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The three-dimensional world of the spleen was explored by scanning electron microscopy on both arterially perfused and nonperfused specimens, as well as on plastic corrosion casts of splenic vasculatures. Of 25 spleens studied, 18 were examples of hypersplenism. These were contrasted to 7 essentially normal spleens taken from children being staged for treatment of Hodgkin's disease whose spleens proved to be uninvolved in the pathologic process. Splenic sinuses in all 25 spleens were typified by a degree of porosity. RBC were caught in the act of entering sinuses from splenic cords. These sinus windows thus represent one end of an “open” circulatiion pathway. In casts of microvasculature, direct arteriovenous connections were demonstrated, thus establishing an anatomical basis for an often disputed “closed” circulation pathway. Spleens from 7 patients with hereditary spherocytosis had a super abundance of red pulp. Splenic cords were thickened and crowded with spherocytes, many of which presented slightly wrinkled membranes, as were also noted on the peripheral blood RBC. It is possible that these membrane features are unique for HS and reflect the intrinsic membrane abnormality in protein composition. The 7 spleens from chronic idiopathic thrombocytopenic purpura had white pulp as the predominant region. Germinal centers were frequent. Lymphocytes and plasma cells with well-developed microvilli were suggestive that relese of antiplatelet antibody might be occurring in white pulp. Platelets were especially notable in peripheral white pulp and marginal zones. Platelet clumps were observed, generally adjacent to splenic macrophages.

The proportion of smooth, disc-shaped platelets (D) in freshly drawn, glutaraldehyde fixed whole blood (WB) and citrated platelet-rich plasma prepared at 37°C is compared for male (N = 55) and female (N = 31) donors. Female donors have significantly more %D than male donors and the variability in %D measured on repeated occasions over a period of 3–69 months is less for female donors. WB and PRP gave similar results. The sex-related differences in %D were not related to hematocrit or to concomitant use of oral contraceptives. There were no significant sex-related differences in platelet mean volume or in platelet plasma membrane surface area as determined by the osmotic spherocyte method. However, the volume of D is smaller for male donors. It is concluded that the sex-related differences in platelet morphology do not represent intrinsic differences in platelet size or measurable total plasma membrane but represent a selective shape change activation of the larger D in the circulation of male donors. The significance of these observations for the sex-related differences in risk of cardiovascular disease and efficacy of anti-thrombotic therapy awaits appropriate prospective epidemiological studies.

Invaginated membrane of the surface-connected canalicular system (SCCS) of the resting platelet can be potentially externalized. We have estimated the SCCS surface area by carefully swelling platelets in hypotonic media. Platelets were osmotically stressed by either interfacing a drop of platelet-rich plasma (PRP) with distilled water on a siliconized glass slide or directly in a test tube by careful additions of 60-80% by volume water. Surface areas of the platelets were estimated from a cinematographic analysis of freely rotating platelets. Within five minutes of the addition of water, large smooth spheres (spherocytes) are formed. Soluble, cytoplasmic lactate dehydrogenase activity is not released and visible membrane damage as estimated from dark-field microscopy observations does not occur for at least one hour. The mean value of the spherocyte diameters was 3.9 ± 0.6 um (3 human donors) and did not change over 5 - 60 minutes. It is estimated that the volume of the spherocyte is ~550% greater than that of the original disc and that the surface area has increased by ~300%. Similar results were obtained for rabbit platelets.Since platelet swelling induced osmotically has been shown to yield large spheres with the disappearance of SCCS (J.G. White, personal communication), it is concluded that: 1) the SCCS of the resting platelet can be easily externalized, and 2) osmotic spherocyte formation allows a quantitative estimation of the SCCS surface area.

The proposition is made that the red cell membrane is a two-dimensional, incompressible material and a general stress-strain law is developed for finite deformations. In the linear form, the character of such a material is analogous to a two-dimensional Mooney material (e.g., rubber), indicating that the molecular structure in the plane of the membrane would consist of long chains, randomly kinked and cross-linked in the natural state. The loose network could be provided by the protein component and the lipid phase could exist interstitially as a liquid bilayer, giving the membrane its two-dimensional incompressibility. The material provides the capability of large deformations exhibited by the discocyte and yet the rigidity associated with the osmotic spherocyte state. It is demonstrated that a membrane of this type can form a sphere at constant area. An illustrative example of the application to single cell discocyte-to-osmotic spherocyte transformations is presented.

Increased mechanical fragility of the sickled red cell is thought to be important in the genesis of the hemolytic process in sickle-cell disease. Sickled cells were observed cinematographically after microincision by a ruby-laser beam. Distortion and charring invariably occurred at the site of injury, and with small injuries there was no further cell change. With larger injuries, variably rapid retraction of spicules occurred accompanied by sphering of the cell. In some cases, progressive loss of hemoglobin accompanied and followed the changes in shape; in others the sphered cell still contained hemoglobin. Regardless of the mecha nisms involved in these changes in vitro, the observations may be applicable to destruction of sickled cells in vivo. We suggest that the cells are subject to avulsion of rigid cellular processes as a result of mechanical injury incurred in normal circulation. Such injured cells may undergo either immediate hemolysis or trans formation into spherocytes which are subject to erythophagocytosis.

The mammalian red blood cell exhibits many morphological states in accordance with different physical and chemical environments. The spectrum of the erythrocyte shape ranges from a discocyte to a spherocyte; among the intermediate states are spiculated varieties known as echinocytes (from the greek: sea urchin) of different degrees. The reversible shape change from a biconcave disk into a crenated sphere, first described in 1895 by Hamburger [13], still remains an unresolved puzzle for many a hematologist and cell physiologist. Despite a wealth of valuable information derived mostly from the work of Ponder [14], investigators are still actively searching for a common underlying mechanism which could explain in a satisfactory manner the fact that a multiplicity of agents or conditions are call capable of inducing this transformation. The mechanics of the shape transformation are specified by the geometrical states and the physical forces involved. The physical forces result from the membrane or interfacial elastic and surface tractions, electrical forces due to induced and bound surface charges, osmotic forces and fluid hydrostatic pressure.

through the 3-llm pore nickel mesh compared with that through the 5-llm pores. It is, therefore, likely that the impaired deformability of HS RBCs arises from the decrease in the cell surface area to volume ratio and the high mean corpuscular hemoglobin concentration. After splenectomy, the dense microspherocytes disappeared, and there was a marked improvement in RBC filterability through 3-llm pores. This suggests that dense cells were microspherocytes that received splenic conditioning in the walls of splenic sinuses. Correspondingly, splenectomy markedly improved clinical manifestations and ameliorated hemolysis. Thus, the laboratory measurement (i.e., the 3-llm filtration test) reflected the patient's clinical state. The use of

Experimental nutritional deficiencies of vitamin E and iron both decrease the filterability of rat red blood cells incubated in vitro but by two completely different mechanisms. The decreased filterability of red cells from vitamin E-deficient rats was accompanied by a discocyte/spherocyte transition and crosslinking of membrane proteins, whereas the decreased filterability of red cells from iron-deficient rats was not associated with any shape change or protein crosslinking. Nutritional deficiencies may provide useful approaches for studying the general problem of red cell deformability.

In the diagnosis of HDN the reticulocyte count is insensitive, inaccurate and assesses red cell production only in the few days prior to testing. In adults it has been reported that EC is elevated in young erythrocytes and as an index of mean cell age is a valuable test for the diagnosis of hemolytic disease. Young cord erythrocytes, isolated by density centrifugation, had EC levels greater than older cells. EC (mg/dl ± SD) was 8.7 ± 1.33 in 7 women, 5.7 ± .64 in 6 men, 10.1 ± 2.97 in 12 fullterm newborns and 10.4 ± 2.57 in 26 premature newborns. In ABO-HDN cord blood EC was elevated (28.9 ± 18) in 9 infants in whom spherocyte counts (glutaraldehyde-fixed cells) were abnormally high and who developed jaundice in the first 24 hours of life. EC was also elevated in Rh disease and in other forms of HDN. These data suggest that in HDN, EC is a valuable diagnostic test. In 19 successive infants with idiopathic hyper-bilirubinemia requiring phototherapy, EC in cord blood was 3.9-10 mg/dl and quantitative assessment of erythrocyte morphology (Am. J. Ped. Hem./Onc. 4, p.45, 1983) was normal in all cases. Thus using accurate assessment of red cell morphology and mean cell age, no evidence of HDN was found in these cases, suggesting that an occult hemolytic process, evident at birth, is an unlikely contributing cause to the pathogenesis of idiopathic hyperbilirubinemia in newborn infants.

To the Editor.— The morphological red blood cell (RBC) abnormalities reported in alcoholism include macrocytes,1macro-ovalocytes,1target cells,2spur cells,3stomatocytes,4polychromasia,5and spherocytes as seen in the Zieve syndrome.6Macrocytes and macro-ovalocytes are usually associated with folic acid deficiency. Target and spur cells are secondary to an alteration in the cholesterol and lecithin in the RBC membrane.2,3Stomatocytes occur as a transient abnormality in acute alcoholics, but are also found in a rare inherited hemolytic disease with a defect in the protein kinase mediated phorphorylation of erythrocyte membrane proteins and associated disturbance in RBC cation content.7The polychromasia results from increased reticulocytosis and the spherocytes are probably related to the presence of abnormal lipids that seem to alter the RBC membrane making them more fragile.8 Five hundred RBC from each of seven alcoholic patients were evaluated and compared with

Introduction Hemolytic disease of the newborn due to ABO blood group incompatibility is a disease entity which is often difficult to diagnose with certainty despite advanced techniques in immunohematology. However, certain specific differences are noted between ABO hemolytic disease (ABO-HD) and hemolytic disease of the newborn due to other blood group incompatibilities, such as the severity of involvement, involvement of the firstborn, frequently negative direct Coombs' test and the presence of spherocytes. The use of more sensitive Coombs' sera has increased the frequency of positive direct Coombs' tests in ABO-HD. 1 On the other hand, false positive direct Coombs' tests have been noted 1 and the possibility of a decreased specificity of these sera must be considered. The presence of spherocytosis in ABO-HD has been reported by many investigators but has neither been accurately defined nor adequately understood. 2-10 The present study was undertaken to delineate the spherocyte as it

It has recently been proposed that the biconcave shape of the human red cell is maintained by inwardly directed electrostatic forces of attrction between the two opposing membrane surfaces of the cell. Treatment of red cells with hypertonic acidified media would be expected to augment these forces by decreasing the distance between the opposing surfaces and increasing the number of centers of opposite charge. When red cells treated in this manner are returned to an isotonic medium, a membranous structure bridging the biconcavity is demonstrable by phase-contrast microscopy. This structure remains intact during osmotic swelling and hemolysis and is clearly visible in the resulting red cell ghosts. Spherocytes (hereditaryspherocytosis) demonstrate no such structure under these conditions, whereas leptocytes (β-thalassemia) show an enhanced tendency to internal bridging in isotonic acidified media. These findings suggest that an increase in the long-range electrostatic forces within the cell promotes the formation of distinct morphologic structure which, under these conditions, contributes to the preservation of the biconcave shape of the cell.