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High-resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used to analyze serum samples and purified immunoglobulins (Ig) obtained from "normal" individuals and from patients diagnosed with monoclonal gammopathies (MG) (n = 47; 5 IgA, 15 IgM, 15 IgG, 4 biclonal IgG, 1 IgD, 7 Bence Jones proteins). Polyclonal and monoclonal heavy (H) chains were located at different restricted gel positions according to their isotype. Monoclonal H chains appeared as sets of spots characterized by charge (pI) and size (M(r)) microheterogeneity. Most of the monoclonal gamma chains were not seen on the gels (12/15). Supplementary polypeptides of 45-48 kDa were detected in serum samples containing monoclonal IgM, but were not seen in MG of other isotypes. However, these polypeptides were not specifically associated with monoclonal IgM because they were also found on protein maps of purified polyclonal IgM. Polyclonal light (L) chains appeared as cloudy bands containing several zones of higher density, whereas monoclonal L chains were usually resolved as single sharp spots. In 6 samples, monoclonal L chains were not seen, and in 9 samples, they appeared as two or more spots, characterized by different pI and/or M(r). In one sample obtained from a patient with a biclonal gammopathy, the L chains were resolved as 4 different spots. Our results confirm that 2-D PAGE is an excellent tool to study Ig. Analysis of the L chain region of the gels was particularly informative. Several monoclonal L chains exhibited heterogeneous two-dimensional spot patterns, suggesting that "subtile" clonal mutations of B-cell lineage and/or posttranslational modifications were involved in their production.

This report describes a mathematical approach for classifying two-dimensional (2D)-protein maps without spot detection or pattern matching. Analysis of electrophoretograms was performed using wave packet decompositions of the signals. The scanned images were automatically decomposed into a set of sub-images organized in a tree structure. Each sub-image contained relevant information such as its energy, or entropy. Moreover the node position itself of the sub-image reflected a frequency localization. A distance was then defined using the tree repartition of these quantities. Finally a statistical clustering on the tree structures was performed, terminating with a classification of the images according to their repartition frequencies. The algorithm has been applied to classify immunoglobulin (Ig) light chain patterns and proved useful to automatically detect monoclonal, oligoclonal or polyclonal Igs.

Plasma/serum proteins of fetal blood samples (N = 88) obtained under ultrasound guidance between the 18th and the 39th week of pregnancy, of blood samples collected from premature infants (N = 19), newborns at term (N = 20) and children of less than 5 years of age (N = 55) were analysed by high-resolution two-dimensional polyacrylamide gel electrophoresis. By comparison with adult 'reference' protein maps, tens of different proteins (and some of their genetic variants) were identified on the electrophoretograms. After the 18th week of gestation, albumin, transferrin, Factor B, glu- and lys-plasminogen, antithrombin III, Gc-globulin, alpha 1-antitrypsin, alpha 2-HS-glycoprotein, several apolipoproteins (apo A-I, A-II, A-IV, C-II, C-III, D, E, J), retinol-binding protein, transthyretin and alpha-fetoprotein could be observed. During intrauterine life, the size of the spots corresponding to alpha-fetoprotein progressively decreased, whereas the protein pattern globally showed an increase in the number and in the size of the spots. These modifications were particularly apparent in the regions of the electrophoretograms restricted to the heavy and light chains of IgG and to alpha 1-antichymotrypsin. In addition, we observed an unidentified fetal polypeptide characterized by an apparent molecular weight (M(r)) of 46 kDa (P46) and a pI of 5.0. P46 was present in all fetuses and all infants of less than 2 years of age.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of prestorage leukocyte reduction was evaluated on platelet concentrates (PCs) obtained by apheresis using the 'surge' technique. Two hours after collection, the PCs were divided into 2 equal units. One unit was filtered through a Sepacell PL-10a, producing a filtered PC (FPC). The second unit constituted a non-filtered PC (NFPC). FPCs and NFPCs were stored at room temperature in 1,400-ml CLX bags on a horizontal agitator up to 7 days. We analyzed platelet samples obtained during storage from NFPCs and FPCs at days 1, 3 and 7. The expression of membrane glycoproteins (GP)Ib and GPIIb/IIIa (assessed by flow cytometry), platelet response to thrombin and ristocetin (aggregometry) and global platelet protein pattern (studied by high-resolution two-dimensional gel electrophoresis) remained stable over the 7 days of storage in NFPCs as well as in FPCs. However, in both preparations, the expression of GMP-140 (flow cytometry) progressively increased during storage. Our in vitro study indicates that early leukocyte reduction by filtration of apheresis PCs does not induce modifications in platelet GPs and protein patterns.

This publication establishes a reference human liver protein map obtained with immobilized pH gradients. By microsequencing, 57 spots or 42 polypeptide chains were identified. By protein map comparison and matching (liver, red blood cell and plasma sample maps), 8 additional proteins were identified. The new polypeptides and previously known proteins are listed in a table and/or labeled on the protein map, thus providing a human liver two-dimensional gel database. This reference map can be used to identify protein spots on other samples such as rectal cancer biopsies.

The clonality pattern of immunoglobulins (Igs) produced after allogeneic bone marrow transplantation (BMT) was studied by high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of serum samples and purified Igs. With this technique, the light (L) chain of a monoclonal Ig usually appears as a single spot. Thus, the degree of clonal diversity of the functional B cells can be appreciated by the electrophoretic pattern of the serum L chains. Furthermore, 2D-PAGE allows a semi-quantitative determination of prominent Ig clones according to the size of L chain spots. We found that serum electrophoretograms of 8/19 patients after BMT (5-9 months) revealed L chain patterns which were similar to those of normal polyclonal Igs, that is, less than five distinguishable small spots among a cloud-like indiscrete L chain spots region ('polyclonal' pattern). A spectrum of clonal abnormalities was observed on the electrophoretograms of 11/19 patients: in five patients, multiple small L chain spots (corresponding to Ig concentrations between 0.2 and 2 g/l) were detected ('oligoclonal' pattern), whereas in six others, 'typical' monoclonal Igs (Ig concentrations > 2 g/l) were observed with (3/19 patients) or without (3/19 patients) multiple small clonal components. Sequential analysis of serum obtained from patients at different times after BMT revealed that imbalanced clonal reconstitution was transient and evolved towards apparently normal polyclonal Ig production. Our observations show that the development of clonal 'gammopathies' after BMT is a frequent, but not obligatory phenomenon. It may reflect a transient restriction of the B cell repertoire either through a limited outgrowth of precursor cells or through selective antigenic pressures.

The reference plasma protein map, obtained with immobilized pH gradients in the first dimension of two-dimensional electrophoresis, is presented. By microsequencing, more than 40 polypeptide chains were identified. The new polypeptides and previously known proteins are listed in a table and labeled on the protein map, thus providing an update of the human plasma two-dimensional gel database.

High-resolution two-dimensional gel electrophoresis (2-DGE) was used to analyse plasma samples and partially purified cold agglutinins (CA) obtained from two selected patients. Both presented an acute hemolytic anemia with CA of high thermal amplitude, normal immunoglobulin levels, no detectable paraproteinemia, and no clinical evidence of a malignant B-cell disorder. The electrophoretograms of their plasma showed evident alternations of the "normal" protein profile, which were directly related to hemolysis (absence of the spots of haptoglobin and in one case of those of hemopexin), but no monoclonal gammopathy. The electrophoretograms of their purified CA revealed two clearly different spot patterns respectively corresponding to a monoclonal IgM and to polyclonal IgM. These results show that the clonality of CA associated with hemolytic anemia can be easily determined by 2-DGE. This technique may be very useful to discriminate chronic cold agglutinin disease in the early phase from "parainfectious" CA.

The occurrence of monoclonal gammopathy in childhood is extremely rare. This report describes the presence of a monoclonal immunoglobulin in a 30 week old premature infant, incidentally discovered by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) during an ongoing study of the plasma/serum protein development. Comparative analysis of the electrophoretogram of the infant with 'reference' protein maps revealed the presence of an 'abnormal' immunoglobulin light chain spot. A spot having an identical apparent molecular weight and isoelectric point was also detected after 2D-PAGE of the mother's plasma and its Protein-A purified immunoglobulin fraction. The observation of a monoclonal gammopathy in a premature infant, most likely transmitted from his mother, highlights the potential usefulness of 2D-PAGE in the clinical laboratory.

A further case of persistent polyclonal B-cell lymphocytosis is reported. This recently identified distinct clinicopathologic entity is frequently associated with the presence of the HLA-DR7 antigen. It follows a benign course and has so far been reported only in women smokers. The disorder is characterized by mild chronic peripheral lymphocytosis, the presence of characteristic binucleate lymphocytes on peripheral blood smears, and a polyclonal increase in serum IgM. In some cases, lymphadenopathies and/or splenomegaly are observed. Surface marker studies of peripheral lymphocytes demonstrate the polyclonal B-cell nature of this entity.

Delayed hemolytic reaction is a well recognized hazard of blood transfusion and occurs mainly in recipients with alloantibodies, due to sensitization to red cell antigens by previous transfusion or pregnancy. Less frequently, such a reaction may be associated with the presence of red cell autoantibodies appearing after alloimmunization. We report the observation of a patient with chronic myelomonocytic leukemia, who presented a transitory episode of delayed hemolysis after multiple transfusions. At first, anti-Kell alloantibodies were identified and, two weeks later, warm "broad-specific" autoantibodies were detected by direct and indirect antiglobulin tests. The anemia was associated with unstable angina. The patient received multiple transfusions and prednisone. After 3 weeks, the transfusion needs diminished and the autoantibodies progressively disappeared from the serum. The direct antiglobulin test was negative 4 months later. This observation illustrates the poorly understood relationships between erythrocytic alloantigen exposure and red cell autoimmunization.

The potential usefulness of an optimalized high-resolution two-dimensional gel electrophoresis (2-DGE) protocol was studied by comparative analysis of plasma/serum obtained from apparently healthy individuals and from patients with a few selected known diseases. Despite their apparent complexity, patient electrophoretograms revealed readily detectable modifications of the 'reference' protein profile for those selected diseases (listed below). Abnormal profiles were characterized by presence or absence of particular spots, by reduction or enlargement of spot size, or by alterations of spot microheterogeneity. Combinations of several modifications enabled different 'disease-associated spot pattern' to be distinguished on the protein maps of patients with: monoclonal gammopathies, hypogammaglobulinemia, hepatic failure, chronic renal failure and hemolytic anemia. This study demonstrates that identification of plasma/serum protein alterations by 2-DGE allows a few selected diseases to be diagnosed solely on the basis of protein map modifications.

We report the case of an 86-year-old female with an unremarkable past history who suddenly developed severe and generalized bleeding. Laboratory work-up revealed prolonged aPTT, a factor VIII level of 7% and the presence of a factor VIII inhibitor. In spite of whole blood, factor VIII concentrates and immunoglobulin administration, the patient died of multiple hemorrhages on the third day after admission. At autopsy no causal or associated pathology could be found. Chromatographic separation of plasma immunoglobulins showed that the anti-factor VIII activity was associated with the patient's IgG. This inhibition was partially corrected in vitro by injectable immunoglobulin preparation. The case report stresses the discrepancy between the measured levels of inhibitor and the severity of the bleeding; our in vitro results may shed light on the mode of action of intravenous immunoglobulins.

Two-dimensional gel electrophoresis (2-DGE) associated with a very sensitive silver staining is one of the most powerful technique available for protein analysis. Proteins are separated in the first dimension by isoelectric focusing in a pH gradient, and in the second dimension by polyacrylamide gel electrophoresis. Proteins are thus separated according to their charge and to their size. The high resolution of 2-DGE allows the analysis of complex mixture of proteins with a great reproducibility. More than hundred different proteins can be individualized after 2-DGE of plasma or serum samples. Using this technique, we analysed samples obtained from normal donors and from patients with different gammopathies. Our results confirm that the heavy chains of immunoglobulins are easily detected and that the alpha, gamma and mu chains are clearly differentiated from one another. The typical polyclonal heavy and light chains appear as indiscrete spots after staining of the gels. On the contrary, the monoclonal chains appear as discrete spots. Monoclonal heavy chains have a restricted charge heterogeneity. We also analysed cold agglutinins, partially purified from plasma of three patients showing evidence of chronic hemolysis, a positive direct antiglobulin test of the complement type and anti-I autoantibodies. The 2-DGE have clearly demonstrated that these cold agglutinins were monoclonal IgM, when these later were not detectable by immunofixation electrophoresis. We think that the 2-DGE is a very usefull specialized aid to the analysis of such particular immunohematological problems.

A case of Wegener's granulomatosis starting as a localized pseudo-tumoral lung lesion and following a fulminating course is reported. The histological diagnosis required thoracotomy. New pulmonary lesions developed post-operatively, together with severe renal failure which was responsible for the patient's death 5 weeks after the thoracotomy. The authors describe the lesions characteristic of the disease and their pathogenesis and discuss the relationship between Wegener's syndrome and other granulomatous angiitis.

Triton X-100 and NaSCN extracts of 18 normal breast and colon tissues and of 20 breast and colon carcinomas were fractionated by SDS-PAGE and plasminogen activators (PA) revealed by a zymographic method. Four different lysis bands, corresponding to MWs of 54,000, 68,000, 95,000 and 110,000 were observed. Using immunoadsorption with specific antisera against urokinase (UK) and tissue PA (t-PA), we found that all normal tissue extracts contained free t-PA (68 kd). Some of these revealed, in addition, a complex (110 kd) of t-PA with a 40-kd component. The latter presumably represents the fast-acting specific inhibitor of t-PA and UK. Most carcinoma extracts contained, in addition to the two t-PA-related lysis bands, the UK-related 54 kd PA, and some a 95 kd complex of UK with the 40 kd component. For each extractant, mean total fibrinolytic activity of normal and tumor tissue was comparable when measured on conventional fibrin plates, but breast and colon carcinomas contained higher concentrations of UK-related PA. PA activity was higher in normal and carcinoma NaSCN extracts than in the corresponding Triton X-100 extracts. In general, Triton X-100 but not NaSCN extracts of malignant tissue contained a high concentration of fibrinolytic inhibitors. Mixing experiments revealed that the inhibitory activity was mainly directed against UK. It was abolished by acidification of the carcinoma extracts. The anti-UK inhibitory activity was absent in extracts of normal breast or colon and appears to be different from the 40 kd fast-acting PA inhibitor. These studies show that malignant transformation of breast and colon is accompanied by important changes of the production of a UK-related PA and of an inhibitory activity directed against UK.

Extramedullary solid tumors of early granulocytic and monocytic precursors are a rare manifestation of acute non lymphoblastic leukemias. They may develop during the course, or be the presenting sign, of leukemia. We describe a case of myelomonocytic tumors involving the skin and the stomach as primary manifestations of leukemia. Two months later the patient developed acute myelomonocytic leukemia with a rapidly fatal course. At autopsy, multiple tumors were found in the skin, the peridural space, the muscles, the peritoneum, the pleura and in several organs, such as in the lungs, the kidneys, the gallbladder and in the whole gastrointestinal tract. The many synonyms of these tumors, the localisation, the chronology of the clinical manifestations and the various problems in the diagnosis are discussed. In the differential diagnosis of histiocytic lymphoma, identification of myelomonocytic differentiation in neoplastic cells should be done, using special techniques, such as immunoperoxidase and cytochemical stains for esterases.

A 61-year-old woman presented with an acute condition involving confusion, abdominal cramps and bloody diarrhea six hours after accidental ingestion of colocynth mistaken for zucchini. Colonoscopic examination revealed pseudomembranous colitis though the patient had no condition known to be associated with pseudomembranous colitis. Within ten days the mental state returned to normal and the colitis resolved completely. It is suggested that the colitis was caused by the ingestion of colocynth.