Hemoglobin profiles and hematologic features of thalassemic newborns: application to screening of [alpha]-thalassemia 1 and hemoglobin E.
Abstract: * Context.--Thalassemia and hemoglobinopathies are major public health problems worldwide. To establish a costeffective screening tool for newborns in regions where the incidence of these disorders is significant, study of the hemoglobin and hematologic features of normal and thalassemic newborns is necessary.

Objective.--To study hemoglobin and hematologic characteristics of normal and various thalassemic newborns and to assess the effectiveness of simple screening methods for [alpha]-thalassemia 1 and hemoglobin E.

Design.--Study was made of 402 cord blood specimens collected from unrelated Thai individuals. Hematologic parameters and hemoglobin profiles were determined. Thalassemia mutations were identified using polymerase chain reaction-related techniques.

Results.--As many as 178 subjects (44.3%) were found to carry thalassemia genes with 18 different genotypes. All forms of [alpha]-thalassemia including double heterozygote for hemoglobin E and [alpha]-thalassemia showed significant reduction in hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin with increasing trend of red blood cell as compared with a non-[alpha]-thalassemic group. Although heterozygous hemoglobin E and [beta]-thalassemia showed no hematologic difference from nonthalassemic group, heterozygous [alpha]-thalassemia 1 including those with hemoglobin E showed significant increase in hemoglobin Bart level.

Conclusions.--Based on these findings, effective primary screening with 100% accuracy for [alpha]-thalassemia 1 and hemoglobin E in newborns in the region could be carried out using mean corpuscular volume less than 95 fL, mean corpuscular hemoglobin less than 30 pg, or hemoglobin Bart greater than 8.0% and hemoglobin E greater than 0.5%, respectively.
Article Type: Report
Subject: Thalassemia (Diagnosis)
Infants (Newborn) (Health aspects)
Infants (Newborn) (Medical examination)
Infants (Newborn) (Methods)
Authors: Tritipsombut, Jaruwan
Sanchaisuriya, Kanokwan
Fucharoen, Supan
Fucharoen, Goonnapa
Siriratmanawong, Nirut
Pinmuang-ngam, Charnchai
Sanchaisuriya, Pattara
Pub Date: 11/01/2008
Publication: Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2008 College of American Pathologists ISSN: 1543-2165
Issue: Date: Nov, 2008 Source Volume: 132 Source Issue: 11
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 230246860
Full Text: Thalassemia is a common inherited hemoglobin (Hb) disorder characterized by an absence or a reduction in globin chain synthesis. There are 2 main types, that is, the [alpha]-and [beta]-thalassemia. High prevalence of both [alpha]-and [beta]-thalassemia as well as other hemoglobinopathies including Hb E, Hb Constant Spring, and Hb Pakse have been documented in Thailand and other southeast Asian countries where interaction of these genes can lead to several complex thalassemia syndromes. (1-3) In adults, although almost all forms of thalassemia could be diagnosed by Hb and hematologic analyses, accurate diagnosis of [alpha]-thalassemia can only be made on DNA analysis. (4,5) This cannot be applied in rural areas where laboratories and facilities are limited. Unlike in an adult, diagnosis of [alpha]-thalassemia in newborns is easier due to the presence of Hb Bart, a homotetramer of excess [gamma]-globin chain ([[gamma].sub.4]). (6) A relationship between Hb Bart in cord blood and the severity of [alpha]-thalassemia has been demonstrated. (7-11) It has also been reported that other hematologic indices such as mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) might also be used for screening of [alpha]-thalassemia in newborns. (12,13) However, in an area where thalassemia and hemoglobinopathies are exceptionally heterogeneous and occur simultaneously, it is questionable whether these hematologic parameters could be applied for primary screening of thalassemia in newborns. In this study, we compare hematologic features and Hb profiles associated with normal and various thalassemic newborns of Thai individuals and establish the cutoff values of MCV, MCH, Hb Bart, and Hb E for use in effective screening of [alpha]-thalassemia 1 and Hb E in the region.

MATERIALS AND METHODS

Subjects

The study protocol was approved by our institutional review board at Khon Kaen University, Thailand (HE490912). Informed consent was obtained from the parents. Cord blood specimens anticoagulated with EDTA were consecutively collected from 402 well babies delivered at the Mother and Child Hospital, Health Promotion Center Region 8, Nakon Sawan province in lower northern Thailand during November 2006 to April 2007. Before blood collection, the umbilical cord was wiped with gauze to reduce maternal blood contamination. Preterm newborns and newborns with others abnormalities were excluded. Maternal gestational ages based on last menstruation period and newborns baseline data (weight, height, and sex) were recorded. The average gestational age at delivery and birth weight were 39.2 [+ or -] 1.3 weeks and 3066.9 [+ or -] 392.9 g, respectively.

[FIGURE 1 OMITTED]

Hb and Hematologic Analyses

Initial hematologic analysis was done immediately after blood collection at the Mother and Child Hospital using an automated blood cell counter (Advia, Bayer Corp, Tarrytown, NY). The remaining blood specimens were then kept at 4[degrees]C and sent on ice to the Centre for Research and Development of Medical Diagnostic Laboratories, Khon Kaen University where further laboratory investigations were performed. Hemoglobin patterns and corresponding levels were determined using the CLC 330 automated high-performance liquid chromatography (HPLC) System (Primus Corp, Kansas City, Mo). A fast peak eluted at relative retention time to Hb F of 0.2 to 0.3 minutes with a level greater than 1.5% was considered Hb Bart, (11) and that eluted at the relative retention time to Hb A of 1.07 to 1.20 with a level greater than 0.5% was reported as Hb E (Figure 1).

DNA Analysis

Genomic DNA was extracted from leukocytes using standard protocol. All common [alpha]-thalassemia mutations, including [alpha]-thalassemia 1 (SEA and THAI deletion), [alpha]-thalassemia 2 (3.7 and 4.2 kb deletions), [[alpha].sup.ConstantSpring],and [[alpha].sup.Pakse] genes were identified by polymerase chain reaction and related methods. (3,14-16) Common [beta]-thalassemia mutations in Thailand and [[beta].sup.E]-globin gene were examined using the allele-specific polymerase chain reaction routinely run in our laboratory. (16-18)

[FIGURE 2 OMITTED]

Statistical Analysis

Descriptive statistics including mean and standard deviation were used to describe hematologic characteristics of each thalassemia genotype. Statistical comparison with Student t test was performed to determine the mean difference of hematologic parameters between normal and thalassemia genotypes with sample sizes of more than 10 with equal variance. To demonstrate a trend of change in hematologic parameters, thalassemia genotypes were divided into 7 groups: 1, nonthalassemia; 2, single [alpha]-gene defect; 3, 2 [alpha]-gene defects; 4, heterozygous Hb E; 5, heterozygous Hb E with single [alpha]-gene defect; 6, heterozygous Hb E with 2 [alpha]-gene defects (including 1 homozygous Hb E with [alpha]-thalassemia 1); and 7, homozygous Hb E. Box plots (median and interquartile range) for each parameter were constructed. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated to determine the effectiveness of MCV, MCH, Hb Bart, and Hb E values for screening of [alpha]-thalassemia 1 and Hb E in newborns.

RESULTS

Of the 402 cord blood samples, 178 (44.3%) were found to carry thalassemia genes with as many as 18 different genotypes. As shown in Table 1, the most prevalent genotype was heterozygous Hb E, which was observed in 63 (15.7%) cases, followed by heterozygous [alpha]-thalassemia 2 (11.0%), heterozygous [alpha]-thalassemia 1 (3.6%), heterozygous Hb Constant Spring (3.0%), double heterozygous HbE and [alpha]-thalassemia 2 (3.0%), and homozygous Hb E (1.2%). Other genotypes found with lower frequencies included compound heterozygous states for [alpha]-thalassemia and double heterozygous states for Hb E and various forms of [alpha]-thalassemia. There were 5 cases with unknown [alpha]-thalassemia 2 and 1 case with uncharacterized Hb variant. Further screening for [beta]-thalassemia mutations identified 1 case of heterozygous [[beta].sup.0]-thalassemia with the codon 17 (A-T) mutation, which is one of the most common [beta]-thalassemia genes in Thailand. (19,20) No thalassemia gene was detected in the remaining 224 cases (55.7%).

Table 2 summarizes hematologic parameters and Hb profiles of each thalassemia genotype as compared with the nonthalassemic group (n = 224). Hematologic data of both male and female babies were pooled together due to the fact that there was no significant difference in hematologic parameters between the 2 sexes in each genotype (data not shown). A box plot of each parameter was constructed and is presented in Figure 2. As compared with nonthalassemic newborns (Figure 2; group 1), all [alpha]-thalassemia genotypes including double heterozygosity for HbE and [alpha]-thalassemia (Figure 2; groups 2, 3, 5, and 6) showed significant reduction in Hb (Figure 2, B), MCV (Figure 2, C), and MCH (Figure 2, D) with increasing trend of red blood cells (Figure 2, A). In contrast, heterozygous Hb E without [alpha]-thalassemia (Figure 2; group 4) and 1 case of a [beta]-thalassemia trait (Table 1) showed no difference from a nonthalassemic group for all parameters. Analysis of Hb-HPLC profiles revealed inconsistency in the presence of Hb Bart among newborns with deletional [alpha]-thalassemia 2 as it may or may not be present in cord blood (Table 2). However, all heterozygous [alpha]-thalassemia 1 (Figure 2; group 3) including those with Hb E (Figure 2; group 6) showed significant increase in Hb Bart level (Figure 2, E). An eluted peak at the Hb [A.sub.2] window with level of greater than 0.5% of total Hb was completely consistent with positive polymerase chain reaction analysis for the [[beta].sup.E]-globin gene.

Based on these data, the appropriate cutoff values of

MCV, MCH, Hb Bart, and Hb E for screening of thalassemia in newborns were determined. It was found that the cutoff values of MCV less than 95 fL, MCH less than 30 pg, and Hb Bart greater than 8.0% could individually provide 100% sensitivity for prediction of [alpha]-thalassemia 1, whereas the Hb E level of greater than 0.5% showed 100% accuracy for diagnosis of Hb E. The sensitivity, specificity, positive predictive value, and negative predictive value of these parameters are comparatively presented in Table 3.

COMMENT

In the present study, we analyzed hematologic features and Hb profiles of 402 cord blood specimens collected at the time of birth. Data were obtained from both normal and various thalassemic newborns, which would be useful for establishment of reference values for use in the region. As for other studies, (21-23) the identification of 18 types of thalassemia in this group of Thai newborns confirms a high prevalence and a diverse heterogeneity of this genetic disorder in southeast Asia and underlines the need for appropriate screening strategy.

As expected, Hb E, which is prevalent among southeast Asian populations, (24) and heterozygous [alpha]-thalassemia were the most common hemoglobinopathies encountered in this group of subjects; they were respectively detected in about 15% of the cases. In contrast, [beta]-thalassemia was clearly less common and was found in only 1 (0.2%) of 402 newborns investigated. The high prevalence of Hb E and [alpha]-thalassemia in this group of newborns was supported by observation of various interactions of Hb E and several forms of [alpha]-thalassemia (Table 1). Hematologic comparison between the nonthalassemic group with various forms of [alpha]-thalassemia revealed decreasing trends for Hb, MCV, and MCH and an increasing trend for red blood cells, which corresponded to the number of [alpha]-globin gene defects (Table 2; Figure 2). This result is in agreement with the study of Kyriacou et al.10 In contrast to the [alpha]-thalassemia, hematologic parameters of heterozygous Hb E and [beta]-thalassemic newborns with no [alpha]-thalassemia showed no significant difference from the nonthalassemic group. This is not surprising as both Hb E and [beta]-thalassemia are caused by defects in [beta]-globin genes, which are not fully expressed in newborns and hence have no effect on hematologic parameters. No difference between hematologic indices of normal and heterozygous [beta]-thalassemia newborns has been documented in other populations. (25) It is therefore not possible to provide accurate diagnosis of heterozygous [beta]-thalassemia in newborns using hematologic analysis alone unless DNA assay is performed. It is noteworthy that coinheritance of Hb E with [alpha]-thalassemia results in similar hematologic changes as various forms of non-Hb E [alpha]-thalassemia. However, the homozygous state of Hb E might be associated with some degree of globin chain imbalance as indicated by slight reduction in Hb, MCV, and MCH values as compared with the nonthalassemic group.

Based on the Hb-HPLC system used in this study, Hb Bart is eluted as a fast peak at a relative retention time to Hb F of 0.2 to 0.3 minutes (Figure 1). We found, however, that almost all cord blood specimens had this fast eluting peak with level ranging from 0.1% to 31.8%. Rugless et al (11) also noted the same findings in their study. The amount rather than an appearance of Hb Bart peak in cord blood is more important for providing accurate diagnosis of [alpha]-thalassemia. We observed the Hb Bart level of less than 1.5% with an average of 0.6 [+ or -] 0.3% for the nonthalassemic group with normal [alpha]-globin gene. Accordingly, the level above 1.5% was considered as Hb Bart. As shown in Table 2, with this diagnostic level, most nonthalassemic newborns, newborns with deletional [alpha]-thalassemia 2, and newborns with heterozygous or homozygous Hb E without [alpha]-thalassemia would be considered as having no Hb Bart. This result indicates that the Hb Bart in cord blood is not a useful marker for identification of a deletional [alpha]-thalassemia 2 carrier. Interestingly, most of the Hb Constant Spring heterozygotes (12/13) had significant level of Hb Bart with an average value of 2.8 [+ or -] 1.3% (Table 2). This result likely indicates a greater globin chain imbalance for a nondeletional [alpha]-thalassemia 2 as compared with the deletional [alpha]-thalassemia 2 although the other hematologic values were not significantly different. Indeed, this is not unexpected as in several [alpha]-thalassemia syndromes, the nondeletional [alpha]-thalassemia 2 is usually associated with a more severe phenotype than that of the deletional [alpha]-thalassemia 2. (1,2,26) In newborns with heterozygous [alpha]-thalassemia 1, compound heterozygous for [alpha]-thalassemia 1/[alpha]-thalassemia 2 and compound heterozygous for [alpha]-thalassemia 2/[[alpha].sup.CS], marked reductions in Hb, MCV, and MCH were clearly observed and the levels of Hb Bart were also remarkably high corresponding to the number of [alpha]-globin defects. Miller et al (27) have also noted the presence of a fast Hb variant (Hb Bart) in 18 black infants with [alpha]-thalassemia 1. These hematologic values and the level of Hb Bart are therefore useful for diagnosis of these clinically more important thalassemias at birth.

Given that a cord blood sample is a noninvasive source for [alpha]-thalassemia screening, we therefore selected the MCV (cutoff, 95 fL), MCH (cutoff, 30 pg), and Hb Bart (cutoff, 8.0%) for evaluating the effectiveness in screening of [alpha]-thalassemia 1 in newborns. As presented in Table 3, all 3 parameters were found to be comparatively sensitive in screening of [alpha]-thalassemia 1. It is noteworthy however that the best predictor for [alpha]-thalassemia 1 in this group of Thai newborns was the MCH. As determined by the positive predictive value, almost 78.3% of newborns with positive MCH screening were predicted correctly, whereas the positive predictive value for Hb Bart was only 70.4%. As for MCH, the MCV also provided a better result than Hb Bart level in our subjects with a corresponding positive predictive value of 75.0%. As compared with the identification of Hb Bart by the Hb-HPLC analysis, MCV and MCH values are more readily available in most laboratories. We recommended therefore that when no Hb-HPLC system is available, MCV and MCH might be used alternatively to Hb Bart (9-11) for effective screening of [alpha]-thalassemia 1 in newborns in the region.

Unlike [alpha]-thalassemia 1, Hb E level (cutoff, 0.5%) was found to be the best predictor for screening of Hb E in newborns. The MCV and MCH values failed to provide acceptable screening results. We have noted previously in adults that heterozygous Hb E is associated with a range of hematologic characteristics ranging from low MCV and MCH to normal red blood cell indices. In addition, an adult with double heterozygote for Hb E and [alpha]-thalassemia 1 always has a lower Hb E level as compared with a pure heterozygous Hb E due to the effect of a decreased availability of [alpha]-chains on the apE subunit formation. (28) This is not the case for newborns as we found that the Hb E level in a double heterozygote for Hb E and [alpha]-thalassemia 1 was not different from the heterozygous Hb E. This might be explained by the similar expression of the [[beta].sup.E] and the [[beta].sup.A]-globin genes in newborns. (29) Based on our results, the Hb E level of greater than 0.5% as determined by HPLC, which provided 100% accuracy for identification of Hb E (Table 3), could be used effectively as a primary screen for Hb E in newborns.

In southeast Asia where thalassemia and hemoglobinopathies are most common, the prime targets of prevention and control of severe thalassemia are homozygous [alpha]-thalassemia 1 (Hb Bart hydrops fetalis syndrome), homozygous [beta]-thalassemia, and [beta]-thalassemia/Hb E disease. The types targeted for screening are therefore [alpha]-thalassemia 1, [beta]-thalassemia, and Hb E. (21,22,30) We have shown that although screening for [beta]-thalassemia at birth is difficult without DNA analysis, screening for [alpha]-thalassemia 1 and Hb E are relatively simple by analysis of cord blood obtained at the time of delivery. The results from this study should facilitate a newborn screening and prevention and control program for thalassemia and hemoglobinopathies in the region.

This work was supported by research grants from Khon Kaen University and the Graduate School, Khon Kaen University, Khon Kaen, Thailand. We thank Helena Thai Laboratories Co, Ltd, Bangkok, Thailand, for help with Hb analysis using the Primus CLC330 HPLC System.

Accepted for publication April 14, 2008.

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Jaruwan Tritipsombut, BSc; Kanokwan Sanchaisuriya, PhD; Supan Fucharoen, DSc; Goonnapa Fucharoen, MSc; Nirut Siriratmanawong, MSc; Charnchai Pinmuang-ngam, MD; Pattara Sanchaisuriya, Dr rer med

From the Graduate School, Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Thailand (Ms Tritipsombut, Drs Sanchaisuriya and S. Fucharoen, and Mrs G. Fucharoen); the Department of Clinical Pathology, Regional Health Center 8, Nakhon Sawan, Thailand (Mr Siriratmanawong and Dr Pinmuang-ngam); and the Department of Nutrition, Faculty of Public Health, Khon Kaen University, Thailand (Dr Sanchaisuriya).

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Supan Fucharoen, DSc, Centre for Research & Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand 40002 (e-mail: supan@kku.ac.th).
Table 1. Thalassemia Genotypes Found in 402
Cord Blood Samples *

Thalassemia Genotypes                       No.     %

Heterozygous Hb E                            63    15.7
Heterozygous [alpha]-thalassemia 2           44    11.0
Heterozygous [alpha]-thalassemia 1           15     3.6
Heterozygous Hb Constant Spring              12     3.0
Double heterozygous Hb E/a-thalassemia 2     12     3.0
Homozygous Hb E                               5     1.2
Compound heterozygous [alpha]-thalassemia
2/Hb Constant Spring                          4     1.0
Double heterozygous Hb E/Hb Constant
Spring                                        4     1.0
Double heterozygous Hb E/[alpha]-             3     0.8
thalassemia 1
Double heterozygous Hb E/homozygous
[alpha]-thalassemia 2                         3     0.8
Compound heterozygous [alpha]-thalassemia
1/[alpha]-thalassemia 2                       2     0.5
Heterozygous Hb Pakse                         1     0.2
Homozygous [alpha]-thalassemia 2              1     0.2
Double heterozygous Hb E/Hb Constant
Spring and [alpha]-thalassemia 2              1     0.2
Homozygous Hb E with                          1     0.2
[alpha]-thalassemia 1
Heterozygous [[beta].sup.0]-thalassemia       1     0.2
Uncharacterized abnormal Hb                   1     0.2
Uncharacterized [alpha]-thalassemia 2         5     1.2
([dagger])
Nonthalassemia                              224    56.0
Total                                       402   100.0

* Hb indicates hemoglobin.

([dagger]) Indicated by Hb Bart > 1.5% but no common a-thalassemia gene
detected.

Table 2. Hematologic Features and Hemoglobin (Hb) Profiles of Cord
Blood Samples According to Thalassemia Genotypes *

Group   Genotypes (n)                      n     RBC, X [10.sup.12]/L

1       Nonthalassemia                     224   4.4 [+ or -] 0.4
2       Single [alpha]-gene defect
        -[alpha]/[alpha][alpha]             34   4.6 ([dagger])
                                                 [+ or -] 0.4
        [alpha][alpha]/[[alpha].sup.cs]     13   4.5 [+ or -] 0.4
        [alpha] (12) and [alpha][alpha]/
        [[alpha].sup.Ps][alpha] (1)
        Unknown                              5   4.8 [+ or -] 0.4
3       Two [alpha]-gene
        defect/Hb H disease
        [alpha]-thalassemia 1 trait         15   5.3 ([dagger])
        (-/[alpha][alpha])                       [+ or -] 0.5
        [alpha]/[[alpha].sup.CS]             5   4.8 [+ or -] 0.6
        [alpha] (4) and
        homozygous a-thalassemia 2 (1)
        --/-[alpha]                          2   5.5, 5.6
4       Heterozygous Hb E                   63   4.4 [+ or -] 0.5
5       Heterozygous Hb E with single
        [alpha]-gene defect
        With [alpha]-thalassemia 2          12   4.6 [+ or -] 0.5
        (-[alpha]/[alpha][alpha])
        With Hb CS ([alpha][alpha]/          4   4.2 [+ or -] 0.8
        [[alpha].sup.cs][alpha])
6       Heterozygous Hb E with 2
        [alpha]-gene defects
        With [alpha]-thalassemia 1           3   5.0 [+ or -] 0.6
        (-/[alpha][alpha])
        With (-[alpha]/-[alpha])             4   5.0 [+ or -] 0.7
        (3) and (-
        [alpha]/[[alpha].sup.CS]
        [alpha]) (1)
7       Homozygous Hb E
        Without a-thalassemia                5   4.6 [+ or -] 0.6
        ([alpha][alpha]/[alpha][alpha])
        With [alpha]-thalassemia 1           1   5.92
        (--/[alpha][alpha])
8       Heterozygous [beta]-thalassemia      1   5.5

Group   Genotypes (n)                      Hb, g/dL

1       Nonthalassemia                     15.5 [+ or -] 1.5
2       Single [alpha]-gene defect
        -[alpha]/[alpha][alpha]            15.0 ([dagger]) [+ or -] 1.3
        [alpha][alpha]/[[alpha].sup.cs]    14.9 [+ or -] 1.3
        [alpha] (12) and [alpha][alpha]/
        [[alpha].sup.Ps][alpha] (1)
        Unknown                            15.3 [+ or -] 0.9
3       Two [alpha]-gene
        defect/Hb H disease
        [alpha]-thalassemia 1 trait        14.3t [+ or -] 1.6
        (-/[alpha][alpha])
        [alpha]/[[alpha].sup.CS]           14.0 [+ or -] 1.5
        [alpha] (4) and
        homozygous a-thalassemia 2 (1)
        --/-[alpha]                        10.7, 11.8
4       Heterozygous Hb E                  15.4 [+ or -] 1.5
5       Heterozygous Hb E with single
        [alpha]-gene defect
        With [alpha]-thalassemia 2         14.7 [+ or -] 1.1
        (-[alpha]/[alpha][alpha])
        With Hb CS ([alpha][alpha]/        13.7 [+ or -] 1.7
        [[alpha].sup.cs][alpha])
6       Heterozygous Hb E with 2
        [alpha]-gene defects
        With [alpha]-thalassemia 1         14.1 [+ or -] 1.1
        (-/[alpha][alpha])
        With (-[alpha]/-[alpha])           14.6 [+ or -] 0.9
        (3) and (-
        [alpha]/[[alpha].sup.CS]
        [alpha]) (1)
7       Homozygous Hb E
        Without a-thalassemia              15.1 [+ or -] 2.3
        ([alpha][alpha]/[alpha][alpha])
        With [alpha]-thalassemia 1         16.3
        (--/[alpha][alpha])
8       Heterozygous [beta]-thalassemia    16.4

Group   Genotypes (n)                      MCV, fL

1       Nonthalassemia                     105.3 [+ or -] 4.9
2       Single [alpha]-gene defect         98.0t [+ or -] 4.8
        -[alpha]/[alpha][alpha]
        [alpha][alpha]/[[alpha].sup.cs]    99.3 [+ or -] 4.0
        [alpha] (12) and [alpha][alpha]/
        [[alpha].sup.Ps][alpha] (1)
        Unknown                            97.4 [+ or -] 6.4
3       Two [alpha]-gene
        defect/Hb H disease
        [alpha]-thalassemia 1 trait        85.7 ([dagger]) [+ or -] 5.3
        (-/[alpha][alpha])
        [alpha]/[[alpha].sup.CS]           91.2 [+ or -] 3.4
        [alpha] (4) and
        homozygous a-thalassemia 2 (1)
        - -/-[alpha]                       73.1, 74.1
4       Heterozygous Hb E                  105.0 [+ or -] 6.1
5       Heterozygous Hb E with single
        [alpha]-gene defect
        With [alpha]-thalassemia 2         95.5 [+ or -] 4.3
        (-[alpha]/[alpha][alpha])
        With Hb CS ([alpha][alpha]/        102.2 [+ or -] 5.4
        [[alpha].sup.cs][alpha])
6       Heterozygous Hb E with 2
        [alpha]-gene defects
        With [alpha]-thalassemia 1         87.2 [+ or -] 1.7
        (-/[alpha][alpha])
        With (-[alpha]/-[alpha])           93.7 [+ or -] 15.2
        (3) and (-
        [alpha]/[[alpha].sup.CS]
        [alpha]) (1)
7       Homozygous Hb E
        Without a-thalassemia              100.0 [+ or -] 3.0
        ([alpha][alpha]/[alpha][alpha])
        With [alpha]-thalassemia 1         86.1
        (--/[alpha][alpha])
8       Heterozygous [beta]-thalassemia    92.8

Group   Genotypes (n)                      MCH, pg

1       Nonthalassemia                     35.2 [+ or -] 1.7
2       Single [alpha]-gene defect         32.6 ([dagger]) [+ or -] 1.6
        -[alpha]/[alpha][alpha]
        [alpha][alpha]/[[alpha].sup.cs]    32.8 [+ or -] 1.3
        [alpha] (12) and [alpha][alpha]/
        [[alpha].sup.Ps][alpha] (1)
        Unknown                            32.2 [+ or -] 1.8
3       Two [alpha]-gene
        defect/Hb H disease
        [alpha]-thalassemia 1 trait        26.9 ([dagger]) [+ or -] 1.4
        (-/[alpha][alpha])
        [alpha]/[[alpha].sup.CS]           29.2 [+ or -] 1.3
        [alpha] (4) and
        homozygous a-thalassemia 2 (1)
        --/-[alpha]                        19.7, 21.0
4       Heterozygous Hb E                  35.1 [+ or -] 1.8
5       Heterozygous Hb E with single
        [alpha]-gene defect
        With [alpha]-thalassemia 2         32.2 [+ or -] 1.6
        (-[alpha]/[alpha][alpha])
        With Hb CS ([alpha][alpha]/        33.0 [+ or -] 2.0
        [[alpha].sup.cs][alpha])
6       Heterozygous Hb E with 2
        [alpha]-gene defects
        With [alpha]-thalassemia 1         28.2 [+ or -] 1.0
        (-/[alpha][alpha])
        With (-[alpha]/-[alpha])           29.7 [+ or -] 3.5
        (3) and (-
        [alpha]/[[alpha].sup.CS]
        [alpha]) (1)
7       Homozygous Hb E
        Without a-thalassemia              32.6 [+ or -] 1.1
        ([alpha][alpha]/[alpha][alpha])
        With [alpha]-thalassemia 1         27.6
        (--/[alpha][alpha])
8       Heterozygous [beta]-thalassemia    31.8

Group   Genotypes (n)                      Hb Type (n)

1       Nonthalassemia                     FA
2       Single [alpha]-gene defect         FA (24)
        -[alpha]/[alpha][alpha]            FAB (10)
        [alpha][alpha]/[[alpha].sup.cs]    FAB (12)
        [alpha] (12) and [alpha][alpha]/
        [[alpha].sup.Ps][alpha] (1)
        Unknown                            FA (1)
3       Two [alpha]-gene
        defect/Hb H disease
        [alpha]-thalassemia 1 trait        FAB
        (-/[alpha][alpha])
        [alpha]/[[alpha].sup.CS]           FAB
        [alpha] (4) and
        homozygous a-thalassemia 2 (1)
        --/-[alpha]                        FAB
4       Heterozygous Hb E                  FAB
5       Heterozygous Hb E with single
        [alpha]-gene defect
        With [alpha]-thalassemia 2         EFA
        (-[alpha]/[alpha][alpha])
        With Hb CS ([alpha][alpha]/        EFA (8)
        [[alpha].sup.cs][alpha])
6       Heterozygous Hb E with 2           EFAB (4)
        [alpha]-gene defects
        With [alpha]-thalassemia 1         EFAB
        (-/[alpha][alpha])
        With (-[alpha]/-[alpha])           EFAB
        (3) and (-
        [alpha]/[[alpha].sup.CS]
        [alpha]) (1)
7       Homozygous Hb E                    EFAB
        Without a-thalassemia              EF
        ([alpha][alpha]/[alpha][alpha])
        With [alpha]-thalassemia 1         EFB
        (--/[alpha][alpha])
8       Heterozygous [beta]-thalassemia    FA

Group   Genotypes (n)                      Hb [A.sub.2]/E, %

1       Nonthalassemia                     None
2       Single [alpha]-gene defect         None
        -[alpha]/[alpha][alpha]            None
        [alpha][alpha]/[[alpha].sup.cs]    None
        [alpha] (12) and [alpha][alpha]/
        [[alpha].sup.Ps][alpha] (1)
        Unknown                            None
3       Two [alpha]-gene
        defect/Hb H disease
        [alpha]-thalassemia 1 trait        None
        (-/[alpha][alpha])
        [alpha]/[[alpha].sup.CS]           None
        [alpha] (4) and
        homozygous a-thalassemia 2 (1)
        --/-[alpha]                        None
4       Heterozygous Hb E                  None
5       Heterozygous Hb E with single
        [alpha]-gene defect
        With [alpha]-thalassemia 2         2.9 [+ or -] 1.2
        (-[alpha]/[alpha][alpha])
        With Hb CS ([alpha][alpha]/        3.9 [+ or -] 1.6
        [[alpha].sup.cs][alpha])
6       Heterozygous Hb E with 2           1.9 [+ or -] 0.7
        [alpha]-gene defects
        With [alpha]-thalassemia 1         2.3 [+ or -] 0.7
        (-/[alpha][alpha])
        With (-[alpha]/-[alpha])           2.2 [+ or -] 0.8
        (3) and (-
        [alpha]/[[alpha].sup.CS]
        [alpha]) (1)
7       Homozygous Hb E                    3.4 [+ or -] 1.3
        Without a-thalassemia              6.6 [+ or -] 2.4
        ([alpha][alpha]/[alpha][alpha])
        With [alpha]-thalassemia 1         12.1
        (--/[alpha][alpha])
8       Heterozygous [beta]-thalassemia    None

Group   Genotypes (n)                      Hb Bart, %

1       Nonthalassemia                     0.6 [+ or -] 0.3
2       Single [alpha]-gene defect         1.0 [+ or -] 0.2
        --[alpha]/[alpha][alpha]           2.0 [+ or -] 0.4
        [alpha][alpha]/[[alpha].sup.cs]    2.8 [+ or -] 1.3
        [alpha] (12) and [alpha][alpha]/
        [[alpha].sup.Ps][alpha] (1)
        Unknown                            1.3
3       Two [alpha]-gene
        defect/Hb H disease
        [alpha]-thalassemia 1 trait        1.8 [+ or -] 0.2
        (-/[alpha][alpha])
        [alpha]/[[alpha].sup.CS]           11.7 [+ or -] 2.0
        [alpha] (4) and
        homozygous a-thalassemia 2 (1)
        --/-[alpha]                        11.0 [+ or -] 2.6
4       Heterozygous Hb E                  31.7, 31.8
5       Heterozygous Hb E with single
        [alpha]-gene defect
        With [alpha]-thalassemia 2         0.5 [+ or -] 0.3
        (-[alpha]/[alpha][alpha])
        With Hb CS ([alpha][alpha]/        0.9 [+ or -] 0.3
        [[alpha].sup.cs][alpha])
6       Heterozygous Hb E with 2           1.9 [+ or -] 0.5
        [alpha]-gene defects
        With [alpha]-thalassemia 1         2.8 [+ or -] 0.6
        (-/[alpha][alpha])
        With (-[alpha]/-[alpha])           11.9 [+ or -] 1.1
        (3) and (-
        [alpha]/[[alpha].sup.CS]
        [alpha]) (1)
7       Homozygous Hb E                    6.1 [+ or -] 3.7
        Without a-thalassemia              0.4 [+ or -] 0.3
        ([alpha][alpha]/[alpha][alpha])
        With [alpha]-thalassemia 1         6.4
        (--/[alpha][alpha])
8       Heterozygous [beta]-thalassemia    0

* Values are presented as mean [+ or -] standard deviation or as raw
data where appropriate. RBC indicates red blood cell; MCV, mean
corpuscular volume; MCH, mean corpuscular hemoglobin; CS, Hb Constant
Spring; Ps, Hb Pakse; and B, Hb Bart.

([dagger]) Statistically significant difference from non-[alpha]-
thalassemia (Student t test, P < .001).

Table 3. Sensitivity, Specificity, Positive Predictive
Value (PPV), and Negative Predictive Value (NPV) of
Mean Corpuscular Volume (MCV), Mean Corpuscular
Hemoglobin (MCH), and Hemoglobin (Hb) Bart for
Prediction of [alpha]-Thalassemia 1, and Hb E Level for
Prediction of Hb E in Newborns

Predicted        Sensitivity,   Specificity,   PPV,   NPV,
Parameters            %              %          %      %

For [alpha]-thalassemia 1

  MCV < 95 fL        100            98.3       75.0   100
  MCH < 30 pg        100            98.7       78.3   100
  Hb Bart > 8%       100            97.9       70.4   100

For Hb E

  Hb E > 0.5%        100            100        100    100
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