Serum proteins analysis by capillary electrophoresis

The purpose of this study was to evaluate the efficacy of multi-capillary electrophoresis instrument in clinical laboratory. An automated clinical capillary electrophoresis system was evaluated for performing serum proteins electrophoresis and immuno-fixation electrophoresis by subtraction. In this study the performance of capillary electrophoresis was compared with the cellulose acetate membrane electrophoresis and agarose gel immunofixation electrophoresis for serum proteins. The results of capillary electrophoresis and cellulose acetate membrane electrophoresis were good (r=0.89∼0.97) for protein fractions and A/G ratio except for β-gobulin fraction (r=0.60). Both within-run and day to day precisions (CVs) of assay results for 5 main fractions and A/G ratio (n=10) were between 0.3∼6.3%. The reference ranges of serum protein fractions obtained from 200 healthy individuals by cellulose acetate membrane electrophoresis were almost equal to that of capillary electrophoresis except for α-1 globulin fraction. No significant difference of electropherograms between cellulose acetate electrophoresis and capillary electrophoresis was observed in the abnormal serum such as presence of bilirubin (<20mg/dl), hemoglobin (<300 mg/dl), lipid (Intralipos <1%) and samples from patients with acute phase response, liver injury, polyclonal hyper gammaglobulinemia or M-proteinemia. The method of capillary immuno-fixation electrophoresis by subtraction showed good agreement with agarose gel immunofixation electrophoresis by subtraction identifying 30 monoclonal gammmopathy patient samples.     

A Case of Light Chain Deposition Disease (LCDD) in a Young Patient

Light chain disease is a variant of multiple myeloma in which the malignant population of marrow cells produces free monoclonal light chains but no heavy chain or complete immunoglobulin. The monoclonal light chains are small enough to be freely filtered by the kidneys and become Bence–Jones protein. Light chain disease comprises about 18% of multiple myeloma patients. Here we present a case report of a 38-year-old man who initially presented with complaints of pain in back and low grade fever off and on. He was found to have collapse of D9 and D12 vertebrae along with ascites and right pleural effusion and massive proteinuria. Multiple myeloma was considered as a differential diagnosis based on the investigations but eventually the patient was lost to follow up. This case is reported here as the light chain variant of multiple myeloma leading to deposition disease is less commonly reported and presents considerable difficulties in diagnosis.     

Spectrum of monoclonal gammapathies in Andhra Pradesh

In the present study, monoclonal gammapathy was identified in a total of 245 patients of plasma cell dyscrasias during period of 1987 to 2000. The monoclonal band was identified in serum by agar gel electrophoresis in all the cases and in urine in a few cases. Characterization of paraprotein (monoclonal immunoglobulin class and light chain type) was carried out by employing immunoelectrophoresis and/or immunofixation electrophoresis using heavy chain specific gamma, alpha, mu, delta and epsilon and light chain specific kappa (K), lambda (λ) antisera. Serum immunoglobulins Ig G, Ig A, and Ig M were estimated by immunoturbidometry. Serum urea, creatinine, uric acid, alkaline phosphatase, total proteins, albumin, calcium and phosphorus were estimated by using routine biochemical methods. Among the 245 cases, 73.1% monoclonal gammapathies were of secretory type and 7.3% were non-secretory. Monoclonal gammapathies were associated with 80.4% of multiple myeloma, 8.9% of solitary plasmacytoma, 4.1% of extra-medullary plasmacytoma, 3.3% of lymphoma and 2.9% of plasma cell leukemia. Classification of secretory monoclonal immunoglobulin revealed monoclonal immunoglobulin Ig G in 74%, Ig A 15% and Ig M in 2.9% cases.     

Gamma heavy chain disease in a patient with rheumatoid arthritis – a laboratory evaluation

Introduction:

Heavy chain diseases (HCD) are neoplastic proliferations of B cells which secrete truncated immunoglobulin heavy chains without associated light chains. Being rare and probably underdiagnosed diseases the aim of this report is to show an additional case of gamma heavy chain disease in a 48 year old female patient with rheumatoid arthritis focusing on the laboratory presentation.

Materials and methods:

Laboratory work-up included agarose gel electrophoresis (AGE), capillary zone electrophoresis (CZE), immunofixation and nephelometrically determined immunoglobulin and immunoglobulin subclasses of the patient’s serum. Urine samples were also subjected to immunofixation and to a SDS-PAGE with consecutive immunoblot.

Results:

Nephelometrically measured elevated IgG concentrations were noted in combination with a decreased gamma globulin region and an increased beta globulin region on AGE. A definite monoclonal spike was not identified on AGE but at least suspected on CZE; finally serum and urine immunofixation demonstrated a monoclonal gamma heavy chain devoid of any corresponding light chains confirming the diagnosis of HCD. Analysis of the gamma heavy chain (HC) with means of SDS-PAGE revealed proteins of 40 kD and 80 kD most likely presenting a truncated HC in its monomeric and dimeric form and possibly leading to the failure of IgG-subclass typing with the applied IgG subclass antisera.

Conclusion:

This case report illustrates a new case of gamma HCD demonstrating variable laboratory manifestations and therefore the need for heightened awareness concerning this disease when confronted with abnormal and discrepant protein profiles in routinely applied laboratory tests.     

An immunological based study of monoclonal gammopathies among suspected individuals in Kashmir region

Serum and urine samples from 513 patients clinically suspected of monoclonal gammopathies over a period of five years (1992–97) were subjected to various immunological procedures viz, electrophoresis, immunoelectrophoresis and immunoglobulin estimations. Laboratory investigations confirmed gammopathies in 10.33%. It was observed that overall age of incidence for monoclonal gammopathies in both sexes was between 42–72 years with a male to female ratio of 1.4∶1. Predominant paraprotein detected was IgG type (75.47%) followed by IgA (16.98%) and Bence Jones proteins (7.55%). Amongst positive patients, 64.16% were having kappa (k) type light chains and 35.84% lambda (δ) type light chains. 69.39% patients with serum M component (IgG and IgA) had Bence Jones proteinuria. Densitometric scanning revealed that majority of IgG type paraprotein was found in the slow gamma globulin region and majority of IgA type paraprotein was found equally distributed between beta and fast gamma globulin regions. Both types had decreased albumin and alpha-2-globulin concentrations as compared to normal controls. Immunoglobulin levels in patients with paraprotein had very high levels of serum IgG (6467.0 mg%) and IgA (2714.0 mg%) in respective types of monoclonal gammopathies; the rest of immunoglobulin classes were either at normal or decreased levels.     

Biochemical studies to confirm a missed case of multiple myeloma in a seventy year old male

Routine investigations of a 70 year old male led to provisional diagnosis of anemia. However further investigations suggested the possibilities of carcinoma of stomach associated with pernicious anemia, multiple myeloma and megaloblastic anemia. Finally serum protein electrophoresis supported by the clinical suspicion confirmed multiple myeloma.     

Haemoglobin electrophoresis in diagnosing a case of sickle cell anaemia associated with β-thalassemia

Alkaline haemoglobin electrophoresis is a useful tool in diagnosing β-thalassemia and sickle-cell anaemia. In this report, using this simple technique, β-thalassemia associated with sickle-cell anaemia is diagnosed. This is the first case we have diagnosed in our laboratory using agarose gel electrophoresis.     

Glycosylated haemoglobin and serum proteins in diabetics

The glycosylated haemoglobin was measured in two different categories of diabetic subjects. In poorly controlled subjects of group II, a significant alteration in serum proteins was observed. Total protein along with albumin concentrations were decreased and α₂ globulin fraction was increased. Insulin therapy resulted in normalisation of blood glucose and gradual decrease in glycosylated haemoglobin in the therapy period of eight weeks. This also resulted in a rise of total protein and albumin concentrations with a decrease in α₂ fraction. The present study indicates that prolonged therapy of insulin is needed to correct the serum protein abnormalities in diabetics.     

Audit of the Prevalence of Noncorrelation of Immunofixation with Protein Electrophoresis and Serum Free Light Chain Assays in Multiple Myeloma in a Tertiary Cancer Care Center

Multiple myeloma (MM) is diagnosed and monitored by correlating panel of test results including serum Protein electrophoresis (SPE), Immunofixation electrophoresis (IFE), serum Free Light chain (sFLC) measurements. This audit is aimed to evaluate the prevalence of non-correlation and discrepancies amongst the three investigations (SPE/IFE/sFLC) for assessment of MM. 106 MM patients were reviewed over 16 months in a tertiary cancer care center by the availability of 3 serum test results (SPE/IFE/sFLC). Patients were divided into 2 groups: group1, newly diagnosed MM patients who were yet to receive myeloma specific treatment (n = 48); and group2, already diagnosed MM patients on treatment and followup (n = 58). Treatment modalities included stem cell transplantation and standard chemotherapy regimens. Non-correlation between the three test results (IFE/SPE/sFLC) was observed (21% in group1 and 45% in group2). Three types of discrepancies were detected as follows: (a) IFE showing less number of restriction bands as compared to SPE (8.6% patients in group2); (b) SPE/IFE negative with an abnormal sFLC ratio (12.5% patients in group1 and 13.7% in group2); (c) SPE/IFE positive but normal sFLC ratio (8% in group1 and 22% in group2). To conclude, IFE may sometimes provide information that does not always correlate with either of the SPE or sFLC results due to different sensitivities, antigen–antibody interactions, or treatment. Hence, SPE plus sFLC may be more useful particularly for patients on follow-up while IFE plus sFLC may help screen the new patients. The judicious selection of the biochemical assays can effectively reduce the treatment cost in a developing country like India.     

Some biochemical markers in various neoplasms: A multivariate analysis

The analysis of biochemical parameters in 162 patients with various neoplastic disorders along with 50 normal subjects showed significant rise in serum alkaline phosphatase and lactate dehydrogenase as compared to normal subjects. 21 patients with other monoclonal gammopathies which include infection and immunological diseases were also studied. Parameters such as serum calcium, uric acid, total protein, albumin and globulin were also analyzed in 42 (26%) cases of multiple myeloma, 27 (17%) cases of gastro-intestinal malignancies, 22 (14%) cases of urogenital malignancies, 11 (6%) cases of carcinoma breast, 4 (2%) cases of bone tumors, 21 (12%) cases of other monoclonal gammopathies, including 7 (4%) cases of infection and 14 (8%) cases of immunological diseases. The results indicate use of enzymes alkaline phosphatase and lactate dehydrogenase in neoplastic disorders.     

Serum Cytokine Profile in Psoriasis-A Case–Control Study in a Tertiary Care Hospital from Northern India

Psoriasis is chronic autoimmune hyperproliferative skin disease with a population prevalence of 1.5–3%. The cause of psoriasis is still not fully understood. It has been hypothesized to be an immune-mediated disorder in which the excessive reproduction of keratinocytes is due to cytokines such as interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha, secreted by infiltrating CD4⁺ and CD8⁺ T cells and natural killer cells. The aim of our study was to determine the serum levels of TNF-α, IL-4, IL-6 & IL-10 in psoriasis patients and compare it with healthy controls. 30 clinically diagnosed psoriasis patients and 30 age and sex matched healthy controls were included in the study. The serum cytokine levels were measured by solid phase sandwich ELISA (DIACLONE Research, France). TNF-α and IL-6 levels were significantly raised in patients and the results were statistically significant (P < 0.001). IL-4 levels were higher in patients than in controls (1.91 ± 4.7 pg/ml in cases & 0.9 ± 0.3 pg/ml in controls) but were not statistically significant. Interestingly, IL-10 levels were found to be higher in controls than in patients but again, it was not statistically significant. Pro-inflammatory cytokines play a pivotal role in the pathogenesis of psoriasis and it is the type 1(TH1) cytokine pattern, i.e., IL-6 & TNF-α, which predominate in the psoriatic T cell response. Further studies on IL-10 levels in psoriasis are recommended to establish their exact role in the pathogenesis of the disease.     

No Evidence for Mutations that Deregulate GARS–AIRS–GART Protein Levels in Children with Down Syndrome

GARS–AIRS–GART is crucial in studies of Down syndrome (DS)-related mental retardation due to its chromosomal location (21q22.1), involvement in de novo purine biosynthesis and over-expression in fetal DS brain postmortem samples. GARS–AIRS–GART regions important for structure–function were screened for mutations that might alter protein levels in DS patients. Mutation screening relied on multiplex/singleplex PCR-based amplification of genomic targets followed by amplicon size determination/fingerprinting. Serum protein samples were resolved by SDS-PAGE and immunoblotted with a GARS–AIRS–GART monoclonal antibody. No variation in amplicon size/fingerprints was observed in regions encoding the ATP-binding, active site residues of GARS, the structurally important glycine-rich loops of AIRS, substrate-binding, flexible and folate-binding loops of GART or the poly-adenylation signal sequences. The de novo occurrence or inheritance of large insertion/deletion/rearrangement-type mutations is therefore excluded. Immunoblots show presence of GARS–AIRS–GART protein in all patient samples, with no change in expression levels with respect to either sex or developmental age.     

Seminal plasma levels of 15-F2α-isoprostane, malondialdehyde and total homocysteine in normozoospermic and asthenozoospermic males

It has been proposed that oxidative stress plays an important role in male infertility. The aims of this study were to compare seminal plasma levels of 15-F2t-isoprostane (8-iso-PGF2α), malondialdehyde (MDA), and total (sum of free and bound) homocysteine (tHcy) from normozoospermic vs. asthenozoospermic men, and to examine the relationships between tHcy and lipid peroxidation products. The study was a case-control study with a simple random sampling. The case group was consisted of 15 asthenozoospermic males. This group was compared with 15 normozoospermic men. Seminal plasma levels of 15-F2α-isoprostane and tHcy were measured using commercially available enzyme immunoassay (EIA) kits. MDA levels were determined by the thiobarbituric acid (TBA) assay. The Mann-Whitney U test was used to compare two groups. Coefficients of correlation were calculated using Spearman’s correlation analysis. All hypothesis tests were two-tailed with statistical significance assessed at the p value <0.05 level. MDA levels were higher in asthenozoospermic subjects than in control subjects (0.72±0.06 μM vs. 0.40±0.06 μM; p<0.05). No differences were seen in 15-F2α-isoprostane levels in asthenozoospermic subjects and controls (65.00±3.20 pg/ml vs. 58.17±4.12 pg/ml; p>0.05). Interestingly, tHcy levels were to be slightly higher in asthenozoospermic subjects than in controls (6.18±1.17 μM vs. 4.8±0.52 μM). Sperm motility was inversely correlated with seminal plasma 15-F2α-isoprostane and MDA levels, respectively (p<0.05). In summary, seminal plasma levels of 15-F2α-isoprostane and tHcy showed no significant difference between normozoospermic and asthenozoospermic men. Sperm motility was not correlated with seminal plasma levels of tHcy. No relationship was found between tHcy and lipid peroxidation.     

Tumor necrosis factor alpha (TNF-α) and estrogen hormone in osteoarthritic female patients

Osteoarthritis of knee joints is a disease of old age in both sex. It is very common after the age of 40 years in elderly females or in postmenopausal phase of females. It is characterized by narrowing of space in joints due to inflammation. The exact mechanism of inflammation in this disease is not yet clear. Tumor necrosis factor alpha (TNF-α) may involve in onset of disease. The present study is being carried out in 130 female subject of age group 40–60 years suffering from osteoarthritis of knee joints and 50 normal healthy control female subjects. A correlation is made between TNF-α and estrogen and found significant inverse correlation (r<0.001), between TNF-α and estrogen hormone in osteoarthritic female patients as compared to normal healthy control female group.     

Spurious Hyperphosphatemia in a Case of Multiple Myeloma

A 50 year old male was admitted in our hospital with anemia and impaired renal function. He was subsequently found to have extremely elevated serum phosphate level (24 mg/dL, reference interval: 2.5–4.5 mg/dL) with normal serum calcium when assayed on a Beckman Coulter AU 480^® analyser. Clinico-biochemical discrepancy led to the suspicion of spurious hyperphosphatemia. Serum total protein was grossly elevated with gross reversal of albumin to globulin ratio. Serum electrophoresis revealed a large M band and was confirmed as Ig G-Kappa type on immunofixation. Subsequently a bone marrow aspiration biopsy confirmed the diagnosis of multiple myeloma. The patient serum was then reassayed for phosphate on a Vitros^® 250 Dry Chemistry platform and the result was within normal reference interval. Paraproteinemias are a common cause of analytical interference in clinical biochemistry laboratories and as multilayered film technology platforms like Vitros^® assay most routine analytes on a protein free filtrate they are unaffected by paraprotein interference. Clinically discordant patient results should always be interpreted keeping such interferences in mind.     

Cinderella in Serum Protein Electrophoresis

Paraproteinemia is characterised by clonal proliferation of plasma cells. A common laboratory finding in paraproteinemia being a monoclonal peak in serum protein electrophoresis (M band). But there are factors which produce a peak similar to M spike in serum protein electrophoresis and these factors are known as pseudoparaproteins. This case report discusses a rare cause of pseudo M spike in a known case of autoimmune hemolytic anaemia due to administration of drug-Rituximab, a monoclonal antibody by itself.     

Investigation of unusual high serum indices for lipemia in clear serum samples on Siemens analysers Dimension

Introduction:

We present our work of monitoring 202 different patients with markedly elevated serum index for lipemia whereby serum samples were clear. We tried to clarify the cause of occurrence of these indices which were detected in the years 2006–2010 on Siemens Dimension analyzers.

Materials and methods:

In samples with unusual lipemia index we measured the concentration of lipids (total cholesterol, triglycerides, HDL and LDL cholesterol, Lp(a), ApoA1, ApoB), total proteins and checked for possible interferents (rheumatoid factor, immunoglobulins). We performed serum protein and immuno- electrophoresis. We investigated the repeatability of unusual lipemia indices during the day and after different time periods and we compared them on four different analyzers (RXL Max, Vista, Hitachi 911 and former Olympus AU640).

Results:

In 87% of 202 samples we found a monoclonal or biclonal peak in serum protein electrophoresis. Different types of paraproteins were confirmed with immunofixation electrophoresis. In the remaining 13%, polyclonal elevated concentrations of immunoglobulins were measured. Other parameters had no influence on appearing of these indices. The repeatability of indices was good during the first day of measurements (P values > 0.05) and markedly lower in the next days or after 3 and 12 months (P values < 0.05). The indices were elevated only on Dimension analyzers, but not on Hitachi and former Olympus analysers.

Conclusion:

A markedly elevated lipemia index in a clear serum sample measured on Siemens analyzers Dimension indicates a high possibility for the presence of a paraprotein in the sample.     

A study of spectrin and lipid peroxidation of red blood cell membrane in thalassaemia carrier

The aim of the present work is to understand the lipid peroxidation of RBC membrane and the spectrin protein content of RBC membrane cytoskeleton of thalassaemic carrier state (trait) of β and hemoglobin E variant (HbE). We have measured the hemoglobin (Hb), malondialdehyde (MDA) and spectrin content of RBC membrane of thalassaemic carrier. The spectrin content (α and β band) of both β and HbE carrier was not changed than normal individuals. However, lipid peroxidation of RBC membrane was significantly increased in both β and HbE trait, and Hb level was also decreased in thalassaemic carrier. It may be assumed that oxidative damage by excess lipid peroxidation may have no role on irreversible membrane damage in β thalassaemia and HbE thalassaemia carrier.