Seudohiperfosfatemia analítica como punto de partida en el diagnóstico de una disproteinemia / Role of pseudo-hyperphosphataemia in the diagnosis of dysproteinaemia

El fósforo es el segundo mineral más abundante en el organismo. Su homeostasis se consigue mantener a través de varios mecanismos mediados principalmente por el riñón, el intestino y el hueso. Se han descrito interferencias en la medición del fósforo que pueden provocar una seudohiperfosfatemia. La causa más frecuente es la presencia de una paraproteína en el suero de los pacientes con mieloma múltiple, macroglobulinemia de Waldenström y gammapatía monoclonal de significado incierto. En los casos de hiperfosfatemia sin causa aparente que la pueda justificar, es importante tener en cuenta la existencia de una seudohiperfosfatemia causada por la presencia de las paraproteínas en sangre en los autoanalizadores de química líquida. El sistema multicapa de Vitros(R) 5600 es un método rápido y fiable para solucionar este problema

Phosphorus is the second most important mineral in the body. Its homeostasis is maintained through several mechanisms mediated mainly by the kidney, intestine, and bone. Interferences have been described in the measurement of phosphorus that could suggest a pseudo-hyperphosphataemia. The most frequent cause was the presence of a paraprotein in the serum of patients with multiple myeloma, Waldenström macroglobulinaemia, or monoclonal gammopathy of uncertain significance, was described as the most frequent cause of interference in phosphorus assay using liquid chemistry autoanalysers. When hyperphosphataemia is present, and no apparent cause can justify it, it is important to consider the possibility of a pseudo-hyperphosphataemia caused mainly by the presence of a paraprotein. The Vitros(R) 5600 multilayer system can be used as a fast and reliable method to avoid this interference

Detection of hereditary bisalbuminemia in bottlenose dolphins (Tursiops truncatus, Montagu 1821): comparison between capillary zone and agarose gel electrophoresis.

BACKGROUND: Hereditary bisalbuminemia is a relatively rare anomaly characterized by the occurrence of two albumin fractions on serum protein separation by electrophoresis. In human medicine, it is usually revealed by chance, is not been clearly associated with a specific disease and the causative genetic alteration is a point mutation of human serum albumin gene inherited in an autosomal codominant pattern. This type of alteration is well recognizable by capillary zone electrophoresis (CZE), whilst agarose gel electrophoresis (AGE) not always produces a clear separation of albumin fractions. The aims of this study is to report the presence of this abnormality in two separate groups of related bottlenose dolphins and to compare the results obtained with capillary zone and agarose gel electrophoresis. RESULTS: Serum samples from 40 bottlenose dolphins kept under human care were analyzed. In 9 samples a double albumin peak was evident in CZE electrophoresis while no double peak was noted in AGE profile. Since only an apparently wider albumin peaks were noted in some AGE electrophoretic profiles, the ratio between base and height (b/h) of the albumin peak was calculated and each point-value recorded in the whole set of data was used to calculate a receiver operating characteristic curve: when the b/h ratio of albumin peak was equal or higher than 0.25, the sensitivity and specificity of AGE to detect bisalbuminemic samples were 87 and 63 %, respectively. The bisalbuminemic dolphins belong to two distinct families: in the first family, all the siblings derived from the same normal sire were bisalbuminemic, whereas in the second family bisalbuminemia was present in a sire and in two out of three siblings. CONCLUSIONS: We report for the first time the presence of hereditary bisalbuminemia in two groups of related bottlenose dolphins identified by means of CZE and we confirm that AGE could fail in the identification of this alteration.

Electrophoretic finding of bisalbuminemia.

BACKGROUND: Bisalbuminemia or alloalbuminemia is a rare inherited or acquired condition characterized by the presence of two albumin fractions during electrophoretical separation of serum proteins. METHODS: Bisalbuminemia was incidentally detected by agarose gel electrophoresis (AGE) during standard laboratory investigation of a 36-year old female patient, referred to our laboratory with the diagnosis of immune thrombocytopenia. RESULTS: The electrophoregram showed dysproteinemia with the presence of two distinct albumin bands. This initiated testing of other family members - mother, father, and son of the patient. CONCLUSIONS: We report a case of inherited bisalbuminemia in a Bulgarian family with two affected members of four investigated. This is the first report of inherited bisalbuminemia in the Plovdiv region and could be of great interest to laboratory practitioners and clinicians providing some new data on the protein evolution and the clinical approach.

Fever of unknown origin (FUO) in an immunocompetent adult due to cytomegalovirus (CMV) with polyclonal gammopathy.

Fever of unknown origin (FUO) may be due to infection, malignancy, collagen vascular/inflammatory disorders, or other causes. Cytomegalovirus (CMV) infection is a rare cause of FUO in immunocompetent adults. We present a case of FUO due to CMV in an immunocompetent adult with polyclonal gammopathy on serum protein electrophoresis (SPEP).

Tissue zinc levels in a child with hypercalprotectinaemia and hyperzincaemia: A case report and a review of the literature.

BACKGROUND: A girl suffering from a rare syndrome of unknown aetiology, termed hypercalprotectinaemia, was evaluated for tissue zinc status, because calprotectin is a protein which chelates Zn at multiple binding-sites, which might have affected the distribution of Zn in her body. METHODS: Measurement of serum, urine, hair and nail zinc (Zn) concentration, complemented with measurement of total Zn in ultrafiltrates of plasma. RESULTS: Her serum Zn concentration was 105-133 µmol/L. Zn levels in her hair (102 µg/g), nail (90 µg/g) and urine (3-12 µmol/L; 20-80 µg/dL) were all at the lower end of the reference intervals described in the sparse literature. Zn concentrations in ultrafiltrates of plasma were below the detection limit (<100 nmol/L). Thus, the elevated serum Zn did not translate into a similarly increased level of Zn in any of the tissues tested, nor in free Zn concentrations. Instead it appeared to be a result of Zn being chelated to binder proteins, most probably calprotectin. CONCLUSION: Her grossly elevated serum calprotectin concentration is probably able to raise circulating total Zn concentrations without raising ionized concentrations, but this Zn remains confined to the circulating blood as well as to excreted body fluids, particularly faeces.

[Clinical characteristics and ALB gene mutation analysis of Korean patients with bisalbuminemia].

BACKGROUND: Bisalbuminemia is a hereditary or an acquired condition characterized by the presence of 2 albumin variants with different mobilities on serum protein electrophoresis (SPE). The clinical significance of bisalbuminemia has not been clearly established. However, some regions of the albumin variant may affect the biochemical analysis of biomolecules such as steroid or thyroid hormones by altering their albumin-binding affinities. In this study, we analyzed the clinical manifestations, genetic variations, and the albumin-binding characteristics in Korean patients with bisalbuminemia. METHODS: We performed SPE for samples from 580 Korean subjects and identified bisalbuminemia on the basis of the results of SPE. The clinical and biochemical characteristics, ALB gene mutations, and the structures of the albumin variants of patients with bisalbuminemia were analyzed. RESULTS: SPE showed bisalbuminemia in 2 patients. One patient showed a genetic variation known as Nagasaki-1 (Asp293Gly) and the other showed a hitherto unreported missense mutation (c.593A>T; Lys198Ile). In both cases, the serum concentrations of the substances with binding affinity for albumin were not affected, and the mutation sites of the albumin were not located with the protein-binding loci. CONCLUSIONS: The 2 Korean patients with bisalbuminemia showed genetic variations, including a novel missense mutation. The ALB gene analysis with 3D modeling is useful for determining the nature of bisalbuminemia and for predicting the effects on the albumin-binding affinity of other biochemical compounds.

Clinical Characteristics and ALB Gene Mutation Analysis of Korean Patients with Bisalbuminemia / 대한진단검사의학회지

BACKGROUND: Bisalbuminemia is a hereditary or an acquired condition characterized by the presence of 2 albumin variants with different mobilities on serum protein electrophoresis (SPE). The clinical significance of bisalbuminemia has not been clearly established. However, some regions of the albumin variant may affect the biochemical analysis of biomolecules such as steroid or thyroid hormones by altering their albumin-binding affinities. In this study, we analyzed the clinical manifestations, genetic variations, and the albumin-binding characteristics in Korean patients with bisalbuminemia. METHODS: We performed SPE for samples from 580 Korean subjects and identified bisalbuminemia on the basis of the results of SPE. The clinical and biochemical characteristics, ALB gene mutations, and the structures of the albumin variants of patients with bisalbuminemia were analyzed. RESULTS: SPE showed bisalbuminemia in 2 patients. One patient showed a genetic variation known as Nagasaki-1 (Asp293Gly) and the other showed a hitherto unreported missense mutation (c.593A>T; Lys198Ile). In both cases, the serum concentrations of the substances with binding affinity for albumin were not affected, and the mutation sites of the albumin were not located with the protein-binding loci. CONCLUSIONS: The 2 Korean patients with bisalbuminemia showed genetic variations, including a novel missense mutation. The ALB gene analysis with 3D modeling is useful for determining the nature of bisalbuminemia and for predicting the effects on the albumin-binding affinity of other biochemical compounds.

Sjögren's syndrome associated with multiple myeloma.

There have been very few reported cases of multiple myeloma (MM) which had Sjögren syndrome (SS) as the first presentation. We report a 63-year-old Moroccan woman with IgA-lambda-type MM presenting as SS and who responded to anti-myeloma treatment. The patient, treated for SS, was admitted to our department for persistent and increasing thoracic pain. Clinical examination was normal. Laboratory investigations showed haemoglobin of 10 g/dL. Erythrocyte sedimentation rate was 80 mm/hr. Monoclonal spike was found in the betaglobulin region of the serum protein electrophoresis. Immunofixation identified it as IgA lambda and the level was 3.7 g/dL. The bone marrow contained 35 percent plasma cells, with atypical features. Radiographs showed diffuse lytic lesions. Treatment with vincristine, adriamycin and dexamethasone (VAD) was started and bisphosphonate was administered regularly. After three cycles of VAD therapy, the MM regressed without any evidence of SS symptoms. The development of MM in the setting of SS is unusual and the aetiopathogenic mechanism still unknown. However, some elements orient toward a common pathway for these two diseases, like the clinical remission of SS after treatment of the MM, such as described in our patient.

Detection and characterization of variant and modified structures of proteins in blood and tissues by mass spectrometry.

Some variant proteins cause diseases, and some diseases result in increases of proteins with abnormally modified structures. The detection, characterization, and estimation of the relative amounts of protein variants and abnormally modified proteins are important for clinical diagnosis and for elucidation of the mechanisms of the pathogenesis of diseases. Analysis of the covalent structures of proteins using matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography-electrospray ionization MS (LC-ESI-MS), which had been developed by the early 1990s, have largely replaced analyses by conventional protein chemistry. Here, we review the detection and characterization of hemoglobin variants, HbA1c measurement, detection of carbohydrate-deficient transferrin, and identification of variants of transthyretin (TTR) and Cu/Zn-superoxide dismutase (SOD-1) using soft ionization MS. We also propose the diagnostic application of the signals of modified forms of TTR, that is, S-sulfonated TTR and S-homocysteinyl TTR. The relative peak height ratio of the abnormal/normal components gives valuable information about the instability of variants and enables the detection of unstable Hb subunits or thalassemia heterozygotes. We found unique modified structures of TTR that suggested changes in amyloid fibrils.

[Dysalbuminemia]. / Les dysalbuminémies.

Albumin is the major circulating protein. It plays a fundamental role maintaining intra-vascular oncotic pressure and carrying many endogenous and exogenous substances. Variations of plasma albumin levels can be physiologic or pathologic and both qualitative and quantitative (more frequent) disorders are regrouped under the name "dysalbuminemia". Although hypoalbuminemia are frequent, analbuminemia exists but is a rare disease. Qualitative disorders, mainly bisalbuminemia, are benign. Detected fortuitously on sera protein electrophoresis, bisalbuminemia could be genetically transmitted, it will then be permanent, or acquired and then be transient. This article proposes to review main kind of dysalbuminemia usually encountered in clinical biology laboratories.

Understanding and interpreting serum protein electrophoresis.

Serum protein electrophoresis is used to identify patients with multiple myeloma and other serum protein disorders. Electrophoresis separates proteins based on their physical properties, and the subsets of these proteins are used in interpreting the results. Plasma protein levels display reasonably predictable changes in response to acute inflammation, malignancy, trauma, necrosis, infarction, burns, and chemical injury. A homogeneous spike-like peak in a focal region of the gamma-globulin zone indicates a monoclonal gammopathy. Monoclonal gammopathies are associated with a clonal process that is malignant or potentially malignant, including multiple myeloma, Waldenstrom's macroglobulinemia, solitary plasmacytoma, smoldering multiple myeloma, monoclonal gammopathy of undetermined significance, plasma cell leukemia, heavy chain disease, and amyloidosis. The quantity of M protein, the results of bone marrow biopsy, and other characteristics can help differentiate multiple myeloma from the other causes of monoclonal gammopathy. In contrast, polyclonal gammopathies may be caused by any reactive or inflammatory process.

The immunoglobulinopathies: from physiopathology to diagnosis.

The diversity of immunoglobulins (Igs) results mainly from recombinations of numerous genes within the heavy (V(Heavy), D, and J(Heavy)) and within the light (V(Light), J(Light)) chain gene loci, and from somatic hypermutations occurring during the immune response of B-cells. Igs production is controlled by complex cellular and humoral mechanisms. Plasma of healthy individuals contains polyclonal Igs. Clonal expansion of cells producing antigen-specific Igs may result from physiological as well as from pathological immune events. Exquisitely sensitive and specific molecular biology techniques have been used to evaluate the clonal diversity of cells producing antigen-specific Ig. However, the application of such techniques is hampered by the necessity to collect the totality of antigen-specific B-cells for subsequent analysis, which is impossible to perform routinely in humans. In addition, these techniques do not provide quantitative information about the concentration of the circulating Igs. It is therefore necessary to use tools allowing study of the quantity of the circulating Igs, and more particularly to detect overproduction of a single homogeneous Ig resulting from the expansion of a B-cell clone secreting Igs. Here, we review the mechanisms of B-cell differentiation and Ig synthesis, discuss the diseases associated with clonal Ig production and review the methods available in the clinical laboratory for Ig analysis.

ProC global: a new automated screening assay for the evaluation of total function of the protein C system.

Protein C (PC) pathway represents a major physiologic inhibitory mechanism regulating the coagulation cascade. A new automated functional screening assay (ProC Global) for the evaluation of the PC-system was tested to define its ability to identify patients with known inherited defects such as factor V (FV) Leiden mutation and PC and protein S (PS) deficiency. A total of 249 patients who were symptomatic or asymptomatic for previous venous thromboembolism (VTE) were evaluated, 50 of whom had FV Leiden mutation, 36 had PC deficiency, and 34 had PS deficiency. One hundred healthy subjects were also tested, as well as 40 blood donors of both sexes in whom coagulation abnormalities were not found. Results of ProC Global test were expressed as normalized ratio (NR) and values below an established cut-off level were consistent with a positive test. ProC Global was positive in all 50 patients with the FV Leiden mutation (mean NR = 0.59; range, 0.37 to 0.69). ProC Global correctly identified 32 of 36 (89%) PC defects (mean NR = 0.63; range, 0.34 to 1.21) and 25 of 34 (73.5%) PS defects (mean NR = 0.76; range, 0.5 to 1.23). Overall, 92.5% of hereditary defects of the PC system considered in this study were identified by ProC Global test. ProC Global exhibited NR above cut-off level in all 40 blood donors without coagulation defects. ProC Global is a new automated screening test with some diagnostic potential in identifying patients with defects of the PC system. However, ProC Global in its current form cannot substitute the assay of each single component of this inhibitory system in the daily screening for thrombophilia.

Laboratory diagnosis of von Willebrand disorder (vWD) and monitoring of DDAVP therapy: efficacy of the PFA-100 and vWF:CBA as combined diagnostic strategies.

We have coevaluated a combination of test processes for diagnosing von Willebrand disease (vWD) and monitoring deamino-delta-D-arginine vasopressin (DDAVP) therapy. Using normal controls (n = 23), closure time (CT) ranges measured by PFA-100(R) were (mean +/- 2SD): (i) collagen/ADP cartridge (C/ADP): 67-127 s (ii) collagen/epinephrine (C/Epi): 94-162 s. From a panel of 125 patients undergoing evaluation for clinical haemostatic defects, 29/30 samples from patients with vWD [17/18 type 1, 1/1 type 3, 3/3 type 2A, 7/7 type 2B and 1/1 pseudo-vWD] gave prolonged CTs using C/Epi. The C/ADP was less sensitive, being normal in 7/18 of the type 1 vWD individuals, with higher sensitivity to more severe vWD. Individuals with haemophilia (six factor VIII-deficient, one factor XI-deficient) gave normal CTs, while those with clinical thrombocytopenia (n=13) gave normal or prolonged CTs, somewhat dependent on platelet count. The PFA-100 was also evaluated as a part of the laboratory monitoring procedure in patients with either vWD or haemophilia undergoing a DDAVP trial as a therapeutic management process. For vWD, correction of an initially prolonged CT by DDAVP, accompanied by normalization of von Willebrand factor (vWF) measurable by von Willebrand factor antigen, vWF collagen binding activity and vWF ristocetin cofactor assays (vWF:Ag, vWF:CBA and vWF:RCof), was achieved in type 1 vWD (n=5). In an individual with type 2A vWD, DDAVP normalized vWF:Ag and vWF:RCof, but had no apparent effect on the baseline maximally prolonged CT. In an individual with type 2B vWD, factor VIII/vWF concentrate also normalized vWF:Ag and vWF:RCof, but similarly had no apparent effect on the baseline maximally prolonged CT. vWF:CBA did not normalize for either of these individuals, potentially suggesting that normalization of vWF:CBA might be required for normalization of CT. This concept is supported by correlation analysis undertaken between CT and various vWF parameters. Among these, vWF:CBA held the strongest relationship in our data set, which showed an inverse progressive rise in CT for falling vWF:CBA. Based on these results, we would conclude that the PFA-100 is highly sensitive to the presence of vWD, and may thus provide a valuable screening test for vWD. Furthermore, the combined utility of the PFA-100 and vWF:CBA as markers of DDAVP responsiveness may prove to be simple, quick but powerful, predictors for its clinical efficacy.

Differential diagnosis of gammopathies by capillary electrophoresis and immunosubtraction: analysis of serum samples problematic by agarose gel electrophoresis.

The capabilities of capillary electrophoresis (CE) for serum protein electrophoresis and immunotyping have been demonstrated. CE-based systems specifically designed for serum protein electrophoresis and immunotyping via immunosubtraction (IS) are now available and are being evaluated for efficiency, specificity and sensitivity by several groups. The use of CE for serum protein electrophoresis and immunotyping (IS) in the clinical laboratory compares well with agarose gel electrophoresis (AGE) and immunofixation (IF) for the detection and characterization of monoclonal proteins. In addition to routine use, this technology is useful for a subset of serum samples that are difficult to interpret with conventional technology. In this study, sera abnormalities difficult to detect/interpret by AGE-IF are subdivided into four categories: (i) patients with polyclonal increases in immunoglobulin, (ii) point of application artifacts, (iii) abnormalities in the beta region, and (iv) patients with free light chains. CE is superior to AGE for evaluating samples characterized by the above abnormalities. Sera containing monoclonal proteins within a polyclonal increase are easier to detect by CE as well as being easier to type by IS than by IF. Point-of-application artifacts, periodically observed with AGE, do not exist on CE since the point of detection is remote from the point of application. Enhanced resolution in the beta region allows for increased detection of monoclonal proteins migrating in this region. Some free light chains are undetected by CE as a result of no apparent abnormalities on the CE serum protein profile and, thus, still require IF for detection. CE detects more serum electrophoretic abnormalities than AGE in this clinically important group of patients with Bence Jones proteinemia.