Clin 8iochem, Vol. 24, pp. 363-374, 1991 Printed in Canada. All rights reserved.

0009-9120/91 $3.00 + .00 Copyright © 1991 The Canadian Society of Clinical Chemists.

Laboratory Diagnosis of Hemoglobinopathies BERTRAM H. LUBIN, H. EWA WITKOWSKA, and KLARA KLEMAN

Children's Hospital Oakland Research Institute, 747 52 Street, Oakland, CA 94609, USA

Diagnostic tests for most common hemogiobinopathies and recent advances in structural analysis of variant hemoglobins are reviewed. Routine and newly introduced methods that apply to the diagnosis of sickle cell anemia, thalassemia and the hemoglobin E disorders are presented. A brief description of the clinical course for each of these disorders is given, and potential pitfalls in diagnosis are discussed. Application of high-performance liquid chromatography and various mass spectrometric techniques (electrospray ionization mass spectrometry, liquid secondary ion mass spectrometry, and tandem mass spectrometry) for evaluation of hemoglobinopathy is presented.

KEY WORDS: hemoglobinopathies; sickle cell anemia; thalassemia; porphyrin metabolism; electrophoresis; chromatography; mass spectrometry.

Introduction he improvement in medical care for patients with hemoglobinopathies has resulted in accepT tance of nationally endorsed screening procedures to detect abnormal hemoglobins (1). As a consequence, hemoglobin variants are now routinely identified in most clinical laboratories. It is currently possible to detect hemoglobinopathies in newborns and provide definitive prenatal diagnosis in "at risk" pregnancies. For the unusual case, sophisticated biochemical and spectroscopic methods are available. This review will provide information on recent technical advances in the diagnosis of commonly encountered hemoglobinopathies. A brief description of clinical and laboratory findings characteristic for sickle cell anemia, thalassemia and the hemoglobin (Hb) E disorders will be given and their diagnostic value discussed. The potential of novel specialized techniques such as high-performance liquid chromatography (HPLC) and mass

Correspondence: Bertram H. Lubin, M.D., Director of Medical Research, Children's Hospital Oakland Research Institute, 747 52 Street, Oakland, CA 94609, USA. Manuscript received January 18, 1991; revised April 1, 1991; accepted April 15, 1991.

CLINICAL BIOCHEMISTRY,VOLUME24, AUGUST 1991

spectrometry for identification of variant hemoglobins will be presented. B a c k g r o u n d a n d genetics The most widely accepted explanation for the worldwide distribution of genes responsible for sickle cell anemia, thalassemia and Hb E disorders is that heterozygote inheritance of these genes results in decreased morbidity and mortality during infection with falciparum malaria (2, 3). Although the mechanisms underlying this resistance are not established, they are likely to involve premature destruction of the parasitized red cell due to abnormal hemoglobin properties. One hypothesis is that the parasite and red cell are oxidized as a consequence of reactions involving intracellular iron released from denatured hemoglobin (4). Although the majority of hemoglobinopathies in the United States are found in people of African, Mediterranean and Asian background, since the geographic distribution of these abnormal hemoglobins is reasonably attributed to malaria and not race, we can expect that screening programs will identify abnormal hemoglobins in a substantial number of ethnic groups. The precise chromosomal location of the genes which produce each globin chain has been established. The 13 globin gene is located on chromosome 11, in close proximity to the ~ and ~ globin genes (5). The genes which determine the switch from ~ to 13 globin production towards the end of fetal development are also located on chromosome 11. When the switch is complete, adult hemoglobin replaces most of the fetal hemoglobin (6). There are two ~ globin genes located on chromosome 16 (7). Point mutations in the m globin genes result in structurally abnormal hemoglobins. However, the most common genetic abnormality involving the ~ globin genes is thalassemia, and this is due to gene deletion not point mutation. Hemoglobinopathies are inherited in an autosomal codominant manner. Patients with sickle cell anemia inherit two sickle cell genes, patients with sickle cell-Hb C disease inherit a sickle gene and a Hb C gene, and patients with 13 thalassemia major inherit two 13 thalassemia genes.

363

LUBIN, WITKOWSKA,AND KLEMAN TABLE 1 Laboratory Test Results in Disorders Producing Microcytosis

Disorder

Serum Iron

Iron Binding

Ferritin

FEP a

Hb A2

Iron deficiency

Chronic disease Thalassemia trait Thalassemia trait

Decreased

Increased

Decreased

Increased

Normal/low

Decreased Normal Normal

Decreased Normal Normal

Increased Normal Normal

Increased Normal Normal

Normal Increased Normal

aFree erythrocyte protoporphyrin. General laboratory procedures The initial suspicion of a hemoglobinopathy may come from the clinical laboratory during interpretation of the complete blood count. Besides the recognition of anemia or variation in the mean cell volume (MCV), characteristic red cell morphologic changes are often noted. All patients who have a suggestion of a hemolytic anemia should be evaluated for a hemoglobinopathy. This would include patients with reticulocytosis or polychromatophilic red cells on their blood smears. Although a hypochromic, microcytic anemia is most likely secondary to iron deficiency, thalassemia trait should especially be considered when the MCV is disproportionately low for the degree of anemia. The employment of appropriate screening techniques for evaluation of patients who have microcytic red cells is likely to save time and money (Table 1). The composition and relative amounts of normal h u m a n hemoglobin are given in Table 2. It is important to recognize that the developmental switch in hemoglobin synthesis, which occurs towards the end of gestation, will affect the relative quantity of the above hemoglobins. This is especially important in the evaluation of newborns and young children (Figure 1).

I

I00 ~

8C

ill

'l

g

Qualitative and quantitative abnormalities ineach of these globin chains have been reported. If only one ~ globin gene is affected by a structural mutation, since there are only two ~ globin genes, slightly less than 50% of the hemoglobin will be altered. In contrast, if only one of the ~ globin genes is affected, the quantity of abnormal hemoglobin will only be 25%. Furthermore, for the structural gene mutations, abnormal hemoglobin A2 and fetal hemoglobin products can be identified. There are several techniques which can be used to screen for abnormal hemoglobins. Thin layer isoelectric focusing (TLIF) is an electrophoretic technique which separates hemoglobins according to their isoelectric points (pI's) on a stable, stationary pH gradient medium in an electric field (8). This method provides excellent resolution of hemoglobins with pI's that differ only by 0.01. We have found TLIF to be an extremely effective technique as our first method to screen blood samples. Hb S is easily identified. Hb E and C, common variants in our patient population, can readily be separated due to their distinct isoelectric points. The costs of materials for this procedure are equivalent to those for cellulose acetate electrophoresis although higher technical skills are required. However, since a confirmatory electrophoretic technique is rarely needed with TLIF, the overall cost is less than that if we required both cellulose acetate and citrate agar electrophoresis to establish a diagnosis. Cellulose acetate electrophoresis using an alkaline pH (8.4) buffer system is frequently used as an initial technique for the detection of hemoglobin variants (9). If the migration pattern differs from normal, additional confirmatory tests are required. TABLE 2

4(?

Human Hemoglobins 20 0

/_

>90

80

3.5

0

0

60-100

5-20

60

3.5

20

0

90-120

5-10

Laboratory diagnosis of hemoglobinopathies.

Diagnostic tests for most common hemoglobinopathies and recent advances in structural analysis of variant hemoglobins are reviewed. Routine and newly ...
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