An Algorithm to Aid in the Investigation of Thalassemia Trait in Multicultural Populations.

The thalassemia syndromes are a group of genetic abnormalities that cause deficient production of the [Alpha]-or [Beta]-globin chains of the hemoglobin (Hb) molecule. These abnormalities are prevalent in patients with Mediterranean, African, Middle Eastern, Asian Indian, Chinese, and Southeast Asian ancestry. In the heterozygous form (thalassemia trait), the syndromes present as a thalassemia minor phenotype characterized by microcytosis with varying degrees of anemia. In the homozygous or compound heterozygous state (thalassemia major), the abnormalities cause severe anemia, resulting in fetal death or a lifelong dependency on transfusion. When investigating a microcytic anemia, the differentiation between thalassemia trait and iron deficiency is an important consideration. This differentiation 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 hemoglobinopathies that present as thalassemia minor in the heterozygous state and as thalassemia major in the homozygous or compound heterozygous state.[1]

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
Hemoglobinopathy      Genotype               Genotype

[Alpha]-Thalassemia   --/[Alpha][Alpha]      --/--

[Beta]-Thalassemia    [[Beta].sup.0]/        [[Beta].sup.0]/
                      [Beta]                 [[Beta].sup.0]

Hemoglobin E          [[Beta].sup.E]/        [[Beta].sup.0]/
                      [Beta]                 [[Beta].sup.E]

Hemoglobin Lepore     [[Beta].sup.Lepore]/   [[Beta].sup.0]/
                      [Beta]                 [[Beta].sup.Lepore] or
                                             [[Beta].sup.Lepore]/
                                             [[Beta].sup.Lepore]

                      Thalassemia Major          Clinical
Hemoglobinopathy      Syndrome                   Significance

[Alpha]-Thalassemia   Hydrops fetalis            Fetal death,
                                                 difficult
                                                 pregnancy

[Beta]-Thalassemia    [Beta]-Thalassemia major   Transfusion
                                                 dependent

Hemoglobin E          [Beta]-Thalassemia major   Transfusion
                                                 dependent

Hemoglobin Lepore     [Beta]-Thalassemia major   Transfusion
                                                 dependent

To differentiate between thalassemia trait and iron deficiency with accuracy requires the use of specialized laboratory procedures, as well as a knowledgeable interpretation of the results.[2] Investigators have long sought a reliable mathematical index based on routine complete blood count results to help differentiate between microcytosis due to iron deficiency and that due to a thalassemia syndrome. Previous work conducted in this laboratory[3] 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 thalassemia 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 minor, a potentially large number of individuals, would be processed for unnecessary tests. These factors raise questions regarding the cost-effectiveness of such a strategy in multicultural populations in which the percentage of individuals with an ethnic back, ground associated with thalassemia 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 [Beta]-thalassemia trait and to present 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 population; the majority of residents are of British, Northern European, North American Aboriginal and Northern Asian ethnic background. The prevalence of [Beta]-thalassemia trait in these ethnic groups is low. An important proportion of the population (23%), however, is of ethnic backgrounds in which [Beta]-thalassemia trait is common. These populations include people of Italian (12%), Portuguese (2.8%), other Mediterranean (1.9%), Chinese (1.6%), Asian Indian (1.3%), African (1.1%), Southeast Asian (1.1%), and Greek (1%) descent.6 The original study consisted of 789 patients 18 years of age or older with an MCV less than 80 fL who were referred for routine complete blood count during a 6-month period. Seventy-seven patients were excluded because of insufficient sample volumes. Forty of the remaining 712 patients were diagnosed with thalassemia minor, 31 with [Beta]-thalassemia trait, 8 with [Alpha]-thalassemia trait, and 1 with HbE trait.[3] This article deals with the [Beta]-thalassemia trait data only, as insufficient numbers of patients with other hemoglobinopathies were identified in the original study. Positive likelihood ratios for [Beta]-thalassemia trait were calculated for various MCV ranges from the previous study data. 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 [Beta]-thalassemia trait is within that MCV range is A/(A + C), and likelihood that a patient without [Beta]-thalassemia trait is within that MCV range is B/(B + D). The positive likelihood ratio for that range is then the ratio of these 2 ratios, the first equation of Table 2. This ratio is independent of the prevalence of [Beta]-thalassemia trait in the group. Subsequent equations in Table 2 combined this neutral ratio with the appropriate prevalence factor for [Beta]-thalassemia trait based on ethnic background to produce the final probability of [Beta]-thalassemia trait for that ethnic group within a given MCV range. These equations represent the application of conditional (Bayesian) probability. Prevalence data for [Beta]-thalassemia trait were obtained from a published compilation of studies.[7] All formulae used for these calculations are outlined in Table 2.[8]

Table 2. Formulae Used for the Calculation of Posttest Probabilities for [Beta]-Thalassemia Trait(*)

                    [Beta]-Thalassemia   [Beta]-Thalassemia
                      Trait Present         Trait Absent

Within mean cell
  volume range              A                    B
Outside mean cell
  volume range              C                    D

(*) Positive Likelihood Ratio = [A/(A + C)]/[B/(B + D)]. Pretest Odds = Pretest Probability/1 - Pretest Probability. Posttest Odds = Pretest Odds x Positive Likelihood Ratio. Posttest Probability = Posttest Odds/1 + Posttest Odds.

RESULTS

The positive likelihood ratios calculated for various MCV ranges are listed in Table 3. Pretest probabilities derived for a variety of ethnic backgrounds are shown in Table 4. They include Italy (various regions), Greece (various regions), Malta, Cyprus, Bulgaria (north and south), Turkey, Lebanon, India (various regions), Pakistan (various regions), China (Kanton), Pacific Islands, Algeria, Liberia, Ghana, Nigeria, and Thailand, as well as the probabilities for Kurdish 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 [Beta]-thalassemia trait for an individual patient can be established from Table 5 using the patient's pretest probability, based on ethnic background (from Table 4) and the patient'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 algorithm for the investigation of microcytosis utilizing posttest probabilities based on ethnic background and MCV.

[Figure ILLUSTRATION OMITTED]

Table 3. Positive Likelihood Ratios for [Beta]-Thalassemia Trait at Various Mean Cell Volume (MCV) Ranges Calculated From the Previous Study Data

                    Positive
MCV Range, fL   Likelihood Ratio

55-59.9              54.19
60-64.9              18.79
65-69.9               3.61
70-74.9               0.34
75-79.9               0.09

Table 4. Prevalence (Pretest Probability) of [Beta]-Thalassemia Trait in Different Populations

Population                     Prevalence, %

African American                    1.4
Algeria                             3.0
Bulgaria
  Northern                          0.1
  Southern                          9.0
China
  Canton Area                       2.0
Cyprus                             15.0
Ghana                               1.5
Greece
  Arta District                    10.0
  Crete                             7.0
  Mainland                          7.0
  Rhodes                           25.0
India
  Ahom                              1.0
  Assam                             5.0
  Bangalen                          3.7
  Bhanishali                       15.0
  Lohana                           13.6
  Saraswat NW                       4.4
  Saraswat West                     3.5
  Sindhi near Bombay                8.0
Italy
  North and Central                 1.3
  Po delta                         13.0
  Sardinia                         12.6
  Sardinia (Southern)              28.5
  Southern (includes Sicily)       10.0
Kurdish
  Jewish population                20.0
Lebanon                             4.0
Liberia                             9.0
Malta                               3.5
Nigeria                             0.8
Pacific Islands
  Lower range                       2.0
  Upper range                      20.0
Pakistan
  Karachi                           1.5
  Patham                            4.0
Thailand
  Northern                         10.0
  Overall                           4.8
Turkey
  Southwest                         1.7
Table 5. Posttest Probability of [Beta]-Thalassemia Trait(*)

Mean Cell                  Pretest Probability
Volume,
fL          1.0%   2.5%   5.0%   7.5%   10.0%   12.5%   15.0%

55-59.9     35.4   58.2   74.0   81.5    85.8    88.6    90.5
60-64.9     16.0   32.5   49.7   60.4    67.6    72.9    76.8
65-69.9      3.5    8.5   16.0   22.6    28.6    34.0    38.9
70-74.9      0.3    0.9    1.8    2.7     3.6     4.6     5.7
75-79.9      0.1    0.2    0.5    0.7     1.0     1.3     1.6

Mean Cell                 Pretest Probability
Volume,
fL          17.5%   20.0%   22.5%   25.0%   27.5%   30.0%

55-59.9      92.0    93.1    94.0    94.8    95.4    95.9
60-64.9      79.9    82.4    84.5    86.2    87.7    89.0
65-69.9      43.4    47.4    51.2    54.6    57.8    60.7
70-74.9       6.7     7.8     9.0    10.2    11.4    12.7
75-79.9       1.9     2.2     2.5     2.9     3.3     3.7

(*) All values reported as percentages.

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 iron deficiency, thalassemia minor resulting from [Alpha]- or [Beta]-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 multicultural population of Hamilton. This can lead to unnecessary medical intervention for iron deficiency and prevent access to prenatal diagnostic services for high-risk patients who are pregnant or contemplating pregnancy. Table 5 demonstrates that the probability of [Beta]-thalassemia trait increases with the patient's pretest probability, based on ethnicity and the degree of microcytosis. The probability of [Beta]-thalassemia trait in patients with a modest pretest 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%. Although posttest probabilities for other thalassemia syndromes were not calculated, the [Beta]-thalassemia trait posttest probabilities demonstrate compelling evidence of the importance of considering a patient's ethnic background in the investigation of microcytosis. The absence of an ethnic background in which thalassemia is common does not exclude thalassemia but makes other causes of microcytosis 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 conceiving an infant with thalassemia major.

In summary, failure to investigate for thalassemia trait in high-risk patients can lead to unnecessary and potentially harmful investigation and therapy for iron deficiency.[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 thalassemia 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 thalassemia is prevalent. The statistical case presented here demonstrates that a high index of suspicion based on ethnic background and low MCV is a relevant approach to the effective investigation of thalassemia trait in these centers. Use of the algorithm presented in the Figure should provide increased sensitivity and specificity for the detection of thalassemia trait in these jurisdictions.

This study was funded by the Department of Laboratory Medicine, St. Joseph's Hospital, Hamilton, Ontario, Canada.

References

[1.] Weatherall DJ. The thalassemias. In: Williams Hematology. 5th ed. New York, NY: McGraw-Hill Inc; 1995:581-615.

[2.] Lee GR. Microcytosis and the anemias associated with impaired hemoglobin synthesis. In: Wintrobe's Clinical Hematology. 9th ed. Philadelphia, Pa: Lea & Febiger; 1993:791-807.

[3.] Lafferty J, Crowther MA, Ali MA, Levine M. The evaluation of various mathematical RBC indices and their efficacy in discriminating between thalassemic and non-thalassemic microcytosis. Am J Clin Pathol. 1996;106:201-205.

[4.] Le Gales C, Galacteros F. Economic analysis of neonatal screening for drepanocytosis in metropolitan France. Epidemiol Sante Publique. 1994;42:478-492.

[5.] Sasi K, Sanderson D, Eydoux P, et al. Prenatal diagnosis for inborn errors of metabolism and haemoglobinopathies: the Montreal Children's Hospital experience. Prenat Diagn. 1997;17:681-685.

[6.] Dickson L, Heale J, Chambers L. Determinants of health: language and ethnicity. In: Fact Book on Health in Hamilton Wentworth. 3rd ed. Hamilton, Ontario, Canada: McMaster University Faculty of Health Sciences; 1994:121-122.

[7.] Weatherall DJ, Clegg JB. The distribution and incidence of beta thalassemia genes in different populations. In: Thalassemia Syndromes. 3rd ed. London, England: Blackwell Scientific Publications; 1981:295-309.

[8.] Fletcher RH, Fletcher SW, Wagner EH. Diagnosis. In: Clinical Epidemiology: The Essentials. 3rd ed. Baltimore, Md: Williams & Wilkins; 1996:43-74.

[9.] Cash JM, Sears DA. The anemia of chronic disease: spectrum of associated diseases in a series of unselected hospitalized patients. Am J Med. 1989;87:638-644.

[10.] Hoffbrand AV, Pettit JE. Erythropoiesis and anemia. In: Essential Hematology 3rd ed. Oxford, England: Blackwell Science; 1993:27-28.

[11.] Wintrobe MM, Lukens JM, Lee GR. The approach to the patient with anemia. In: Wintrobe's Clinical Hematology. 9th ed. Philadelphia, Pa: Lea & Febiger; 1993:715-744.

[12.] Ravel R. Basic hematologic tests and classification of anemia. In: Clinical Laboratory Medicine. 6th ed. St Louis, Mo: Mosby; 1995:9-21.

[13.] Ali M. Hemoglobinopathies in the Hamilton region, II: thalassemia trait and iron therapy. Can Med Assoc J. 1975;32:701-702.

[14.] Lafferty J. The laboratory diagnosis of [Alpha] thalassemia. Can J Med Lab Sci. 1998;60:183-189.

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, Ontario, Canada (Dr Ali and Mr Lafferty); and Father Sean O'Sullivan Research Centre, St Joseph's Hospital, Hamilton, Ontario, Canada (Dr Levine).

Reprints: John Lafferty, ART, Hemoglobinopathy Laboratory, St Joseph's Hospital, 50 Charlton Ave E, Hamilton, Ontario, Canada L8N 4A6.