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.     

Impact of Ultra-Sensitive technology and contemporary therapy on laboratory results

The introduction of ultra-sensitive labels for immunoassays has exposed some of the limitations inherent in them. Thus, for instance a major problem with acridinium esters used, as chemiluminescence label is the formation of the so called “pseudobases” at an alkaline pH, which if not suppressed can affect the rate of the chemiluminescence reaction. Chemiluminescence labels such as luminol can also be problematic when attached to antibodies and small molecules to the extent that the sensitivity of the assay can be reduced by the decrease in the intensity of chemiluminescence. The increased use of molecular methods such as polymerase chain reaction (PCR) and post PCR methods to study mutations have various pittalls, which if unrecognized and uncontrolled can lead to incorrect results and misinterpretation. Patients who are exposed to mouse immunoglobulins through imaging or therapeutic techniques can develop antibodies to mouse immunoglobulins (human anti-mouse antibodies or HAMA) which can be a major problem for non optimized immunoassays using murine monoclonal antibodies. The types of therapy such as anticoagulant used in anticoagulant therapy, blood substitute and drug therapy can impact on the measurements of some of the biochemical and other analytes. One must separate the transient, physiological effects introduced by therapy from long-term biochemical alterations due to disease.     

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.     

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.     

Plasma proteins and proteinuria in gestational malaria

The plasma concentrations of total protein, albumin, immunoglobulins IgG, IgA and IgM and urinary protein were assayed in 250 pregnant Nigerian women with malaria and compared with 250 healthy pregnant women which served as controls. The mean values of plasma total proteins, albumin, IgG and IgA were significantly lowered (P<0.05) while a slight increase in IgM was observed in the malaria patients. Urinary proteins value of 23.10±0.50 mg/dl was obtained for the pregnant women with malaria, this was significantly higher (P<0.05) than that of the controls with the corresponding value of 15.32±0.09 mg/dl. This study has therefore demonstrated elevations of the urinary and decrease in plasma proteins in gestational malaria. These findings suggest that the protein profile should be considered in the diagnosis and treatment of malaria.     

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.     

Detection of immunoglobulins in a laser induced fluorescence system utilizing polydimethysiloxane microchips with advanced surface and optical properties

We developed an automated laser induced fluorescence system utilizing microfluidic chips for detection and quantification of immunoglobulins. Microchips were fabricated from polydimethysiloxane (PDMS) using the so-called "prepolymerization technique." The microchip structure helped minimize the effects of PDMS autofluorescence and light scattering. Furthermore, a thin and uniform PDMS layer forming the top of the microchip enabled proper focusing and collection of the excitation beam and the emitted fluorescence, respectively. The developed system was tested for the detection of mouse immunoglobulins. The capturing antibodies were immobilized on internal microchannel walls in the form of a polyelectrolyte. We clearly show that this immobilization technique, if correctly realized, gives results with high reproducibility. After sample incubation and washing, secondary antibodies labeled by fluorescein isothiocyanate were introduced into microchannels to build a detectable complex. We show that mouse antibodies can be quantified in a wide concentration range, 0.01-100 μg ml(-1). The lower detection limit was below 0.001 μg ml(-1) (6.7 pM). The developed laser induced fluorescence (LIF) apparatus is relatively cheap and easy to construct. The total cost of the developed LIF detector is lower than a typical price of plate readers. If compared to classical ELISA (enzyme linked immunosorbent assay) plate systems, the detection of immunoglobulins or other proteins in the developed PDMS microfluidic device brings other important benefits such as reduced time demands (10 min incubation) and low reagent consumption (less than 1 μl). The cost of the developed PDMS chips is comparable with the price of commercial ELISA plates. The main troubleshooting related to the apparatus development is also discussed in order to help potential constructors.     

Herceptin functionalized microfluidic polydimethylsiloxane devices for the capture of human epidermal growth factor receptor 2 positive circulating breast cancer cells

Building on recent breakthroughs in the field of microfluidic-based capture of rare cancer cells circulating in the blood, the present article reports on the use of Herceptin functionalized PDMS devices designed to efficiently capture from blood cancer cells, overexpressing the tyrosine kinase human epidermal growth factor receptor (HER2). The identification of patients overexpressing HER2 is critical as it typically associates with an aggressive disease course in breast cancer and poor prognosis. Importantly, HER2 positive patients have been found to significantly benefit from Herceptin (Trastuzumab), a humanized monoclonal antibody (MAb) against HER2. Disposable PDMS devices prepared using standard soft lithography were functionalized by the plasma polymerization of an epoxy-containing monomer. The epoxy-rich thin film (AGEpp) thus created could be conjugated with Herceptin either directly or through a polyethylene glycol interlayer. The properties and reactivity toward the monoclonal antibody conjugation of these coatings were determined using x-ray photoelectron spectroscopy; direct conjugation provided a good compromise in reactivity and resistance to biologically nonspecific fouling and was selected. Using the breast cancer cell line SK-BR-3 as a model for cells overexpressing HER2, the immunocapture efficacy of the Herceptin functionalized PDMS was demonstrated in model studies. Validation studies confirmed the ability of the device to efficiently capture (～80% capture yield) HER2 positive cells from full blood.     

Immunodiagnosis of human lymphatic filariasis: A review

A monoclonal antibody-based antigen detection system was used to detect the levels of circulating antigen in filarial patients before and after treatment with DEC and in normal individuals living in an area endemic forW. bancrofti infection in Chennai, India. The present study was to show the use of this assay as a means of efficient screening for filariásis in an endemic area where blood was absorbed onto the filter paper by finger prick during day time. The results of the antigen levels collected onto filter strips correlated with their corresponding plasma antigen levels (r=0.83). In microfilaraemics, DEC treatment did not alter the levels of circulating antigens upto a period of one month. We conclude that this monoclonal antibody based ELISA using filter strips may be used in daytime and can replace the existing routine night blood survey.     

Efficacy of a Fish Protein Hydrolysate in Malnourished Children

Protein hydrolysates are good nutritional supplements as their bioactive ingredients can be easily absorbed and utilized for various metabolic activities. A fish protein hydrolysate (Amizate), prepared by a unique process of hydrolysis has the advantage of high di/tri peptide content (<10 kDa) along with essential and non essential amino acids, micronutrients and vitamins. The effect of Amizate on malnourished children (6–8 years, a total of 438) of Grade I and II (Gomez’s classification) with respect to immunoglobulins, CD4/CD8 ratios and hemoglobin was examined. Measurement of these parameters during the user trial study (at the beginning and the end after 4 months) indicated that the levels of the immunological parameters were not significantly altered by the Amizate treatment. The values of immunoglobulins and CD4/CD8 ratios of malnourished children (India) are in the normal range and are in accordance with the reported values of various ethnic groups.     

Phaseguide-assisted blood separation microfluidic device for point-of-care applications

We propose a blood separation microfluidic device suitable for point-of-care (POC) applications. By utilizing the high gas permeability of polydimethylsiloxane (PDMS) and phaseguide structures, a simple blood separation device is presented. The device consists of two main parts. A separation chamber with the phaseguide structures, where a sample inlet, a tape-sealed outlet, and a dead-end ring channel are connected, and pneumatic chambers, in which manually operating syringes are plugged. The separation chamber and pneumatic chambers are isolated by a thin PDMS wall. By manually pulling out the plunger of the syringe, a negative pressure is instantaneously generated inside the pneumatic chamber. Due to the gas diffusion from the separation chamber to the neighboring pneumatic chamber through the thin permeable PDMS wall, low pressure can be generated, and then the whole blood at the sample inlets starts to be drawn into the separation chamber and separated through the phaseguide structures. Reversely, after removing the tape at the outlet and manually pushing in the plunger of the syringe, a positive pressure will be created which will cause the air to diffuse back into the ring channel, and therefore allow the separated plasma to be recovered at the outlet on demand. In this paper, we focused on the study of the plasma separation and associated design parameters, such as the PDMS wall thickness, the air permeable overlap area between the separation and pneumatic chambers, and the geometry of the phaseguides. The device required only 2 μl of whole blood but yielding approximately 0.38 μl of separated plasma within 12 min. Without any of the requirements of sophisticated equipment or dilution techniques, we can not only separate the plasma from the whole blood for on-chip analysis but also can push out only the separated plasma to the outlet for off-chip analysis.     

Comparative analysis of RBC membrane fatty acids, proteins and glycophorin in patients with heterozygous beta thalassemia and iron deficiency anemia

Membrane lipid and protein composition was compared in erythrocytes from iron deficiency anemia (IDA) and heterozygous beta thalassemia patients. The study was planned to correlate the influence of iron deficiency with the intrinsic defect of the heterozygous condition on the membrane structural integrity as well as to investigate whether there are differences in membrane changes between the two conditions. Results indicate high levels of saturated fatty acids and low unsaturated fatty acids in both disorders although arachidonic acid and the unsaturation index were lower in heterozygous thalassemia than IDA. Nevertheless, neither of the conditions provoked any alterations in membrane protein or glycophorin suggesting alterations in the lipid moiety only. Present findings indicate that irrespective to the etiology, both, iron deficiency and the heterozygous condition show a common pattern of lipid derangement, which may in turn result in increased membrane rigidity and decreased cellular deformability.     

Detection of L1 CAM mutation in a male child with mental retardation

Recent studies have presented evidence for the involvement of L1CAM gene mutations in various X-linked mental retardation syndromes. The neural cell adhesion molecule, L1CAM is a transmembrane protein belonging to the super family of the immunoglobulins that play a key role in embryonic development of the nervous system and is involved in memory and learning. No studies were carried out from India on L1 CAM gene in X-linked mental retardation syndromes. Hence, an investigation was taken up to delineate the role of L1CAM gene in mental retardation. Two families (Family I and Family II) having only two members affected with mental retardation in each family were studied for mutations in L1CAM gene. In family II, the younger sibling showed deletion involving region between the nucleotide 13,773 (intron 25) and 14,158 (intron 27) region. The mutation what we observed in younger sibling of the family II is a novel mutation which was not hitherto reported in the world literature.     

Enhanced expression of HMG-Y proteins in proliferating tissues

The high mobility group (HMG) proteins I and Y are well characterised non-histone chromosomal proteins which bind to A-T rich regions of DNA and regulate gene expression and/or DNA replication. A correlation has been demonstrated between the increased expression of HMG-Y proteins and malignancy. However, it is not known whether the expression of HMGs particularly, the Y group, is a function of proliferation rate. In the present study, we have used normal tissues of calf testes, thymus and liver. The results show distinctly high expression of HMG-Y proteins in testes than in thymus and the expression was practically undetectable in liver. The results suggest that even in normal tissues there is a direct correlation between the proliferation rate and the expression of the HMG-Y proteins, which can partly explain its increased expression in cancer.     

Microfluidic point-of-care blood panel based on a novel technique: Reversible electroosmotic flow

A wide range of diseases and conditions are monitored or diagnosed from blood plasma, but the ability to analyze a whole blood sample with the requirements for a point-of-care device, such as robustness, user-friendliness, and simple handling, remains unmet. Microfluidics technology offers the possibility not only to work fresh thumb-pricked whole blood but also to maximize the amount of the obtained plasma from the initial sample and therefore the possibility to implement multiple tests in a single cartridge. The microfluidic design presented in this paper is a combination of cross-flow filtration with a reversible electroosmotic flow that prevents clogging at the filter entrance and maximizes the amount of separated plasma. The main advantage of this design is its efficiency, since from a small amount of sample (a single droplet  ～ 10 μl) almost 10% of this (approx 1 μl) is extracted and collected with high purity (more than 99%) in a reasonable time (5–8 min). To validate the quality and quantity of the separated plasma and to show its potential as a clinical tool, the microfluidic chip has been combined with lateral flow immunochromatography technology to perform a qualitative detection of the thyroid-stimulating hormone and a blood panel for measuring cardiac Troponin and Creatine Kinase MB. The results from the microfluidic system are comparable to previous commercial lateral flow assays that required more sample for implementing fewer tests.     

Evaluation of serum zinc level and plasma SOD activity in senile cataract patients under oxidative stress

An imbalance in the systemic redox status leading to oxidative stress has been an important factor in development of senile cataracts, which is reflected by an increase in serum TBARS and a decrease in plasma SOD activity. Zinc has been an important cofactor required for structural stability of SOD. In the present study the role of serum zinc level and plasma SOD activity was analyzed in senile cataract patients showing significant oxidative stress. Serum TBARS, plasma SOD and serum zinc level was measured in thirty randomly selected senile cataract patients against properly matched controls. Although, the analysis of means showed a significant increase in serum TBARS and decrease in plasma SOD and serum zinc level in cases, but plasma SOD was found to be just significantly correlated (p=0.05) with the serum zinc only in the cases. The results of partial correlation studies and multiple regression analysis, also, showed only a significant correlation and predictable dependence between serum TBARS and plasma SOD, excluding any role of serum zinc level. The present study concludes that it is chiefly the plasma SOD activity, but not the serum zinc level, that determines the proneness of the patients for development of senile cataract.     

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.     

Stability considerations in tumor marker measurements

Approaches to the stabilization of tumor markers vary depending on the lability of the tumor marker. Thus while merely freezing the serum promptly at ?70°C may be adequate for some analytes such as gastrin, some other polypeptide hormones such as vasoactive intestinal peptide require collection of blood in EDTA and prompt freezing of plasma to protect the analyte from metal oxidation. In addition to collection of blood in EDTA an addition of a proteolytic enzyme inhibitor such as aprotinin may be necessary to preserve labile polypeptide hormones such as ACTH, somatostatin etc. Analytes such as parathyroid hormone related protein (PTH-rp) are so labile that additives such as leupeptin and pepstatin have to be added to EDTA and aprotinin and the plasma refrigerated to achieve stability for up to 24 hrs. Catecholamines also require stabilization with additives such as EGTA and glutathione and prompt freezing of plasma at ?70°C. Special precautions are required in handling tumor tissue intended for hormone receptor measurements since they are not only extremely heat labile but are also subjected to alteration during specimen preparation.     

Multiple Myeloma: A Case of Atypical Presentation on Protein Electrophoresis

Multiple myeloma is a group of B-cell disorders resulting in the secretion of a specific and unique monoclonal immunoglobulin (M-protein). Protein electrophoresis is advised whenever multiple myeloma is suspected. The monoclonal protein migrates as a single entity in the electric field and is detected by the non-specific protein stain as a more intensely stained band superimposed on the usual protein pattern. The M-protein usually migrates in the gamma or beta region of the normal protein pattern; very rarely it may appear in the α2 or even in α1 region. Here we have given an atypical case presentation where the patient with multiple myeloma presented with two M-spike one each in α2 and β-globulin region on agarose gel protein electrophoresis with hypoglobulinemia but with reversed A:G ratio.