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.     

Separation of serum LD isoenzymes by capillary electrophoresis

Separation and quantitative estimation of the isoenzymes of lactate dehydrogenase(LD) in serum were accomplished with capillary electrophoresis system. An uncoated fused silica capillary column 50 cm long, 75μm I.D. and substrate containing running buffer including L-lactic acid and NAD⁺ were used for the separation of serum LD isoenzymes. The resulting product of “NADH” was detected at 340 nm. Injection of 10 nL of five fold diluted serum sample were performed by pressure injection within 2 seconds. The isoenzymes were separated at 10 kV of voltage for 5 min, by turning off the voltage applied for 30 min incubation at 24°C for reaction between substrate and isoenzymes, and applying voltage of 30 min. Under these conditions, the isoenzymes of LD were detected by a NADH generated as isoenzyme of LD-5 emerged at 20 min, LD-1 peak at 23.5 min with close to baseline separation of the other isoenzymes which emerge between LD-5 to LD-1, after the emergence to LD-1 peak, followed another peak, termed “sample shock”: The results obtained by the proposed method correlated well with those by gel electrophoresis systemes (r=0.92∼0.98) for each five LD isoenzymes, respectively. Within-run precision CVs for 5 replicate analysis were 3.01 (LD-3, mean 14.6%)%∼7.82% (LD-4, mean 4.22%.), respectively.     

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.     

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.     

Serum glycoconjugates and ceruloplasmin in cancer of uterine cervix

The present study was done to assess the levels of glycoconjugates and ceruloplasmin in sera of patients with cervical cancer. Serum hexoses, hexosamines, sialic acid and fucose are elevated in a variety of inflammatory and neoplastic conditions. All the glycoconjugates, except fucose were increased in serum of patients compared to controls. Also, hexoses and sialic acid levels were high in patients with later stages of cancer compared to patients with early stage disease (P=<0.0001, P=0.03). Serum ceruloplasmin was increased in patients with early stage cancer (51.5mg/dl) and with late stage cancer (61mg/dl) compared to controls (38mg/dl). The elevated glycoconjugates may be the result of inflammatory reaction associated with neoplasia, as serum ceruloplasmin (an acute phase reactant) is also increased in these patients.     

A preliminary report on the role of yoga asanas on oxidative stress in non-insulin dependent diabetes mellitus

Nineteen subjects of non-insulin dependent diabetes mellitus (NIDDM) between the age group of 30–60 yrs were studied to see the effect of specific yoga asanas on fasting and postprandial blood glucose (FBG, PPG), serum malondialdehyde (MDA) and glycosylated hemoglobin (HbA₁) in addition to drug treatment and diet control. The duration of diabetes ranged from 1–10 years. Patients with renal, cardiac and proliferative retinal diseases were excluded from the study. The same patients served as their own control. Subjects were called in the morning to the cardio-respiratory laboratory and were given training by a yoga expert. Yoga asanas included Suryanamskar, Tadasan, TriKonasan, Padmasan, Pranayam, Paschimottanasan, Ardhmatsyendrasan, Pavanmukthasan, Sarpasan and Shavasan. The asanas were done every day for 40 days for 30–40 min. FBG, PPG, serum MDA and HbA₁ were estimated before and after 40 days of yoga asanas regimen. Significant reduction was seen in FBG from 220 mg/dl to 162 mg/dl, PPG from 311 mg/dl to 255 mg/dl, MDA from 6 nmol/l to 3 nmol/l and HbA₁, from 8.8% to 6.4%. Subjects felt better and were relieved of their stresses and had an improvement in their day to day performance. The decrease was statistically significant (p<0.0001 for FBG and PPG, p<0.001 for MDA and for HbA₁).     

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.     

Abnormal gel flotation in a patient with apperant pneumonia diagnosis: a case report

Introduction:

Serum blood collection tubes with separator gel are widely used by many laboratories for chemistry analyses. We describe a case of a primary blood collection tube filled with blood sample and a floating separator gel.

Materials and methods:

The blood sample was collected from a 51 years old female in intensive care unit with the diagnosis of pneumonia into a BD Vacutainer SST tube (Becton Dickinson, NJ, USA) containing serum separator gel and conveyed to the core laboratory of Marmara University Hospital within 30 minutes from collection. Sample was immediately centrifuged at room temperature at 1500 × g for 10 minutes.

Results:

The analyses revealed a highly increased total protein concentration of 145 g/L (reference interval 64–83 g/L). The nephelometric analyses showed an elevated serum IgG concentration of 108 g/L (reference interval 6.5–16 g/L) and IgG lambda monoclonal band was determined by serum immunofixation electrophoresis.

Conclusion:

Limitation of the separator gel tubes in patients with a high plasma density and its possible effects on test results and laboratory costs should be remembered. The clinical diagnosis stated in the information system should also reveal known comorbid conditions besides the apparent admission reason. This information would avoid resampling, additional testing, and communication efforts with the clinicians.     

Higher alleles of apolipoprotein B gene 3′ VNTR: Risk for gallstone disease

Background: Imbalance in cholesterol homeostasis may lead to gallstone disease. Apolipoprotein B is sole component of low-density lipoprotein and plays an important role in cholesterol metabolism. The present study was carried out to explore the association of APOB 3′ VNTR, exon 26 XbaI and signal peptide insertion/ deletion polymorphisms with gallstone disease. 214 ultrasonographically proven gallstone patients and 322 healthy, age and sex matched controls were taken for the study. Genotyping was done using PCR followed by polyacrylamide gel electrophoresis for VNTR and insertion/ deletion analysis. For APOB XbaI polymorphism PCR product was digested with XbaI restriction enzyme, followed by agarose gel electrophoresis. All statistical analyses were done using SPSS v11.5. Higher repeat alleles of APOB 3′ VNTR polymorphism were more frequent in gallstone patients than in controls. Alleles with more than 57 repeats were present only in patient group. Long (L) alleles with repeat higher than 49, were significantly higher (P=0.000; OR=3.705, 95% CI 2.577–5.326) and medium (M) alleles were lower (P=0.000; OR=0.406, 95% CI 0.304–0.542) in patients than in controls. To nullify the effect of gender, data was further stratified into male and female population. APOB 3′ VNTR, L alleles were imposing risk and M alleles were protective in both male and female population. APOB XbaI and insertion/deletion polymorphisms were not found to be associated with the gallstone disease. Longer alleles of APOB 3′ VNTR occur more frequently in gallstone patients, and may be an important risk factor for the development of gallstone disease. APOB XbaI and signal peptide insertion/deletion polymorphisms may not be contributing to the risk for gallstone disease.     

Three Cases of Lead Toxicity Associated with Consumption of Ayurvedic Medicines

Ayurveda is a traditional form of medicine used by majority of the Indians. Here we report three cases of lead toxicity, following intake of Ayurvedic medicines. Three patients presented with blood lead levels (BLLs) of 122.4, 115 and 42.8 μg/dl respectively at the time of hospitalization. The first case was chelated with D- penicillamine, the second with calcium disodium ethylene diamino tetra acetate (EDTA) and the third with environmental intervention and education. Associated Ayurvedic products were collected from patients and analyzed for metallic concentration. Cessation of Ayurvedic medication along with chelation, nutritional intervention and education, reduced the BLL to 27.4 μg/dl in the first case after 1 year, 21.1 μg/dl after 9 months in the second and 18.2 μg/dl after 6 months in the third case.     

Standardization of a colorimetric method for the determination of fructose using o-cresol: Sulphuric acid reagent

Fructose developed a pinkish orange chromogen on treatment with o-cresol: 70% sulphuric acid at 32°C for 15 minutes with a λ max of 500nm. Fructose was 185 times more chromogenic than glucose. Total carbohydrate and fructose values in protein-free filtrate of normal serum samples were in the range, 55.4–86.3 mg/dl and 1.55–3.29 mg/dl, respectively. In diabetes, the observed values were 197–354 mg/dl and 2.91–6.81 mg/dl, respectively.     

Impact of Chronic Lead Exposure on Selected Biological Markers

Lead poisoning remains a major problem in India due to the lack of awareness of its ill effects among the clinical community. Blood lead, δ-aminolevulinic acid dehydratase (δ-ALAD) and zinc protoporphyrin (ZPP) concentrations are widely used as biomarkers for lead toxicity The present study was designed to determine the impact of chronic lead exposure on selected biological markers. A total of 250 subjects, of both sexes, ranging in age from 20 to 70 years, were recruited. On the basis of BLLs, the subjects were categorized into four groups: Group A (BLL: 0–10 μg/dl), Group B (BLL: 10–20 μg/dl). Group C (BLL: 20–30 μg/dl) and Group D (BLL: 30–40 μg/dl) having BLLs of 3.60 ± 2.71 μg/dl, 15.21 ± 2.65 μg/dl, 26.82 ± 2.53 μg/dl and 36.38 ± 2.83 μg/dl, respectively. Significant changes in biological markers due to elevated BLLs were noted. The relation of BLL and biological markers to demographic characteristics such as sex, habits, diet and substances abuse (smoking effect) were also studied in the present investigation. Males, urban population, non-vegetarians, and smokers had higher blood lead levels. δ-ALAD activity was found to be significantly lower with increased BLL (P < 0.001), while the ZPP level was significantly higher with increased BLL (P < 0.001). Further, BLL showed a negative correlation with δ-ALAD (r = −0.425, P < 0.001, N = 250) and a positive correlations with ZPP (r = 0.669, P < 0.001, N = 250). Chronic lead exposure affects the prooxidant-antioxidant equilibrium leading to cellular oxidative stress.     

Serum lipids in hypothyroidism: Our experience

In order to determine whether the screening of lipid profile is justified in patients with hypothyroidism we estimated serum lipids in cases having different levels of serum TSH. 60 patients of hypothyroidism in the age group of 20 to 60 yrs were studied for thyroid profile over a period of one year. On the basis of serum TSH level the cases were divided into three groups: In the first group TSH concentration was 8.8±2.99 μlU/ml, 95% confidence interval (Cl) 8.8±1.07, whereas serum total cholesterol and LDL-chol levels were 196±37.22 and 126±29.17 mg/dl respectively. The statistical analysis of these two groups showed a significant correlation between raised TSH levels and serum total cholesterol and LDL-chol (P<0.05 & P<0.01) respectively. We conclude that hypothyrodism is associated with changes in lipid profile.     

Interference of anti-T3 autoantibodies in the measurement of total and free T3 in serum

A case of anti-trilodothyronine autoantibodies is presented in this report. The thyroid hormone profile of a hypertensive patient, with multinodular goiter and history of exogenous thyroid hormone therapy, was found to be highly ambiguous. The total as well as free T3 levels were consistently high (out of range) whereas the T4 (total and free) values were always within normal limits. The thyrotropin was found to be partially suppressed. Very low T3-Uptake indicated some kind of interference in the immunoassays. We incubated the sera with the radio-iodine labelled T3 and observed that the patient's serum bound about ten times more radioactivity than a control run in parallel. On further resolving the serum proteins on cellulose acetate electrophoresis, the radioactivity was detected in the γ-globulin band. Therefore it was established that the patient's serum carried the antibodies reactive with T3 which were interfering in the immunoassays. Elevated anti-thyroid microsomal antibodies were also present in the patient's serum. The anti-T3 antibodies were highly specific for T3 and did not show any cross reactivity with the T4 or its analogues used in the estimation of free T4.     

Blood zinc protoporphyrin, serum total protein, and total cholesterol levels in automobile workshop workers in relation to lead toxicity: Our experience

Blood zinc protoporphyrin (ZPP), serum total protein (TP), and total cholesterol (TC) levels in automobile workshop workers in relation to lead toxicity were analysed. In the present study, automobile workshop workers (healthy male workers at an age between 28 and 35 from four major automobile workshops in Kottayam, Kerala State, India) and the control (male healthy adults at an age between 28 and 35 residing at Aymanam, a distant village at Kottayam District, Kerala having reduced or no chance of lead exposure) displayed significant difference in blood lead (BPb) and blood ZZP (BZPP) level. The mean value of BPb in automobile workshop workers was 15.76±0.33 μg/dl, while in the control it was 8.20±0.15 μg/dl. In automobile workshop workers, the mean value of BZPP was 34.2±0.62 μg/dl. The control group exhibited a mean of 11.5±0.22 μg/dl. Automobile workshop workers exhibited significant increase in BZPP was corresponding to the increase in BPb level. The total protein levels estimated in automobile workshop workers showed significant decrease compared to control individuals, but was within the reference range of healthy individuals. The mean value of TP level in automobile workshop workers and control was 6.9±0.13 g/dl and 7.71±0.18 g/dl, respectively. There was no significant difference in blood haemoglobin (BHb) level among the automobile workshop workers and control. The serum TC level in automobile workshop workers showed significant decrease compared to the control individuals, but was with in the reference range of healthy individuals. The mean level of serum TC in automobile workshop workers was 162.00±3.44 mg/dl and the same in control was 172.86±4.32 mg/dl. The present study affirms occupational lead toxicity in automobile workshop workers and its effect on serum protein and cholesterol levels.     

Interference of anti-T4 autoantibodies in the single step free T4 radioimmunoassays

The thyroid hormone profile of a hypothyroid patient, with moderate sized diffuse goiter and history of exogenous thyroid hormone therapy, was found to be highly ambiguous. The total as well as free T4 levels were consistently high (out of range) with suppressed T3 (total and free) and elevated thyrotropin levels. We incubated the serum with the radio-iodine labelled T4 and observed that the patient’s serum bound about 50.5% of the total radioactivity resulting in 85.3% interference in the radioimmunoassay. On resolving the serum proteins by cellulose acetate electrophoresis, the radioactivity was detected in the gamma globulin band. The protein profile showed mildly raised levels of IgG. Therefore it was established that the patient’s serum carried T4 reactive antibodies (IgG) which were interfering in the immunoassays. Elevated anti-thyroid microsomal antibodies were also present in the patient’s serum. The anti-T4 antibodies were highly specific for T4 and did not show any cross reactivity with the T3 or its analogues used in the estimation of free T3. We stripped the serum by precipitating the immunoglobulins and were successful in estimating the levels of free T4 in the stripped serum by single step radioimmunoassy.     

Interleukin-1 receptor antagonist gene polymorphism and obesity: A pilot study from north India

The main adverse consequences of excess bodyweight are cardiovascular disease, type II diabetes, and several cancers, IL-1Ra serum concentration has been reported earlier to increase in human obesity and it is therefore assumed that the polymorphism of IL-1Ra may influence cytokine production. We designed this study to investigate whether the IL-1Ra polymorphism was associated with obesity. A total number of 103 individuals; 19 lean (BMI<25 Kg/m²), 51 overweight (BMI 25–29.9 Kg/m²) and 33 obese (BMI≥30.0 Kg/m²) were enrolled in this study. Genotyping was performed using a polymerase chain reaction PCR amplification of the intron-2 fragment harboring a variable number of tandem repeat (VNTR) nucleotide sequences 86 pb of tandem repeat. The PCR products were separated on 2% agarose gel. Statistical analysis was performed using SPSS software (version 11.5). We found no significant difference in genotype and allele frequencies between the three groups; lean vs. overweight and lean vs. obese (p=0.323; 0.202; 0.123 and 0.068 resp). However, an increased risk for obesity had a propensity to be higher in those having genotype II/II. This genotype has been reported to be a ‘high producer’ of IL-1Ra. Although no statistically significant relationship between IL-1Ra polymorphism and BMI was observed, however, a trend towards an increase of allele^*II in overweight and obese group was observed. This may suggest that IL-1Ra appears to be induced by inflammatory stimuli as well as obesity-associated factors. This is relatively a pilot study: but nevertheless, may assist in identifying the pathophysiological cause for obesity.     

Comparison of two methods of estimation of low density lipoprotein cholesterol, the direct versus friedewald estimation

Current recommendations of the Adult Treatment Panel and Adolescents Treatment Panel of National Cholesterol Education Program make the low-density lipoprotein cholesterol (LDL-C) levels in serum the basis of classification and management of hypercholesterolemia. A number of direct homogenous assays based on surfactant/solubility principles have evolved in the recent past. This has made LDL-C estimation less cumbersome than the earlier used methods. Here we compared one of the direct homogenous assays with the widely used Friedewald’s method of estimation of LDL-C to see the differences and correlation. We used direct homogenous assay kit to estimate serum LDL-C and high-density lipoprotein cholesterol (HDL-C). Serum Triglyceride (TG) and Total Cholesterol (TC) was estimated and using Friedewald’s formula LDL-C was calculated. The LDL-C level obtained by both methods in 893 fasting serum samples were compared. The statistical methods used were paired t-test and Pearson’s correlation. There was significant difference in the mean LDL-C levels obtained by the two methods at the TG levels <200 mg/dl (p<0.02) and TC levels >150 mg% (p<0.001). The correlation coefficient (r) between Friedewald’s and direct assay estimation was 0.88. Friedewald’s method classified 23.5 % of patients as high cardiac risk whereas there were 17.58% by direct assay. Both had good correlation even though the serum triglyceride and total cholesterol levels affect the difference in LDL-C estimated by both methods. Taking into account the cost and performance, Friedewald’s method is as good or even better for classifying and managing patients.     

Association of plasma level of thiobarbituric acid reactive substances with extent of hepatocellular injury in preterm infants with cholestatic jaundice

The present study was designed to determine the association between extent of hepatocellular injury and plasma level of thiobarbituric acid reactive substances (TBARS) in pre term infants with cholestasis. Preterm infants (<35 weeks gestation) admitted to the neonatal intensive care unit were enrolled (with their parents informed consent) in either the ‘cholestasis’ group (if their direct bilirubin was >2 mg/dl) (n=25) or in the control group (n=16). Blood samples for measurement of TBARS, direct bilirubin and transaminases were obtained with-in 24 hours of enrollment. The cholestasis and control groups were comparable with respect to gestational age, birth weight and Apgar score. Serum direct bilirubin, SGOT (EC 2.6.1.1) and SGPT (EC 2.6.1.2) levels were significantly high in the cholestasis group. Plasma levels of TBARS in cholestasis group were correlated with SGOT (F=276.92; P<0.0001) and SGPT (F=355.17; P<0.0001) and differed significantly between cholestatic and control infants. Our findings suggest that oxidative stress in preterm infants with cholestasis is associated with hepatocellular injury.