Hematopathology / SCREENING FOR THE (-- SEA ) ALPHA 0 -THALASSEMIA DELETION

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1 Hematopathology / SCREENING FOR THE (-- SEA ) ALPHA 0 -THALASSEMIA DELETION A Reliable Screening Test to Identify Adult Carriers of the (-- SEA ) alpha 0 -Thalassemia Deletion Detection of Embryonic zeta-globin Chains by Enzyme-Linked Immunosorbent Assay John D. Lafferty, ART, 1 Mark A. Crowther, MD, 1,2 John S. Waye, PhD, 1,2 and David H.K. Chui, MD 1,2 Key Words: Microcytosis; Thalassemia; alpha Thalassemia; Hydrops fetalis; Hemoglobin H; Hemoglobin Bart; Thalassemia testing Abstract Homozygous (-- SEA ) alpha 0 -thalassemia deletion, the cause of up to 80% of fetal hydrops in Southeast Asia, is encountered in many other countries. Heterozygous carrier rates of the deletion in Southeast Asian populations range from 4% to 14%. The laboratory screening for adult carriers of (-- SEA ) and other alpha 0 -thalassemia deletions currently rests primarily with microscopic detection of hemoglobin H inclusion bodies within erythrocytes (Hb H screen). This test is laborious and observer dependent and has poor sensitivity. We assessed a colorimetric enzymelinked immunosorbent assay (ELISA) to detect embryonic zeta-globin chains in adult hemolysates as an alternative to detect (-- SEA ) alpha 0 -thalassemia deletion carriers. Blood samples from 221 adults with a mean corpuscular volume less than 80 µm 3 (80 fl) were studied prospectively by currently accepted hemoglobin screening tests and ELISA. Suspected cases of alpha-thalassemia were confirmed by DNA-based diagnostics. ELISA was highly sensitive (1.0) and specific (0.94) for the detection of adult carriers of (-- SEA ) alpha 0 -thalassemia deletion. The hemoglobin H screen had a sensitivity of 0.47 and specificity of The zeta-globin ELISA proved simple to perform, rapid, and applicable to high volume or population-based screening programs. People with a normal genotype have 4 alpha-globin genes, 2 adjacent to each other, ie, in cis, on each chromosome 16p13.3. Alpha-thalassemia is the result of decreased production of the alpha-globin chain of hemoglobin (Hb) caused by a mutation of one or more of the alpha-globin genes. The clinical severity of alpha-thalassemia is directly proportional to the number of alpha-globin genes affected. Deletional mutations are the most common cause of alphathalassemia; however, point mutations also have been described. People with a single alpha-globin gene deletion (-α/αα) have normal Hb levels with a normocytic or slightly microcytic blood picture and are described as silent carriers of alpha-thalassemia. Deletion of 2 alpha-globin genes (--/αα) or (-α/-α) causes microcytosis with Hb levels indicating normal to very mild anemia. Mutations involving 3 alphaglobin genes (--/-α), ie, Hb H disease, give rise to moderate microcytic anemia that is usually not transfusion dependent. Deletion of all 4 alpha-genes (--/--) causes Hb Bart hydrops fetalis, a condition that results in fetal death and is associated with serious maternal complications, such as preeclampsia, hemorrhage, and dystocia. People with the 2 alpha-gene deletion in cis, ie, (--/αα) or (--/-α), are carriers of Hb Bart hydrops fetalis. 1,2 The Southeast Asian (SEA) alpha 0 -thalassemia mutation (-- SEA ) deletes both alpha-globin genes in cis and is the most common cause of Hb Bart hydrops fetalis. The heterozygous carrier frequencies of this mutation in SEA populations range from 4.5% in Hong Kong to 14% in northern Thailand. 2-4 With recent global migration patterns, this carrier form of alpha-thalassemia is now found in all parts of the world and is often the cause of microcytosis encountered in clinical Am J Clin Pathol 2000;114:

2 Lafferty et al / SCREENING FOR THE (-- SEA ) ALPHA 0 -THALASSEMIA DELETION laboratories. Couples in which both members are heterozygous for the (-- SEA ) deletion or another extensive alpha 0 - thalassemia deletion, such as the Filipino (-- FIL ) or Thai (-- THAI ) deletion, have a 25% risk in each pregnancy of conceiving a fetus with Hb Bart hydrops fetalis. 1,2 Laboratory screening for the (-- SEA ) deletion is problematic. The most reliable method of detection is by DNA-based genotypic analyses; however, these techniques are available only in highly specialized laboratories. At present, the only non DNA-based test for alpha-thalassemia is the Hb H inclusion body screen (Hb H screen), which is laborious and observer dependent and has poor sensitivity. The sensitivity of the Hb H screen can be improved by first excluding other causes of microcytosis, eg, iron deficiency or betathalassemia trait, followed by an exhaustive search for Hb H inclusion bodies. 5 When necessary after this, as for genetic counseling purposes, samples with unexplained microcytosis can be referred for definitive DNA-based genotypic analysis. 6-8 Neither DNA-based screening nor the current exclusion strategy are suitable for high-volume screening programs. Previous studies have shown that minute amounts, eg, less than 0.5%, of embryonic zeta-globin chains are present in erythrocytes of adult carriers of the (-- SEA ) alpha 0 - thalassemia deletion Further studies found that immunocytochemical staining techniques or enzyme-linked immunosorbent assays (ELISA) designed to detect embryonic zeta-globin chains were sensitive and specific for the identification of carriers of the (-- SEA ) alpha 0 -thalassemia deletion The objective of the present study was to assess a newly developed zeta-globin ELISA kit, comparing its effectiveness with the current exclusion strategy using the Hb H screen. Materials and Methods Cohort Study A consecutive cohort study of adult blood samples with a mean corpuscular volume less than 80 µm 3 (80 fl) referred for hemoglobinopathy investigation was performed. Samples in the cohort study were evaluated using the zeta-globin ELISA and the current diagnostic strategy, including Hb H screen. The sensitivity and specificity for the detection of the (-- SEA ) deletion and all other forms of alpha-thalassemia encountered were calculated for the zeta-globin ELISA and for the Hb H screen. 15 Blood Samples Adult blood samples submitted for hemoglobinopathy diagnosis were used in the study. Blood samples were drawn into EDTA using standard venipuncture techniques, shipped at room temperature, and analyzed within 48 hours of receipt in the laboratory. For zeta-globin ELISA, samples were batched and run at biweekly intervals. Samples used for the precision analyses (see Precision Analyses ) were previously analyzed blood specimens that had been stored at 4 C for 1 to 6 weeks. Current Diagnostic Strategy For all blood samples in the cohort study, findings were available from the CBC, hemoglobin electrophoresis by isoelectric focusing, Hb A 2 quantitation, Hb H screen, and serum ferritin determination. Hb F quantitation by alkali denaturation was performed whenever measurable Hb F levels were detected by isoelectric focusing. Samples referred for genetic counseling workup, with positive Hb H screen, or with no detectable cause for microcytosis were referred for alpha-globin genotyping. Genotyping was done by Southern blot analysis or gap polymerase chain reaction methods. 2,6,16 zeta-globin ELISA The zeta-globin ELISA test kits were provided by United Biotech, Mountain View, CA. The kits contain microtiter plates coated with murine antihuman zeta-globin chain monoclonal antibodies. Hemolysate was dispensed into each microtiter plate well, incubated, and washed according to manufacturer s instructions. Subsequently, murine antihuman zeta-globin chain monoclonal antibodies conjugated with horseradish peroxidase were added, followed by chromogenic substrate. In the present study, the color developed from the reaction was first assessed macroscopically as positive, weakly positive, or negative compared with appropriate control samples. The optical density of the color reaction then was determined spectrophotometrically at 450 nm (OD 450nm ) by an ELISA reader (Microelisa model MR580). An OD 450nm greater than 0.25 absorbance units (AU) is recommended by the manufacturer to represent a positive result. Precision Analyses To determine the reproducibility of the ELISA, precision analyses were undertaken in which blood samples with or without known alpha-thalassemia mutations were analyzed under different conditions. Samples were analyzed on 4 to 8 occasions during a 2-week period. Precision was assessed by paired analysis of samples run in duplicate with the same reagents on the same day, samples run with the same reagents 7 days apart, samples run in duplicate on the same day with different lots of enzyme conjugates, and samples run in duplicate on the same day with different lots of microtiter plates. 928 Am J Clin Pathol 2000;114:

3 Hematopathology / ORIGINAL ARTICLE Results Cohort Study We included 221 adult blood samples with microcytosis in the cohort study. Of these, 108 had a diagnosis other than thalassemia, eg, iron deficiency. Of the remaining, there were 59 with beta-thalassemia trait, 2 with delta-beta-thalassemia trait, 1 with Hb Lepore trait, and 51 with possible alphathalassemia trait. Alpha-globin genotypes were determined in 65 blood samples. Two were found to have Hb H disease (-α 3.7 /-- SEA ), 17 (-- SEA /αα), 2 (-- FIL /αα), 7 (-α 3.7 /-α 3.7 ), 10 (-α 3.7 /αα), 3 (-α 4.2 /αα), and 24 (αα/αα). ELISA was performed on all 221 blood samples. Thirtyone blood samples yielded OD 450nm readings higher than 0.25 Table 1. They included 2 (-α 3.7 /-- SEA ), 17 (-- SEA /αα), 4 (-α 3.7 /-α 3.7 ), 2 (-α 3.7 /αα), 1 (-α 4.2 /αα), 2 (αα/αα) with betathalassemia trait, and 3 with iron deficiency (Table 1). All 19 carriers of the (-- SEA ) deletion were positive by ELISA, as determined macroscopically and by ELISA reader with an OD 450nm ranging from 0.51 to more than 2.0 AU. The Hb H screen was positive in 9 of 19 carriers of the (-- SEA ) deletion. The sensitivity and specificity of the ELISA for the detection Table 1 Samples With Positive Enzyme-Linked Immunosorbent Assay (ELISA) Results of the (-- SEA ) deletion were 1.0 and 0.94, respectively. By increasing the OD 450nm cutoff to 0.5 AU, the sensitivity and specificity of the ELISA were 1.0 and In contrast, the Hb H screen had sensitivity and specificity for the detection of the (-- SEA ) deletion of 0.47 and 0.99, respectively. Seven blood samples with alpha-thalassemia other than the (-- SEA ) deletion had positive ELISA results with OD 450nm ranging from 0.25 to more than 1.3 AU. For all forms of alpha-thalassemia, the ELISA had sensitivity and specificity of 0.62 and 0.97, respectively, compared with 0.29 and 1.0, respectively, for the Hb H screen. The 2 blood samples from (-- FIL ) carriers did not have detectable zeta-globin chains, consistent with a previously published report. 12 One of the (-- FIL ) carriers had a positive Hb H screen result. Precision Analyses Blood samples from people with the following genotypes were included in the precision analyses: (-α 3.7 /-- SEA ), 1; (-- SEA /αα), 4; (-- MED /αα), 2; (-α 3.7 /-α 3.7 ), 2; (-α 3.7 /αα), 2; and (αα/αα), 2. Their OD 450nm results are given in Table 2. No significant variation was observed between samples run in duplicate with the same reagents on the same day (P =.79). Significant variations were observed between paired alpha-globin ELISA Macroscopic Hemoglobin H Diagnosis Genotype OD 450nm Reading Inclusion Bodies alpha-thalassemia trait -- SEA /αα >1.5 Positive Negative alpha-thalassemia trait -- SEA /αα >1.4 Positive Negative Hemoglobin C trait with alpha thalassemia -α 3.7 /-α 3.7 >1.3 Positive Negative alpha-thalassemia trait -- SEA /αα >1.2 Positive Occasional Hemoglobin E trait with alpha-thalassemia trait -- SEA /αα >1.2 Positive Negative alpha-thalassemia trait -- SEA /αα 1.18 Positive Occasional beta-thalassemia trait with alpha thalassemia -α 4.2 /αα >1.0 Positive Negative alpha-thalassemia trait -- SEA /αα 0.9 Positive Negative Hemoglobin H disease -α 3.7 /-- SEA 0.67 Positive Numerous alpha thalassemia -α 3.7 /-α Positive Negative Hemoglobin H disease -α 3.7 /-- SEA 0.51 Positive Numerous alpha thalassemia -α 3.7 /αα 0.48 Weakly positive Negative Iron deficiency anemia Not available 0.46 Positive Negative Iron deficiency anemia Not available 0.45 Weakly positive Negative alpha thalassemia -α 3.7 /-α Weakly positive Negative Homozygous hemoglobin E with alpha thalassemia -α 3.7 /αα 0.34 Positive Negative beta-thalassemia trait αα/αα 0.33 Weakly positive Negative Iron deficiency anemia Not available 0.31 Weakly positive Negative beta-thalassemia trait αα/αα 0.3 Weakly positive Not available alpha thalassemia -α 3.7 /-α Weakly positive Negative OD, optical density. Am J Clin Pathol 2000;114:

4 Lafferty et al / SCREENING FOR THE (-- SEA ) ALPHA 0 -THALASSEMIA DELETION Table 2 Results of the Precision Study on the zeta-globin Enzyme- Linked Immunosorbent Assay Kit Genotype No. of Samples Range of OD 450nm -α 3.7 /-- SEA SEA /αα > MED /αα >1.40 -α 3.7 /-α α 3.7 /αα αα/αα OD, optical density. samples tested 7 days apart (P =.01), paired analyses using different lots of enzyme conjugates (P =.02), and paired analyses using different lots of microtiter plates (P =.01). Both (-α 3.7 /-- SEA ) blood samples gave positive ELISA results (OD 450nm higher than 0.5 AU) in the cohort study. However, during the precision analyses, 6 of the 14 analyses on the 1 (-α 3.7 /-- SEA ) blood sample gave OD 450nm values less than 0.25 AU. Discussion This prospective cohort study demonstrates that the zeta-globin ELISA kit tested has a high sensitivity and specificity for identifying adult carriers of the (-- SEA ) alpha 0 - thalassemia deletion. Compared with the Hb H screen, the ELISA is more reliable, less laborious, and amenable to the development of high throughput automation systems, which will facilitate large-scale screening programs. Two blood samples with the (-- MED /αα) genotype also gave positive ELISA results, as has been previously reported. 11 More blood samples are needed for testing to document the sensitivity and specificity of the ELISA to detect (-- MED ) alpha 0 - thalassemia deletion carriers. In contrast to other studies, the ELISA test results were positive in several people with (-α 3.7 /-α 3.7 ), (-α 3.7 /αα), (-α 4.2 /αα), and (αα/αα) genotypes in the present study. 12,17 Precision analyses showed highly variable results in these cases. These variations effectively limit the usefulness of this assay to screening for (-- SEA ) alpha 0 -thalassemia deletion carriers whose hemolysates yield markedly elevated OD 450nm readings (Table 1). The ELISA is a screening test and does not obviate the need for definitive diagnosis by DNA-based genotyping for genetic counseling purposes or when clinically indicated. Results of the present study demonstrated that the ELISA could be effectively used as a macroscopically read semiquantitative screening test (Table 1). Results based on macroscopic readings in the cohort study provided equal sensitivity for the detection of the (-- SEA ) deletion, comparable with those with OD 450nm readings (Table 1). This simplified approach has the advantage of not requiring an ELISA reader. Blood samples from patients with Hb H disease (-α 3.7 / -- SEA ) gave OD 450nm readings less than that of heterozygous carriers (-- SEA /αα). In the precision study, 6 of 14 OD 450nm readings from an (-α 3.7 /-- SEA ) blood sample were below 0.25 AU. In contrast, other immunocytologic tests have shown that erythrocytes from patients with (-α 3.7 /-- SEA ) contain elevated proportions of zeta-globin chains. 10,11 The cause of this apparent discrepancy is not clear. One possibility is that in these erythrocytes, zeta-globin chains are attached to the plasma membrane and not readily available for detection by ELISA. Carriers of alpha 0 -thalassemia deletions involving the total zeta-alpha-globin gene cluster, such as the (-- FIL ) deletion, do not have detectable zeta-globin chains in their erythrocytes. 12 Hb H screen still can be positive in these cases. The Hb H screen is always strongly positive in Hb H disease, as in patients with (-α 3.7 /-- SEA ). These observations underscore the importance of using the zeta-globin ELISA in conjunction with other relevant clinical and laboratory findings for proper hemoglobinopathy diagnosis. Conclusions The present study confirms that the zeta-globin ELISA is a simple, rapid, and reliable screening test to identify adult carriers of the (-- SEA ) alpha 0 -thalassemia deletion. It performs better than the Hb H screen and can reduce the need for DNA-based studies for alpha-thalassemia. Unlike the exclusion strategy using Hb H screen, the sensitivity of the zeta-globin ELISA is not affected by the presence of other causes of microcytosis. Furthermore, the zeta-globin ELISA is well suited to use in high-volume screening programs to identify couples at risk of conceiving fetuses with Hb Bart hydrops fetalis. From the 1 Provincial Hemoglobinopathy Laboratory, Hamilton Regional Laboratory Medicine Program, and the 2 Department of Pathology and Molecular Medicine, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada. Address reprint requests to Mr Lafferty: Provincial Hemoglobinopathy Laboratory, St Joseph s Hospital, 50 Charlton Ave East, Hamilton, ON, Canada L8N 4A6. Acknowledgments: We thank Andrew McFarlane, ART, Mila Vacovsky, MLT, Linda Halchuk, MLT, Judy Ireland, MLT, and Millie Danek, MLT, for performing the hemoglobinopathy screening tests; Barry Eng, ART, and Margaret Patterson, MLT, for performing DNA analyses of the globin genes; and Wuan Lu, PhD, United Biotech, Mountain View, CA, for providing the ELISA kits used in this study. 930 Am J Clin Pathol 2000;114:

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