Fluorescence of Erythrocytes after a Chemical Conversion of Haemoglobin

The auto‐fluorescence of erythrocytes is very weak and erythrocytes are not stained by ordinarily used fluorochromes (as e.g. acridin‐orange). Within the haemoglobin molecule some of the conjugated double bonds of haematoporphyrin are bound to Fe‐atoms, whereby the ability of haematoporphyrin to fluoresce ceases. Heller showed (1916) that it was possible to split of the Fe‐atoms from haemoglobin by means of strong acids without destroying the porphyrin ring or the fluorescence. In this report is described how this method was used to get erythrocytes to fluoresce Venous blood was collected with acid dextrose citrate as anti‐coagulant and diluted by saline to get a 10 per cent suspension of erythrocytes. Smears prepared of this suspension were dried and fixed in aethanol for 10 min. One drop of 20 per cent HCl was placed on the glass slide and covered with a coverslip. The slides were examined using a Reichert Binolux fluorescence microscope (primary filter: Erreger filter BG 12/3 mm; secondary filter GG 9/1 mm + OG 1/1.5 mm; dark field condensor). After ultraviolet illumination in about 15 seconds all the erythrocytes showed a vivid red fluorescence while all the leucocytes and thrombocytes showed no fluorescence at all The method is suitable to detect traces of haemoglobin and can be used to show the remaining quantity of haemoglobin by preparing erythrocyte ghosts The method can also be used to show the existence of erythrocytes containing haemoglobin of an abnormal type, if haemoglobin‐A can be removed. We have demonstrated foetal erythrocytes in a mixture of foetal and adult erythrocytes by preparing the smears with Kleihauer et al. 's acid elution (1957) after fixation. Further investigations of this application of the method will be published.