A new density system for the separation of human red blood cells by density-gradient centrifugation is described. The gradient medium is made with colloidal silica particles coated with polyvinylpyrrolidone suspended in aqueous solution of meglamine diatrizoate. By this method, more than 10 red-cell fractions can be separated. These show different ages (by creatine content and 59Fe in vivo labelling) and different characteristics (ie, potassium content). A 40-fold enrichment in reticulocytes can be obtained in the top layers, with a great improvement of specific activity in labelling procedures of newly synthesized globin chains. The method is simple, rapid, inexpensive, reliable, nontoxic for erythrocytes, and is suitable for globin synthesis and other studies of erythrocyte metabolism.

A new test for the laboratory diagnosis of spherocytosis, conventionally called ‘Pink test’, is presented. This test, semi-quantitatively or quantitatively, determines the hemolysis of small blood samples in a solution containing glycerol (135 mmol/l), NaCl (25 mmol/l), NaN₃ (1.5 mmol/l), buffered to pH 6.66 with Bis-Tris (70 mmol/l) and HC1. ‘Pink test’, as well as ‘acidified’ glycerol lysis test, were positive in 100% of 42 patients suffering from hereditary spherocytosis, and optimally discriminated them from healthy subjects, showing a diagnostic sensitivity greater than ‘standard’ glycerol lysis test and osmotic fragility in hypotonic saline solutions of fresh or incubated blood. ‘Pink test’ was also positive in some cases of renal failure, immunohemolytic anemia, chronic hemoproliferative disorders, normal pregnant women, and negative in other microcytic anemias (beta-thalassemia, iron deficiency anemia). The results do not critically depend on pH of the solution (differently from those obtained with ‘acidified’ glycerol lysis test), and for this reason they show a good reproducibility.

The effects of lead on red blood cell (RBC) membrane proteins were studied in two groups of workers with different lead exposure levels: Group I (6 subjects employed in a battery plant) with a mean blood lead of 40.1 (SD = 3.7) micrograms/100 ml; Group II (5 workers employed in different industries) with a mean blood lead of 60.6 (SD = 8.0) micrograms/100 ml, compared with a control group with mean blood lead of 15.6 (SD = 9.3) micrograms/100 ml. The analysis of RBC membrane polypeptides was carried out by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and by using a densitometer for percentage measurement of the bands corresponding to protein fractions. The results show a very significant decrease in Band 3 (anion channel) and 4.1 in more exposed workers (Group II) only. The effects of lead on RBC membrane proteins seem to be evident at blood-lead levels higher (greater than 50 micrograms/100 ml) than those previously reported in literature. These results confirm the effects of lead on membrane proteins, even if the exact mechanism, particularly the influence of proteolysis and the meaning of the interference, still needs to be investigated thoroughly.

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