Cryohemolysis test is for what's causing the spherocytosis

Abstract

Hereditary spherocytosis (HS) is an inherited disease causing usually a mild to moderate hemolytic anemia that is basically related to the spherical shape of erythrocytes. The type of inheritance is mostly autosomal dominant, and the underlying molecular defects are well recognized.Diagnosis of hereditary spherocytosis should be relatively straightforward in patients with a family history of HS, relevant clinical presentation, as anemia, jaundice and splenomegaly, and laboratory findings, as presence of typical spherocytes in peripheral blood smear, raised mean cell hemoglobin concentration (MCHC) and increased reticulocytes. However, if the family history, clinical presentation and/or laboratory tests do not show clear findings, then a screening test with high positive predictive value for HS is necessary, and hence, the recommended screening tests by the BCSH are either hypertonic cryohemolysis (HCH) or flow cytometry EMA binding. 1 A direct Coombs' test is not a requisite in the guidelines, but in our center, it is always performed with polyspecific reagent looking for the negative result to exclude possibility of AIHA. Previously, we used to perform the osmotic fragility test, which is not specific nor sensitive for HS, but in the right clinical and laboratory settings is considered to be a useful confirmation that the cells seen in the peripheral blood smear have decreased surface area/volume ratio, that is, mostly spherocytes.The HCH test is currently in use in practice in our laboratory to confirm the provisional diagnosis of HS and is performed according to the technique in Dacie and Lewis practical hematology, 2 which is based on observation that HS red cells are much more susceptible to temperature changes than normal cells while suspended in hypertonic solutions. 3 The steps of HCH test are as follows:• Washing the red cells three times with 4 °C cold 9 g/l NaCl, then making a suspension of 50-70% cells in the saline, and keeping it on ice.• Preparing 2 ml volumes of buffered 0.7 mol/l sucrose reagent and leaving it for 10 min in a 37 °C water bath to equilibrate temperature.• Pipetting 50 μl of the red cell suspension into each of two tubes of the warmed sucrose reagent, then vortex immediately for a few seconds, and incubate for exactly 10 min at 37 °C.• Transferring the tubes to an ice bath for another 10 min, then vortex for a few seconds, and centrifuge to sediment the remaining cells. Transfer some of the supernatant to a clean tube. • Prepare a 100% hemolysate solution by pipetting 50 μl of the original sample into 2 ml of water. Centrifuge and dilute 200 μl of the supernatant in 4 ml of water, resulting in 21 times dilution. • Read absorbance (A) at 540 nm of the test and the 100% lysis samples. % cryohemolysis = {A of test/A of hemolysate * 21} * 100.This procedure is in practice in our laboratory since the publication of the BCSH guidelines 1 in 2011 that recommended its use, and although some publications may not totally agree with the guidelines, 4 but still we are obliged to use it as the ...