Relative iron deficiency in hereditary spherocytosis

Seventy-three patients with hereditary spherocytosis (HS) (58 nonsplenectomized, 15 splenectomized) were studied to evaluate iron status and the adequacy of iron availability for erythropoiesis. Splenectomized patients, who had hemoglobin levels in the normal or upper normal range, had higher levels of serum iron, transferrin saturation, and serum ferritin than normal matched controls and normal zinc protoporphyrin (ZnPP) levels. On the contrary, nonsplenectomized patients presenting with mild to severe anemia had higher red cell ZnPP concentrations than both splenectomized subjects and matched normal controls. ZnPP in nonsplenectomized patients correlated inversely with Hb concentration, mean corpuscular volume (MCV), mean red cell hemoglobin concentration (MCHC), transferrin saturation, and serum iron, and directly with reticulocyte count. At multiple regression analysis only Hb concentration was a significant explanatory variable for high ZnPP. The authors conclude that a number of nonsplenectomized HS patients have relative iron deficiency primarily because of expansion of erythropoiesis caused by anemia.     

Serum acute-phase protein level as indicator for liver failure after liver resection

BACKGROUND/AIMS: We investigated the relationship between postoperative liver failure and serum acute-phase protein level before and after liver resection. METHODOLOGY: Thirty-four patients who underwent liver resection were prospectively included. Serum concentrations of negative (albumin, prealbumin and retinol-binding protein) and positive (orosomucoid, haptoglobin and C-reactive protein) acute-phase proteins were assayed prior to surgery (baseline) and on postoperative day 3, 12 and 45. Postoperative liver failure was defined as serum bilirubin more than 50 micromol/L or prothrombin time less than 50% on postoperative day 7. Univariate analysis was performed to compare patients who did and did not present postoperative liver failure. RESULTS: Postoperative liver failure occurred in 8 cases and was correlated with: 1) higher negative and lower positive acute-phase protein levels (p<0.04) at baseline, 2) lower negative and lower positive acute-phase protein levels on postoperative day 3, 12 or 45 (p< or =0.05). CONCLUSIONS: Early onset of inflammatory serum protein profile was correlated with absence of postoperative liver failure. Serum acute-phase protein could be used as predictor as well as early postoperative diagnosis marker of postoperative liver failure. Relationship between preoperative inflammation and postoperative liver failure warrants further investigations because of potential therapeutic consequences.     

Impaired erythrocyte calcium homeostasis in beta-thalassemia

Intracellular calcium (Ca) concentration in erythrocytes (RBCs) is controlled by a low passive influx through a relatively impermeable membrane and by active efflux catalyzed by Ca2+,Mg2+-ATPase. Since precipitation of alpha-globin chains in thalassemic RBCs may interfere with normal membrane function, we studied the RBC intracellular Ca content and the RBC membrane Ca2+,Mg2+-ATPase activity in two groups of patients with nonsplenectomized (n = 9) and splenectomized (n = 9) beta- thalassemia intermedia and in two groups of matched controls. The mean +/- SD Ca concentration in the nonsplenectomized (n = 12) and splenectomized (n = 6) controls were 6.1 +/- 6.0 and 5.8 +/- 3.4 mumol Ca per liter of RBCs, respectively, compared with 26.0 +/- 7.6 (P less than .001) and 85 +/- 24.4 (P less than .001) in the nonsplenectomized and splenectomized thalassemia patients, respectively. The mean +/- SD Ca2+,Mg2+-ATPase activity in the eight nonsplenectomized patients was 0.77 +/- 0.58 mumol inorganic phosphate (Pi) per milligram of protein per hour compared with 0.66 +/- 0.41 in the controls (P = NS). Similar values were obtained for the splenectomized patients and their controls. No correlation was found between either the intracellular Ca content or the Ca2+,Mg2+-ATPase activity with the peripheral nucleated RBC count. These findings suggest that there is a major defect in the membrane of the thalassemic RBC leading to an increased Ca content that is more pronounced in splenectomized patients.     

Platelet aggregation and activation in thalassemia major patients in Indonesia

Thromboembolic events and hypercoagulable state have been reported in patients with thalassemia. As platelets play an important role in the pathogenesis of thrombosis, the authors aimed to find the pattern of changes in platelet count, function and activation, and evidence of coagulation activation in patients with thalassemia major in Indonesia. A total of 31 patients with splenectomized and 35 patients with nonsplenectomized thalassemia major were enrolled in this study. Platelet count, platelet aggregation, beta-thromboglobulin, and D-dimer levels were measured. All measured parameters were significantly higher in splenectomized than in nonsplenectomized patients. beta-thromboglobulin level was increased, but D-dimer level was within normal range. The authors concluded that there was an increase in platelet activation in patients with beta-thalassemia major. Platelet activation was higher in splenectomized than in nonsplenectomized patients.     

Sialic acid level reflects the disturbances of glycosylation and acute-phase reaction in rheumatic diseases

In the rheumatic diseases, the changes in the carbohydrate part of serum glycoproteins occur and these abnormalities can be monitored by serum level of total and free sialic acid. The aim of this study was to evaluate the total and free sialic acid level as a marker of inflammation activity (TSA) and the changes in glycosylation of blood glycoproteins (FSA) in rheumatoid arthritis (RA), systemic sclerosis (SSc) and systemic lupus erythematosus (SLE). Studies were carried out in 50 patients with RA, 24 with SLE and 32 with SSc. TSA concentration was measured with an enzymatic, colorimetric method and FSA with a thiobarbituric method. The serum levels of TSA in RA and SLE patients were significantly increased compared to controls and in RA patients were higher than that in SSc patients. The mean serum level of FSA in RA patients was significantly higher, but in SSc patients significantly lower than that in the controls, and in RA patients was significantly higher than in SLE and in SSc patients. All acute-phase proteins were changed: Positive acute-phase proteins were elevated, and the negative protein was decreased. The positive acute-phase proteins positively correlated with the levels of TSA and FSA in RA and SSc patients. In SLE patients, TSA positively correlated with haptoglobin and α1-antitrypsin. In RA patients, there was the positive correlation of TSA and FSA with DAS 28. The changes in the serum levels of TSA and FSA in the course of rheumatic diseases could reflect the abnormalities in glycosylation/sialylation patterns of glycoproteins induced by acute-phase response.     

Hairy cell leukemia: a retrospective study of 235 cases by the Italian Cooperative Group (ICGHCL) according to Jansen's clinical staging system

This is a report from a cooperative study on hairy cell leukemia (HCL) involving 20 Hematology Departments in Italy. Data for the patients was collected between January 1967 and December 1981 and included 235 cases of which 203 could be evaluated; 160 were males (78.8%) and 43 females (21.2%) with an M:F ratio of about 3:1; mean age was 54 years (range 26-82 yrs). The diagnostic criteria of admission were: typical aspect of hairy cells, in peripheral blood and bone marrow smears, tartrate resistant acid phosphatase (TRAP) positivity, typical bone marrow, spleen, liver and/or lymph node histology, and/or electronmicroscopy. On the basis of hemoglobin level and spleen size at the time of diagnosis, three stages could be distinguished according to Jansen: 51 patients, 27 of which splenectomized, were in stage I; 67 patients, of which 44 splenectomized, were in stage II; 85 patients of which 60 splenectomized, were in stage III. The actuarial survival curves of these patients showed clear distinction between the three stages. In the first stage the difference in survival, between splenectomized and nonsplenectomized groups, was not statistically significant (p less than 0.5): on the contrary, in stages II and III the difference in survival was statistically significant (stages II and III; p less than 0.01).     

Transforming growth factor beta 1 regulates production of acute-phase proteins.

We explored the possible role of transforming growth factor beta 1 (TGF-beta), a cytokine that appears to be an important modulator of inflammation and tissue repair, in regulation of human plasma protein synthesis during the acute-phase response. In Hep 3B cells, TGF-beta led to increased secretion of the positive acute-phase proteins alpha 1-protease inhibitor and alpha 1-antichymotrypsin and decreased secretion of the negative acute-phase protein albumin. In Hep G2 cells, after incubation with TGF-beta, the same changes in secretion of alpha 1-protease inhibitor, alpha 1-antichymotrypsin, and albumin were observed, as well as decreased secretion of both the negative acute-phase protein alpha-fetoprotein and the positive acute-phase protein fibrinogen. In addition, TGF-beta modulated the effects of interleukin 6; these cytokines, in combination, were additive in inducing synthesis and secretion of alpha 1-protease inhibitor and alpha 1-antichymotrypsin and in decreasing secretion of albumin and alpha-fetoprotein. TGF-beta inhibited the induction of fibrinogen caused by interleukin 6. The effects on alpha 1-protease inhibitor were confirmed by metabolic labeling in Hep 3B cells and by demonstrating increased accumulation of specific mRNA in Hep G2 cells, and the effects on fibrinogen were confirmed in Hep 3B cells by studies of mRNA for the alpha chain of fibrinogen. TGF-beta had no effect on haptoglobin or alpha 1-acid glycoprotein secretion, either directly or in the presence of interleukin 6, which is capable of inducing these proteins. These studies demonstrate that TGF-beta can affect hepatic synthesis and secretion of a subset of acute-phase proteins, both directly and by modulating the effect of interleukin 6. The affected group of plasma proteins is distinct from those affected by other recognized acute-phase protein-inducing cytokines. These findings support the view that combinations of cytokines mediate the response of the hepatocyte to inflammatory stimuli.     

Assessment of splenic and RES function of patients with thalassemia major long after partial splenic embolization: in vivo clearance study

The activity of the remaining reticuloendothelial system (RES) and the function of the splenic remnants was estimated in 5 thalassemic patients who had undergone successful partial splenic embolization (PSE) 6 yr previously. The kinetics of 125I-heat-denatured human albumin as well as that of 51Cr-heat-damaged homologous red blood cells were applied for this purpose and the parameters derived were compared to those of nonsplenectomized as well as splenectomized thalassemics with the following results: (a) The parameters of splenic function in embolized thalassemics were found to be within the limits observed in nonsplenectomized patients. (b) Their maximum phagocytic capacity was significantly lower, not only than that found in nonsplenectomized, but also than in thalassemic patients splenectomized at about the same time. It is concluded that, 6 yr after PSE has been performed, a reorientation of the altered circulatory dynamics has taken place in the splenic remnants allowing previously blockaded areas to gain normal function. It therefore seems that, despite the continuing hemolytic stimulus, RES hyperplasia is prevented, resulting in the stable, low-level transfusion requirements that have been observed in embolized thalassemics.     

Hypocholesterolemia in hairy cell leukemia: A marker for proliferative activity

Hypocholesterolemia is a well-documented phenomenon associated with a variety of hematological malignancies and nonmalignant disorders associated with splenomegaly. To determine the incidence of hypocholesterolemia in patients with hairy cell leukemia (HCL), we measured the serum cholesterol levels before and after a single cycle of 2-chlorodeoxyadenosine (2-CdA) in 46 patients. The mean pre-treatment serum cholesterol level was 152.8 mg/dl (range, 60 to 293 mg/dl). The mean post-treatment serum cholesterol level was 190.0 mg/dl. This was significantly higher than the pre-treatment values (P < 0.0001). Twelve patients who had previously undergone splenectomy showed a similar response to treatment, with a pre-treatment value of 180.0 mg/dl and a post-treatment value of 219.8 mg/dl (P < 0.0001). However, there was a significant difference in the pre-treatment serum cholesterol levels in the nonsplenectomized patients (143.0 mg/dl) compared to the splenectomized patients (180.0 mg/dl) (P < 0.03). The pre-treatment serum cholesterol did not correlate with the pre-treatment splenic index (correlation coefficient = −0.39, P < 0.065). Similarly, there was no correlation between the change in splenic index and the change in serum cholesterol level post-treatment. These findings suggest that hypocholesterolemia in HCL is related to tumor burden and not to splenomegaly alone. Since cholesterol is critical to hairy cell metabolism and structure, treatment strategies interfering with cholesterol synthesis may be productive. Am. J. Hematol. 55:129–133 1997. © 1997 Wiley-Liss, Inc.     

Apolipoproteins A-IV and A-V are acute-phase proteins in mouse HDL

BACKGROUND: Infection and inflammation are associated with atherosclerosis. During infection and inflammation, HDL decreases and there are changes in the levels of several HDL-associated proteins. To identify changes in the protein composition of HDL during infection and inflammation, a proteomic approach was utilized. METHODS AND RESULTS: Using two-dimensional gel electrophoresis and mass spectrometry, we found the expected increases in apolipoprotein (apo) SAA and apo E, as well as a decrease in apo A-I on HDL isolated from mice injected with endotoxin. We identified apo A-IV and apo A-V as positive acute-phase proteins in mouse HDL. We also found an increase in hepatic mRNA levels of apo A-IV and apo A-V after injection of endotoxin. Interleukin-6 increased apo A-IV and apo A-V mRNA levels in Hep3B cells. Additionally, we demonstrated that the protein levels of apo A-II in acute-phase HDL and the hepatic mRNA levels of apo A-II were decreased. CONCLUSIONS: Apo A-IV and A-V are positive acute-phase proteins that increase in the serum during inflammation while apo A-II is a negative acute-phase protein in mice. Similar to other positive and negative acute-phase proteins, changes in hepatic production account for the changes in serum levels. However, the changes in apo A-IV and apo A-V, two apolipoproteins whose activities are not fully understood, may serve functions other than regulating lipid metabolism during the acute-phase response (APR). Coupled with the other changes in HDL proteins that occur, these changes are likely to alter the functional properties of HDL perhaps increasing the risk of atherosclerosis.     

Splenectomy for hemocytopenia in systemic lupus erythematosus. A controlled appraisal.

We compared 15 patients with systemic lupus erythematosus (SLE) treated with splenectomy for thrombocytopenic purpura and/or hemolytic anemia to 15 similar SLE patients treated only medically. There was no significant difference between the splenectomized and the nonsplenectomized patients when their entire course, as well as the presplenectomy and postsplenectomy or their equivalent control periods, were compared by means of an overall severity index. Splenectomized patients, however, had a significantly higher frequency of cutaneous vasculitis after splenectomy than in their own presplenectomy period and a significantly higher frequency of cutaneous vasculitis than the nonsplenectomized patients. Serious infections were more frequent in the postsplenectomy period than in an equivalent period in the nonsplenectomized patients. Splenectomy produced only short-term benefit in the management of hemocytopenic episodes in SLE and seems only warranted as an emergency procedure in patients unresponsive to medical treatment.     

Increased levels of acute-phase serum proteins in diabetes

Serum viscosity's increase in diabetes has been linked to the presence of microvascular sequelae and to changes in serum protein composition. The major change is a decline in albumin and an increase in the levels of acute-phase proteins. In this study, albumin and five acute phase proteins--alpha-1 acid glycoprotein, alpha-1 antitrypsin, haptoglobin, ceruloplasmin, and C-reactive protein--were measured. Levels in adult diabetes (principally type II) were compared with those in both subjects with glucose intolerance and control subjects (healthy subjects and nondiabetic ambulatory patients). Haptoglobin, alpha-1 acid glycoprotein, and C-reactive protein increased markedly in both diabetes and glucose intolerance; ceruloplasmin and alpha-1 antitrypsin increased more marginally. Serum albumin level decreased more strikingly as hyperglycemia advanced. Acute-phase proteins also increased in advanced glucose intolerance as in established diabetes. The acute-phase protein elevation did not differ with degree of control or duration of diabetes. When diabetics were divided into those with and without clinically detectable evidence of microvascular sequelae, elevation of haptoglobin, C-reactive protein and alpha-1 acid glycoprotein, and depression of albumin were found to progress with number of sequelae. The levels of these proteins, particularly haptoglobin, were also highly correlated with serum viscosity expressed as viscosity number. Mild serum albumin depression and a more striking acute-phase protein elevation are greater in diabetes with microangiopathy, develop in glucose intolerance, and contribute substantially to elevated plasma viscosity in diabetes.     

Intensive iron chelation therapy in beta-thalassemia major: some effects on iron metabolism and blood transfusion dependence

Subcutaneous infusions of Desferal (DF) rarely induce negative iron balance in thalassemia major patients less than 6 years old. In nonsplenectomized patients the requirements for blood transfusions increase slightly. Urinary iron excretion decreases during the first days following a blood transfusion. An average of 5.8 mg/day equivalent to 30% of the total iron excretion is eliminated with the feces after subcutaneous infusions of DF. Serum ferritin does not decrease significantly after 18-24 months of therapy. The effectiveness of long-term therapy progressively increases in the splenectomized patients, while it decreases appreciably in the course of the treatment in the nonsplenectomized ones.     

Cytokines and acute-phase response markers derangements in patients with obstructive jaundice

BACKGROUND/AIMS: Prolonged acute-phase response and increase of cytokines have been associated with higher mortality and surgical complications. This study investigated the status of cytokines and acute-phase response markers in patients with obstructive jaundice. METHODOLOGY: Forty-one patients were investigated. Endotoxin, tumor necrosis factor-alpha, interleukin-6, nitric oxide, C-reactive protein, liver enzymes, albumin and percentage of weight loss were determined at admission. RESULTS: Endotoxin, interleukin-6 and C-reactive protein were significantly elevated in both benign and malignant obstructive jaundice. Increased plasma levels of tumor necrosis factor-alpha were only detected in malignant tumors (68 vs. 24 pg/mL; P < 0.001). Patients with positive acute-phase response (C-reactive protein > mean + 2 SD of controls) had greater weight loss (P = 0.02), endotoxin (P = 0.03) and interleukin-6 plasma levels (P = 0.05) than those with no inflammatory response. Prolonged biliary obstruction (> 10 days) was associated with higher weight loss (P = 0.04), tumor necrosis factor-alpha (P = 0.003) and interleukin-6 (P = 0.05) plasma levels. CONCLUSIONS: A prolonged high-grade biliary tract obstruction prompted an increase in endotoxin levels, associated with a positive acute-phase response and cytokine elevation.     

Alterations in serum protein levels in patients with Cushing's syndrome before and after successful treatment

Alteration in serum protein concentration is used commonly in clinical practice as a nonspecific indicator of underlying disease or to monitor disease activity. Although hypercortisolemia may affect protein metabolism directly or indirectly, data regarding alterations of levels of serum protein in a large series of patients with Cushing's syndrome (CS) have been lacking. We have now evaluated, retrospectively, the levels of circulating serum albumin, globulins, total proteins, and the albumin to globulin ratio in 99 patients with endogenous CS before, immediately after, and 3, 6, and 12 months following successful treatment. Subjects with concomitant infections or other chronic diseases were excluded from the analysis. Although mean serum albumin and total protein levels were within the normal reference ranges, in general, they gradually increased after treatment with maximal values being reached at 12 months after normalization of hypercortisolemia (P < 0.0001 for both); there were no significant changes in serum globulin levels or in the albumin to globulin ratio. Patients with CS as a whole showed a weak but significant negative correlation between serum albumin and 0900 h cortisol level (r = -0.303; P = 0.0035). In conclusion, our data suggest that CS is associated with a small but significant reduction in circulating serum protein levels, which are restored following treatment of hypercortisolemia, although these changes occur within the reference range. Thus, extreme alterations in serum total protein or albumin levels in patients with CS should alert physicians to the presence of concomitant pathology, and additional specific investigation should be undertaken to elucidate the cause.     

Correlation of serum IgD level with clinical and histologic parameters in Hodgkin disease

The concentration of serum IgD was measured in 100 patients with Hodgkin disease; 65 had 3--50-fold increases in IgD levels. Several parameters were found to influence serum IgD concentration: IgD level in older patients (greater than 50 yr) was significantly lower than in the younger patients (P less than 18 yr). IgD concentration was significantly higher in splenectomized than in nonsplenectomized patients (p less than 0.005). Therapy was found to depress IgD concentration, which fell to a value significantly lower than in untreated patients (p less than 0.05). An interesting correlation was found between IgD levels and histologic type of the disease, lower levels being preferentially associated with the lymphocyte predominance type and higher with the lymphocyte dipletion type. The logarithms of the means of these two groups were significantly different from the overall mean of the disease (p less than 0.05 and p less than 0.0005, respectively). Mixed cellularity and nodular sclerosing showed intermediate values.     

Clinical implications of increased plasma levels of CD8 in patients with hairy cell leukemia

Plasma levels of soluble T-suppressor/cytotoxic antigen (sCD8) were measured at diagnosis or before systemic treatment in 69 patients with hairy cell leukemia (HCL). The 49 nonsplenectomized patients were characterized by high concentrations of sCD8 antigen as compared with 17 controls (P less than .0001). The median sCD8 level in non- splenectomized patients was 1,050 U/mL (range: 160 to 2,400 U/mL) and was significantly higher (P less than .0001) than the median of 275 U/mL (range: 20 to 1,080 U/mL) in splenectomized patients. The relationship of sCD8 to clinical response to subsequent interferon alpha (IFN alpha) treatment was analyzed. Patients who showed subsequent hematologic response with normalization of all blood counts had significantly lower levels of sCD8 concentrations at diagnosis than those who did not (P = .0056). Furthermore, normalization of sCD8 during IFN alpha treatment paralleled the achievement of normal counts in peripheral blood, whereas soluble interleukin-2 receptor (sIL-2R) levels remained high in most patients after 12 to 15 months of treatment. We speculate that activation of suppressor/cytotoxic T cells might play a role in myelosuppression, and its modulation during treatment with IFN alpha correlates with normalization in peripheral blood counts.     

Development and validation of a real-time quantification assay to detect and monitor BRAFV600E mutations in hairy cell leukemia

The BRAFV600E mutation was recently detected in hairy cell leukemia (HCL) by whole exome sequencing. To make use of this new marker for diagnosis and follow-up of HCL, we developed a BRAFV600Emut-specific quantitative real-time PCR assay and validated it in 117 HCL patients and 102 non-HCL/BRAFwt patients. The cut-off level to discriminate BRAFV600E-positive/-negative cases was set at 0.023% BRAFV600E/BRAFwt. A total of 115 of 117 HCL (98.3%) demonstrated percentage BRAFV600E/BRAFwt above the cut-off (mean, 29.6 ± 41.1). The remaining 2 of 117 HCL with lower percentage BRAFV600E/BRAFwt ratios were also BRAFwt by deep-sequencing technology. Sixteen HCL-variant patients showed percentage BRAFV600E/BRAFwt values corresponding to "non-HCL." Follow-up studies in 19 HCL cases demonstrated a decrease of percentage BRAFV600E/BRAFwt during therapy. The log-reductions as determined by RT-PCR and immunophenotyping correlated significantly (P < .001). In conclusion, we confirmed BRAFmut as a useful marker in HCL, its absence in HCL variant, and developed an RT-PCR-based assay to monitor minimal residual disease in HCL.     

Iron absorption in patients with beta-thalassaemia/haemoglobin E disease and the effect of splenectomy

Intestinal 59Fe absorption was measured in 16 patients with beta-thalassaemia/haemoglobin E disease and in 5 normal controls, using a total-body counting technique. The average iron absorption in the patients was 62% in contrast to 16.5% in the normal controls. 6 of the 9 splenectomized patients had absorption values above 65%, while only 2 of the 7 nonsplenectomized patients had comparably high values.     

Phosphorylation in erythrocyte membranes from abnormally shaped cells.

Erythrocyte protein phosphorylation was examined in membrane preparations of 25 patients with hereditary spherocytosis (HS). Reduced phosphorylation in substrate polypeptides was observed in 22 HS erythrocyte membranes for both splenectomized and nonsplenectomized patients. A reduction in labeling was also observed in a polypeptide which was labeled only in the presence of cAMP. No reduction was observed in membranes from immunologically acquired spherocytosis cells or from cells from patients with hereditary elliptocytosis. Heating membranes to 45 degrees C caused negligible inhibition of membrane protein phosphorylation, while heating to 50 degrees C extensively inhibited phosphorylation. Dephosphorylation of membrane protein that occurred in isolated membranes was not dependent upon cAMP.