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Laboratory diagnosis of HB CC-α-thalassemia
Clin. Biochem. 12 (5) 157-158 (1979)
Laboratory Diagnosis of HB CC- -Thalassemia S. C. WONG and M. A. M. ALI Departments of Laboratory Medicine and Pathology, St. Joseph's Hospital, McMaster University, Hamilton, Ontario L8N 1Y4 (Accepted May 22, 1979)
Departments of Laboratory Medicine and Pathology, St. Joseph's Hospital, McMaster University, Hamilton, Ontario L8N 1Y4
electrophoresis at pH 9 using Tris-EDTA-borate buffer and by citrate-agar electrophoresis at pH 6.0 (a'4). Hb A2 was quantitated by column chromatography after its separation from Hb C in CM-cellulose cation exchanger (s). Hb F was measured by the Betke method and its distribution in the red cells by the Kleihauer techniqueCe'T~; and a / B hemoglobin chain synthesis studies were made following the procedure described by I-Iuisman(~.
LABORATORY DIAGNOSIS OF HB CC-a- THALASSEMIA
RESULTS
CLBIA, 12 (5) 157-158 (1979) Clin. Biochcm. Wong, S.C. and All, M.A.M.
Routine laboratory investigations of hemoglobinopathies include Hb electrophoresis for abnormal hemoglobins, determination of Hb A2 (a2~2) for fithalassemia traits, staining for Hb H (fi4) inclusions for a-thalassemia traits and estimation of Hb F (a2%) for the presence of hereditary persistence of fetal hemoglobin genes ( H P F H ) . Frequently, analytical column chromatography and a//~ hemoglobin chain synthesis are used in the studies of more complicated hemoglobinopathies. This communication outlines the procedures used in this laboratory for the diagnosis of a case of Hb CC-athalassemia. MATERIALS AND METHODS
Two tubes of 10ml blood sample were obtained by venipuncture. One tube was for tests outlined in Stage 1; the other tube was for synthesis studies in Stage II. The following is a protocol of the tests performed.
Stage I: EDTA Whole Blood (A) CBC (B) Blood smear (C) Reticulocyte counts and Hb H inclusion bodies (D) Free erythrocyte protoporphyrin (FEP) (E) Hb F staining
Hemolysate (F) Starch gel electrophoresis (G) Acid-citrate-agar electrophoresis (H)Hb A2 determination by column chromatography (I) Hb F determination by alkali denaturation
Stage II: EDTA whole blood ---*a/13 hemoglobin chain synthesis studies. Hematological data from the whole blood sample was obtained with standard methods'. Reticulocytes and Hb H inclusion bodies were estimated in smears prepared from whole blood after incubation with brilliant cresyl blue at 37°C for 90 minutes. Free erythrocyte protoporphyrin was measured by a spectrofluorometric method2. Hemolysate was prepared from thrice saline-washed red cells by the addition of an equal volume of distilled water and the removal of the cellular debris in half volume of carbon tetrachloride by centrifugation. Hemoglobin analysis of the hemolysate was made by starch gel Correspondence: Dr. S.C. Wong Hemoglobinopathy Laboratory St. Joseph's Hospital 50 Charlton Ave. East Hamilton, Ontario L8N 1Y4.
Our p a t i e n t (J.B.) is a 31-year-old m a l e N i g e r i a n s u m m e r s t u d e n t at M c M a s t e r U n i v e r s i t y . Red cell m o r p h o l o g y showed h y p o c h r o m i a and m i c r o c y t o s i s with m a r k e d n u m b e r s of t a r g e t cells. H e m a t o l o g i c a l d a t a w e r e : H b = 1 2 . 5 g / d l , R B C = 6 . 3 x 10'~/1, P C V = 0.371/1, M C V = 5 9 f l , M C H = 2 0 p g , M C H C = 3 4 g / d l , r e t i c u l o c y t e s = 4 . 4 % and F E P = 1 8 / ~ g / d l , RBC (normal=15-50/~g/dl). S t a r c h gel e l e c t r o p h o r e s i s ( F i g . 1) showed only one m a j o r Hb b a n d in the C position (Hb C r u n s tog e t h e r with Hb A2) and a m i n o r Hb b a n d in t h e F position. No Hb A was detected. A c i d - c i t r a t e - a g a r e l e c t r o p h o r e s i s c o n f i r m e d the p r e s e n c e of Hb C a n d the a b s e n c e of A. Hb A~ was s e p a r a t e d f r o m Hb C by CM-cellulose column c h r o m a t o g r a p h y and q u a n t i t a t e d as 3.5% ( F i g . 2) Hb F was 3.7% and heterog e n e o u s l y d i s t r i b u t e d in the red cells. No Hb H (rid i n c l u s i o n bodies were d e t e c t e d by t h e b r i l l i a n t c r e s y l b l u e stain. C h r o m a t o g r a p h i c s e p a r a t i o n of the v a r i o u s hemoglobin c h a i n s is shown in F i g . 3. D e t e r m i n a t i o n of the r a d i o a c t i v i t i e s of t h e a and tic c h a i n s y i e l d e d an a/ti c r a t i o of 0.75. DISCUSSION T h i s c o m m u n i c a t i o n r e p o r t s t h e a p p l i c a t i o n of v a r i o u s l a b o r a t o r y p r o c e d u r e s in o r d e r to a r r i v e at t h e p r o p e r d i a g n o s i s of a case of Hb C C - a - t h a l a s semia. A h y p o c h r o m i c and m i c r o c y t i c blood p i c t u r e m a y be due to one or a c o m b i n a t i o n of t h e f o l l o w i n g c o n d i t i o n s : (1) iron deficiency, (2) f l - t h a l a s s e m i a t r a i t and (3) a - t h a l a s s e m i a t r a i t . This case is comp l i c a t e d by the h o m o z y g o s i t y of Hb C r e s u l t i n g in m a r k e d n u m b e r s of t a r g e t cells. F a m i l y m e m b e r s were not a v a i l a b l e f o r studies. Iron d e f i c i e n c y was excluded in o u r p a t i e n t u s i n g his n o r m a l f r e e e r y t h r o c y t e p r o t o p o r p h y r i n level. O r d i n a r i l y , an e l e v a t e d Hb A2 v a l u e is i n d i c a t i v e of f l - t h a l a s s e m i a t r a i t , while the p r e s e n c e of Hb H (rid i n c l u s i o n bodies is d i a g n o s t i c of a - t h a l a s semia t r a i t . However, t h e p r e s e n c e of Hb C in o u r p a t i e n t m a d e p r o p e r d i a g n o s i s d i f f i c u l t . Hb C has e l e c t r o p h o r e t i c and a n i o n - c h r o m a t o g r a p h i c m o b i l i ties s i m i l a r to Hb A2, m a k i n g it i m p o s s i b l e to q u a n t i t a t e Hb A~ by the routine DE-52 column chromato-
158
WONG AND A L I
Hb A Hb F
Fig. 1 at p H buffer.
-
S t a r c h gel electrophoresis 9.0 u s i n g T r i s - E D T A - b o r a t e -
:~.
Hb C + A 2 •..:- .-f,
)
Adults
Propositus I ~
:,
I Cord
1.20
1.00
1.0(
25oo
0.8¢
2OO0
E e,
ID Z
84,4% C
0.80
Bc
0.60
1500 er
to
U Z
< as @
0.6(
as ¢¢
o
I-
0.~
,<
0.4~
o(J
< 0.2f
~
8.4%
_
)
40
80
120
160
F R A C T I O N N U M B E R S (4 m l / 1 5
J C.
200
0.20
240
500
;
280
minutes)
Fig. 2 - - CM-cellulose column c h r o m a t o g r a p h i c s e p a r a tion o f H b A~ f r o m Hb C w i t h p h o s p h a t e developers.
FRACTION NUMBERS
(6 m1/15 m i n u t e s )
Fig. 3 - - CM-cellulose column c h r o m a t o g r a p h i c s e p a r a t i o n of tic and a globin chains w i t h a linear Na + g r a d i e n t in 8 M urea.
RF_,FF~ENCES
g r a p h y ~9'. Also the lack of normal fl chains in a CC homozygote renders the staining for Hb H (rio inclusion bodies unreliable. In our case, Hb A2 was separated from Hb C by cation-exchange column c h r o m a t o g r a p h y and was found to be normal, excluding t - t h a l a s s e m i a trait (Hb A ~ 2 . 8 - 5 . 4 % in CC homozygotes, Ref. 5). Finally, a-thalassemia trait in o u r p a t i e n t w a s c o n f i r m e d by an i m b a l a n c e d a / t i c c h a i n s y n t h e s i s r a t i o of 0.75 ( n o r m a l a / f l r a t i o = 0.91-1.10). ACKNOWLEDGEMENTS
Appreciations are extended to Ms. A. Quinlan and Mrs. S. Boyadjian for excellent technical assistance, and to the St. Joseph's Hospital Foundation for continual financial support.
1. Dacie, J.V. and Lewis, S.M. (1975). Practical Hematology, 5th Edition, Churchill Livingstone, Edinburgh/ London/New York. 2. Piomelli, S. (1973). J. Lab. Clin. Med., 81, 932-40. 3. Efremov, G.D., Huisman, T.H.J., Smith, L.L., Wilson, J.B., Kitchens J.L., Wrightstone, R.N., and Adams, H.R. (1969). J. Biol. Chem., 244, 6105-16. 4. Robinson, A.R., Robson, M., Harrison, A.P., and Zuelzer, W.W. (1957). J. Lab. Clin. Med., 50, 745-52. 5. Huisman, T.H.J., (1972). Clin. Chim. A c t a . , 40, 15963. 6. Betke, K., Marti, H.R., and Schlicht, L. (1959). N a t u r e 184, 1877-78. 7. Kleihauer, E. (]974). The detection of hemoglobinopathies. (R:M. Schmidt, T.H.J. Huisman, H. Lehmann, eds.), CRC Press, Inc., Cleveland, 1974. 8. Huisman, T.H.J. and Jonxis, T.H.P. {1976). The hemoglobinopathies. Techniques for identification. Marcel Dekkar, Inc., New York. 9. Efremov, G.D., Huisman, T.H.J., Bowman, K., Wrightstone, R.N., and Schroeder, W.A. (1974). J. Lab. Clin Med., 83, 657-64.
Laboratory Diagnosis of HB CC- -Thalassemia S. C. WONG and M. A. M. ALI Departments of Laboratory Medicine and Pathology, St. Joseph's Hospital, McMaster University, Hamilton, Ontario L8N 1Y4 (Accepted May 22, 1979)
Departments of Laboratory Medicine and Pathology, St. Joseph's Hospital, McMaster University, Hamilton, Ontario L8N 1Y4
electrophoresis at pH 9 using Tris-EDTA-borate buffer and by citrate-agar electrophoresis at pH 6.0 (a'4). Hb A2 was quantitated by column chromatography after its separation from Hb C in CM-cellulose cation exchanger (s). Hb F was measured by the Betke method and its distribution in the red cells by the Kleihauer techniqueCe'T~; and a / B hemoglobin chain synthesis studies were made following the procedure described by I-Iuisman(~.
LABORATORY DIAGNOSIS OF HB CC-a- THALASSEMIA
RESULTS
CLBIA, 12 (5) 157-158 (1979) Clin. Biochcm. Wong, S.C. and All, M.A.M.
Routine laboratory investigations of hemoglobinopathies include Hb electrophoresis for abnormal hemoglobins, determination of Hb A2 (a2~2) for fithalassemia traits, staining for Hb H (fi4) inclusions for a-thalassemia traits and estimation of Hb F (a2%) for the presence of hereditary persistence of fetal hemoglobin genes ( H P F H ) . Frequently, analytical column chromatography and a//~ hemoglobin chain synthesis are used in the studies of more complicated hemoglobinopathies. This communication outlines the procedures used in this laboratory for the diagnosis of a case of Hb CC-athalassemia. MATERIALS AND METHODS
Two tubes of 10ml blood sample were obtained by venipuncture. One tube was for tests outlined in Stage 1; the other tube was for synthesis studies in Stage II. The following is a protocol of the tests performed.
Stage I: EDTA Whole Blood (A) CBC (B) Blood smear (C) Reticulocyte counts and Hb H inclusion bodies (D) Free erythrocyte protoporphyrin (FEP) (E) Hb F staining
Hemolysate (F) Starch gel electrophoresis (G) Acid-citrate-agar electrophoresis (H)Hb A2 determination by column chromatography (I) Hb F determination by alkali denaturation
Stage II: EDTA whole blood ---*a/13 hemoglobin chain synthesis studies. Hematological data from the whole blood sample was obtained with standard methods'. Reticulocytes and Hb H inclusion bodies were estimated in smears prepared from whole blood after incubation with brilliant cresyl blue at 37°C for 90 minutes. Free erythrocyte protoporphyrin was measured by a spectrofluorometric method2. Hemolysate was prepared from thrice saline-washed red cells by the addition of an equal volume of distilled water and the removal of the cellular debris in half volume of carbon tetrachloride by centrifugation. Hemoglobin analysis of the hemolysate was made by starch gel Correspondence: Dr. S.C. Wong Hemoglobinopathy Laboratory St. Joseph's Hospital 50 Charlton Ave. East Hamilton, Ontario L8N 1Y4.
Our p a t i e n t (J.B.) is a 31-year-old m a l e N i g e r i a n s u m m e r s t u d e n t at M c M a s t e r U n i v e r s i t y . Red cell m o r p h o l o g y showed h y p o c h r o m i a and m i c r o c y t o s i s with m a r k e d n u m b e r s of t a r g e t cells. H e m a t o l o g i c a l d a t a w e r e : H b = 1 2 . 5 g / d l , R B C = 6 . 3 x 10'~/1, P C V = 0.371/1, M C V = 5 9 f l , M C H = 2 0 p g , M C H C = 3 4 g / d l , r e t i c u l o c y t e s = 4 . 4 % and F E P = 1 8 / ~ g / d l , RBC (normal=15-50/~g/dl). S t a r c h gel e l e c t r o p h o r e s i s ( F i g . 1) showed only one m a j o r Hb b a n d in the C position (Hb C r u n s tog e t h e r with Hb A2) and a m i n o r Hb b a n d in t h e F position. No Hb A was detected. A c i d - c i t r a t e - a g a r e l e c t r o p h o r e s i s c o n f i r m e d the p r e s e n c e of Hb C a n d the a b s e n c e of A. Hb A~ was s e p a r a t e d f r o m Hb C by CM-cellulose column c h r o m a t o g r a p h y and q u a n t i t a t e d as 3.5% ( F i g . 2) Hb F was 3.7% and heterog e n e o u s l y d i s t r i b u t e d in the red cells. No Hb H (rid i n c l u s i o n bodies were d e t e c t e d by t h e b r i l l i a n t c r e s y l b l u e stain. C h r o m a t o g r a p h i c s e p a r a t i o n of the v a r i o u s hemoglobin c h a i n s is shown in F i g . 3. D e t e r m i n a t i o n of the r a d i o a c t i v i t i e s of t h e a and tic c h a i n s y i e l d e d an a/ti c r a t i o of 0.75. DISCUSSION T h i s c o m m u n i c a t i o n r e p o r t s t h e a p p l i c a t i o n of v a r i o u s l a b o r a t o r y p r o c e d u r e s in o r d e r to a r r i v e at t h e p r o p e r d i a g n o s i s of a case of Hb C C - a - t h a l a s semia. A h y p o c h r o m i c and m i c r o c y t i c blood p i c t u r e m a y be due to one or a c o m b i n a t i o n of t h e f o l l o w i n g c o n d i t i o n s : (1) iron deficiency, (2) f l - t h a l a s s e m i a t r a i t and (3) a - t h a l a s s e m i a t r a i t . This case is comp l i c a t e d by the h o m o z y g o s i t y of Hb C r e s u l t i n g in m a r k e d n u m b e r s of t a r g e t cells. F a m i l y m e m b e r s were not a v a i l a b l e f o r studies. Iron d e f i c i e n c y was excluded in o u r p a t i e n t u s i n g his n o r m a l f r e e e r y t h r o c y t e p r o t o p o r p h y r i n level. O r d i n a r i l y , an e l e v a t e d Hb A2 v a l u e is i n d i c a t i v e of f l - t h a l a s s e m i a t r a i t , while the p r e s e n c e of Hb H (rid i n c l u s i o n bodies is d i a g n o s t i c of a - t h a l a s semia t r a i t . However, t h e p r e s e n c e of Hb C in o u r p a t i e n t m a d e p r o p e r d i a g n o s i s d i f f i c u l t . Hb C has e l e c t r o p h o r e t i c and a n i o n - c h r o m a t o g r a p h i c m o b i l i ties s i m i l a r to Hb A2, m a k i n g it i m p o s s i b l e to q u a n t i t a t e Hb A~ by the routine DE-52 column chromato-
158
WONG AND A L I
Hb A Hb F
Fig. 1 at p H buffer.
-
S t a r c h gel electrophoresis 9.0 u s i n g T r i s - E D T A - b o r a t e -
:~.
Hb C + A 2 •..:- .-f,
)
Adults
Propositus I ~
:,
I Cord
1.20
1.00
1.0(
25oo
0.8¢
2OO0
E e,
ID Z
84,4% C
0.80
Bc
0.60
1500 er
to
U Z
< as @
0.6(
as ¢¢
o
I-
0.~
,<
0.4~
o(J
< 0.2f
~
8.4%
_
)
40
80
120
160
F R A C T I O N N U M B E R S (4 m l / 1 5
J C.
200
0.20
240
500
;
280
minutes)
Fig. 2 - - CM-cellulose column c h r o m a t o g r a p h i c s e p a r a tion o f H b A~ f r o m Hb C w i t h p h o s p h a t e developers.
FRACTION NUMBERS
(6 m1/15 m i n u t e s )
Fig. 3 - - CM-cellulose column c h r o m a t o g r a p h i c s e p a r a t i o n of tic and a globin chains w i t h a linear Na + g r a d i e n t in 8 M urea.
RF_,FF~ENCES
g r a p h y ~9'. Also the lack of normal fl chains in a CC homozygote renders the staining for Hb H (rio inclusion bodies unreliable. In our case, Hb A2 was separated from Hb C by cation-exchange column c h r o m a t o g r a p h y and was found to be normal, excluding t - t h a l a s s e m i a trait (Hb A ~ 2 . 8 - 5 . 4 % in CC homozygotes, Ref. 5). Finally, a-thalassemia trait in o u r p a t i e n t w a s c o n f i r m e d by an i m b a l a n c e d a / t i c c h a i n s y n t h e s i s r a t i o of 0.75 ( n o r m a l a / f l r a t i o = 0.91-1.10). ACKNOWLEDGEMENTS
Appreciations are extended to Ms. A. Quinlan and Mrs. S. Boyadjian for excellent technical assistance, and to the St. Joseph's Hospital Foundation for continual financial support.
1. Dacie, J.V. and Lewis, S.M. (1975). Practical Hematology, 5th Edition, Churchill Livingstone, Edinburgh/ London/New York. 2. Piomelli, S. (1973). J. Lab. Clin. Med., 81, 932-40. 3. Efremov, G.D., Huisman, T.H.J., Smith, L.L., Wilson, J.B., Kitchens J.L., Wrightstone, R.N., and Adams, H.R. (1969). J. Biol. Chem., 244, 6105-16. 4. Robinson, A.R., Robson, M., Harrison, A.P., and Zuelzer, W.W. (1957). J. Lab. Clin. Med., 50, 745-52. 5. Huisman, T.H.J., (1972). Clin. Chim. A c t a . , 40, 15963. 6. Betke, K., Marti, H.R., and Schlicht, L. (1959). N a t u r e 184, 1877-78. 7. Kleihauer, E. (]974). The detection of hemoglobinopathies. (R:M. Schmidt, T.H.J. Huisman, H. Lehmann, eds.), CRC Press, Inc., Cleveland, 1974. 8. Huisman, T.H.J. and Jonxis, T.H.P. {1976). The hemoglobinopathies. Techniques for identification. Marcel Dekkar, Inc., New York. 9. Efremov, G.D., Huisman, T.H.J., Bowman, K., Wrightstone, R.N., and Schroeder, W.A. (1974). J. Lab. Clin Med., 83, 657-64.