Splenic Red Cell Pooling: A Diagnostic Feature in Polycythaemia

S ummary . Total red cell volumes and splenic red cell pools were measured in 31 patients with polycythaemia. 22 had polycythaemia vera (PV), 12 of whom had clinically detectable splenomegaly, and nine patients had secondary polycythaemia (PS). The mean red cell pool was 192.8ml (SD 126.6) in PV (all cases), and 130.9 ml (SD 28.4ml) in PV without splenomegaly; it was 61.1 ml (SD 8.3 ml) in PS. When expressed relative to spleen size (in cm), differences were even more striking: PV (all cases)—mean 13.7 ml/cm (SD 4.3); PV without splenomegaly—mean 12.7 ml/cm (SD 2.2); PS—mean 6.6ml/cm (SD 1.2). Measurement of splenic red cell pool thus appears to be valuable diagnostic tool for distinguishing between PV and PS. The findings point to the presence in PV of a splenic structural abnormality which is not simply an effect of the inceased circulating red cell mass.     

Studies of Splenic Function in the Myeloproliferative Disorders and Generalized Malignant Lymphomas

The relative importance of splenic red-cell pooling, sequestration and cell destruction in the causation of anaemia has been studied in 29 patients—16 with generalized lymphoproliferative disease, 12 with myeloproliferative disease and one with idiopathic autoimmune haemolytic anaemia.
A scanning method with [¹¹C]carbon monoxide was used for direct in vivo measurement of splenic red-cell volume, and the spleen was delineated by-a scan after injection of ⁸¹Rb-labelled red cells, damaged with non-radioactive 1-mercuri-2-hydroxypropane (MHP). The clearance time of the damaged cells from the circulation was used as an index of splenic function. The fraction of red cells in the spleen varied from 2.9% to 32% and the splenic red-cell volume ranged from 38 to 1000 ml. In patients with lymphoproliferative disorders their spleens contained a smaller proportion of red cells, relative to splenic size, than patients with myeloproliferative disease. Clearance of cells damaged with 1-mercuri-2-hydroxypropane (MHP) was 30–60 min in normal subjects. Slow clearances were found in some patients with lymphosarcoma; fastest clearances occurred in patients with obvious haemolytic anaemia. No clear relationship was noted between the rate of clearance and splenic size or splenic red-cell volume.     

Haematological Effects of the Idiopathic Splenomegaly Seen in Uganda

The haematological effects of idiopathic splenomegaly have been studied in a group of 15 Ugandan patients. The results of the main investigations were compared with those obtained in five patients with miscellaneous diseases and without palpable splenomegaly.
The splenic enlargement was mainly due to reticuloendothelial hyperplasia and not to sinusoidal congestion.
A normochromic anaemia was present in almost all the patients and a proportion had leucopenia and thrombocytopenia. One patient presented with a crisis of anaemia; she was later shown to have red cell G6PD deficiency which was believed to be coincidental.
Three aetiological factors were found in relation to the anaemia; reduction of red cell survival time, present in all the patients, haemodilution from expansion of the plasma volume, and the exclusion of a proportion of the red cell mass from the general circulation by the spleen. Six to 39 per cent of the red cell volume, the amount varying with spleen size, was sequestered in a slow mixing compartment, believed to be the extrasinusoidal spaces (the spleen pool) in the patients with splenomegaly. No spleen pool could be shown in any of the patients without splenomegaly.
Intrasplenic red cell destruction was not conspicuous and it has been suggested that the red cells, after damage from repeated stagnation in the spleen pool, are destroyed widely throughout the body.
The reasons for expansion of the plasma volume is not known. It relates to spleen size but cannot wholly be explained by plasma in the spleen pool.
A good result from splenectomy was obtained in six out of seven patients in whom this was undertaken. Two months after operation the red cell survival time was corrected in the patients in whom this was measured. The rise in the PCV could be explained only by substantial reduction in plasma volume.     

Splenic haematocrit and the splenic plasma pool

A method was established for estimation of plasma volume and splenic plasma pool by quantitative scanning using 113mIn labelled transferrin (TF). The method was used in 12 patients with various haematological disorders and degrees of splenomegaly. 113mIn-TF consistently gave about 6% over-estimation of plasma volume by comparison with the standard 125I HSA method. The splenic plasma pool ranged from 1.2% to 11.4% of the total plasma volume. By concurrent measurement of splenic red cell pool the splenic haematocrit (SHct) was obtained: mean 0.51, SD 0.08; the SHct/PCV ratio was 1.21 (SD 0.31) and the SHct/body Hct ratio was 1.30 (SD 0.30). SHct was independent of PCV and body Hct but there was a trend to a lower SHct in cases where splenomegaly was more marked. Direct measurement of splenic plasma pool may help to elucidate the cause of increased total plasma volume in such patients.     

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.     

Assessment of the sites of red cell destruction using quantitative measurements of splenic and hepatic red cell destruction

Red cell survival, surface counting indices, the splenic and hepatic contribution to red cell destruction and the rate of splenic and hepatic red cell destruction were measured in 29 patients. Splenectomy was performed in 14. No correlation could be found between the splenic excess count index and both the amount and rate of red cell destruction in the spleen, but the rate of splenic and hepatic red cell destruction was related to the rate of disappearance of red cells from the circulation. The mean fractions of red cell destruction in spleen and liver were 46.1%± 20.5 (SD) and 11.7%± 4.2 (SD) respectively. After splenectomy, the haematocrit returned to normal in all patients despite fractions of red cell destruction in the spleen not exceeding 60%. Although the measurements of the splenic red cell destruction rate and of the fraction of red cell destruction in the spleen provide more precise information on the role of the spleen in red cell destruction, their prognostic value in patients who underwent splenectomy was not obvious.     

Effects of forced diving on the spleen and hepatic sinus in northern elephant seal pups

In phocid seals, an increase in hematocrit (Hct) accompanies diving and periods of apnea. The variability of phocid Hct suggests that the total red cell mass is not always in circulation, leading researchers to speculate on the means of blood volume partitioning. The histology and disproportionate size of the phocid spleen implicates it as the likely site for RBC storage. We used magnetic resonance imaging on Northern elephant seals to demonstrate a rapid contraction of the spleen and a simultaneous filling of the hepatic sinus during forced dives (P < 0.0001, R(2) = 0.97). The resulting images are clear evidence demonstrating a functional relationship between the spleen and hepatic sinus. The transfer of blood from the spleen to the sinus provides an explanation for the disparity between the timing of diving-induced splenic contraction ( approximately 1-3 min) and the occurrence of peak Hct (15-25 min). Facial immersion was accompanied by an immediate and profound splenic contraction, with no further significant decrease in splenic volume after min 2 (Tukey-Kramer HSD, P = 0.05). At the conclusion of the dive, the spleen had contracted to 16% of its predive volume (mean resting splenic volume = 3,141 ml +/- 68.01 ml; 3.54% of body mass). In the postdive period, the spleen required 18-22 min to achieve resting volume, indicating that this species may not have sufficient time to refill the spleen when routinely diving at sea, which is virtually continuous with interdive surface intervals between 1 and 3 min.     

Red cell survival in patients with nonalcoholic liver cirrhosis before and after distal splenorenal shunt

We previously reported severe hemolysis in one patient immediately after distal splenorenal shunt (DSRS). The purpose of the present study was to evaluate changes in red cell survival after DSRS. In ten patients with nonalcoholic cirrhosis in whom DSRS was performed for esophageal varices, red cell survival and splenic quantitative hemodynamic studies were performed before and after DSRS. The splenic venous blood flow per unit volume (flow/volume ratio) was calculated. The red cell survival was significantly (P<0.05) shortened after DSRS; the apparent half-life survival time (T_1/2) before and after DSRS was 24.6±5.9 (mean±SD) and 16.3±8.5 days, respectively. After DSRS, the spleen volume was significantly (P<0.05) decreased, whereas the splenic venous blood flow was slightly increased. The spleen flow/volume ratio was significantly (P<0.05) increased after DSRS. There was a significant and negative correlation (r=−0.684,P<0.05) between the postoperative percentage change in T_1/2 and the spleen flow/volume ratio. These findings suggest that the red cell survival period is significantly decreased after DSRS in patients with nonalcoholic cirrhosis, and that the increased splenic blood flow per unit spleen volume after DSRS may play an important role in the hemolytic reaction in the spleen after this procedure.     

In vitro and in vivo behavior of 111In-labelled platelets: An experimental study of healthy male volunteers

The aim of this study was to obtain a critical evaluation of a simple method for labelling platelets with ¹¹¹In-oxine. All experiments were carried out on healthy volunteers. 65 ± 7 (SD) % of the platelets in collected blood were labelled and reinjected. As compared to control experiments, only in response to a low final ADP concentration (1.0 μmol/l) did ¹¹¹In-labelled platelets show reduced in vitro aggregability. The mean platelet volume for ¹¹¹In-labelled platelets was slightly lower than the mean platelet volume in whole blood. The results for initial platelet recovery and platelet mean lifespan closely agreed with those of other studies in which considerably higher platelet extraction from whole blood was obtained. After injection, the splenic uptake and blood disappearance of ¹¹¹In-labelled platelets followed a monoexponential function with almost identical rate constants. By compartmental analysis of the equilibration of platelets between blood and spleen, the splenic blood flow was estimated to be 4.8 ± 1.9 (SD) % of the total blood volume/min; the intrasplenic platelet transit time was 9.7 ± 1.6 (SD) min, and the exchangeable splenic platelet pool 31 ± 8 (SD) %. Highly significant relationships were present between the splenic blood flow and the splenic platelet pool size, as well as between the splenic blood flow and the initial platelet recovery. It is concluded that the requirements for adequate interpretation of platelet kinetics are well met with the present method for harvesting and labelling of platelets.     

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.     

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.     

Quantitative Determination of Splenic Red Blood Cell Destruction in Patients with Splenomegaly

In order to evaluate a method permitting quantitation of splenic red blood cell destruction, a model of erythrocyte destruction in enlarged spleens was created: Erythrocytes are destroyed in the splenic erythrocyte pool at a constant rate, producing in labelling studies hyperhaemolysis due to random destruction. A mathematical analysis of the model shows that the splenic destruction rate can be calculated with great accuracy from quantitation of the initial excess radioactivity, measured over the spleen during the first days after infusion of ⁵¹Cr-labelled autologous erythrocytes. 18 patients with splenomegaly (479–4700 g) were investigated. The splenic erythrocyte destruction rate was estimated to be between 0.5–4.4% of the total erythrocyte mass per day, increasing significantly with increasing splenic weight. The results indicate that erythrocyte destruction takes place almost exclusively in the enlarged spleen in cases of predominant splenomegaly without complicating immunohaemolysis.     

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.     

Erythrocyte Pooling and Sequestration in Enlarged Spleens: Estimations of Splenic Erythrocyte and Fiasma Volume in Splenomegalic Patients

Erythrocyte pooling and sequestration in the spleen were studied in 73 patients with splenomegaly due to various haematological disorders using ⁵¹Cr-labelled autologous erythrocytes and surface counting. Arbitrary values for the accumulation of activity in spleens of different sizes were obtained by correcting measured splenic surface activities (correction factors have been experimentally estimated). Absolute values for the splenic content of ⁵¹Cr and ¹²⁵I were obtained by comparative surface activity measurements over the enlarged spleen and a model of the spleen charged with a known amount of activity. In this way the splenic erythrocyte and plasma volume may be estimated. In cases of pronounced splenomegaly without complicating immunohaemolysis the splenic erythrocyte pool (splenic erythrocyte volume/total erythrocyte volume) increases with increasing splenic weight, apparently independently of the underlying haematological disorder, the splenic erythrocyte content varying only with body haematocrit. Hyperhaemolysis is a regular finding in enlarged spleens. The splenic erythrocyte sequestration rate increases with increasing splenic erythrocyte pool and is much higher in myeloproliferative than in lymphoproliferative disorders. The total plasma volume is expanded in splenomegalic patients, but the splenic plasma volume constitutes only a minor part of this increment. The anaemia of splenomegalic patients is a consequence of splenic erythrocyte concentration and hypersequestration combined with expansion of the plasma volume. Frequently the total erythropoietic capacity is reduced. Splenectomy is regularly followed by an increased venous haemoglobin concentration.     

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.     

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.     

Morphologic and morphometric light and electron microscopic studies of the spleen in patients with hereditary spherocytosis and autoimmune haemolytic anaemia

With the aim of contributing to a better understanding of the haemolytic function of the spleen, a morphologic and morphometric study of this organ fixed by arterial perfusion was performed in nine patients with hereditary spherocytosis (HS), three with autoimmune haemolytic anaemia (AHA) and six with Hodgkin's disease without splenic involvement (controls). The spleen weight in HS and AHA (621 +/- 429 g, mean +/- SD) was significantly increased with respect to controls (168 +/- 36 g) (P = 0.003). In HS the red cell retention in the cords of Billroth was significantly increased (203 +/- 68 per 10(4) microns 2) with respect to the cases with AHA (93 +/- 35 per 10(4) microns 2) and to the controls (57 +/- 28 per 10(4) microns 2) (P = 0.004). In HS and AHA the number of macrophages per 10(4) microns 2 of red pulp was significantly increased (5.41 +/- 1.10 and 7.52 +/- 2.91, respectively) with respect to the controls (3.25 +/- 0.58) (P less than 0.003). There was no statistically significant difference between the number of macrophages in HS and AHA. The transmission (TEM) and scanning electron microscopy (SEM) studies demonstrated predominantly red cell retention in the cords of HS spleens, red cell phagocytosis by cordal macrophages in AHA spleens and in a lesser intensity in HS spleens, and phagocytosis of haematic corpuscles by sinus endothelial cells (SEC) in the cases of HS. These quantitative studies allow a better understanding of splenic red cell destruction in haemolytic syndromes.     

A new method for studying splenic reticuloendothelial dysfunction in sickle cell disease patients and its clinical application: a brief report.

Differential interference contrast (DIC) microscopy (Nomarsky optics) readily demonstrates the formation of "pits" or crater-like depressions in red cell membranes of splenectomized individuals. Splenic reticuloendothelial dysfunction characteristic of many patients with sickle cell disease (SCD) can be demonstrated by technetium spleen scans, but this technique is expensive, requires injection of radioactive material into children, and is cumbersome to perform at regular intervals. However, pit formation in red cells, which also appears to reflect splenic dysfunction, can readily be quantitated in a finger-stick blood sample using DIC microscopy. In this study, the degree of red cell pitting was compared with results of technetium spleen scans and measurements of Howell-Jolly bodies in individuals with sickle cell disease. The average pitted cell percentage in the control population was 0.5% +/- 0.5 (range 0.0-2.6) and 30.5% +/- 13.9 in the SCD population (range 2.4-71.1) (less than 0.001). Of the individuals studied with SCD, 12 also had technetium (99mTc) sulfur colloid scans and measurements of Howell-Jolly bodies. The percentage of Howell-Jolly bodies was low and did not correlate well with the degree of splenic visualization. However, there was an excellent correlation between pit count and splenic dysfunction as measured by spleen scan. Determination of red cell pitting, therefore, appears to offer a simple means for clinical evaluation of splenic reticuloendothelial function in patients with SCD.     

Blood cell kinetics in the spleen

The mean intrasplenic blood cell transit time (STT), splenic blood cell volume (SV) and the slow mixing splenic blood cell volume (SSV) have been measured in normal subjects and patients with congestive and infiltrative splenomegaly. The normal red cell SV was 0.47 +/- 0.14 (mean +/- SD) % of the circulating red cell volume (RCV). In patients with congestive and infiltrative splenomegaly, SV was 9.82 +/- 3.55% and 0.96 +/- 0.71% of RCV, respectively. The SSV was 69.1 +/- 8.4% of the SV in normal subjects, but in patients with splenomegaly, its value was lower than that of normal SSV. The STT was 0.43 +/- 0.11 min. in normal subjects. In the patients with splenomegaly, it prolonged. But, the STT/SV and STT/SSV were rapid in patients with congestive splenomegaly and low in infiltrative splenomegaly. It seemed that SST/SV and STT/SSV were useful to distinguish between congestive and infiltrative splenomegaly. In the intrasplenic granulocyte kinetics, SV was similar to that of red cell. The STT was longer than that of red cell, in normal subjects and patients with splenomegaly. In the intrasplenic platelet kinetics, the ratio of cellular splenic volume to general circulation was the largest in platelets. The STT of platelet was the slowest in blood cells.     

The interpretation of platelet kinetic studies for the identification of sites of abnormal platelet destruction

The kinetics of platelets labelled with 111In have been studied in a series of 175 subjects including 18 normal volunteers, and 12 patients with idiopathic thrombocytopenic purpura (ITP), but excluding patients in whom there was scintigraphic evidence of intravascular platelet consumption. From analysis of the kinetics, the following parameters were calculated: splenic blood flow (SBF), intrasplenic platelet transit time (t-), splenic platelet pool capacity (expressed as a percentage of the total circulating platelet population), the fraction of the dose of labelled platelets ultimately destroyed in the spleen and the mean platelet life span (MPLS). SBF increased with increasing spleen size up to values of 25% total blood volume (TBV) per min. Some patients with immune complex related diseases were identified with elevated SBF (up to 24% TBV min-1) but without significant splenomegaly. Patients with cardiac decompensation had reduced SBF relative to spleen size. t- showed no relationship with spleen size. It tended to fall in patients who had high SBF relative to spleen size and to rise in those with low SBF relative to spleen size; i.e. it was inversely related to splenic perfusion (flow per unit tissue volume). The splenic platelet pool capacity is dependent on platelet input (SBF) and splenic platelet clearance (reciprocal of t-), and showed a close relationship with spleen size. When all subjects except those with ITP were considered, splenic platelet destruction showed a good correlation (r = 0.70, n = 42, P less than 0.001) with the splenic platelet pooling capacity. The ratio of the fraction of platelets destroyed in the spleen to the fraction pooling there, the D/P ratio, was approximately unity and did not appear to vary with MPLS, spleen size or the patient's condition, except in ITP where it varied between about 0.5 and 2. This variation in ITP was thought to be the result of an immune mediated re-direction of reticulo-endothelial platelet destruction. It is suggested that the D/P ratio, rather than the absolute quantity of 111In labelled platelets destroyed in the spleen, may be a more useful predictor of response to splenectomy since it takes into account the observed, appropriate, tendency for the spleen to destroy platelets in proportion to its platelet pooling capacity.