[Analysis of serum free light chains κ/λ ratio and heavy/light chain pairs of immunoglobulin to the stratification of multiple myeloma according to Mayo Stratification of Myeloma and Revised International Staging System]. / Analýza vztahu volných lehkých retezcu κ/λ a páru tezkých/lehkých retezcu imunoglobulinu ke stratifikaci mnohocetného myelomu podle Mayo Stratification of Myeloma and Revised International Staging System.

INTRODUCTION: Assessment of serum levels of free light chains (FLC-κ and FLC-λ) and recently heavy/light chain pairs of immunoglobulin (HLC-κ and HLC-λ) and their ratio (FLC-r and HLC-r) has significantly enriched traditional algorithm of multiple myeloma (MM) evaluation. The aim of the presented study was to assess the relationship of classical prognostic parameters of MM, standard FLC-κ/λ and HLC-κ/λ ratio ((s)FLC-r and (s)HLC-r), modified ratio of "involved/uninvolved" FLC and HLC ((m)FLC-r and (m)HLC-r ), the difference between "involved - uninvolved" FLC and HLC (FLC-dif. and HLC-dif.) to current stratification models of MM based on the result of cytogenetic analysis. PATIENTS AND METHODS: In a group of 97 patients with MM we assessed serum levels of FLC by FreeliteTM method, and we calculated (s)FLC-r, (m)FLC-r and FLC-dif. indices by HevyliteTM method. For cytogenetic analysis we used FICTION (fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms). For MM stratification we used standard staging systems according to Durie-Salmon (D-S) and International Staging System (ISS) as well as novel stratification systems based on the results of cytogenetic analysis, ie. "Mayo Stratification of Myeloma and Risk-Adapted Therapy" (mSMART) and "Revised International Staging System" (R-ISS). RESULTS: Stratification mSMART and R-ISS has significantly different representation of "standard" or "low-risk" (71, 15.5, 11.3 a 29.9 %), "intermediate risk" (15.5, 53.6, 34 a 33 %) and "high risk" patients (13.4, 30.9, 54.7 a 37.1 %) compared to standard staging systems. mSMART stratification was compared to prognostic factors of MM (Hb, albumin, ß(2)-M, creatinine and LDH), and the only significant relationship was found in the case of ß(2)-M, R-ISS had relationship only to Hb and creatinine. In the case of D-S staging we found significant relationship of stages 1-3 and substages A and B to the levels of (m)FLC-r, FLC-dif. and (m)HLC-r, ISS had moreover relationship to k HLC-dif. and MIg concentration. Analysis of mSMART stratification showed primarily significant relationship of risk categories 1-3 to (m)FLC-r and (s)HLC-r indices, and R-ISS to (m)HLC-r index and MIg concentration. In both cytogenetics-based stratifications there was a lack of relationship to (s)FLC-r, FLC-dif. and HLC-dif. indices. CONCLUSION: Comparison of the results of standard staging systems according to D-S and ISS with cytogenetics based models mSMART and R-ISS showed different representation of risk groups, and significantly different relationship to classical prognostic factors together with original relationship of sMART stratification to (m)FLC-r and (s)HLC-r, and R-ISS to (m)HLC-r and MIg concentration.

Relationship of differences in immunoglobulin heavy/light chain pairs (Hevylite), selected laboratory parameters and stratification systems in different immunochemical types of multiple myeloma.

BACKGROUND AND AIMS: Advances in the diagnosis and treatment of multiple myeloma (MM), place increasing demands on accurate stratification of patients as the starting point for optimal individualized therapy. The present study focused on assessing the association between HLC levels and the HLC-r to parameters of MM activity, prognosis and tumor mass volume.The objective was to assess the correlation of immunoglobulin (Ig), heavy/light chain (HLC) pairs (IgG-κ and-λ, IgA-κ and -λ HLC) and the ratio of monoclonal involved-HLC (i-HLC) to polyclonal uninvolved (u-HLC) Ig concentrations assessed by the Hevylite(TM) method with the free light chain κ/λ ratio (FLC-r), selected prognostic laboratory parameters i.e. Hb, platelets, albumin, ß2-microglobulin (ß2-M), Ca, lactate dehydrogenase (LDH), creatinine and the Durie-Salmon (D-S) and International Staging System (ISS), stages (1-3) for MM. METHODS: Hevylite assays were done on the sera of 132 MM patients at the time of diagnosis (IgG 94, IgA 38). HLC-r was calculated in the case of i-HLC-κ from the i-HLC-κ/u-HLC-λ ratio and for i-HLC-λ from the i-HLC-λ/u-HLC-κ ratio. D-S and ISS stages were evenly distributed. RESULTS: Md IgG-κ HLC-r was 64.8 (2.7-2222) and of IgG-λ HLC-r 49.6 (0.7-465.1), in the case of IgA-κ, Md HLC-r was 408.9 (3.4-3966) and for IgA-λ HLC-r the Md was 180.0 (0.1-3110). Normal levels of HLC pairs and HLC-r did not always rule out the diagnosis of MM. HLC-r correlated with FLC-r in IgG (r = 0.244, P = 0.018), but not in the IgA type. For IgG, HLC-r values were significantly different in patients with abnormal vs normal levels of Hb (P < 0.0001), albumin (P < 0.043), ß2-M (P < 0.0001) and creatinine (P = 0.034) but not thrombocyte count, Ca or LDH. For the IgA isotype, we found a significant difference in HLC-r values only for thrombocyte count (P = 0.026) and ß2-M (P = 0.016) but not for Hb, albumin, Ca, LDH or creatinine. For the IgG isotype there was a significant relationship of HLC-r index to stages 1-3 (P = 0.038) and substage A vs B (P = 0.048) according to D-S, and with high significance to stages 1-3 according to ISS (P = 0.005) and between stages 1 vs 3 (P = 0.001). For the IgA isotype, we found significant differences in HLC-r only between stages 1-3 (P = 0.025) according to D-S but not in the case of ISS. There were no significant correlations between i-HLC Ig levels and D-S or ISS stages in both IgG-κ and λ and IgA-κ and λ. Exceptions were significant differences for stages 1 vs 3 (P = 0.012) and 2 vs 3 (P = 0.017) for the IgG-λ isotype. There were no correlations of the HLC-r and u-HLC levels for either D-S or ISS stratifications in all HLC isotypes. CONCLUSION: We found a significant positive contribution of HLC-r using the i-HLC/u-HLC ratio even in the case of i-HLC-λ i.e. i-HLC-λ/u-HLC-κ. Variable results for the relationship of important laboratory parameters and D-S and ISS stratifications (stage 1-3) to HLC-r values in IgG and IgA isotypes make separate interpretation of the Hevylite method results necessary in clinical practice.

[Analysis of serum levels of Dickkopf-1 (DKK-1) in monoclonal gammopathy of undetermined significance and multiple myeloma]. / Analýza sérových hladin Dickkopf-1 (DKK-1) u monoklonální gamapatie nejistého významu a mnohocetného myelomu.

BACKGROUND: Several recent studies aim at the detection of biological parameters that enable more precise diagnostics and stratification of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). The objective of our study was to assess the potential contribution of serum levels of Dickkopf-1 (DKK-1) in MGUS and MM from the point of more specific differentiation of both conditions, and the relationship of DKK-1 to selected laboratory parameters, individual forms and clinical stages of both conditions. METHODS AND RESULTS: The analyzed cohort consisted of 46 individuals with MGUS and 152 patients with MM at the time of diagnosis. For the assessment of serum levels of DKK-1 we used ELISA method. We assessed also serum levels of free light chains (FLC) κ and λ using the Freelite system, and ß2-microglobulin (ß2-M) using the Immulite 1000 method. For statistical estimation we used: Pearson χ2-test, U-test according to Mann-Whitney and Kruskal-Wallis test. Our analysis revealed that there was no significant difference between the levels of DKK-1 in MGUS risk groups (0-3) and between the states with different FLC concentration including the κ/λ index of monoclonality. In MM there was a significant relationship of DKK-1 to the level of hemoglobin (p<0.008) but not to the levels of FLC, creatinine or ß2-microglobulin. Within the Durie-Salmon staging system, there were significant differences of DKK-1 between the stages I vs. III (p=0.001) and I vs. II+III (p=0.002). In the International Staging System (ISS) there were significant differences only between stages 1 vs. 2+3 (p=0.045). Although there was no overall significant difference of DKK-1 levels between MGUS and MM, there was a difference between MGUS vs stage III (p=0.001) and II+III (p=0.001) according to Durie-Salmon, and also MGUS vs. stage 2 (p=0.005) and vs. stages 2+3 (p=0,012) according to ISS. There were no significant differences in DKK-1 between MGUS and initial/asymptomatic form of MM (stage I). CONCLUSION: Although there was a significant difference of serum levels of DKK-1 between MGUS and initial/asymptomatic stage of MM when compared to advanced stage MM, and in patients with different Hb levels, we do not find the evaluation of serum levels of DKK-1 useful for routine discrimination of MGUS and MM, and for the specification of temporary stratification systems.

[Analysis of the relationship of heavy/light chain pairs of immunoglobulin (Hevylite) to the results of gel electrophoresis and nefelometric examination of serum proteins at the time of multiple myeloma diagnosis]. / Analýza vztahu sérových hladin páru tezkých/lehkých retezcu imunoglobulinu (Hevylite) k výsledkum standardní gelové elektroforézy a nefelometrického vysetrení bílkovin séra pri diagnóze mnohocetného myelomu.

BACKGROUND: The diagnostics and treatment of multiple myeloma (MM) requires precise analysis of serum immunoglobulins, which might be limited by the sensitivity of standard examination methods. Hevylite method enables quantitative analysis of heavy/light chain pairs (HLC) of normal and tumor IgG and IgA immunoglobulin and their ratio (HLC-r). The aim of the study was to assess the contribution of Hevylite method in the diagnostics of MM in comparison with nephelometry (NEF), standard protein electrophoresis (SPE), immunofixation electrophoresis (IFE) and the examination of serum free light chains (FLC) of immunoglobulin using Freelite test and heavy/light chain pairs of immunoglobulin (HLC) using Hevylite. METHODS: Using the methods Hevylite, NEF, SPE, IFE and Freelite, we examined a cohort of 134 individuals fulfilling the International Myeloma Working Group (IMWG) criteria. 96 patients were of IgG and 38 of IgA type. RESULTS: The levels of HLC-kappa (K) and HLC-lambda (L), as well as HLC-r were independent of age and gender. Abnormal HLC levels were present in 84-100%, pathological HLC-r was in 92-100% cases based on MIg isotype. We found strong positive correlation between IgG and IgA (NEF) and the sum of HLC IgG-K + IgG-L (Hevylite) (r = 0.80, p < 0.0001) and HLC IgA-K + IgA-L (r = 0.75, p < 0.0001). Very strong positive correlation was between the concentration of MIg (SPE) and the levels of HLC (Hevylite) in IgG-K (r = 0.73), IgG-L (r = 0.76), IgA-K (r = 0.70) and IgA-L (r = 0.89), p < 0,0001. Systematic difference between Hevylite vs. MIg (SPE) was confirmed by Bland-Altmann test in the case of HLC IgA-K and IgA-L (not HLC IgG-K and IgG-L), and in the correlation of HLC with IgG and IgA (NEF). The most significant correlation between SPE (patients with < 15 g/L) vs. Hevylite was found within the analysis of HLC IgG-K+ IgA-K (r = 0.85, p < 0.0001), and in the whole cohort of MM patients, i.e. IgG + IgA-kappa and lambda (r = 0.76, p < 0.0001), confirmed by Bland-Altmann test. Tight positive correlation was between HLC-r and index of monoclonality FLC-K/L in MM of IgG and IgA type MM (p < 0.0001). CONCLUSION: Hevylite method, especially the assessment of HLC-r of IgA type MM is more sensitive in comparison with SPE evaluated by NEF, and increases the diagnostic sensitivity and the extent of tumor mass examination. Despite its limitation in the case of high levels of IgG type MIg, Hevylite technique has a promising potential to enrich the standard analytic tools as it enables to assess the concentration and ratio of the levels of both tumor and physiological immunoglobulins e.g. depth of immunoparesis, valid especially in MM with low levels of MIg.

Interlaboratory study of free monoclonal immunoglobulin light chain quantification.

BACKGROUND: Quantification of monoclonal immunoglobulin free light chains (FLCs) in serum is used increasingly in clinical practice for the diagnosis, prognostic assessment, and treatment monitoring of monoclonal gammopathies. It is used as an adjunct to standard serum protein electrophoresis and immunofixation. However, methods for FLC quantification need further standardization and validation. METHODS: The Czech Myeloma Group and the Czech Society of Clinical Biochemistry have initiated an interlaboratory study where six laboratories collaborating with the primary myeloma treatment centres measured FLC concentrations in 12 serum samples from patients with monoclonal gammopathies. RESULTS: Repeatability of the measurements in five laboratories was calculated based on differences between the results of duplicate measurements. We found that repeatability depended more on the laboratory than on the device used for measurement. CONCLUSIONS: The study revealed several weak points in the methodology, including the need for a uniform sample dilution procedure. Interlaboratory reproducibility was comparable with values achieved in the NEQAS programme. Because the κ/λ ratio cannot be measured with high precision, κ and λ FLC concentrations should be used where possible. Due to its impact on the clinical management of patients with gammopathy, FLC quantification needs to become a part of the regular quality control cycle in myeloma centres.

The correlation of serum immunoglobulin free light chain levels and selected biological markers in multiple myeloma.

AIMS: the presented study is aimed at the evaluation of correlation of free light chains serum levels - kappa, lambda and their relation (K/L ratio) and serum levels of selected biological markers in a group of patients with multiple myeloma examined at the time of the diagnosis. METHODS: 102 patients with multiple myeloma were included in this prospective study. Free light chains serum levels were determined by Freelite Binding Site system, for determination of serum levels of selected parameters the following methods were used: REA thymidinekinase (TK), RIA (beta(2)microglobulin (beta(2)m), ICTP, PINP), enzymoimmunoassay (sIL-6R, sVCAM-1, sICAM-1, sOPG) and quantitative enzymatic immunoassay (sHGF, sVEGF, sSyndecan-1 (sSyn-1) a sFas). RESULTS: There was found a correlation in the kappa-group of the dominant kappa chain and serum readings of beta(2)m (r = 0.344, p = 0.005), TK (r = 0.263, p = 0.035), ICTP (r = 0.402, p = 0.001), PINP (r = 0.264, p = 0.039), sOPG (r = 0.328, p = 0.028), sSyn-1 (r = 0.255, p = 0.046) and sFas (r = 0.418, p = 0.001). In case of K/L ratio there was found a statistically significant association of levels of beta(2)m (r = 0.316, p = 0.01), TK (r = 0.274, p = 0.027), ICTP (r = 0.346, p = 0.006), PINP (r = 0.261, p = 0.042), sSyn-1 (r = 0.283, p = 0.026) and sFas (r = 0.377, p = 0.002). In the lambda-group the analysis confirmed mutual dependence of the dominant lambda chain levels on beta(2)m (r = 0.476, p = 0.003), ICTP (r = 0.375, p = 0.022), sVCAM-1 (r = 0.383, p = 0.019), sHGF (r = 0.441, p = 0.006) and sFas (r = 0.334, p = 0.040). In addition we ascertained a correlation of L/K ratio and levels of beta(2)m (r = -0.473, p = 0.003), TK (r = -0.412, p = 0.011), ICTP (r = -0.331, p = 0.045), PINP (r = -0.409, p = 0.012), sHGF (r = -0.357, p = 0.028), sSyn-1 (r = -0.449, p = 0.005) a sFas (r = - 0.371, p = 0.022). CONCLUSIONS: The study confirmed mutual correlation of FLC serum levels and the levels of several selected biological markers, in particular beta(2)m, TK, ICTP, PINP, sSyn-1 a sFas at time of the diagnosis. It referred to the mutual relation of bone marrow microenvironment, biological qualities of clonal plasmocytes and the intensity of the free light chains production by the tumour cell population.

High-throughput capillary electrophoretic method for determination of total aminothiols in plasma and urine.

Increased interest in the analysis of aminothiols in body fluids during the last years results in a request for high-throughput analytical methods for their determination. We report here a novel, high-throughput method for the determination of total concentrations of biogenous aminothiols - homocysteine, cysteine, glutathione, cysteinylglycine, gamma-glutamylcysteine, and of penicilamine, mercaptopropionylglycine, and cysteamine, three compounds used to treat disorders of aminothiol metabolism in plasma and urine. Samples were reduced with tris(carboxyethyl)phosphine and labeled with 5-(bromomethyl)fluorescein. Capillary electrophoretic separations were performed in 60 mmol/L borate - 15 mmol/L sodium dodecyl sulfate - 2-amino-2-methyl-1-propanol, pH 10.0, with laser-induced fluorescence detection. Analysis time was less than 2 min. The assay is linear (r > 0.999) up to 500 micromol/L. Reproducibilities of migration times (coefficient of variation, CV) were < 0.5%. Interassay repeatabilities (CV, n = 10) were 5.08% and 6.09% for 5 micromol/L addition of homocysteine and 0.60% and 3.78% for 100 micromol/L addition of cysteine in plasma and urine, respectively. Recovery values were within 94-106% and sensitivity was better than 0.19 micromol/L for all analyzed compounds. Results agreed well with a standard high-performance liquid chromatography (HPLC) method. The diagnostic usefulness of the method has been proven on 79 samples of cystinuric patients and 12 samples of homocystinuric patients. We report here a novel method for the determination of aminothiols in body fluids by capillary electrophoresis (CE). Determination is fast and sensitive enough for diagnostic purposes.

Screening method for inherited disorders of purine and pyrimidine metabolism by capillary electrophoresis with reversed electroosmotic flow.

Capillary electrophoresis with electroosmotic flow reversed by cationic surfactant for diagnosis of purine and pyrimidine inherited enzyme deficiencies is reported. Final separation conditions consist of 45 mM borate, 55 mM N-tris[hydroxymethyl]methylglycine, 10 mM tartrate, 1 mM cetyltrimethylammonium bromide and 0.44% tetrabutylammonium hydroxide-2-amino-2-methyl-1,3-propanediol (pH 8.6). Average sensitivity (2.51 microM), reproducibility of migration times (run-to-run C.V. < or = 0.6%, day-to-day C.V. < or = 2.5%), linearity (R2>0.994) and imprecision (mean intra-assay RSD 4.7% and inter-assay RSD 6.6%) of the method are acceptable for diagnostic purposes. Applicability of the method is demonstrated on urine samples from patients with enzymatically proven enzyme deficiencies.