/83/ $01.20 Institute for Clinical Science, Inc.

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1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 13, No , Institute for Clinical Science, Inc. Hematologic Findings in Southeast Asian Immigrants with Particular Reference to Hemoglobin E *t$ W ILLIAM L. MARSH, JR., M.D. 1f ZORA R. ROGERS, B.A.J D EN N IS P. NELSON, P hd. 1 and THOMAS S. VEDVICK, P hd. Department of Laboratory Medicine, Naval Regional Medical Center, San Diego, CA Department of Pathology, School of Medicine, University of California, San Diego, San Diego, CA Clinical Investigation Department, Naval Hospital, San Diego, CA ABSTRACT Recent immigrants from Southeast Asia w ere screened for hematologic abnorm alities using a m ultichannel cell counter (Coulter S), peripheral smear, free erythrocyte protoporphyrin (FEP), isoelectric focusing, and a qualitative screen for glucose-6-phosphate dehydrogenase deficiency. H e matologic abnormalities were further defined by hemoglobin electrophoresis, globin electrophoresis, HbA2 levels, and H bf levels. O f the 189 adults studied, 68 (36 percent) w ere hematologically abnormal, including 28 hem oglobin E (HbE) heterozygotes, six H be homozygotes, 14 with a- thalassemia minor, and 10 with presum ptive iron deficiency. O f the 54 people with microcytic (MCV < 80fl) red blood cells (RBC), 52 had evidence of H be or thalassemia and two had iron deficiency alone; five had both iron deficiency and a hemoglobinopathy. Homozygosity for HbE results in an asymptomatic condition similar to thalassemia minor with microcytic RBC, large num bers of target cells, normal or slightly reduced hem atocrit and > 90 p ercen t H be. People heterozygous for H be are asymptomatic and have hematologic findings similar to thalassemia m inor w ith slightly reduced or low normal MCV and 25 to 35 percent H be. * Supported in part by the National Heart, Lung, + The opinions or assertions expressed in this paper and Blood Institute Short Term Research Training are those of the authors and are not to be construed Grant #HL as official or as necessarily reflecting the views of the t Support in part by the Bureau of Medicine and Department of the Navy or the naval service at large. Surgery Clinical Investigation Program, Project # /83/ $01.20 Institute for Clinical Science, Inc.

2 3 00 M A R SH, R O G E R S, N E L S O N, A N D V E D V IC K Introduction Hematologic abnorm alities are common in people of Southeast (SE) Asian origin. F req u en tly en countered conditions are the thalassem ias and hem o globin E (H be). The possible survival advantage of people w ith thalassem ia, H be, or glucose-6-phosphate dehydrogenase (G -G PD ) deficiency in areas of endemic falciparum malaria has been discussed elsew here Most SE Asians with H be or thalassemia syndromes are asymptomatic and require no treatm ent; however, they often have microcytic red blood cells (RBC) and in the United States may be misdiagnosed as iron deficient. The a-thalassemia syndromes are characterized by decreased synthesis of the a-globin chain of hem oglobin and occur with frequencies of up to 20 to 40 percent in certain areas of SE Asia ' ,42,43,46 M ost of the ct-thalassem ia syndromes occurring in SE Asians are attributed to deletion of one or more of the four a-genes normally occurring on chromosome '46 The (3-thalassemia syndromes are characterized by decreased synthesis of the (3-chain of hem oglobin and occur with frequencies of up to 10 to 15 percent in SE Asia '30'33 The common (3-thalassemia gene in SE Asia, (3, produces no detectable (3-chains Hemoglobin E is a (3-chain variant ((3 26 Glu» Lys) which was first described in Hemoglobin E is common in people originating from SE Asia or parts of India but occurs only sporadically in other populations. Hemoglobin E is associated w ith th e au stro asiatic (M on- Khmer) language group and occurs with frequencies as high as 20 to 50 percent, especially in areas of Thailand, C am bodia, and Laos Both heterozygotes (H ba E) and hom ozygotes (HbEE), even with coexistent 1 or 2 a- gene deletions, are asym ptom atic and have no apparent survival disadvantage. D ouble heterozygosity for H be and (3 thalassemia produces m oderate to severe anem ia Approximately 400,000 SE Asians have im m igrated to the U nited States in the last few years with m ore than one-third settling in C alifornia.6 Physicians m ust becom e increasingly aware of the clinical and laboratory features of the hem atologic syndrom es prevalent in SE Asian populations in order to make the correct diagnosis and provide th e appropriate counselling or treatm ent. The hem atologic findings in 189 recent immigrants from SE Asia are reported, with particular reference to HbE. M ethods Im m igrants from Southeast Asia attending English classes offered by the Indochinese Education Project at San Diego City College w ere asked both in English and in th eir native language (Vietnamese, Cambodian, or Laotian) to participate in this study. This oral consent approach was approved by the Hum an Subjects C om m ittee of the University of California at San Diego. Approximately five ml of venous blood, anticoagulated with ethylenediam inetetraacetic acid (EDTA), w ere obtained from each of the 189 Indochinese volunteers. P eripheral sm ears w ere p re pared and a hem ogram was obtained on a cell counter,* calibrated w ith whole blood analyzed by standard methods; the m icro h em ato crit was co rre c ted for trap p ed plasm a. F ree erythrocyte p ro toporphyrin (FEP) was d eterm in ed in duplicate on all blood samples using a hem atofluorom eter (ESA #4000). E levated FE Ps w ere confirmed by Piomelli s extraction m eth o d.20 A qualitative assay for G-6PD was perform ed on all sam ples.3 * Coulter counter, model S series, Coulter Electronics, Inc., Hialeah, FL.

3 H E M A T O L O G IC F IN D IN G S IN S O U T H E A S T A SIA N IM M IG R A N T S 301 T he co m p o n en t hem oglobins of all sam ples w ere analyzed by isoelectric focusing on 20 cm six percent polyacrylam ide gels at 400 volts for 18 hours at 4 C and quantitated with a spectrophotometer. t 37 In addition, all samples with mean cell volum e (MCV) less than 80 fl or an abnormal hem oglobin on isoelectric focusing had the following determ inations: HbA2 by m icrocolum n chrom a- tographyi, H bf by Singer alkalai denaturation, and H b electrophoresis on cellulose acetate at p H 8.6. All samples with abnormal hemoglobins were also analyzed by H b electrophoresis on citrate agar at ph 6.0 to 6.2 and globin electrophoresis at both alkaline and acid ph. All people w ith hem atologic abnormalities received w ritten reports of the findings for th eir m edical record. Patien ts w ith iron-d eficien cy anem ia or unexplained anem ia w ere referred to a physician. Seventeen additional SE Asians with H be w ere also studied. Ten are relatives of three of the m en in the original study group of 189. Seven are patients recently seen at NRMC, San Diego. Results The hematologic data are summarized in tables I, II, and III. The original study population consisted of 154 m en and 35 women who appeared healthy and were unrelated to each other except for four w om en. O ne h u n d re d m en and 21 women w ere classified as controls on the basis of essentially norm al hem atologic values: H ct > 0.41 in m en or >0.36 in women, MCV 5* 80 fl, F E P < 40 meg per dl whole blood at H ct = 0.35, and normal hemoglobin electrophoresis. Two men with slightly elevated MCV and one f Gilford, model 240, Gilford Instrument Laboratories, Inc., Oberlin, OH. $ Helena Laboratories, Beaumont, TX. TABLE I Hematologic Findings in an Immigrant Indochinese Population V i e t - C am b- L a o - L a o t i a n - n a m e s e b o d i a n t i a n Hmong T o t a l Total Population Men Women Total Controls (HbAA,* FEPf <40, MCV$ >80, Hct >0.41 in men or >0.35 in women) Men Women Total Hemoglobin E (HbAE or HbEE) Men 2 10 Women _0_ 2 Total 2 12 Thalassemia minor (14 alpha, and 2 beta) Men 2 3 Women _0 1 Total 2 4 Iron Deficiency (presumptive, based on elevated FEP) Men Women _0 2_ 0 Total *Adult hemoglobin ffree erythrocyte protoporphyrin _ 1 _ _1 _ possible alpha, _5 _ jmean cell volume Hematocrit man with slightly elevated H ct and MCV were, however, included in the controls. Hematologic abnormalities w ere found in 68 people (36 percent of total population), including 12 p ercen t of V ietnamese, 21 percent of Laotian-Hmong, and 48 percent of Cambodians. A total of 80 diagnoses were made in the 68 people: H ba E (28), H b E E (6), a-th alassem ia minor (14), possible a-thalassemia minor (7), (3-thalassemia minor (2), probable iron deficiency (10), anemia with normal FEP (9), G-6PD deficiency (3), and abnormal fast hem oglobin (1). O ne p a tie n t had th re e diagnoses, 10 p a tien ts had two diagnoses, and 57 patients had one diagnosis. The diagnosis of H be was established in 34 people (28 heterozygotes and six homozygotes) on the basis of an isoelectric focusing band at the same level as a

4 302 MARSH, ROGERS, NELSON, AND VEDVICK TABLE I I Selected Hematologic Data in an Immigrant Indochinese Population R e d B lo o d C e l l C o u n t ( x l O ^ / m c l ) M ean C e l l V o lu m e (fl) H e m a t o c r i t ( P e r c e n t ) Control Group Men (100) 5.36 ( ) 88.7 (80-104) 47.2 ( ) Women (21) 4.62 ( ) 90.4 (85-99) 41.5 ( ) HbAE (24 men) 5.81 ( ) 77.9 (66-87) 45.2 ( ) HbEE (6 men) 6.44 ( ) 63.7 (57-70) 41.0 ( ) Alpha thalassemia minor (11 men) 6.43 ( ) 67.5 (62-75) 43.6 ( ) F or re d b lo o d c e l l c o u n t, mean c e l l volume, and h e m a to c rit, th e mean v a lu e i s follo w ed by th e range in p a re n th e s e s. known H be E control and on the basis of an electrophoretic band which migrated w ith H bc on alkaline Hb electrophoresis, with HbA on acid citrate agar Hb electrophoresis and, similarly, to the 13- chain of H bc on both acid and alkaline globin electrophoresis. H em oglobin E was found in 32 percent of Cambodians, 27 percent of Laotians, 4 percent of Vietnamese, and none of Laotian-Hmongs. Of the 28 heterozygotes for H be, one man and one wom an had concom itant iron deficiency and one woman had a possible concom itant 3 a-gene deletion with MCV of 53 fl, Hct of 22.8, FEP of A g e / S e x TABLE I I I Hematologic Values in Eight Southeast Asian Adults with Homozygous Hemoglobin E C o u n tr y o f O r ig in RBC MCV H ct MCHC HbF FEP 32M Laos M Laos M Cambodia ? M Cambodia M Cambodia M Laos F Thailand F Thailand RBC = Red blood cells (x 106/mcl) MCV = Mean cell volume (fl) HcT = Hematocrit (percent) MCHC = Mean corpuscular hemoglobin concentration HbF = Fetal hemoglobin (percent) FEP = Free erythrocyte protoporphyrin (mcg/dl) 37, and 14 percent H be. Hematologic findings in the 24 men with uncom plicated HbAE w ere as follows: MCV of 66 to 87 fl w ith eight having MCV 2» 80 fl; RBC of 4.66 to 7.01 x 106 per mcl, five with RBC < 5.5 and two with RBC <5.0; H ct of , two with H ct <0.41; H bf < 2 percent except for one person w ith 2.3 p e rc e n t; and p e rc e n t H be ranging from 24 to 35 percent with mean of 30 percent. The person with 24 percent H be also had the MCV of 66 fl and may have a concomitant deletion of 1 or 2 ct-genes. Peripheral smear exam in 24 men with uncom plicated HbAE revealed that 18 had findings suggestive of hem o globinopathy (target cells, m icrocytes, ovalocytes, hypochrom ic cells); the remaining six m en had minimal RBC abnormalities. Six m en, three Cambodians and three Laotians, had hom ozygous H be. H e matologic values for these six m en and two healthy wom en with H be E recently seen at the Naval Hospital, San Diego, CA are liste d in tab le III. P erip h eral smears in all eight people with H be E show ed large num bers of target cells, w ith m icrocytes and scattered ovalocytes. Fifteen additional SE Asians with heterozygous H be w ere studied. Thirteen were relatives of five of the people with

5 H E M A T O L O G IC F IN D IN G S IN S O U T H E A S T ASIAN IM M IG R A N T S 3 03 H b E E, in clu d in g 11 c h ild ren, one brother, and one m other. All 15 H be heterozygotes had low norm al or re duced MCV, low norm al or slightly reduced Hct, norm al FE P, and 26 to 35 percent H be. Ten of the 15 had RBC morphology suggesting hem oglobinopathy (primarily targets and microcytes); the rem ainder had minimal RBC abnormalities. A presum ptive diagnosis of a-thalassemia m inor was m ade on 11 m en and three women on the basis of MCV < 76 fl, elevated or high norm al RBC count, normal FEP, normal HbA2 and H bf, and norm al H b electrophoresis. The RBC count was > 6.0 in all b u t one man and >5.3 in the three women. T here was a range from 62 to 75 fl for MCV, and H ct was below normal in only one of the 14 people. All 14 people had blood smears typical of thalassemia minor with microcytes, ovalocytes, hypochrom ia, and scattered target cells. An additional seven people w ere classified as possible a-thalassemia m inor on the basis of MCV of 77 to 79 fl, high norm al to increased RBC count, norm al F E P, norm al HbA 2 and HbF, and normal Hb electrophoresis. One man and one woman, both Laotian, w ere classified as 3-thalassem ia minor on the basis of elevated RBC count, decreased MCV (<65 fl), normal FEP, and elevated H ba 2. P eripheral sm ears were typical of thalassemia minor. A diagnosis of presum ptive iron deficiency was m ade in six m en and four women on the basis of low or low normal MCV and an elevated FEP. One person had a borderline low Hct, and the remainder had mild anemia. Three men had a disproportionately low MCV and probably had concom itant thalassemia minor. One man and one woman had concomitant HbAE. Nine people had anem ia with a normal FEP. T hree had H be E, two had HbAE, one had p-thalassemia minor, one had a- thalassemia minor, and two had HbAA. The anem ia was of m ild degree except in one woman with 14 percent H be (see previous data). Three people had G-6PD deficiency. All three w ere m en of Laotian extraction. One had concomitant HbE, one had concom itant H be E, and the third had no other hematologic abnormality. One individual (of Cambodian extraction) had an abnorm al hemoglobin which m igrated anodal to HbA on alkaline Hb electrophoresis, with HbA on acid citrate agar H b electrophoresis, and similar to H bj on globin electro p h o resis. T he Center for Disease Control confirmed the findings and classified the abnormal hemoglobin as a (3-chain variant with electrophoretic mobility similar to Hb New York.3147 Discussion Evaluation of 189 SE Asian immigrants by clinical laboratory m ethods revealed a 36 percent incidence of hematologic abnormalities. Of the 68 people with hematologic abnormalities, 34 had HbE and 23 had thalassemia minor. Ten people req u ired fu rth er evaluation for probable iron deficiency and two required further evaluation for normocytic anemia. One patient had a moderately severe microcytic anem ia associated w ith 14 percent H be. T hree people had G -6PD deficiency by qualitative screening. The reported incidence of G-6PD deficiency in SE Asia is variable, w ith frequencies up to 24 percent Since the study population is small and non-random, it may not reflect the true incidence of these hematologic abnorm alities in the population. The percentage of HbE in heterozygotes is usually 25 to 35 percent, as compared to the 35 to 45 percent usually seen in heterozygotes for stable (3-chain variants The unexpectedly low percentage of H be in heterozygotes has been attributed to both instability of HbE

6 3 04 M A R SH, R O G E R S, N E L S O N, A N D V E D V IC K and decreased synthesis of H be ' 34,35,45 Recent evidence suggests that the low percentage of H be in heterozygotes is d u e to d ecre a sed synthesis of 3EmRNA.35 Concom itant iron deficiency or a-gene deletions may further reduce the percentage of H b E.3943 Separation of the effects of H be from those of a-gene deletions is difficult by routine m ethods and requires family studies or sophisticated molecular studies.11 One study reported 24 to 30 p e rc e n t H b E and m icrocytic RBC in a small num ber of H be heterozygotes with four normal a-genes (by deoxyribonucleic acid mapping).35 D e sp ite th e com m on o ccu rren ce of H be in certain SE Asian populations, relatively few hematologic data are available on large numbers of HbE heterozygotes stu d ie d w ith m odern cell counting m ethods ,27,39 The reported values for MCV in H be heterozygotes have varied widely. The greatest variation is in the older literature7 936 and may be due, in part, to the inaccuracies of hem acytom eter RBC counts Microhematocrit readings are also falsely elevated by trap p ed plasm a, particularly with increased aniso- and poikilocytosis. M ore recent studies on relatively small num bers of people suggest that almost all p eo p le w ith H ba E have m icrocytic RBC However, of 24 adult men and one w om an w ith u n com plicated HbAE in the present study, 17 had a d e creased MCV and eight had a low normal MCV. Since gene mapping was not performed, the possible effect of a concomitant 1 or 2 a-gene deletion on the MCV of people with HbAE cannot be evaluated. People heterozygous for H be have an asymptomatic mild thalassemia-like syndrom e owing to decreased production of (3E-globin. The MCV is usually slightly reduced b u t may be in the low normal range. T he RBC c o u n t is norm al or slightly increased. The hematocrit is usually normal but may be slightly reduced. The peripheral sm ear usually shows mild to m oderate RBC abnormalities (targets, m icrocytes, ovalocytes) b u t may show only minimal RBC abnormalities. Most H be heterozygotes are readily detectable because of microcytic RBC and abnorm al blood sm ear. H ow ever, those H be heterozygotes with both MCV and blood sm ear w ithin normal limits will be detected only by electrophoretic screening or family studies. People with homozygous H be have an asymptomatic mild thalassemia-like condition characterized by microcytic RBC, num erous target cells, normal or slightly reduced hem atocrit, >90 percent H be, and <10 percent H be Although some o ld e r stu d ies show norm al MCVs in people who are apparently homozygous for H be, m ore recent studies dem onstrate that people with H bee have microcytic RBC w ith n u m ero u s ta rg et cells.25 In th e p resent study, the eight people w ith H be E have MCVs ranging from 57 to 70 fl and all have large num bers of target cells. M ost older studies state that people with homozygous H be may have anemia. Using a restrictive definition of H be E w hich re q u ire d confirm atory fam ily studies, Fairbanks could find only seven acceptable cases of H bee in the literatu re, all of w hich had norm al or only slightly reduced hem atocrits.54 W ith the present-day clinical laboratory evaluation of hem oglobinopathies, it is unlikely that such a restrictive definition of H be E is necessary for clinical purposes. The only other known condition which could be confused with homozygous HbE is double heterozygosity for H be and p -thalassemia ((3E/(3 ). The distinction should be m ade readily in m ost p a tie n ts since p e o p le w ith p E/(3 h av e m o d e ra te to severe anemia and H bf > 10 percent. ^a In the present study, blood was available from family members of five of th e e ig h t ad u lts w ith H b E E and showed confirmatory evidence of HbE.

7 H E M A T O L O G IC F IN D IN G S IN S O U T H E A S T ASIAN IM M IG R A N T S Of the eight people with H be E, four had normal hem atocrits and four had slightly reduced hematocrits; all eight had normal FEPs. Owing to the increasing num ber of SE Asian im m igrants and the increasingly routine use of electronic cell counters which detect microcytic RBC, physicians should be aware of the clinical and laboratory features of HbE and the thalassemia syndromes. In this study, 54 (29 percent) of SE Asian immigrants had microcytic RBC (MCV < 80 fl); 52 had evidence of H be or thalassemia minor; two had iron deficiency alone; and five had evidence of both iron deficiency and thalassemia minor. People with H be or thalassem ia m inor are asym ptom atic, b u t they must be correctly diagnosed in order to receive appropriate counselling and avoid unnecessary treatm ent for iron deficiency. The diagnosis of HbE or thalassemia can usually be established by examination of blood smear, Hb electrop h o re s is (a lk a lin e a n d acid), an d d e te rm in a tio n of RBC in d ices, F E P, HbA 2, and HbF. People from SE Asia with microcytic RBC should not receive iron therapy until a diagnosis of iron deficiency is established by tests such as F E P, saturation p ercent of transferrin, serum ferritin, or bone marrow iron. R eferences 1. A l i, M. A. M., Q u i n l a n, A., and W o n g, S. C.: Identification of hemoglobin E by the isopropanol solubility test. Clin. Biochem. i3: , A l t a y, C., N ia z i, G. A., and H u i s m a n, T. H. J.: The combination of HbS and HbE in a black female. Hemoglobin 1: , B e u t l e r, E. and M i t c h e l l, M.: Special modification of the fluorescent screening method for G- 6PD. Blood 32: , B l a c k w e l l, R. Q., H u a n g, J. T - H., and C h i e n, L-C.: Hemoglobin E in Vietnamese. Nature 207:768, B o w m a n, J. E., C a r s o n, P. E., F r i s c h e r, H., e t a l : Hemoglobin and red cell enzyme variation in some populations of the Republic of Vietnam with comments on the malaria hypothesis. Amer. J. Phys. Anthropol. 34: , C e n t e r f o r D i s e a s e C o n t r o l. T u b e r c u l o s is - C a l - i f o r n i a. MMWR 30: , C h a t t e r j e a, J. B.: Some aspects of haemoglobin E and its genetic interaction with thalassemia. Ind. J. Med. Res. 53: , C h e r n o f f, A. I., M i n n i c h, V., and C h o n g c h a r - e o n s u k, S.: Hemoglobin E, a hereditary abnormality of human hemoglobin. Science 120: , C h e r n o f f, A. I., M i n n i c h, V., N a - N a k o r n, S., e t a l : Studies in hemoglobin E I. The clinical, hematologic and genetic characteristics of the hemoglobin E syndrome. J. Lab. Clin. Med. 47: , C u n n i n g h a m, T. C. : Hemoglobin E in Indochinese refugees. West. J. Med. 737: , F a ir b a n k s, V. F., G i l c h r i s t, G. S., B r i m h a l l, B., e t a l : Hemoglobin E trait reexamined: A cause of microcytosis and erythrocytosis. Blood 53: , F a ir b a n k s, V. F., O l i v e r o s, R., B r a n d a b u r, J. H., e t a l : Homozygous hemoglobin E mimics betathalassemia minor without anemia or hemolysis. Amer. J. Hematol. 8: , F la tz, G.: Hemoglobin E: Distribution and population dynamics. Humangenetik 3: , F l a t z, G., P i k, C., andsringam, S.: H e m o g lo b in E a n d b e ta -th a la sse m ia : t h e ir d is trib u tio n in T hailand A nn. H um. G enet. 29: , F o r t n e r, A., T a e t l e, R., R a n n e y, H., e t a l : Clinical and laboratory manifestations of the hemoglobin E syndromes. Blood 58:53a, F r i s c h e r, H. and B o w m a n, J.: Hemoglobin E, an oxidatively unstable hemoglobin. J. Lab. Clin. Med. 85: , H a r l e y, J. D., C h r i s t o p h e r, P., G r u c a, M. A., e t a l : Inherited red cell defects in Indochinese immigrants. Med. J. Austral. 2:437, I t a n o, H. A., B e r g r e n, W. R., and S t u r g e o n, P.: Identification of a fourth abnormal human hemoglobin. Amer. Chem. Soc. J. 76:2278, L i e - I n j o, L. E.: Distribution of genetic red cell defects in Southeast Asia. Trans. Roy. Soc. Trop. Med. Hyg. 63: , M a r s h, W. L. and K o e n i g, H. M.: The laboratory evaluation of microcytic red blood cells. CRC Crit. Rev. Clin. Lab. Sci. i6: , M e h t a, B. C., A g a r w a l, M. B., V a r a n d a n i, D. G., e t a l : Hemoglobin E-thalassemia: A study of 16 cases. Acta. Haemat. 64: , M o n z o n, C. M., F a ir b a n k s, V. F., B u r g e r t, E. O., e t a l : Hematologic and genetic disorders of SE Asian refugees. Blood 58:55a, N a - N a k o r n, S., M i n n i c h, V., and C h e r n o f f, A. I.: Studies on hemoglobin E II. The incidence of hemoglobin E in Thailand. J. Lab. Clin. Med. 47: , N a - N a k o r n S, W a s i P : Alpha-thalassemia in northern Thailand. Amer. J. Hum. Genet. 22: , P a g n i e r, J., W a j c m a n, H., and L a b i e, D.: Defect in hemoglobin synthesis possibly due to disturbed association. FEBS Lett. 45: , P o o t r a k u l, S., A s s a w a m u n k o n g, S., a n d N a - N a -

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