More than one year passed since Itano’s arrival at Caltech and in early 1948, he had produced no conclusive experimental results demonstrating the difference between sickle cell and normal hemoglobin, but he had completed some other important investigations. He reproduced experiments that hindered and induced sickling, which acquainted him with the literature and phenomena. He also attempted two unsuccessful experiments that built upon Pauling’s previous work with hemoglobin.

At Pauling’s request Itano analyzed the absorption spectrum of the compounds formed by combining hemoglobin with cyanate and thiocyanate. It was known that a spectrometer gives different values for hemoglobin and its derivatives of oxyhemoglobin and carbonmonoxyhemoglobin. However, normal and sickle cell hemoglobin yielded the same spectrum, and therefore produced no conclusive results. Pauling most likely suggested this procedure because in 1943 he aided the war effort by developing a spectrophotometer that determined the amount of carbon monoxide in the air based on the concentration of carbon monoxide in a sample of blood.

Itano also examined the magnetic susceptibility of hemoglobin in order to ascertain whether the iron bonds of normal and sickle cell hemoglobin differ. However, a microsusceptometer had to be constructed before conducting this experiment. Ultimately, Itano wanted to determine if the kind of bond formed (ionic or covalent) was responsible for the different shapes observed in deoxygenated normal and sickle cell hemoglobin. As mentioned above, Pauling and Coryell had used magnetism in the 1930s to learn whether covalent or ionic bonds formed in hemoglobin and its derivatives and had found that, depending on the substance, a different kind of bond forms in the same location. The iron in hemoglobin forms ionic bonds with nitrogen and the globin, while oxyhemoglobin and carbonmonoxyhemoglobin form covalent bonds.

Although his attempts failed to yield successful results in detecting a difference between sickle cell and normal hemoglobin, Itano had made headway in other directions. He developed a procedure that quickly indicated whether a blood sample sickled. Itano and Pauling published “A Rapid Diagnostic Test for Sickle Cell Anemia” in January 1949. Their method used sodium dithionite to cleave oxygen from a blood sample and induce sickling. This procedure was especially effective because it took only fifteen to thirty minutes, rather than hours, to observe sickled cells. Others developed similar methods for reducing oxyhemoglobin at this same time including William B. Castle and Geneva A. Daland, a hematology laboratory technologist. They proposed using sodium bisulfite as the reducing agent and their procedure received more acknowledgment than Itano and Pauling’s method because their reagent was more stable.