Red pulp in splenomegaly syndrome: morphometric light and electron microscopy studies

From a series of consecutively studied spleens, perfusion-fixed and investigated according to a standardized procedure allowing morphometric investigations at the light and electron microscopic levels, 33 spleens causing splenomegaly syndrome (12 lymphoproliferative diseases, 10 hairy cell leukaemia, 11 myeloproliferative diseases) were compared with data in controls and autoimmune haemolytic anaemia and hereditary spherocytosis from previous studies. In splenomegaly syndrome, especially in hairy cell leukaemia and myeloproliferative diseases, less so in lymphoproliferative diseases, there is a disproportionate increase in the volume of pulp cords in the red pulp. Values for erythrocyte volume density are of the same order as splenic erythrocyte concentration determined by scintigraphic kinetic methods. Cases with complicating immunohaemolysis show a rather high proportion of erythrocyte profiles with nearly spheric shape as in autoimmune haemolytic anaemia and hereditary spherocytosis.     

The spleen and haemolysis: evaluation of the intrasplenic transit time

The mean intrasplenic red cell transit time (STT) and the slow mixing splenic red cell volume (SSV) have been measured in patients with hereditary spherocytosis (HS), autoimmune haemolytic anaemia (AIHA) and lymphoproliferative disease (LD). There was an inverse relationship between the mean red cell life span (MRCLS) and the STT in HS (r = -0.96, P less than 0.001) and in AIHA (r = -0.90, P less than 0.001). No such relationship existed in LD. The size of the spleen and the SSV were not related to the severity of haemolysis. Our data offer strong evidence for the conditioning effect of the spleen on HS- and AIHA red cells and suggest that the STT is an index of the adverse effect of the spleen on red cells in patients with HS or AIHA.     

Splenic Red Cell Pooling in Hairy Cell Leukaemia

The splenic red cell volume has been measured directly by an isotope method with quantitative scanning in 10 patients with leukaemic reticuloendotheliosis (hairy cell leukaemia). The volume ranged between 211 and 726 ml (mean 410 ml, SD 158) and this constituted 15–48% (mean 28.1%, SD 9.5) of the total circulating red cell volume. This is an exceptionally large pool when compared with that found in myeloproliferative and lymphoproliferative disorders with the same degree of splenomegaly. It is consistent with the histological features which show marked red cell accumulation in the splenic cord areas. The red cell pooling in the spleen thus appears to be a significant factor in the anaemia and there was fairly good correlation between the percentage of improvement in the anaemia and the percentage of red cell volume contained in the spleen. By direct measurement of the splenic red cell pool, it is possible to predict the extent to which splenectomy will benefit the anaemia and this may also provide an indirect measure of the extent of bone marrow dysfunction in the causation of the anaemia.     

Studies of the Anaemia in an Acute Rat Leukaemia

S ummary . Possible reasons for the anaemia which develops in the acute myelomono-cytic leukaemia, L 5222, in rats were investigated. The reduction in erythropoietic precursors in the bone marrow leads to a decreased output of erythrocytes to the blood, as demonstrated by ⁵⁹Fe-labelling, but this reduced cell production is probably of only minor importance for the anaemia during the short course of the disease. Cross transfusion of ⁵¹Cr-labelled erythrocytes between normal and leukaemic donors, and also of ⁵⁹Fe-labelled erythrocytes newly produced during the leukaemia, showed no evidence for an intrinsic red cell defect as a cause of anaemia, but indicated that the rapid loss of cells in the leukaemic animals was due to extrinsic factors. Since splenectomy did not alleviate the shortened red-cell survival in leukaemic animals, it is assumed that the grossly enlarged spleen is not responsible for the rapid loss of cells. Comparison of the distribution pattern of ⁵¹Cr in various organs of leukaemic animals with the patterns found after treatment of healthy rats with phenylhydrazine, to produce anaemia by haemolysis, or whole body irradiation, to produce anaemia mainly by bleeding, suggested that both haemolysis and haemorrhage occur in the leukaemia. An unexpected finding was the high amount of ⁵¹Cr deposited in the liver, suggesting that this organ must be considered a major site of red-cell destruction in this rat leukaemia.     

Sites of Red Cell Destruction in the Haemolytic Anaemia of Adoptively Immunized NZB Mice

autoimmune haemolytic anaemia in the NZB strain of mice was first described by Bielchowsky, Helyer and Howie (1959) and a positive direct Coombs test (D.C.T.) in this condition was first reported by Holmes, Gorrie and Burnet (1961). The presence of another red-cell autoantibody which cross-reacts with human red cells (A.H.R.) has recently been observed in these mice (Holborow, Barnes and Tuffrey, 1965). It seems likely that the spleen also plays a part in this disease process. Splenomegaly has been noted and, in addition, splenectomy in young animals seems to reduce haemolysis in spontaneously diseased NZB mice (Helyer and Howie, 1963). Diminution of the effects of haemolytic disease following splenectomy in young male animals has been confirmed by Holmes and Burnet (1963). We have recently succeeded in inducing the formation of both D.C.T. and A.H.R. antibodies in young NZB mice by adoptively immunizing these animals with spleen cells from older animals possessing these antibodies. It appears that in such animals, the occurrence of the A.H.R. antibody enhances the increased red-cell destruction associated with a positive D.C.T. (Barnes and Tuffrey, 1966).
Using a ⁵¹Cr labelled red-cell method in such an experimental model, we have now tried to relate the occurrence of these two antibodies to the haematological disturbance, paying particular attention to the role of the liver and spleen in the haemolytic process.     

Regional Variations in the Proportion of Red Cells in the Blood in Man

Surface counting techniques following the intravenous injection of ⁵¹Cr labelled red cells and ¹³¹I labelled human serum albumin have been used to investigate the regional haematocrit in normal subjects, subjects with splenic enlargement and after splenectomy. Regional variations in the proportions of red cells and plasma were demonstrated, the red-cell concentration being highest at the precordium and lowest in the hepatic region. Intermediate values were obtained for the lumbar and splenic regions in normal subjects.
The enlarged spleen has a definite but variable tendency to be associated with high local haematocrit values when compared with the venous haematocrit. Raised whole-body to venous haematocrit ratios result from the pool of red cells in the spleen which shows delayed mixing with the cells of the extrasplenic circulation. The raised whole-body to venous haematocrit ratio associated with splenomegaly is more dependent on the concentration of the pooled cells relative to the venous haematocrit than on the volume of pooled cells and spleen size.
The significance of variations in regional to venous haematocrit and whole-body to venous haematocrit ratios is discussed.     

Hypersplenism and splenectomy in lymphoproliferative and myeloproliferative disorders

Splenic pooling of red cells and an expanded plasma volume are considered to be among the major mechanisms responsible for the anaemia in hypersplenism. In those conditions in which massive splenomegaly is associated with various degrees of marrow failure, diagnosis of the cause of anaemia may be difficult. A simple technique was used to estimate the degree of hypersplenism, from red cell mass data, in 94 patients with unequivocal lymphoproliferative or myeloproliferative disorders. The splenic effect was found to correlate well with both the size of the spleen (r = 0.75-0.90) and the actual red cell mass (0.79), and was abolished by splenectomy. Clinical data is also presented on 43 of these patients who underwent splenectomy. The incidence and type of complications, survival figures, and possible criteria for patient selection are discussed.     

Anaemia in myelofibrosis: its value in prognosis

S ummary . Forty-four patients with myelofibrosis were investigated in our hospital in the period 1971–81. Their clinical, laboratory and radioisotope parameters were analysed. The direct correlation between plasma volume and splenic red cell pool has highlighted the role of the spleen in the dilutional anaemia seen in myelofibrosis. ⁵²Fe quantitation enabled us to show that the bone marrow contributes relatively more to effective erythropoiesis than the extramedullary sites. The prognostic value of changes in plasma volume and bone marrow ⁵²Fe activity has been demonstrated. We have shown that the Hb: reticulocyte relationship at diagnosis can be used to recognize probable stages of the disease and provides a useful prognostic determinant.     

The Removal of Heat-damaged and 51Cr-labelled Red Cells in Haemoglobinopathies

S ummary . Fifty-seven individuals, all living in Ghana, and having Hb types AA (controls), AS, S-thalassaemia, CC, SC, and SS, were studied using ⁵¹Cr-labelled heatdamaged red cells. Investigations were designed to show the effect of heating on red cell morphology, G6PD activity, osmotic and mechanical fragility, ESR, sickling, and the clearance of the cells from the circulation after injection. In transfusion experiments, damaged cells were given to recipients with differing haemoglobin types and the cell disappearance and radioactive uptake by the liver and spleen were measured. In AS, S-thalassaemia and CC patients the clearance and splenic uptake were similar to those of the AA subjects. In some of the SC and in all of the SS patients, the spleen did not sequester damaged cells, although they were cleared fairly rapidly from the circulation in a few cases. It was concluded that intracorpuscular factors cannot be ignored in the removal of heat damaged cells from the circulation, even though extracorpuscular mechanisms play the major part.     

Quantitative Studies of Splenic Erythropoiesis in Polycythaemia Vera and Myelofibrosis

S ummary . A quantitative scanning method employing cyclotron-produced ⁵²Fe has been developed to assess splenic erythropoiesis in patients with myeloproliferative disorders. In 12 patients with myelofibrosis splenic uptake of ⁵²Fe was from 5.0% to 48% of the injected dose. Although a single patient with classical polycythaemia vera had a minor uptake of 2.8% six other patients with this diagnosis showed no concentration of isotope in the splenic area. The fraction of ⁵²Fe in the spleen of four patients with 'transitional'myeloproliferative disorders characterized by a high red cell mass, hypercellular bone marrow and a leucoerythroblastic blood film varied from 5% to 41%. No clear relationship was noted between the degree of splenic erythropoiesis as defined by this technique and the level of haemoglobin, the degree of splenomegaly, the effectiveness of erythropoiesis or traditional ⁵⁹Fe surface counting. If splenectomy is considered in patients with myelofibrosis splenic ⁵²Fe quantitation will provide more precise data on the contribution of splenic erythropoiesis than ⁵⁹Fe surface counting alone.     

Blood Volume Changes in Splenomegaly

S ummary . Blood volume changes have been measured in 65 patients with splenomegaly due to a miscellany of causes. The red-cell mass is often normal despite the fact that anaemia is present, and the anaemia is in part due to sequestration of red cells in a splenic pool and haemodilution of the red cells in an expanded plasma volume. Both factors may be relieved by splenectomy although the ultimate prognosis is dependent on the primary disease present.     

Fibrinogen Catabolism in Microangiopathic Haemolytic Anaemia

S ummary . Fibrinogen catabolism has been studied in six patients with microangiopathic haemolytic anaemia, three patients with fragmentary haemolytic anaemia following the insertion of valve prostheses into the heart and ten control patients. Increased rates of catabolism of ¹³¹ I-fibrinogen were found in the patients with microangiopathic haemolytic anaemia. Evidence of enhanced fibrinolysis was not present.
This finding suggests that intravascular coagulation is a feature of some cases of microangiopathic haemolytic anaemia and supports the hypothesis that the interaction of red cells with fibrin may result in the characteristic morphological appearances in these cases.     

The Anaemia of Erythropoietic Porphyria

A case of congenital erythropoietic porphyria was found to have an intermittent haemolytic anaemia. During the haemolytic phase, red-cell lysates from the patient made type I coproporphyrin on incubation with ALA. In the phase of non-haemolysis, red-cell lysates behaved as normal cells, only type III coproporphyrin being produced. There is thus a relationship between haemolytic anaemia in this disease and the disturbance in the uroporphyrinogen isomerase-porphobilinogen deaminase enzyme complex in the red cell. Red-cell glucose utilization, ATP stability on incubation of red cells, glutathione and glutathione stability, and glutathione reductase activities showed no abnormalities. The glucose-6-phosphatase dehydrogenase activity was slightly increased.     

Measurement of Red Cell Volume and Splenic Red Cell Pool using 113mIndium

S ummary . Using the lipophilic chelating agent, acetylacetone, red cells have been radiolabelled with the short-lived, generator-produced isotope, ^113mIn. Following re-injection of these labelled cells, red cell volume has been measured and compared with corresponding values using ^99mTc labelled red cells in 18 patients, and with ⁵¹Cr labelled red cells in five patients. ^99mTc slightly overestimated red cell volume in relation to ^113mIn, but ⁵¹Cr values were identical to ^113mIn values. There was a close correlation between splenic red cell pool measured with ^99mTc and with ^113mIn. It was concluded that the intracellular stability and gamma emission of ^113mIn make this isotope a superior alternative to ^99mTc and ⁵¹Cr in measurements of red cell volume and splenic red cell pool.     

Ferrokinetics and Erythropoiesis in Man: Red-Cell Production and Destruction in Normal and Anaemic Subjects

Recent advances in the analysis of plasma ⁵⁹Fe clearance have produced a unified method for measuring effective and ineffective erythropoiesis (Ricketts et al , 1975). We have used this method to investigate the balance between red-cell production and destruction in normal subjects and in patients with megaloblastic anaemia, iron deficiency anaemia and refractory hypoplastic anaemia. The results show that the normal marrow can maintain an appropriate red-cell mass by altering red-cell production to match destruction. In the anaemias we have studied there is an increased rate of either intra- or extra-medullary red-cell destruction. The response of the marrow may be limited by iron supply, by defective nuclear maturation or by some intrinsic marrow defect.     

Splenic Function in Adult Coeliac Disease

S ummary . The rate of clearance of ¹⁵Cr-labelled, heat-damaged red cells from the circulation has been measured in 18 patients with adult coeliac disease. This has been combined with scintillation scanning of the spleen using a colour scanning method. Only two of the patients had clearance times within normal limits. Five had a peripheral blood picture suggestive of splenic atrophy. In these the half time of clearance was greater than 50 min and the scintillation scan showed either no evidence of functioning splenic tissue or only minimal localization of radioactivity suggesting marked hyposplenism. Nine patients had a somewhat prolonged clearance time, the half time of clearance varying between 19 and 44 min. The scan, however, showed an apparently normal spleen and the characteristic changes of splenic atrophy were not present in the peripheral blood film. Two patients had clearance rates faster than that found in control subjects. Both of these patients had enlarged spleens. In one this was associated with hepatic cirrhosis and in the other with an unexplained neutropenia.     

Inhibition of hexose monophosphate shunt in young erythrocytes by pyrimidine nucleotides in hereditary pyrimidine 5' nucleotidase deficiency

Recent reports have suggested that haemolytic anaemia in pyrimidine 5' nucleotidase (P5'N) deficiency might be due to impaired erythrocyte hexose monophosphate shunt (HMS). To investigate the relationship between pyrimidine accumulation, HMS impairment and shortened red-cell survival, we tested glucose 6-phosphate dehydrogenase (G-6PD), HMS, P5'N activities and the UV spectrum in whole red cells and in red cells of different age from 2 P5'N-deficient patients with different degrees of haemolytic anaemia. In whole red cells we found a reduction of both G-6PD and stimulated HMS activity in the presence of a variable amount of pyrimidine nucleotides (37.79 and 17.88 mumol/gHb respectively). A drastic inhibition of stimulated HMS activity was already present in the lightest red-cell fractions from patient 1, who presented a more severe haemolytic anaemia. The variable degree of pyrimidines found among red cell fractions, with a minor accumulation in the older red cells, supports the hypothesis that pyrimidine accumulation and HMS impairment occur in the younger erythrocytes of P5'N-deficient patients.     

Cold haemagglutinin disease with severe anaemia, reticulocytopenia and erythroid bone marrow

A quantitative assessment of total, effective and ineffective erythropoiesis, and mean red cell life-span was performed in 2 patients with idiopathic cold-haemagglutinin disease (CHAD) who had severe anaemia, reticulocytopenia and erythroid bone marrow. In both cases, ineffective erythropoiesis represented the major factor in the pathogenesis of the anaemia. The most likely explanation of this was that cold antibodies had some effect on the maturing red-cell precursors. Ineffective erythropoiesis has already been shown in patients affected by auto-immune haemolytic anaemia due to warm antibodies with reticulocytopenia and erythroid bone marrow. Therefore, it is apparent that both warm and cold antibodies may cause not only peripheral destruction of mature red cells but also intramedullary death of red-cell precursors.     

The splenomegaly of myeloproliferative and lymphoproliferative disorders: splenic cellularity and vascularity

Employing radionuclide scanning, the volume of the spleen, its red cell pool and plasma pool have been measured in vivo, and the relative proportions of cellularity and vascularity of the spleen have been calculated in 51 patients with myeloproliferative and lymphoproliferative disorders. In primary proliferative polycythaemia (polycythaemia vera), the increase of spleen size was attributed mainly to the increase of splenic vascularity; in myelofibrosis and in hairy cell leukaemia, the increase of spleen size was associated with increase in both splenic vascularity and cellularity, whilst in CGL and CLL the increase was attributed more to cellularity than to vascularity.     

The Anaemia of Chronic Disorders: Studies of Iron Reutilization in the Anaemia of Experimental Malignancy and Chronic Inflammation

The purpose of this study was to evaluate the contribution of defective reutilization of iron in the pathogenesis of the anaemia of chronic disease (ACD). Normal rats with or without splenectomy and rats with Walker 256 carcinosarcoma with or without splenectomy were studied. The reutilization of ⁵⁹Fe labelled heat-damaged red cells (⁵⁹Fe DRBC), the sequestration of ⁵⁹Fe DRBC in major organs, red cell mass, ⁵¹Cr red cell survival, serum iron, plasma erythropoietin and routine haematological parameters were measured.
Three weeks after tumour implantation in rats a moderate degree of anaemia was noted. The maximum ⁵⁹Fe reutilization in the tumour group and tumour with splenectomy group (63 ± 6% and 56 ± 9%, respectively) was not significantly different than observed in the normal group (64 ± 6%) or normal with splenectomy group (53 ± 7%). Excessive iron sequestration in liver and spleen was not observed in animals with cancer. Red cell survival in the tumour group was slightly decreased as compared to normal. Plasma erythropoietin levels were appropriately increased for the degree of anaemia in rats with cancer. This type of experiment was repeated in a second group of anaemic animals with turpentine induced abscesses. Reutilization of ⁵⁹Fe DRBC in rats with inflammation was normal.
These results indicate that adult rats with anaemia of malignancy or chronic inflammation do not have a decreased reutilization of red cell iron. Thus RE blockade of iron is not a major factor in anaemia of chronic disease in this experimental model. Furthermore, these experiments confirm the importance of decreased red cell production in the anaemia of malignancy.