Made in United States of America Reprinted from BLOOD, The Journal of Hematology Vol. IV, No. 1, January, 1949 A RAPID DIAGNOSTIC TEST FOR SICKLE CELL ANEMIA By HARVEY A. ITANO, M.D., AND LINUS PAULING, PH.D. S ICKLE cell anemia is a congenital chronic hemolytic type of anemia char- acterized hematologically by the development of oat-shaped and sickle-shaped erythrocytes. Other cellular abnormalities which are due to excessive blood de-' struction and active blood formation are also seen in blood smears. Six to IO per cent of Negroes possess the sickle trait 2p 3; their red blood cells have the capacity to sickle, but most of these individuals do not develop anemia. The course of the sickling process as observed under the microscope has been described in detail by several investigators, 4* j* 6* lo but little is known about the physical processes involved in sickling. It has been established, however, that the erythrocytes of individuals with sickle cell anemia and sickle cell trait become sickled when the hemoglobin is reduced. 8* I4 When the hemoglobin is combined with oxygen or carbon monoxide, the cells are indistinguishable in form from normal erythrocytes. The term promeniscocyte has been applied to the latter form and meniscocyte to the former. l1 Hahn and Gillespie8 and Sherman'" obtained sickling physically by reducing the partial pressure of oxygen over suspensions of promeniscocytes. They were able to reverse the process by passing oxygen or carbon monoxide over meniscocytes. When oxygen is removed from promeniscocytes, their hemoglobin aggregates in one or more foci within the cells, and the cell membrane collapses. When oxygen is `added to these cells, they resume their normal contour, and hemoglobin appears to be distributed uniformly through- out their interior. Meniscocytes are strongly birefringent under the polarizing microscope14 while promeniscocytes are not. When a drop of blood is sealed between a cover slip and a slide, the decline in oxygen tension due to oxidative processes in the blood cells leads to sickling.' This is the common diagnostic test for sickle cell anemia and sickle cell trait used in clinical laboratories. Sherman found that increase in temperature, high leuko- cyte count, and bacterial contamination, all of which increase the rate of oxygen consumption, accelerated the sickling process. In another method, a saline citrate suspension of blood is allowed to stand in a test tube under a layer of paraffin oil until sickling takes place.' In employing any of the common diagnostic tests for sickling it is desirable to obtain blood which has a low.fraction of oxyhemoglobin. Thus, the moist stasis method, l3 in which blood is obtained from a patient's finger after its circulation has been occluded for five minutes, gives the most rapid and consistent results. Even with this method it is sometimes necessary to observe the preparation for several hours before the result is conclusive.5 In order to find a more convenient and rapid method of produing meniscocytes Contribution No. 1186 from the Gates and Crellin Laboratories of Chemistry, California Institute of Technology, Pasadena, Calif. This work was done under a grant from the U. S. Public Health Service and was briefly reported by Dr. Dan H. Campbell at the Hematology Symposium of the U. S. Public Health Service held at St. Louis, MO., on February L, 1948. 66 HARVEY A. ITANO AND LINUS PAULING 67 we turned to chemical reducing agents. Sodium dithionite, Na2S204, rapidly reduces oxyhemoglobin to reduced hemoglobin, and this property suggested its use in testing erythrocytes for sickling. When a solution of sodium dithionite was added to promeniscocytes, nearly all of the cells showed sickling or the early changes in the sickling process within a few seconds. Dithionite ion tends to decompose to thiosulfate and sulfite with formation of hydrogen ion9 so that solutions made up from commercial preparations of sodium dithionite are often strongly acid in reaction; but by adding Na2HP04 to the solutions it is possible to increase the pH and at the same time provide a buffering medium. Hahn and Gillespie found that sickling .was obtained most consistently if cell suspensions were buffered at a slightly acid pH. We have prepared a satisfactory reagent by adding 0.114 M aqueous Na2HP04 to 0.114 M aqueous Na&O, until the final pH was 6.8. The ratio of the volumes of Na2HP04 and Na&O, necessary to obtain this pH was about three to two. The blood used in the following experiments was obtained from 6 different cases of sickle cell anemia, 3 of whom were being treated for exacerbations and 3 of whom were in remission. An excess of the dithionite reagent was added to pro- meniscocytes on a microscope slide; almost immediately changes were evident in the erythrocytes. Typical crescentic forms did not appear in large numbers, pre- sumably because of the time required for the reduced hemoglobin molecules to become oriented in what Ponder calls the paracrystalline state." However, nearly all of the cells underwent changes in contour, and other changes described by earlier observers took place at an accelerated rate. The forms of many of these cells cor- responded to the "holly wreath" cells of Sherman and cells classified as "ab- normal" by Reinhard and his co-workers. l2 After about fifteen to thirty minutes the aggregates of hemoglobin in many of the cells became birefringent. The pres- ence of so many holly wreath cells is in accord with Sherman's observation that this form appears in large numbers when the rate of removal of oxygen is rapid. Since dithionite does not react with carbon monoxide, promeniscocytes saturated with carbon monoxide would not be expected to undergo changes in contour upon addition of this reducing agent. This is indeed the case. Although no sickle cell trait blood was available to us for study, there is good reason to believe that such blood would behave in the same manner as sickle cell anemia blood.* METHOD The rapidity and simplicity of this test suggests that it would be useful as a clinical laboratory procedure for diagnosing sickle cell anemia and sickle cell trait. No special precautions are necessary in collecting the blood for this test; oxygenated cells may be used since an excess of reducing agent can always be added. The test works equally well with oxalated blood or fingertip puncture specimens and may be applied in several ways. (I) About 0.03 ml. of reagent may be added to a very small drop (about 0.01 ml.) of blood on a slide. A cover slip is then laid over * A brief note by da Silva (Science 107, ZLI (1948)) which appeared since the preparation of this paper indicates that he has successfully identified sicklemia (sickle cell trait) by a procedure similar to method (I) below. 68 DIAGNOSTIC TEST FOR SICKLE CELL ANEMIA the mixture and cells observed under a microscope. (2) An excess of reagent may be added to a small volume of blood in a test tube and a drop of the mixture observed. (3) A convenient method for studying the entire process of sickling in a short period of time involves the use of a hemocytometer counting chamber. The cham- ber is half filled with a dilute saline suspension of promeniscocytes; the reagent is then added to fill the rest of the chamber. The erythrocytes may be observed as the reducing agent diffuses into the part of the charl)er which they occupy. Since the dithionite reagent is unstable as mentioned above, its reducing PO er should be tested frequently by the addition of a test portion to a dilute suspensi B n of oxygenated erythrocytes. If the reagent is satisfactory, a change from the CO r of oxyhemoglobin to that of reduced hemoglobin should be observed. A la&e volume of stock Na2HP0, solution may be prepared, but it is desirable to make up the Nat&O4 solution daily. ACKNOWLEDGMENTS We are indebted to Dr. Edward K. Evans and D. Travis Winsor for their aid in obtaining the blood used in these experiments. REFERENCES 1 BECK, J. S. P., AND HERTZ, C. S.: Am. J. Clin. Path. I: 3x3, 1935. 2 Camozo, W. W. : Arch. Int. Med. 60: 623, 1937. a DIGGS, L. W., AHMANN, C. F., AND Bme, J. : Ann. Int. Med. 7: 769, 1933. 4 __ AND BIBB, J.: J. A. M. A. 111. 695, 1939. 5 ~ AND PETTIT, V. D. : J. Lab. & Clin. Med. 2~: 1106, 1940. 6 GRAHAM, G. S., AND MCCARTY, S. H.: J. Lab. & Clin. Med. IL; 536, 1927. 7 HAHN, E. V.: Am. J. M. SC. 171: 206, 1918. s -- AND GILLESPIE, E. B. : Arch. Int. Med. 39: 233, 19~7. 9 LATIMBR, W. M.: The Oxidation States of the Elements and their Potentials in Aqueous Solutions. Prentice-Hall, Inc., 1938. 10 MURPHY, R. C., AND SHAPIRO, S.: Arch. Int. Med. 71: 18, `944. 11 PONDER, E.: Ann. N. Y. Acad. SC. 48; 579, 1947. 12 REINHARD, E. H., MOORE, C. V., DUBACH, R., AND WADE, L. J.: J. Clin. Investigation ~9: 682, 1944. 13 Scnwen, J. B., AND WAUGH, T. R.; Canad. M. A. J., 23: 375, 1930. 14 SHERMAN, I. J. : Bull. Johns Hopkins Hosp. 67; 309, 1940.