An Algorithm to Aid in the Investigation of Thalassemia
Trait in Multicultural Populations
Thomas L. Kiss, MD, FRCPC; Mahmoud A. M. Ali, PhD, FRCPC; Mitchell Levine, MD, MSc, FRCPC; John D. Lafferty, ART
● Context.—The differentiation between iron deficiency
and a thalassemia syndrome is an important consideration
in the investigation of microcytic anemia.
Objective.—An established statistical method was used
to demonstrate the importance of considering ethnic back-
ground in combination with mean cell volume (MCV) in
the investigation of b-thalassemia trait in a multicultural
urban population.
Design.—Posttest probabilities for b-thalassemia trait
were calculated using likelihood ratios for various micro-
cytic MCV ranges in conjunction with published pretest
probabilities for b-thalassemia trait based on ethnic back-
ground.
Setting.—Regional hemoglobinopathy laboratory, St Jo-
seph’s Hospital, Hamilton, Ontario, Canada.
Patients.—Patient data were derived from a previously
T he thalassemia syndromes are a group of genetic ab-
normalities that cause deficient production of the a-
or b-globin chains of the hemoglobin (Hb) molecule. These
abnormalities are prevalent in patients with Mediterra-
nean, African, Middle Eastern, Asian Indian, Chinese, and
Southeast Asian ancestry. In the heterozygous form (thal-
assemia trait), the syndromes present as a thalassemia mi-
nor phenotype characterized by microcytosis with varying
degrees of anemia. In the homozygous or compound het-
erozygous state (thalassemia major), the abnormalities
cause severe anemia, resulting in fetal death or a lifelong
dependency on transfusion. When investigating a micro-
cytic anemia, the differentiation between thalassemia trait
and iron deficiency is an important consideration. This dif-
ferentiation takes on added importance in patients who
are pregnant or planning a pregnancy, since thalassemia
trait is the carrier form for the more severe thalassemia
major phenotypes. Table 1 lists common hemoglobinopa-
thies that present as thalassemia minor in the heterozy-
gous state and as thalassemia major in the homozygous
or compound heterozygous state.1
To differentiate between thalassemia trait and iron de-
Accepted for publication March 31, 2000.
From the University Health Network/The Princess Margaret Hospital,
Toronto, Ontario, Canada (Dr Kiss); St Joseph’s Hospital, Hamilton, On-
tario, Canada (Dr Ali and Mr Lafferty); and Father Sean O’Sullivan Re-
search Centre, St Joseph’s Hospital, Hamilton, Ontario, Canada (Dr
Levine).
Reprints: John Lafferty, ART, Hemoglobinopathy Laboratory, St Jo-
seph’s Hospital, 50 Charlton Ave E, Hamilton, Ontario, Canada L8N
4A6.
1320 Arch Pathol Lab Med—Vol 124, September 2000
published study. The original study cohort consisted of 789
patients aged 18 years or older who had an MCV less than
80 fL and were referred for routine complete blood count
during a 6-month period.
Main Outcome Measures.—Posttest probabilities.
Results.—Simplified tables for the determination of post-
test probabilities for b-thalassemia trait in individual pa-
tients based on ethnic background and MCV are provided.
An algorithm to assist in determining when thalassemia in-
vestigations are indicated is presented.
Conclusions.—A high index of suspicion based on ethnic
background and low MCV can provide increased sensitivity
and specificity for the detection of thalassemia trait in cen-
ters with multicultural populations similar to the study
population.
(Arch Pathol Lab Med. 2000;124:1320–1323)
ficiency with accuracy requires the use of specialized lab-
oratory procedures, as well as a knowledgeable interpre-
tation of the results.2 Investigators have long sought a re-
liable mathematical index based on routine complete
blood count results to help differentiate between micro-
cytosis due to iron deficiency and that due to a thalasse-
mia syndrome. Previous work conducted in this labora-
tory3 established that the use of the mean cell volume
(MCV) alone was as reliable or more reliable than any
other published index. This study demonstrated that the
use of an MCV less than 72 fL as a predictor of thalasse-
mia trait resulted in a sensitivity of 0.88 and a specificity
of 0.84.3 The clinical usefulness of this approach, however,
is limited by the fact that 12% of patients with thalassemia
minor will be missed, while 16% without thalassemia mi-
nor, a potentially large number of individuals, would be
processed for unnecessary tests. These factors raise ques-
tions regarding the cost-effectiveness of such a strategy in
multicultural populations in which the percentage of in-
dividuals with an ethnic background associated with thal-
assemia is relatively small.4 This situation exists in many
jurisdictions and suggests that a more specific approach
to the detection of thalassemia trait is indicated here.5 The
purposes of this article are to demonstrate the importance
of considering ethnic background in combination with
MCV in the investigation of b-thalassemia trait and to pre-
sent an algorithm to assist in determining when specific
thalassemia investigations are indicated in multicultural
populations.
METHODS
Data used come from a previous study conducted in Hamilton,
Ontario, Canada. This region has a broad-based multicultural
MCV and Ethnic Background in Thalassemia—Kiss et al
 Table 1. Common Hemoglobinopathies, Which Present as Thalassemia Minor in the Heterozygous State and as
Thalassemia Major in Homozygous or Compound Heterozygous States
Thalassemia Trait Thalassemia Major Thalassemia Major
Hemoglobinopathy Genotype Genotype Syndrome Clinical Significance
a-Thalassemia 22/aa 22/22 Hydrops fetalis Fetal death, difficult
pregnancy
b-Thalassemia b0/b b0/b0 b-Thalassemia major Transfusion dependent
Hemoglobin E bE/b b0/bE b-Thalassemia major Transfusion dependent
Hemoglobin Lepore bLepore/b b0/bLepore or bLepore/bLepore b-Thalassemia major Transfusion dependent

Table 2. Formulae Used for the Calculation of Table 4. Prevalence (Pretest Probability) of b-
Posttest Probabilities for b-Thalassemia Trait* Thalassemia Trait in Different Populations
b-Thalassemia b-Thalassemia Population Prevalence, %
Trait Present Trait Absent
African American 1.4
Within mean cell Algeria 3.0
volume range A B Bulgaria
Outside mean cell Northern 0.1
volume range C D Southern 9.0
* Positive Likelihood Ratio 5 [A/(A 1 C)]/[B/(B 1 D)]. China
Pretest Odds 5 Pretest Probability/1 2 Pretest Probability. Canton Area 2.0
Posttest Odds 5 Pretest Odds 3 Positive Likelihood Ratio. Cyprus 15.0
Posttest Probability 5 Posttest Odds/1 1 Posttest Odds. Ghana 1.5
Greece
Arta District 10.0
Crete 7.0
Table 3. Positive Likelihood Ratios for b-Thalassemia Mainland 7.0
Trait at Various Mean Cell Volume (MCV) Ranges Rhodes 25.0
Calculated From the Previous Study Data India
Positive Ahom 1.0
MCV Range, fL Likelihood Ratio Assam 5.0
Bangalen 3.7
55–59.9 54.19 Bhanishali 15.0
60–64.9 18.79 Lohana 13.6
65–69.9 3.61 Saraswat NW 4.4
70–74.9 0.34 Saraswat West 3.5
75–79.9 0.09 Sindhi near Bombay 8.0
Italy
North and Central 1.3
Po delta 13.0
population; the majority of residents are of British, Northern Eu-
Sardinia 12.6
ropean, North American Aboriginal, and Northern Asian ethnic Sardinia (Southern) 28.5
background. The prevalence of b-thalassemia trait in these ethnic Southern (includes Sicily) 10.0
groups is low. An important proportion of the population (23%), Kurdish
however, is of ethnic backgrounds in which b-thalassemia trait Jewish population 20.0
is common. These populations include people of Italian (12%), Lebanon 4.0
Portuguese (2.8%), other Mediterranean (1.9%), Chinese (1.6%), Liberia 9.0
Asian Indian (1.3%), African (1.1%), Southeast Asian (1.1%), and Malta 3.5
Greek (1%) descent.6 The original study consisted of 789 patients Nigeria 0.8
18 years of age or older with an MCV less than 80 fL who were Pacific Islands
referred for routine complete blood count during a 6-month pe- Lower range 2.0
riod. Seventy-seven patients were excluded because of insufficient Upper range 20.0
sample volumes. Forty of the remaining 712 patients were diag- Pakistan
nosed with thalassemia minor, 31 with b-thalassemia trait, 8 with Karachi 1.5
a-thalassemia trait, and 1 with HbE trait.3 This article deals with Patham 4.0
the b-thalassemia trait data only, as insufficient numbers of pa- Thailand
tients with other hemoglobinopathies were identified in the orig- Northern 10.0
inal study. Positive likelihood ratios for b-thalassemia trait were Overall 4.8
Turkey
calculated for various MCV ranges from the previous study data.
Southwest 1.7
Relative to any range of MCV values, a group of subjects may be
divided into 4 mutually exclusive subgroups, A through D (Table
2). Likelihood that a patient with b-thalassemia trait is within
that MCV range is A/(A 1 C), and likelihood that a patient with- These equations represent the application of conditional (Bayes-
out b-thalassemia trait is within that MCV range is B/(B 1 D). ian) probability. Prevalence data for b-thalassemia trait were ob-
The positive likelihood ratio for that range is then the ratio of tained from a published compilation of studies.7 All formulae
these 2 ratios, the first equation of Table 2. This ratio is indepen- used for these calculations are outlined in Table 2.8
dent of the prevalence of b-thalassemia trait in the group. Sub- RESULTS
sequent equations in Table 2 combined this neutral ratio with the
appropriate prevalence factor for b-thalassemia trait based on The positive likelihood ratios calculated for various MCV
ethnic background to produce the final probability of b-thalas- ranges are listed in Table 3. Pretest probabilities derived for
semia trait for that ethnic group within a given MCV range. a variety of ethnic backgrounds are shown in Table 4. They
Arch Pathol Lab Med—Vol 124, September 2000 MCV and Ethnic Background in Thalassemia—Kiss et al 1321
 Table 5. Posttest Probability of b-Thalassemia Trait*
Mean Cell
Pretest Probability
Volume,
fL 1.0% 2.5% 5.0% 7.5% 10.0% 12.5% 15.0% 17.5% 20.0% 22.5% 25.0% 27.5% 30.0%
55–59.9 35.4 58.2 74.0 81.5 85.8 88.6 90.5 92.0 93.1 94.0 94.8 95.4 95.9
60–64.9 16.0 32.5 49.7 60.4 67.6 72.9 76.8 79.9 82.4 84.5 86.2 87.7 89.0
65–69.9 3.5 8.5 16.0 22.6 28.6 34.0 38.9 43.4 47.4 51.2 54.6 57.8 60.7
70–74.9 0.3 0.9 1.8 2.7 3.6 4.6 5.7 6.7 7.8 9.0 10.2 11.4 12.7
75–79.9 0.1 0.2 0.5 0.7 1.0 1.3 1.6 1.9 2.2 2.5 2.9 3.3 3.7
* All values reported as percentages.

An algorithm for the investigation of micro-

cytosis utilizing posttest probabilities that are
based on ethnic background and mean cell
volume (MCV). 1In microcytic patients who
are pregnant, the requirement for rapid turn-
around of test results may indicate the need
for a thalassemia investigation regardless of
posttest probability. 2Thalassemia investiga-
tion includes hemoglobin (Hb) A2 level, Hb
electrophoresis, HbF level, HbH inclusion
body screen, and serum ferritin. Interpretation
of these tests is reviewed elsewhere.14

include Italy (various regions), Greece (various regions),
Malta, Cyprus, Bulgaria (north and south), Turkey, Leba-
non, India (various regions), Pakistan (various regions),
China (Kanton), Pacific Islands, Algeria, Liberia, Ghana,
Nigeria, and Thailand, as well as the probabilities for Kurd-
ish Jews and African Americans. These probabilities include
many of the ethnic backgrounds encountered in the study
population. The posttest probabilities calculated using the
pretest probability, likelihood ratio, and MCV are listed in
Table 5. For the purpose of simplification, MCV values are
divided into ranges of 4.9 fL, while the pretest probabilities
are rounded to the nearest 2.5%. The posttest probability
of b-thalassemia trait for an individual patient can be es-
tablished from Table 5 using the patient’s pretest probabil-
ity, based on ethnic background (from Table 4) and the pa-
tient’s MCV. For example, an Italian patient with a Sardinian
background and an MCV of 63 fL has a pretest probability
of 12.6% and a posttest probability of 72.9%. With an MCV
of 76 fL, the same patient would have a greatly reduced
posttest probability of 1.3%. The Figure presents an algo-
rithm for the investigation of microcytosis utilizing posttest
probabilities based on ethnic background and MCV.
COMMENT
Microcytic anemias are among the most common types
of anemia encountered by physicians in general hospitals
and outpatient clinics.9 The differential diagnosis includes
1322 Arch Pathol Lab Med—Vol 124, September 2000
iron deficiency, thalassemia minor resulting from a- or b-
thalassemia trait or from variant hemoglobins such as
HbE and Hb Lepore, anemia of inflammation, congenital
sideroblastic anemia, and lead poisoning.10,11 The 2 most
common causes of microcytic anemia are iron deficiency
and thalassemia minor.12 It is imperative to distinguish
between these 2 diagnoses appropriately. Iron deficiency
requires a diagnostic workup to determine the underlying
cause of the deficiency, medical intervention to correct it,
and replacement iron therapy. Thalassemia minor, on the
other hand, does not require the aforementioned medical
intervention but has implications for prenatal diagnosis
and genetic counseling.1 The experience of this laboratory
is that an investigation for thalassemia trait is often not
considered when investigating microcytosis in the multi-
cultural population of Hamilton. This can lead to unnec-
essary medical intervention for iron deficiency and pre-
vent access to prenatal diagnostic services for high-risk
patients who are pregnant or contemplating pregnancy.
Table 5 demonstrates that the probability of b-thalassemia
trait increases with the patient’s pretest probability, based
on ethnicity and the degree of microcytosis. The proba-
bility of b-thalassemia trait in patients with a modest pre-
test probability of 5% increases to almost 50% with an
MCV of 60 to 64.9 fL. In some patients from high-risk
ethnic backgrounds, the presence of an MCV of 55 to 59.9
fL results in a posttest probability of greater than 90%.
MCV and Ethnic Background in Thalassemia—Kiss et al
Although posttest probabilities for other thalassemia syn-
dromes were not calculated, the b-thalassemia trait post-
test probabilities demonstrate compelling evidence of the
importance of considering a patient’s ethnic background
in the investigation of microcytosis. The absence of an eth-
nic background in which thalassemia is common does not
exclude thalassemia but makes other causes of microcy-
tosis more likely. The combination of a low MCV and an
ethnic background in which thalassemia trait is prevalent
strongly indicates the need for specific thalassemia testing.
This is especially true in patients who are pregnant or are
planning a pregnancy, as these patients are candidates for
nondirective genetic counseling if they are at risk of con-
ceiving an infant with thalassemia major.
In summary, failure to investigate for thalassemia trait
in high-risk patients can lead to unnecessary and poten-
tially harmful investigation and therapy for iron deficien-
cy.13 In patients who are pregnant or are contemplating
pregnancy, a failure to investigate for thalassemia trait can
prevent access to prenatal diagnostic services for thalas-
semia major. In Hamilton, the majority of the population
consists of ethnic backgrounds in which thalassemia trait
is uncommon, and an investigation for thalassemia trait
in these patients reduces effective laboratory utilization.
Many cities have multicultural populations similar to the
population of Hamilton, where an important minority of
the population includes ethnic backgrounds in which thal-
assemia is prevalent. The statistical case presented here
demonstrates that a high index of suspicion based on eth-
nic background and low MCV is a relevant approach to
the effective investigation of thalassemia trait in these cen-
ters. Use of the algorithm presented in the Figure should
Arch Pathol Lab Med—Vol 124, September 2000
provide increased sensitivity and specificity for the detec-
tion of thalassemia trait in these jurisdictions.
This study was funded by the Department of Laboratory Med-
icine, St. Joseph’s Hospital, Hamilton, Ontario, Canada.
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