A novel approach for more precise quantification of M-protein using variables derived from immunosubtraction electropherogram and associated biochemistry analytes

IntroductionDue to limitations in currently used methodologies, the widely acknowledged approach for quantifying M-protein (MP) is not available. If employed as a source of quantitative data, the immunosubtraction electropherogram (IS-EPG), a qualitative analysis of MP, has the potential to overcome known analytical issues. The aim of this study is to explore measured and derived variables obtained from immunosubtraction electropherogram as a tool for quantifying MP and to compare the derived results to currently available methods. Materials and methodsMeasurands were amplitudes of MP and albumin fractions. Assessed derived variables included also immunoglobulin (Ig) G, IgA, IgM and total protein data. Capillary electrophoresis was used for determination of MP (in % of total protein concentration, or concentration of MP in g/L) by perpendicular drop and tangent skimming method. ResultsPassing-Bablok analysis showed the most comparable results in D1Ig and D1nIg variables, and the largest discrepancies in AD1nIg and AD2nIg variables. The background presence had greater impact on D1nIg comparison results than did on D1Ig results. The contribution of albumin fraction data did not improve the comparability of the results. The coefficients of variation of derived variables were lower (maximum 3.1%) than those obtained by densitometric measurements, regardless of MP concentration, polyclonal background, or migration pattern (2.3-37.7%). ConclusionThe amplitude of MP spike in IS-EPG is an valuable measurand to compute derived variables for quantifying MP. The most comparable results were achieved with the D1Ig variable. Patients with monoclonal gammopathy can benefit from increased precision employing an objective and background independent measurand, especially during longitudinal follow-up

Reference Intervals in Laboratory Medicine

Laboratorijski nalazi imaju široku primjenu u medicini, od postavljanja dijagnoze bolesti, praćenja tijeka bolesti do praćenja uspješnosti terapije. Za racionalnu interpretaciju laboratorijskih nalaza potrebno je poznavanje referentnih intervala. Referentni interval obuhvaća vrijednost između gornje i donje referentne granice, uključujući i vrijednost samih granica. Najčešće je upotrebljavani i preporučeni oblik referentnog intervala 95-postotni interval koji obuhvaća 95 % središnjih vrijednosti omeđenih 2,5. i 97,5. percentilom. Za izradu referentnih vrijednosti treba odabrati: referentne osobe koje čine referentnu populaciju, referentni uzorak u kojem se određuju referentne vrijednosti koje pokazuju referentnu distribuciju iz koje se zatim izračunavaju donje i gornje referentne granice koje omeđuju referentne intervale. Određivanje referentnih vrijednosti i intervala treba provesti na velikom broju zdravih ispitanika, što predstavlja dugotrajan i skup proces. Zato se u laboratorijskoj praksi upotrebljavaju uglavnom referentne vrijednosti i intervali koje navodi proizvođač korištenih testova. Prema preporukama struke, svaki laboratorij mora verificirati referentne intervale proizvođača kako bi provjerio jesu li su prikladni za namijenjenu populaciju bolesnika. Referentne intervale moguće je provjeriti na nekoliko načina i važno je da se provjera ponavlja periodično odnosno jednom godišnje ili pri promjeni bilo kojih uvjeta koji mogu znatno utjecati na primjenjivani referentni interval. Od nekoliko preporučenih postupaka najčešće se primjenjuje provjera na 20 uzoraka zdravih ispitanika.Laboratory findings are widely used in medicine; from the diagnosis monitoring the disease to tracking success of therapy. For a rational interpretation of laboratory results it is necessary to be familiar with reference intervals. Reference interval includes the value between the upper and lower reference limits, including the values of the limits. The most commonly used and recommended form of the reference interval is 95 % interval which covers 95% of the central values, confining with 2.5. and 97.5. percentiles. To create the reference values should be selected: the reference persons comprising the reference population, reference samples in which is determined reference values. Reference values shows the reference distribution from which then are calculated lower and upper reference limits delimiting reference intervals. Determination of reference values and intervals must be carried out on a large number of healthy subjects which is a lengthy and expensive process. Therefore, in laboratory practice are used mainly baseline and intervals specified by the manufacturer. According to the recommendations of the profession each laboratory must verify manufacturer reference intervals to confirm that they are suitable for the intended patient population. It is possible to verify reference interval using few procedures and is important to repeat verification periodically once a year or by changing any conditions that may have a significant influence on the applied reference interval. Of the few recommended procedures most often is used the one which includes 20 healty subjects

Immunoglobulin heavy/light chain analysis enhances the detection of residual disease and monitoring of multiple myeloma patients

Aim To evaluate the clinical utility of incorporating a novel heavy/light chain immunoassay (HLC) into the existing methods for the assessment of multiple myeloma (MM) patients. Methods Convenience sera samples from 90 previously treated IgG and IgA MM patients in different disease stages were analyzed. The study was conducted in Clinical Hospital Center Zagreb between 2011 and 2013. The collected sera were analyzed by standard laboratory techniques (serum protein electrophoresis, quantification of total immunoglobulins, serum immunofixation, serum free light chain [FLC] assay) and HLC assay. Results HLC ratios outside the normal range were found in 58 of 90 patients, including 28 out of 61 patients with total immunoglobulin measurements within the normal range and 5 out of 23 patients in complete response. Both elevated HLC isotype level and abnormal HLC ratio correlated with the parameters of tumor burden, including percentage of plasma cells in the bone marrow (P < 0.001 and P = 0.002, respectively) and an abnormal serum FLC ratio (for both P < 0.001). In addition, abnormal HLC isotype level correlated with serum beta-2-microglobulin level (P = 0.038). In terms of prognosis, abnormal HLC isotype level and abnormal HLC ratio were significantly associated with shorter overall survival (P < 0.001 and P = 0.002, respectively). Interestingly, suppression of the uninvolved (polyclonal) isotype pair, but not other non-myeloma immunoglobulin isotypes, was also associated with a shorter overall survival (P = 0.021). In a multivariate analysis, an abnormal HLC ratio and β2-microglobulin level >3.5mg/L were independent risk factors for survival. Conclusion The new HLC assay has greater sensitivity in detecting monoclonal protein, correlates with tumor burden markers, and affects patients’ outcome

Aberrant glycosylation of Igg heavy chain in multiple myeloma

Although the majority of eukaryotic proteins are glycosylated, there is a dearth of knowledge regarding protein sugar moieties and their changes in disease. Most multiple myeloma cases are characterized by production of monoclonal immunoglobulins (Ig). We studied galactosylation and sialylation of IgG heavy chains in 16 patients with IgG myeloma using lectin blotting and densitometry. In comparison to age and sex matched controls, galactosylation was reduced in multiple myeloma (median 317 vs. 362, range 153-410 vs. 309-447 relative units, p = 0.015, Student's t-test). Sialylation was stage dependent; samples from patients with stage IIA had lowest amounts of sialic acid, IIIA intermediate and IIIB highest (142.6 vs. 185.9 vs. 248.5 relative units, correlation coefficient r = 0.55). Both galactosylation and sialylation levels were independent of age, sex, treatment type, response to treatment, disease duration and IgG and b2 microglobulin concentration. These data indicate that multiple myeloma is characterized by aberrant immunoglobulin glycosylation

Acute Phase Proteins in Psoriasis

Psoriasis is a chronic, relapsing, inflammatory and hyperproliferative skin disorder. The levels of acute phase proteins are suggested to be elevated in those patients with acute disease. The expression of selected acute phase proteins as the parameters of humoral immunity has been analyzed in 70 patients with acute psoriasis and 40 healthy controls. The aim was to evaluate the inflammatory response in the exacerbation of psoriasis and to determine the correlation of these objective inflammatory parameters with clinical features of disease activity. Main demographic and epidemiologic features were, as well, analysed. The plasma levels of C-reactive protein (CRP) (c2 = 23.61; p<0.005), and alpha1-acid glycoprotein (a1-AGP) (c2 = 7.,42; p<0.01) were significantly increased in those patients with acute phase of the disease compared to healthy controls. Our results suggest that psoriasis is an inflammatory disease and therefore its worsening seemed to be linked to the increase in the inflammatory response. It seems that CRP and al-AGP levels could serve as important prognostic factors for the worsening of psoriasis

Unusual pattern in haemoglobin electrophoresis in Croatian population: a case report

Haemoglobinopathies are hereditary disorders of globin chain synthesis and are the most common inherited diseases worldwide. Haemoglobin E is a structural haemoglobin variant characteristic for South East Asian population. We present a rare and unusual finding of haemoglobin E detected in University Hospital Centre Zagreb by capillary zone electrophoresis. Detection of haemoglobin structural variant helped to avoid misdiagnosis of sideropenic anemia and thus potentially harmful therapeutic intervention. In today’s European multiethnic population haemoglobinopathies are a public health issue and Croatian laboratory professionals should be aware of a possibility of finding an unusual haemoglobin pattern