13.6 38.1 74.4 26.6 35.7 5.1 12.9 −
VIII Case 11 17/M Microcytosis 40.4 14.3 43.3 76 25.1 33 5.7 16.7 −
Case 12 18/F Family study 42.4 12.6 36.2 81.9 28.6 34.9 4.4 14.3 −
IX Case 13 15/F Microcytosis 39 13.8 39.3 78.1 27.4 35.1 5.1 12.7 −
Case 14 39/F Family study 39 11.9 35.3 80.6 27.1 33.7 4.4 14.3 −
X Case 15 30/M Premarital screening 36.1 15.3 46 84 27.9 33.3 5.5 13.5 −
XI Case 16 31/F Premarital screening 33 14.6 43.6 81.3 27.2 33.5 5.4 13.3 +
XII Case 17 36/F Premarital screening 38 13.5 39.3 89.5 30.8 34.4 4.4 13.6 −
XIII Case 18 45/M Family study 39.4 16.1 46.1 84 29.3 34.9 5.5 13.2 NA
XIV Case 19 25/F Microcytosis 40 13.4 39.7 76.3 25.8 33.8 5.2 13.6 −
XV Case 20 29/M Erythrocytosis 43 17.1 51.9 84.6 27.8 32.9 6.1 12.7 −
XVI Case 21 26/F Premarital screening 42.1 12.4 37.2 82.3 27.4 33.3 4.5 13.8 −
*Values after ID therapy.
F indicates female; Hb D-LA, hemoglobin D-Los Angeles; HCT, hematocrit; ID, iron deficiency; M, male; MCH, mean corpuscular hemoglobin; MCHC, mean
corpuscular hemoglobin concentration; MCV, mean corpuscular volume; NA, not available; RBC, red blood cell; RDW, red cell distribution width; +, present; −, absent.

5×1012/L and also significantly different from that of healthy in different populations and have been nomenclated
controls (RBC: 5.2 vs. 4.7×1012/L, P = 0.002) (Table 2). according to the regions in which they were described. As
they have the same chemical composition as the first one
discovered, Hb D-LA,2 Hb D is known either by this initial
DISCUSSION name or the name of the most prevalent region, Hb
Mutations in genes encoding Hb chains can affect the D-Punjab, Northwest India. Because of migration and
production rate of globin chains and cause thalassemia, or intermarriage, it is also seen in other populations, including
they can modify molecular structure and generate Hb white Northern Europeans.10–15
variants.2 Hemoglobin variants are usually the consequence There are 3 types of normal Hb flowing in erythrocytes:
of single amino acid substitutions caused by point mutations adult (Hb A), fetal (Hb F), and minor adult (Hb A2). Hb D
in those genes, resulting in a tetramer with different phys- is the inherited variant of Hb A resulting from the
icochemical characteristics. Hb D, despite being clinically replacement of the amino acid glutamate with glutamine at
silent in itself, becomes significant when coinherited with Hb position 121 of the β-chain (β 121[GH4]Glu > Gln; HBB:
S or β0-thalassemia.4–9 After its discovery in a mixed British c.364G > C).1 In Hb D trait, erythrocytes contain both
and American family of Indian origin from the Los Angeles normal Hb A and variant Hb D. This condition is consid-
area,3 other Hbs with similar patterns have been discovered ered to be completely silent, as the percentage of Hb D is

TABLE 2. The Mean ± SD Values of Erythrocyte Parameters in the Groups and Statistical Comparisons Between the Groups
P

Hb D-LA Healthy Control BTT Hb D-LA vs. Healthy Hb D-LA Healthy Control
(n = 21) (n = 47) (n = 35) Control vs. BTT vs. BTT
Hb (g/dL) 14.1 ± 1.6 13.4 ± 1.1 11.5 ± 1.3 0.055 0.000 0.000
HCT (%) 41.5 ± 4.9 39.6 ± 3.3 36.2 ± 3.6 0.110 0.000 0.000
MCV (fL) 80.7 ± 5.5 83.5 ± 2.4 62.3 ± 4.3 0.038 0.000 0.000
MCH (pg) 27.5 ± 1.9 28.2 ± 0.9 19.8 ± 1.7 0.144 0.000 0.000
MCHC (g/dL) 34.0 ± 0.9 33.7 ± 0.8 31.8 ± 0.9 0.300 0.000 0.000
RBC (×1012/L) 5.2 ± 0.5 4.7 ± 0.4 5.8 ± 0.5 0.002 0.000 0.000
RDW (%) 14.1 ± 1.3 13.2 ± 0.8 16.6 ± 2.6 0.090 0.000 0.000
BTT indicates β-thalassemia minor; Hb, hemoglobin; Hb D-LA, hemoglobin D-Los Angeles; HCT, hematocrit; MCH, mean corpuscular hemoglobin;
MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; RBC, red blood cell; RDW, red cell distribution width.

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 J Pediatr Hematol Oncol  Volume 00, Number 00, ’’ 2019 Hemoglobin D Trait and Erythrocyte Parameters

lower than that of Hb A (40%), there is no reduced rate of in accordance, we detected a significant rate of microcytosis.
production of Hb D β-chains, and Hb D is a normally A closer look at the study by Mahdavi et al25 also revealed
functioning oxygen carrier. However, as the substitution is some cases with low MCV and high RBC. Therefore, re-
at a critical point for tetramer assembly,1,2 the resultant Hb evaluation of this variant in new large population studies in
is slightly unstable, and hematological changes are likely which raw data are presented might be helpful.
to occur. In conclusion, on the basis of our clinical experience,
To date, several compound heterozygous cases of Hb we suggest that Hb D-LA in the heterozygous state may not
D have been reported, with a special emphasis on clinical be silent, and the value of CBC should not be under-
features,4,7,16,17 as laboratory features, variant Hbs, and estimated. Abnormal CBC results of microcytosis and
causative mutations have been presented. There is no report erythrocytosis may be seen.
specifically evaluating erythrocyte parameters in Hb D,
neither in the heterozygous nor in the homozygous form.
Unlike previous studies, we primarily and directly describe ACKNOWLEDGMENTS
CBC results, and further compare them with those of pos-
The authors are grateful to Dr Elif Keleş for data collection
itive and negative control groups.
from patient records and Corinne Logue Can for her language
All of our cases were molecularly confirmed hetero-
editing.
zygotes. Intergroup comparisons showed that results in D
trait were similar to those in healthy controls at a global
level and significantly different from BTT (Table 2). However, a
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