Thalassemia Minor: Routine Erythrocyte Measurements and Differentiation from Iron Deficiency

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1 Thalassemia Minor: Routine Erythrocyte Measurements and Dierentiation rom Iron Deiciency CAGE S. JOHNSON, M.D., CONSTANTINE TEGOS, M.D., AND ERNEST BEUTLER, M.D. The clinical dierentiation o the causes o microcytosis is diicult because o the lack o a method or the diagnosis o alpha thalassemia. A number o laboratory tests have been proposed or the dierentiation o alpha thalassemia rom iron deiciency, including decision unctions based on the red blood cell indices generated by electronic cell counters. The accuracy o these screening methods was assessed in 9 patients with microcytosis known to be secondary to either iron deiciency or beta thalassemia minor and, prospectively, in 2 patients with microcytosis in whom globin chain synthesis ratio was used to diagnose thalassemia. The unctions evaluated were: RBC volume distribution curve; osmotic ragility; erythrocyte count; discriminant unction = MCV - (5 X Hgb) - RBC -.; ratio o MCH/RBC; ratio o MCV/RBC; and.1 X MCH X (MCV) 2. A simpliied method o measuring anisocytosis using the RBC volume distribution curve was signiicantly more accurate (P <.1) in distinguishing iron deiciency rom thalassemia than any o the other decision unctions. Analysis o red blood cell volume distribution, although not suiciently accurate or deinitive diagnosis, appears to be a useul technic in the initial screening o patients with microcytosis and in determining which additional testing should be done. (Key words: Thalassemia; Erythrocyte indices; Iron deiciency) Am J Clin Pathol 19; : 1- MICROCYTOSIS IS A COMMON CLINICAL FIND ING, occurring in nearly % o admissions to metropolitan general hospitals. 17 A common cause o microcytic anemia and a signiicant public health problem worldwide is iron deiciency. The thalassemia syndromes are another cause o microcytosis, and although severe thalassemias generally are easily recognized, milder orms o both alpha and beta thalassemia may be misdiagnosed and treated as iron deiciency. ',2 The diagnosis o beta thalassemia minor is made readily by demonstration o elevated hemoglobin A 2 levels in the absence o concomitant iron deiciency anemia that may result in normal hemoglobin A 2 levels. However, there is no clinically available test or alpha thalassemia types 1 and 2, conditions that are now recognized to be more common than beta thalassemia, 71 nor or those cases o beta thalasemia minor with normal A 2 levels. These Received March 1, 192; received revised manuscript and accepted or publication January 2, 19. Supported in part by Grants No. HL 1512 and 25552, NHLBI and GCRC RR-, Division o Research Resources, NIH. Address reprint requests to Dr. Johnson: Department o Medicine, University o Southern Caliornia School o Medicine, 225 Zonal Avenue, Los Angeles, Caliornia 9. Department o Medicine, University o Southern Caliornia, Los Angeles County-USC-Medical Center, Los Angeles, and Department o Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla, Caliornia orms o thalassemia may be responsible or the large number o unexplained cases o microcytosis ound in population surveys. 17 Because o the clinical need to dierentiate iron deiciency rom thalassemia and o the diiculty o establishing a deinitive diagnosis o alpha thalassemia, a number o screening methods adapted rom the generation o red blood cell indices by electronic counters have been proposed " 2 These calculations are based on the act that microcytosis is usually more prominent than anemia in mild thalassemia. They have been useul in the evaluation o uncomplicated cases 121 but less deinitive in hospitalized patients. 117 ' 2 Another approach to this problem has been the use o red blood cell volume distribution analysis,9 which has been shown to be eective in making this distinction and is becoming more available. It is the purpose o this study to assess the clinical utility o these approaches to the dierentiation o iron deiciency rom heterozygous thalassemia using globin chain synthesis as the reerence diagnostic technic. Materials and Methods As part o a diagnostic evaluation o microcytosis, red blood cell size distribution curves were obtained on blood rom 9 patients reerred to the hemoglobinopathy screening clinic in whom the microcytosis was explained by either iron deiciency or beta thalassemia alone. These 9 patients generally were ree o complicating illnesses; 5 their hematologic values are given in Table 1. During a separate study on the prevalence o alpha thalassemia in black Americans 1 blood was obtained, with inormed consent, rom 1 black adults at the Los Angeles County-University o Southern Caliornia Medical Center or globin chain synthesis. Control samples were obtained simultaneously in 2 white persons. Seventy-two cases rom this population and all 2 o the 2-917//7/1 $1.1 American Society o Clinical Pathologists 1

2 2 JOHNSON, TEGOS, AND BEUTLER A.J.C.P. July 19 Table 1. Comparison o Clinical Data in Those with ' Iron Deiciency and Beta Thalassemia Minor rom the Screening Clinic and in the Normal Subgroup rom those in Whom Globin Synthesis was Measured* Diagnosis Hgb (g/dl) Hct (%) MCVa (L) MCH (pg) MCHC (g/dl) RBC(X1 12 /L) MCVd (L) EVR 5 (L) HbA 2 (%) Ferritin (ng/l) 95% CI a/i ratio Iron Deicient 1.2 ± 1. 2 ± to ± ±. 7 ± ±. 7 2 to 19 2 to 2 ND* / Thalassemia ± 1.7 ± 5 ±7 5 to ± ± ±.72t ± 22±2 5.2 ±.7 97t 27 to 7 2 to 2 ND Normal ± 9 + to ± to 2 2 to Data are expressed as means ± I SD. except or erritin, where geometric mean,, and 95% conidence interval are shown. t P <.1. % ND = not done. white controls had normal hemoglobin levels, greater than 1 g/dl in males, greater than 12.5 g/dl in emales, normal alpha/beta globin synthesis ratios, and were ree o diseases o the liver, kidney, or bone marrow known to aect red blood cell production; these were considered hematologically normal, 5 and their clinical characteristics also are given in Table 1. Twenty-six patients rom the population prospectively studied with globin chain synthesis had microcytosis (MCV less than 1 L) and ormed a second study group with microcytosis complicated by a variety o acute and chronic illnesses as opposed to the clinic group that generally lacked a second illness. The hematologic data on these 2 patients is given in Table. The remaining black patients had either normocytic or macrocytic anemia and were excluded rom urther analysis. Hematologic measurements were perormed on a Coulter Model S electronic cell counter that was standardized daily using a commercial control. Hemoglobin electrophoresis was perormed on cellulose acetate at ph. and, when indicated, on citrate agar gel at p\\.2. Hemoglobin F was measured by the Betke method o alkali denaturation and hemoglobin A 2 by DEAE cellulose microchromatography. Other determinations, including hemoglobin H preparation, serum iron, iron binding capacity, erritin and olic acid, were perormed by standard methods. Synthetic ratios were determined on globin chains separated on Cellogel, as previously reported. 21 Osmotic ragility screening was perormed in a.% salt solution. 1 Blood samples were collected in EDTA, and within our hours, red blood cell volume distribution curves were generated on a Coulter Counter, Model ZBi (aperture width 7 micrometers, aperture current 1 milliamp, ampliication setting 1/2) and a Channelyzer C- 1 (edit switch on, base channel threshold 5, window width 1). The instrument was calibrated against polystyrene spheres o known size, Coulter C control, and the MCV obtained rom a simultaneous blood sample 1 r 1 > o z OJ o UJ cc u. FIG. 1. Red blood cell volume distribution curves in two cases with a similar degree o microcytosis. The EVR 5 is the width in emotoliters between the ascending and descending limbs o the curve at a requency o 5%; the MCVd is the midpoint o the EVR 5. (A) a thalassemia: MCVd 75 L, EVR 5 2 L, a/p ratio.7. (B) Iron deiciency; MCVd 7 L, EVR 5 L, a// ratio 1.. VOLUME (l) VOLUME (l)

3 Vol. No. 1 THALASSEMIA MINOR Table 2. Screening Methods in the Evaluation o Microcytosis Method Formula Thalassemia Iron Deiciency Reerences Discriminant unction RBC count Osmotic ragility Volume * Coeicient o variation* Volume distribution curve MCV - (5 X Hgb) - RBC -. MCH -5- RBC MCV -r RBC.1 X MCH X (MCV) 2 Per cent hemolysis EXP(^+ 1.9<T)-EXPU-- 1.9<r) EXP (ain) X 1 EVR 5 <1 <. <1 <15 >5. X 1 I2 /L <95% <5L <1% <2 L >1 >. >1 >15 >5. X 1,2 /L >95% >55L >1% >27L, ' Where *i = log median cell volume, a = log standard deviation. on a Coulter Counter Model S (MCVa). This calibration resulted in a value o emtoliter (L) per channel. Blood samples were diluted in Isoton to produce a concentration o X 1 per liter. The degree o anisocytosis was assessed by measuring the erythrocyte volume (EVR 5 ) in emtoliters at the hal maximum o the curve as shown in Figure 1. The mean cell volume (MCVd) was determined as the midpoint o the EVR 5. Statistical analysis was perormed at the 5% level o signiicance using Student's Mest, the chi-square test, or the proportion test. Results Thirty-six o the 9 cases evaluated in the screening clinic proved to have microcytosis caused by iron deiciency, as shown by low serum erritin and a subsequent response to iron therapy. Beta thalassemia minor was diagnosed in 57 patients in whom serum erritin levels were normal but hemoglobin A 2 levels were elevated. The clinical characteristics o these cases and the hematologically normal group are given in Table 1. Those with beta thalassemia were less anemic when compared with those with iron deiciency and had higher RBC counts and a greater degree o microcytosis; however, the MCHC was lower in iron deiciency. These dierences were statistically signiicant. Anisocytosis, as measured by the EVR 5, was signiicantly less in thalassemia (mean ± 1 SD = 22 ± 2 L) than in iron deiciency, (mean = ± L) (P <.1). Moreover, there was complete separation o these groups as shown by the inding that EVR 5 was less than 2 L in all 57 cases o thalassemia and greater than 27 L in all cases o iron deiciency. Anisocytosis, as measured by the volume distribution curve methods, the osmotic ragility screen, and the ive numerical decision unctions or separating iron deiciency rom thalassemia, was tested in the 9 cases rom the clinic with a known cause o microcytosis. The calculations and diagnostic criteria are given in Table 2. The EVR 5 method o assessing anisocytosis was compared with the volume method o England and associates 9 " and with the coeicient o variation (CV) method o Bessman and Feinstein (Table ). There was no signiicant dierence in the accuracy o classiication between the volume and CV methods (Z = 1, P >.). However, the EVR 5 method o analysis was signiicantly more accurate than the other methods (Z =., P <.1). When the ive numerical unctions reported to distinguish thalassemia rom iron deiciency were tested in these patients, to 7% were classiied correctly. There was no signiicant dierence in the perormance o these unctions. Anisocytosis measured by the EVR 5 was correctly classiied all 9 cases, and its accuracy was signiicantly better than any o the ive numerical unctions (Z =., P <.1). In the 2 patients with MCV less than 1 L rom the population studied by globin chain synthesis (Table ), the microcytosis was determined to be caused by iron deiciency alone in eight patients on the basis o low serum iron, high iron binding capacity, and low erritin, and by thalassemia alone in eight patients because o an abnormal globin chain synthesis ratio, the absence o Table. o Correctly Identiied Cases by the Screening Methods in the 9 Cases o Microcytosis rom the Screening Clinic Method MCV - (5 X Hgb) - RBC -. MCH -r RBC MCV -r RBC.1 X MCH X (MCV) 2 RBC Volume Coeicient o variation EVR 5 Correct in Iron Deicients Correct in 57 Thalassemias % Correct

4 JOHNSON, TEGOS, AND BEUTLER A.J.C.P.- July 19 Table. Clinical Data in the 2 Cases in Whom Globin Chain Synthesis Was Measured* Diagnosis Thalassemia Iron Deicient Both ACD Hgb (g/dl) Hct (%) MCVa (L) MCH (pg) MCHC (g/dl) RBC(X1 I2 /L) MCVd (L) EVR 5 (L) HbA 2 (%)% Ferritin (jig/l) 95% CI a/ ratio 12.7 ± 1. ± 7 ± 71 to ± ±.5.91 ±.9 77 ± 5 25 ±.5 ± to 9 21 to 51 5 a.77 ±.5 J 1.71 ± ± 1.9 ± 75 ± 1 to gl 2. ± ±.7.7 ±.7 75 ± 7 2 ±5 2. ±.5 1 to 1 to 1.99 ±. 9. ± ±9 77 ± 5 7 to 1 2. ± ±.7. ± ± 7 1 ±9.2 ± to 17 to 2 a.7 ± ± 1.9 ± 5 79 ± 7togl 2. ± ±..5 ±.91 g ± 2g±2 2. ±. 17 5g to 1 17 to ±. Data are expressed as means ± 1 SD except or erritin, where geometric mean,, and 95% conidence interval are shown. t ACD = anemia o chronic disease. X Three with beta thalassemia. One with beta thalassemia. Table 5. Results o the Screening Methods Applied to the 2 Cases o Microcytosis in Whom Globin Chain Synthesis Was Measured Diagnosis Method MCV - (5 X Hgb) - RBC -. MCH -r RBC MCV -r RBC.1 X MCH X (MCV) 2 RBC Osmotic ragility EVR 5 iron deiciency, and elevated hemoglobin A 2 or F levels in three cases with beta thalassemia minor. Four patients had both iron deiciency and thalassemia (one beta), and six patients had the anemia o chronic disease as shown by low serum iron, low binding capacity, normal or elevated erritin level, and the presence o a chronic disease. 5 There was little dierence in the laboratory eatures o these 2 patients when grouped by diagnosis. Serum erritin levels were signiicantly lower in those with iron deiciency than in the other two subgroups (P <.1). Hemoglobin and MCHC values in the group with both iron deiciency and thalassemia were lower than in the other three groups, but reached statistical signiicance only when compared with the eight patients with thal- Thalassemia n = tt * Iron Deiciency n = t i n ACD = anemia o chronic disease. 11 = indicated thalassemia, = indicated iron deiciency. tx /><.5. Both n = t t ACD* n = assemia alone (P <.); small numbers were probably responsible or the lack o signiicance vs. the other subgroups. Interestingly, the combination o iron deiciency and thalassemia produced a signiicant reduction in the red blood cell count (P <.) as compared with the eight patients with thalassemia. The EVR 5 was signiicantly less in the thalassemic groups (P <.1) as compared with those with iron deiciency, and the values were similar to those seen in the clinic population. The two groups with iron deiciency had values or EVR 5 similar to that or uncomplicated iron deiciency, while the EVR 5 was in the normal or those with the anemia o chronic disease. The ive numerical unctions, the osmotic ragility, and the red blood cell volume distribution curve were applied to these 2 cases to determine their ability to distinguish iron deiciency rom thalassemia (Table 5). The accuracy o the arithmetic manipulations o the red blood cell indices in the eight cases o thalassemia was uniormly poor. The erythrocyte count and osmotic ragility screen were somewhat more accurate, whereas the distribution curve correctly identiied 75% o these cases (x 2 =.1, P <.5). The mathematical unctions were more sensitive in detecting iron deiciency but were surpassed again by the accuracy o the RBC volume distribution curve. In the our patients with both iron deiciency and thalassemia, the numerical calculations and the erythrocyte count indicated iron deiciency as did the distribution curve in three o our cases; the osmotic ragility indicated thalassemia in three o the our cases. In the six patients with the anemia o chronic disease and microcytosis, all o the indicators avored iron deiciency.

5 Vol. No. I THALASSEMIA MINOR 5 Discussion Microcytosis is a classical laboratory eature o the disorders o hemoglobin synthesis such as iron deiciency or the thalassemias and may be seen in the anemias o chronic disorders, plumbism, and other rare types o anemia. It is encountered commonly in hospital populations 17 and is presumably as requent in outpatients. Although clinical eatures, iron studies, and hemoglobin electrophoresis can be useul in determining the cause o microcytosis, the diagnosis o heterozygous alpha thalassemia is clinically diicult. This diagnosis usually is made by the exclusion o known causes o microcytosis or by amily studies showing that microcytosis is inherited. 1 A hemoglobin H preparation is deinitive but is rarely positive in heterozygotes, 12 ' 22,2 and a negative test is o little value. Reerence technics or this diagnosis, such as globin chain synthesis or restriction endonuclease studies, are only available in the research laboratory. The widespread use o electronic cell counters has generated intense interest in the interpretation o microcytosis as evidenced by the large number o papers addressing this subject that have appeared in recent years. The appreciation that alpha thalassemia is more common than previously believed, particularly in the black population where its prevalence may reach %, 711 has stimulated search or a simple, inexpensive method o making a positive diagnosis. Many investigators ' 2 proposed numerical manipulations o the red blood cell indices that were eective in distinguishing microcytosis caused by iron deiciency rom that resulting rom thalassemia in uncomplicated, outpatient cases, 121 and permitted a presumptive diagnosis o alpha thalassemia to be made in some cases. In the study o Okuno and Chou, 17 % o their cases o microcytosis had normal iron studies and normal levels o hemoglobins A 2 and F and had indings similar to the cases o alpha thalassemia described by Pearson and associates. 1 The discriminant unction o England and Fraser was helpul in less than hal o these cases. Cunningham and Rising studied a group o patients with moderate to severe microcytosis and noted that nearly one-quarter o their patients with iron deiciency had a discriminant unction indicating thalassemia. They suggested the discriminant unction was useul in uncomplicated thalassemia but was less helpul in complicated cases. Ten per cent o their patients had microcytosis o uncertain cause and, in 7% o these, the discriminant unction indicated iron deiciency that could not be substantiated by simultaneous iron studies. KJee and associates investigated the use o the discriminant unction 1 and Srivastava's 2 and Mentzer's ratios" in the evaluation o microcytosis and compared these with the use o the erythrocyte count alone. They noted that the numerical unctions perormed better in the uncomplicated cases but, overall, none was more accurate than the erythrocyte count in distinguishing iron deiciency rom thalassemia. Red blood cell volume distribution curves can be generated easily by electronic cell counters and have been shown to reliably distinguish microcytosis caused by iron deiciency rom that caused by heterozygous thalassemia, - 9 because iron deiciency produces increased anisocytosis, while thalassemia minor is characterized by little anisocytosis (Fig. 1). The distribution curve is the most sensitive indicator o iron deiciency" and can separate the microcytosis caused by partially treated iron deiciency rom that caused by thalassemia. 1 Furthermore, the volume distribution curve is an accurate tool in hospitalized patients. This test has the advantage o being automated and available with the CBC in the new generation o electronic cell counters. However, the calculations proposed or quantiying the degree o anisocytosis by both England and associates 9 " and by Bessman and Feinstein are tedious to perorm. In this paper, we have simpliied the method o calculating anisocytosis by measuring the erythrocyte volume as suggested by England and Down 9 at an arbitrary point on the curve, as originally suggested by Bessman and Johnson. This simpliied method o analysis was signiicantly more accurate in the evaluation o both uncomplicated and hospitalized patients with microcytosis than either o the two statistical methods. The erythrocyte count was superior to the numerical decision unctions but did not approach the accuracy o the distribution curve. In the cases with both iron deiciency and thalassemia, the decision unctions universally indicated iron deiciency which seems appropriate; ater conirmation o the diagnosis and ollowing iron therapy, continuing microcytosis should prompt reevaluation, whereupon a repeat distribution curve is likely to indicate the presence o thalassemia. 2 In the anemia o chronic disease, the decision unctions again indicated iron deiciency; anisocytosis seems likely because o the abnormal iron metabolism in these disorders, and determination o serum iron studies should be characteristic o this situation. 5 Analysis o red blood cell volume distribution, although not suiciently accurate or deinitive diagnosis, appears to be a useul technic in the initial screening o patients with microcytosis and in determining which additional testing should be done. A low erritin level or serum iron and a large E VR 5 suggests iron deiciency, whereas a normal erritin level with a small EVR 5 suggests thalassemia; an elevated Hb A 2 and/or F level would conirm the diagnosis o beta thalassemia, while normal Hb A 2 and F levels would support a presumptive

6 JOHNSON, TEGOS, AND BEUTLER A.J.C.P. July 19 diagnosis o alpha thalassemia. The identiication o patients with alpha thalassemia may prevent their inappropriate treatment with iron,22,2 and can be helpul in genetic counseling. Reerences 1. Bessman JD: microcytic polycythemia. Frequency o nonthalassemic causes. JAMA 1977; 2: Bessman D: Erythropoiesis during recovery rom iron deiciency: Normocytes and macrocytes. Blood 1977; 5: Bessman JD, Feinstein DI: Quantitative anisocytosis as a discriminant between iron deiciency and thalassemia minor. Blood 1979; 5:2-29. Bessman JD, Johnson RK: Erythrocyte volume distribution in normal and abnormal subjects. Blood 1975; : Cartwright GE, Lee GR: The anaemia o chronic disorders. Br J Haematol 1971;21: Cunningham LO, Rising JA: Erythrocytic microcytosis: Clinical implications in 1 patients. Am J Med Sci 1977; 27: Dozy AM, Kan VW, Embury SH, et al: a-globin gene organisation in blacks precludes the severe orm o a-thalassemia. Nature 19; 2:5-7. England JM, Bain BJ, Fraser PM: Dierentiation o iron deiciency rom thalassemia trait. Lancet 197; 1: England JM, Down MC: Red-cell-volume distribution curves and the measurement o anisocytosis. Lancet 197; 1: England JM, Fraser PM: Dierentiation o iron deiciency rom thalassemia trait by routine blood-count. Lancet 197; 1: England JM, Ward SM, Down MC: Microcytosis, anisocytosis and the red cell indices in iron deiciency. Br J Haematol 197; : Hedge UM, White JM, Hart GH, et al: Diagnosis o a-thalassaemia trait rom coulter counter 'S' indices. J Clin Pathol 1977; :-9 1. Johnson CS, Tegos C, Beutler E: a-thalassemia: Prevalence and hematologic indings in American blacks. Arch Intern Med 192; 12: Klee GG, Fairbanks VF, Pierre RV, et al: Routine erythrocyte measurements in diagnosis o iron-deiciency anemia and thalassemia minor. Am J Clin Pathol 197; : Mentzer WC: Dierentiation o iron deiciency rom thalassemia trait. Lancet 197; 1:2 1. Miale JB: Laboratory methods, 5th ed. St. Louis, CV Mosby, Okuno T, Chou A: The signiicance o small erythrocytes. Am J Clin Pathol 1975; : Pearson HA, O'Brien RT, Mcintosh S: Screening or thalassemia trait by electronic measurement o mean corpuscular volume. N Engl J Med 197; 2: Shine I, Lai S: A strategy to detect a-thalassemia minor. Lancet 1977; 1: Srivastava PC: Dierentiation o thalassaemia minor rom iron deiciency. Lancet 197; 2: Tegos C, Beutler E: A simpliied method or studies o haemoglobin biosynthesis. Clin Lab Haemat 19; 2: Walord DM: a-thalassemia in the United Kingdom. Br J Haematol 1977; 5: Walord DM, Deacon R; Alpha-thalassemia trait in various racial groups in the United Kingdom: Characterization o a variant o alpha-thalassemia in Indians. Br J Haematol 197; : 19-2