SECTION I CELLULAR PHYSIOLOGY Hereditary Spherocytosis A 20-year-old wonan suffers from anenia and occasional jaundice.A thorough review of her medical records reveals that over the past 10 years she has had episodes of more severe anemia,usually after periods of febrile illness.The patient has a markedly emlarged spleen.Microscopic examination of the patient's blood showed a large number of microspherocytes (red blood cells [RBCs]that are round and somewhat smaller than erythrocytes).The osmotic fragility (measured by putting RBCs in hypotonic solutions)was ruch greater than that of RBCs fron bealthy individuals. When the patient's erythrocytes were incubated in a buffer solution at 37 C under sterile conditions,the fraction of the RBCs that were hemolyzed was much larger than the hemolyzed fraction from a healthy individual.This 'autohemolysis"could be greatly diminished by including glucose and adenosine triphosphate (ATP)in the RBC incubation solution.RBCs from fresh blood had a normal content of Na+and K+. The permeabilities of the patient's erythrocyte mombranes to Na+and K+were found to be about three times normal.The level of Na+.K+-ATPase in the patient's RBC merbranes was also about three times the level in RBCs from healthy individuals. The average life span of the patient's erythrocytes was well below the normal life span.When an aliquot of the patient's RBCs was labeled and injected intravenously into a bealthy individual,the patient's RBCs had a markedly reduced survival time compared with mormal RBCs.When labeled RBCs from a healthy individual were infused into the patient,the survival time of the normal RBCs was comparable with their survival time in the domor.The patient's spleen was removed,and after the splenectomy.the patient's anemia was largely aneliorated. 1.Thy should the patient's erythrocytes have a greater osnotic fragility than RBCs from healthy individuals?
SECTION I CELLULAR PHYSIOLOGY Hereditary Spherocytosis A 20-year-old woman suffers from anemia and occasional jaundice. A thorough review of her medical records reveals that over the past 10 years she has had episodes of more severe anemia, usually after periods of febrile illness. The patient has a markedly enlarged spleen. Microscopic examination of the patient's blood showed a large number of microspherocytes (red blood cells [RBCs] that are round and somewhat smaller than erythrocytes). The osmotic fragility (measured by putting RBCs in hypotonic solutions) was much greater than that of RBCs from healthy individuals. When the patient's erythrocytes were incubated in a buffer solution at 37°C under sterile conditions, the fraction of the RBCs that were hemolyzed was much larger than the hemolyzed fraction from a healthy individual. This "autohemolysis" could be greatly diminished by including glucose and adenosine triphosphate (ATP) in the RBC incubation solution. RBCs from fresh blood had a normal content of Na+ and K+. The permeabilities of the patient's erythrocyte membranes to Na+ and K+ were found to be about three times normal. The level of Na+, K+-ATPase in the patient's RBC membranes was also about three times the level in RBCs from healthy individuals. The average life span of the patient's erythrocytes was well below the normal life span. When an aliquot of the patient's RBCs was labeled and injected intravenously into a healthy individual, the patient's RBCs had a markedly reduced survival time compared with normal RBCs. When labeled RBCs from a healthy individual were infused into the patient, the survival time of the normal RBCs was comparable with their survival time in the donor. The patient's spleen was removed, and after the splenectomy, the patient's anemia was largely ameliorated. l. Why should the patient's erythrocytes have a greater osmotic fragility than RBCs from healthy individuals?
2.Why might the patient's RBCs 'autohemolyze"more rapidly than normal erythrocytes when they are incubated at 37'C under sterile conditions? 3.Why should including glucose and ATP in the incubation mixture diminish the extent of autohemolysis? 4.Why should the patient's RBCs have a reduced life span?What might the spleen have to do with this? 5.What is proved by the observations that the patient's RBCs have a reduced life span in the circulation of a healthy individual and that the RBCs of a healthy individual have a pormal life span in the patient's circulation? 6.Why might the patient have more severe episodes of anemia following febrile illnesses? 7.Why should splenectory largely correct the patient's anemia? ANSVER 1.The spherical shape of the patient's red blood cells (RBCs)would be expected to lead to greater osmotic fragility.When a normal RBC hemolyzes.it first swells to an approxinately spherical shape.Any attempt to increase its volune beyond this point causes hemolysis because membranes cannot stretch appreciably (although they can bend).In hemolysis focal nicroscopic "breaks"in the nenbrane allow the internal contents of the RBC to equilibrate with the extracellular fluid.The patient's RBCs are already spherical,so they cannot first swell to become spherical when placed in a hypotonic solution.The spberocytes thus hemolyze more readily when put in hypotonie solutions. 2.A major way in which cells prevent osmotic lysis is by purping Nat out of the cell by the Nat,K+ATPase.The Nat gradient belps to counterbalance the osnotic gradient in the other direction because of intracellular impermeant substances and permeant ions that are in equilibrium across the merbrane.When RBCs are incubated at 37'C,in the absence of substrates for producing energy,ATP levels in the cell
2. Why might the patient's RBCs "autohemolyze" more rapidly than normal erythrocytes when they are incubated at 37° C under sterile conditions? 3. Why should including glucose and ATP in the incubation mixture diminish the extent of autohemolysis? 4. Why should the patient's RBCs have a reduced life span? What might the spleen have to do with this? 5. What is proved by the observations that the patient's RBCs have a reduced life span in the circulation of a healthy individual and that the RBCs of a healthy individual have a normal life span in the patient's circulation? 6. Why might the patient have more severe episodes of anemia following febrile illnesses? 7. Why should splenectomy largely correct the patient's anemia? ANSWER 1. The spherical shape of the patient's red blood cells (RBCs) would be expected to lead to greater osmotic fragility. When a normal RBC hemolyzes, it first swells to an approximately spherical shape. Any attempt to increase its volume beyond this point causes hemolysis because membranes cannot stretch appreciably (although they can bend). In hemolysis focal microscopic "breaks" in the membrane allow the internal contents of the RBC to equilibrate with the extracellular fluid. The patient's RBCs are already spherical, so they cannot first swell to become spherical when placed in a hypotonic solution. The spherocytes thus hemolyze more readily when put in hypotonic solutions. 2. A major way in which cells prevent osmotic lysis is by pumping Na+ out of the cell by the Na+, K+ATPase. The Na+ gradient helps to counterbalance the osmotic gradient in the other direction because of intracellular impermeant substances and permeant ions that are in equilibrium across the membrane. When RBCs are incubated at 37° C, in the absence of substrates for producing energy, ATP levels in the cell
decrease.Eventually the pumping rates of Na+and K+diminish.Na+leaking into the cell down its electrochemical gradient transfers osnotic equivalent to the RBC interior.This process leads to swelling.and ultirately to lysis,of the RBC. Because the patient's RBCs are three tines more permeable to Na+than normal,the swelling and osnotic lysis occur more rapidly.Moreover.the hemolysis of the spherical RBCs occurs more readily than hemolysis of normal RBCs for the reasoas discussed in the answer to question 1. 3.The patient's RBCs are aided in resisting swelling and benolysis by having an elevated level of Na+,K+-ATPase in their plasma menbranes.Thus when Na+leaks into the cells at an elevated rate (compared with normal cells),it can be punped back out of the cell at a sinilarly elevated rate by the greater nunber of Nat, K+-ATPase molecules.When the ATP level falls during incubation,the Na+,K+-ATPase is no longer able to keep up with Na+influx.Cell swelling and lysis result. Providing the RBCs with ATP and with glucose (fron which the cells can make ATP) allows the Na+,K+-ATPase molecules to keep pumping Na+out of the cell at elevated rates that compensate for the elevated rate of Na+leak into the cell.In this way glucose and ATP help to prolong the time of incubation that the patient's cells can undergo before the onset of appreciable hemolysis.The ability of glucose and ATP to prevent an elevated rate of autohemolysis is one of the best diagnostic criteria of the disease known as hereditary spherocytosis.This criterion helps to distinguish hereditary spherocytosis fron other microcytic anemias. 4.Flexibility of erythrocytes is required for them to deform sufficiently to pass through the narrow slit in the basement membrane that separates the splenic cords from the venous sinuses of the spleen.The patient's spherical RBCs are less deformable than normal discoidal RBCs.The patient's RBCs are thus retained in the splenic cords to a greater extent than normal.Response to this engorgement of splenic cords is believed to contribute to the splenomegaly observed in patients with hereditary spherocytosis.While the patient's RBCs are delayed in the splenic cords,they tend to deplete their glucose and then their ATP levels.which results
decrease. Eventually the pumping rates of Na+ and K+ diminish. Na+ leaking into the cell down its electrochemical gradient transfers osmotic equivalent to the RBC interior. This process leads to swelling, and ultimately to lysis, of the RBC. Because the patient's RBCs are three times more permeable to Na+ than normal, the swelling and osmotic lysis occur more rapidly. Moreover, the hemolysis of the spherical RBCs occurs more readily than hemolysis of normal RBCs for the reasons discussed in the answer to question 1. 3. The patient's RBCs are aided in resisting swelling and hemolysis by having an elevated level of Na+, K+-ATPase in their plasma membranes. Thus when Na+ leaks into the cells at an elevated rate (compared with normal cells), it can be pumped back out of the cell at a similarly elevated rate by the greater number of Na+, K+-ATPase molecules. When the ATP level falls during incubation, the Na+, K+-ATPase is no longer able to keep up with Na+ influx. Cell swelling and lysis result. Providing the RBCs with ATP and with glucose (from which the cells can make ATP) allows the Na+, K+-ATPase molecules to keep pumping Na+ out of the cell at elevated rates that compensate for the elevated rate of Na+ leak into the cell. In this way glucose and ATP help to prolong the time of incubation that the patient's cells can undergo before the onset of appreciable hemolysis. The ability of glucose and ATP to prevent an elevated rate of autohemolysis is one of the best diagnostic criteria of the disease known as hereditary spherocytosis. This criterion helps to distinguish hereditary spherocytosis from other microcytic anemias. 4. Flexibility of erythrocytes is required for them to deform sufficiently to pass through the narrow slit in the basement membrane that separates the splenic cords from the venous sinuses of the spleen. The patient's spherical RBCs are less deformable than normal discoidal RBCs. The patient's RBCs are thus retained in the splenic cords to a greater extent than normal. Response to this engorgement of splenic cords is believed to contribute to the splenomegaly observed in patients with hereditary spherocytosis. While the patient's RBCs are delayed in the splenic cords, they tend to deplete their glucose and then their ATP levels, which results
in osnotic hemolysis by the mechanisms described carlier.Moreover,splenic macrophages engulf and destroy kCs retained in the splenic cords. 5.These observations prove that there is no defect in the patient's spleen but rather that the defect is in the patient's RBCs. 6.The patient's decreased RBC life span,and the resultant anenia,are partially compensated by an inereased rate of erythropoiesis.In a febrile illness,the rate of erythropoiesis is decreased.leading to transiently increased anemia. 7.A major contributor to the shortened life span of the patient's RBCs is the increased rate of destruction of RBCs in the patient's spleen,for the reasons discussed in the answer to question 4.Renoving the patient's spleem dramatically prolongs the life of the average nicrospherocyte and thus leads to a marked improvement in the patient's anemia
in osmotic hemolysis by the mechanisms described earlier. Moreover, splenic macrophages engulf and destroy RBCs retained in the splenic cords. 5. These observations prove that there is no defect in the patient's spleen but rather that the defect is in the patient's RBCs. 6. The patient's decreased RBC life span, and the resultant anemia, are partially compensated by an increased rate of erythropoiesis. In a febrile illness, the rate of erythropoiesis is decreased, leading to transiently increased anemia. 7. A major contributor to the shortened life span of the patient's RBCs is the increased rate of destruction of RBCs in the patient's spleen, for the reasons discussed in the answer to question 4. Removing the patient's spleen dramatically prolongs the life of the average microspherocyte and thus leads to a marked improvement in the patient's anemia