ALTERATIONS IN OSMOTIC AND MECHANICAL FRAGILITY RELATED TO IN VIVO ERYTHROCYTE AGING AND SPLENIC SEQUESTRATION IN HEREDITARY SPHEROCYTOSIS Robert C. Griggs, … , Russell Weisman Jr., John W. Harris J Clin Invest. 1960;39(1):89-101. https://doi.org/10.1172/JCI104032. Research Article Find the latest version: https://jci.me/104032/pdf ALTERATIONS IN OSMOTIC AND MECHANICAL FRAGILITY RELATED TO IN VIVO ERYTHROCYTE AGING AND SPLENIC SEQUESTRA- TION IN HEREDITARY SPHEROCYTOSIS * t By ROBERT C. GRIGGS3 RUSSELL WEISMAN, JR. AND JOHN W. HARRIS § (From the Department of Medicine, Western Reserve University School of Medicine at Cleve- land Metropolitan General Hospital, and University Hospitals of Cleveland, Ohio) (Submitted for publication July 20, 1959; accepted August 20, 1959) Increased osmotic and mechanical fragility of the acteristics to those of the general population. The erythrocytes is a consistent finding in patients with changes in osmotic and mechanical fragility oc- hereditary spherocytosis, although in a few indi- curring in these labeled red cells during in vivo viduals it is necessary to incubate the cells at 370 aging and splenic sequestration were followed. In C. for 24 hours before the otherwise latent ab- addition, erythrocytes were recovered from the normality becomes demonstrable. Splenectomy spleen at the time of splenectomy and reinjected will correct the hemolytic process of hereditary into the patient's peripheral circulation. The spherocytosis despite the continuing production of post-splenectomy survival and changes in osmotic abnormally fragile cells by the patient. The spleen and mechanical fragility of these cells, labeled with is, therefore, essential in the destruction of the in- Cr51, were followed and distinguished from the trinsically defective erythrocytes. In the periph- cells of the general population. eral blood of some individuals with this disease a "double population" of cells exists. One com- METHODS ponent consists of more markedly fragile cells that are not present and Routine hematologic studies were performed by stand- after splenectomy must, there- ard methods (1). Autohemolysis was determined by the fore, have been related to the presence of the method of Selwyn and Dacie (2) as modified by Young, spleen. Izzo, Altman and Swisher (3). Erythrocyte osmotic The present study was undertaken to investi- fragility tests were done as described by Emerson and gate the changes in osmotic and mechanical fra- associates (4) and the osmotic fragility of the labeled gility accompanying in vivo erythrocyte aging and cells was distinguished from that of the general popula- tion by the following modification of this method. The splenic sequestration and to elucidate the source volume of each saline solution was increased to 3 ml., and fate of the more markedly fragile group of red and 0.3 ml. of defibrinated whole blood was added to each cells found in some patients. tube. These were then centrifuged, decanted and the Advantage was taken of the increased erythro- hemoglobin content of 0.5 ml. of the supernatant solu- poiesis in patients with hereditary spherocytosis tion determined in a Beckman (Model B) spectropho- tometer. The remainder of the supernatant was trans- prior to splenectomy to label in vivo with Fe59 the ferred to a calibrated isotope counting tube and the new red cells produced during a short time span, radioactivity determined in a low background, well-type thereby obtaining an identifiable group of cells of scintillation counter (Tracerlab). Sufficient counts were known age. Using techniques that will be de- obtained to give a statistical error of ± 3 per cent or scribed, it was possible to differentiate these iso- less. As a blank, 0.3 ml. of the defibrinated whole blood was suspended in hypertonic saline (1.25 Gm. NaCl per topically-labeled cells from the other cells in the 100 ml.) and the radioactivity found in this supernatant peripheral circulation and to compare their char- after centrifugation was subtracted from that obtained for the other samples. The value for radioactivity thus * A preliminary report of this work appeared in ab- obtained represented isotope released by the red cells stract form (J. clin. Invest. 1958, 37, 899). lysed at each concentration of sodium chloride. The t This study was supported by a grant from the Web- amount of radioactivity in each sample was calculated ster-Underhill Fund and Public Health Service Grant as a percentage of that released into the supernatant when No. A-745. 0.3 ml. of the defibrinated whole blood had undergone + Webster-Underhill Fellow, Western Reserve Uni- complete hemolysis by addition to 3 ml. distilled water. versity School of Medicine. The results were plotted as two curves, one showing the § Markle Scholar in Medical Science, Western Reserve percentage of hemolysis measured as hemoglobin (the University School of Medicine. osmotic fragility of all the cells in the sample) and the 89 90 ROBERT C. GRIGGS, RUSSELL WEISMAN, JR. AND JOHN W. HARRIS other curve representing the percentage of radioactivity Scintillation counting over the body surface was done released at each concentration of sodium chloride (the by the methods described by Huff and co-workers (7) and osmotic fragility of only the isotopically labeled cells). Jandl, Greenberg, Yonemoto and Castle (9). The count- Control studies demonstrated that no appreciable Fe' ing rates over the liver and spleen are plotted as ratios was present in the stroma of the lysed cells and that relative to the counting rate over the precordium. osmotic fragility curves obtained by this isotope method on our patients after general distribution of the Fe' RESULTS through the red cell population were identical with those obtained by the usual method in which hemoglobin from Three unrelated adults who were referred to lysed red cells is measured. this hospital for investigation of anemia or jaundice The mechanical fragility of the red cells was deter- were studied. All had splenomegaly and showed mined by a modification of the method of Shen, Castle and Fleming (5). The hematocrit of a sample of defibri- spherocytosis on peripheral blood smears; appro- nated venous blood was adjusted to 35 per cent and 0.5 priate laboratory tests confirmed the diagnosis of ml. subjected to the standardized trauma produced by hereditary spherocytosis. Family history in all rotating it for 90 minutes in a 50 ml. Erlenmeyer flask three was negative for anemia, jaundice or spleno- at 30 rpm with 10 uniform 4 mm. selected glass beads. megaly. The mother of Case 2 was the only rela- To obtain an adequate sample for the isotope counting procedures, two flasks were used for each determination tive available for examination and her blood stud- and combined after rotation. The results were corrected ies, including osmotic and mechanical fragility, by a blank consisting of a similar blood sample in hy- were normal. Identifying data and laboratory pertonic saline (1.25 Gm. NaCl per 100 ml.) and ex- findings are recorded in Table I. Presplenectomy pressed as percentage of hemolysis compared to hemoly- studies with Fe59 were performed on Cases 1 and sis in distilled water. Ten /Ac. of Fe' in the form of ferric chloride (Abbott) 2 and post-splenectomy studies with Cr5' on Cases (3 to 10 /Ag. of elemental iron) was incubated with 20 ml. 2 and 3. of normal compatible heparinized plasma and adminis- tered intravenously to each patient. Since patients with Presplenectomy studies hemolytic anemias have increased saturation of their iron-binding protein, normal compatible plasma was used Case 1 showed evidence of a marked hemolytic instead of the patient's. The plasma Fe" clearance and process. A Cr5' autosurvival had been performed the red cell Fe" utilization studies were performed ac- in our laboratory on this individual one year prior cording to the method of Huff and associates (6, 7). to the present study and demonstrated a red cell Red cell survival was determined by the method of half-life of eight days compared to a normal of at Ebaugh, Emerson and Ross (8) using 120 Ac. of Cr'. least 28 days for this method. During the present In each case the patient's own red cells, obtained from study he showed a marked hyperbilirubinemia either peripheral vein or splenic pulp, were used. In- (indirect reacting) and sustained reticulocytosis tact red cells were recovered from the spleen at opera- (Table I). Both the spleen and liver were en- tion. Using sterile technique, this was accomplished by larged. The administered Fe59 was rapidly cleared making a number of large incisions into the surgical specimen immediately after removal and allowing the from the plasma, with a half-time of 15 minutes, formerly entrapped blood to flow into a glass flask where contrasted to a normal of 60 to 120 minutes. The it was defibrinated by rotating with glass beads. utilization of Fe59 for red cell formation and the TABLE I Laboratory data on patients in study Erythro- Erythrocyte % Autohemol- cyte mechanical ysis 48 hours Plasma survival Age Erythrocyte fragility clearance Cr'l Race Bilirubin osmotic No With Fe"9 half- Case Sex Hgb.* Hct. Retics. direct total fragility 0 hrs. 24 hrs. glucose glucose half-time time Gm.% % % mg./100 mi. % % min. days Normal 1-5 9-15 0-3 0-1 60-120 28 + range 1 68 W M 10 30 15 1.4 5.1 Increased 22 37 15 8 2 32 N F 9.5 32 4 0.7 1.1 Increased 18 37 12.4 8.3 31 3 54 W M 9.5 29 3 0.5 1.0 Increased 7 17 5.7 1.5 16 * Hgb. = hemoglobin; Hct. = hematocrit; Retics. = reticulocytes. ERYTHROCYTE FRAGILITY IN HEREDITARY SPHEROCYTOSIS 91 DISTRIBUTION OF FE59 IN HEREDITARY SPHEROCYTOSIS I-. 4 BODY SURFACE COUNTS ,iaM t 3 0 i 4M.0I- kL 2- o0 a ,{ ~~~~~~~~~~~~LIVER x41wo Il _- -* An - 9n 0.