| | Re: RNA purification from reticulocytes
Reticulocytes manual count and classification Dispense in tube, 100 µl of brilliant cresyl blue (0.5% BCB in 0.8% NaCl, 0.7% trisodium citrate in dH2O and filter before to use) and to add 200-250 µl of whole EDTA fresh blood samples, mix and incubated at 37 °C for 20-25 min. The blood volume to be added to the dye depends on the volume of packed red blood cells volume (PCV), 200 µl for PCV equal or superior to 30% and 250 µl for less than 30% PVC. At the end of incubation, mix again the contents of the tube well and place a small drop of the mixture on a clean glass slide and prepare a thin smear. Counterstain with Wright's stain, if desired. Allow smear to air-dry and reticulocytes are counted, for up to 72 hours. Place the slide on the microscope stage and, using the low power objective, locate the thin portion of the smear in which the red cells are evenly distributed and are not touching each other. Switch to oil immersion magnification and count the number of reticulocytes in 5 fields of 200 RBCs. For to determine Ret% calculate dividing number reticulocytes counted/10. When Hct value of the sample is different to Hct-normal people, about 45, is need to correct reticulocytes value obtained using the following formula : Ret% x Hct-patient/45.
Reticulocytes are counted such as young (group I, II) or mature (group III, IV) cells, according to the amount and distribution of stained reticulum. Based on the classification of Heilmeyer and Westhauser (Heilmeyer L. and Westhauser R.: Studies on the ripening of surviving reticulocytes in vitro and their significance in the evaluation of daily hemoglobin production in vivo. Zitschr. Klin. Med. 121: 361-379, 1932) and of Seip (Seip M.: Reticulocyte Studies. Acta Med. Scandinav. Suppl. 282: 9-164, 1953), group I reticulocytes have a dense coherent reticulum filling the greater part of the cell (0-4%). Group II reticulocytes have an extended reticulum, which is looser than that in group I because of occasional breaks in the network (6-22%). Group III reticulocytes have some small bits of reticulum (6- 18%), and group IV cells have only a few blue granules and are essentially erythrocytes (normal value : 78-92%).