In parallel with the clinical and scientific observations of the role of Bence Jones protein, electrophoretic techniques for protein separation were evolving and eventually entered clinical laboratories. Perlzweig et al.  were the first to report hyperproteinaemia in MM in 1928. Shortly after in 1930, Tiselius reported the homogeneity of certain serum globulins using a technique he devised, termed moving boundary electrophoresis  In this technique, proteins moved through a U-shaped electrophoretic cell and the detection system was based on the refraction of light by proteins as they moved through the tube. Tiselius found that there was a difference in the refractive index of light at boundary interfaces between major protein fractions, and in 1937 he described how serum globulins could be separated into alpha, beta and gamma components . In 1939, Tiselius and Kabat  demonstrated antibody activity in the gamma globulin fraction. Moving boundary electrophoresis became commercially available, but was very labour-intensive and the technique was limited in its ability to detect subtle abnormalities . Using this technique, in 1939, Longsworth et al.  recognised tall, narrow-based, “church spire” peaks in the sera of patients with multiple myeloma (MM). Electrophoresis was subsequently improved by using filter paper as a support, which allowed the separation of protein into discrete zones that could be visualised with dyes . The method was further improved using cellulose acetate and agarose gels in the 1950s and 1960s, and subsequently automated using capillary zone electrophoresis in the 1990s (Chapter 4) . Finally, immunofixation electrophoresis was first introduced in 1964 by Wilson et al.  and became established in the 1980s .
Jan Waldenström , in a seminal lecture series in 1961, was the first to describe the key concept of monoclonal vs. polyclonal gammopathy. Importantly, he distinguished monoclonal proteins (characterised by a narrow band of gammaglobulin on electrophoresis, often associated with MM or macroglobulinaemia) from hypergammaglobulinaemia (characterised by a broad band, and regarded as a polyclonal increase in proteins, that was associated with an inflammatory or reactive process).