Određivanje razreda i tipa monoklonskog proteina metodom imunosuptrakcije

Cilj Za detekciju monoklonskog proteina (MP) važno je koristiti osjetljivu metodu budući da asimptomatski bolesnici s dokazanim MP razviju multipli mijelom ili sličan mijeloproliferativan poremećaj s godišnjom stopom od 1-2%. Cilj ovog rada je usporednim analizama i interpretacijama rezultata imunofiksacije i imunosuptrakcije utvrditi prednosti i nedostatke imunosuptrakcije te procijeniti može li ova metoda u skorijoj budućnosti zamijeniti imunofiksaciju u detekciji i tipizaciji MP. Ispitanici i metode Obrađeno je 50 uzoraka seruma bolesnika Zavoda za hematologiju Interne klinike KBC Zagreb. Izabrani su bolesnici kod kojih postoji sumnja na monoklonsku gamapatiju odnosno na prisutnost stanja karakteriziranog prisutnošću MP. Elektroforeza proteina u serumu u svim je uzorcima provedena kapilarnom zonskom elektroforezom na uređaju Capillarys 2 Sebia. Koncentracija imunoglobulina A, G i M određena je turbidimetrijski na Roche Cobas 6000cee analizatoru. Imunofiksacija je provedena na Sebia Hydrasys poluautomatskom sustavu koristeći Hydragel 2/4 agarozne gelove. Imunosuptrakcija je izvođena na Capillarys 2 uređaju. Imunofiksacija se temelji na detekciji uske monoklonske vrpce na agaroznom gelu nakon elektroforeze i imunoprecipitacije dok se imunosuptrakcija temelji na usporedbi referentne slike elferograma s elferogramom nakon imunoreakcije sa specifičnim protutijelom vezanim na kuglice Sepharose. Rezultati U 44/50 uzorka jednostavno je i jednoznačno tipiziran MP objema metodama. U preostalih 6 uzoraka (biklonalna gamapatija i zadržana poliklonska sinteza neuključenih imunoglobulina) jednostavnije je bilo opisati rezultat imunofiksacije nego imunosuptrakcije što se djelomično može objasniti većim iskustvom u očitavanju rezultata imunofiksacije. Zaključak Imunosuptrakcija je dobra alternativa za imunotipizaciju monoklonskog proteina u kliničkom laboratoriju. Metoda je brza, potpuno automatizirana, ali u usporedbi s imunofiksacijom manje osjetljivosti u detekciji MP. Stoga su iskusni i dobro educirani laboratorijski stručnjaci neophodni za interpretaciju rezultata imunosuptrakcije.Objectives It is important to use a sensitive method to detect monoclonal protein (MP) because asymptomatic patients with MP develop multiple myeloma or similar myeloproliferative disorder at the rate of 1-2% a year. The aim of this study was with comparative analyzes and interpretations of results immunofixation electrophoresis (IFE) and immunosubtraction electophoresis (ISE) determine the advantages and disadvantages of ISE and also assess whether this method can replace IFE in the detection and immunotyping of MP in the near future. Patients and Methods Processed are 50 serum patient samples from the Department of Hematology, Clinic for Internal Medicine, University Hospital Centre Zagreb. Patients are selected due to suspicion of monoclonal gammopathy or a condition characterized by the presence of MP. Serum protein electrophoresis was perform in all samples by capillary zone electrophoresis (CZE) method using Capillarys 2 Sebia system. Concentration of immunoglobulin G, A and M was measured on Roche Cobas 6000cee by turbidimetric procedure. IFE was perform on Sebia Hydrasys using Hydragel 2/4 IF gels. ISE was perform on Capillarys2. IFE is based on detecting appearance of monoclonal band after agarose gel electrophoresis and immunoprecipitation. The principle of ISE involves comparision of electrophoretic reference pattern with specific electrophoretic pattern obtained after immunoreaction with specific antibody bound to Sepharose beads. Results In 44/50 samples obtained results were equal and not diffucult to interpret by both methods. In the remaining 6 samples (biclonal gammopathy and reserved polyclonal synthesis of uninvolved immunoglobulin) the obtained results by IFE were easier to describe than obtained by ISE which can be explained by greater experience in interpreting immunofixation results. Conclusion The ISE is a good alternative for MP immunotyping in clinical laboratory. The method is fast, fully automated, but comparing to IFE lower sensitivity in detecting MP. Therefore, experienced and well trained laboratory professionals are necessary to interpret results of ISE

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

Quantification of serum M-protein by immunosubstraction method

Prisutnost M-proteina u serumu karakterizira stanje monoklonske gamapatije. Koncentracija M-proteina izravno korelira s veličinom tumorske mase, osim u vrlo rijetkim slučajevima nesekrecijske bolesti. Detekcija, tipizacija i kvantifikacija M-proteina nužne su za postavljanje dijagnoze, procjenu rizika progresije i praćenje uspješnosti terapije. Ograničenja postojećih metoda kvantifikacije serumskoga M-proteina (denzitometrijska i imunokemijska) razlog su kontinuiranih istraživačkih napora u osmišljavanju novih postupaka koji bi unaprijedili dijagnostiku i praćenje kliničkoga stanja bolesnika s monoklonskom gamapatijom. Cilj je ovoga istraživanja bio razviti i evaluirati osmišljeni računski postupak za kvantifikaciju serumskoga M-proteina primjenom laboratorijskih rezultata kvalitativne metode imunosuptrakcije. Istraživanje je obuhvatilo 133 uzorka bolesnika s monoklonskom gamapatijom klinički praćenih u Kliničkom bolničkom centru Zagreb. U svim je uzorcima provedena elektroforeza serumskih proteina, imunofiksacija i imunosuptrakcija te određena koncentracija ukupnih proteina i pojedinih razreda imunoglobulina G, A i M. Amplituda vrška M-proteina nativnoga uzorka i globulinske krivulje nakon imunoprecipitacije prepoznate su kao ključni parametri u laboratorijskim rezultatima imunosuptrakcije. Podatci o amplitudi krivulja su, uz koncentraciju ukupnih proteina, razreda imunoglobulina uključenoga u sintezu M-proteina i globulina, poslužili kao temelj osmišljenih računskih modela. Utvrđena je vrlo dobra povezanost deriviranih varijabli AD1nIg, D1Ig, D2Ig i D1nIg temeljenih na podacima iz imunosuptrakcijskog elferograma s rezultatima uspoređivanih metoda. Podatci iz imunosuptrakcijskog elferograma o albuminskoj frakciji nisu pridonijeli jačini povezanosti rezultata deriviranih varijabli s rezultatima postojećih metoda. Dokazana je veća preciznost određivanja koncentracije M-proteina primjenom deriviranih varijabli u odnosu na denzitometrijsku metodu i nije ovisila o koncentraciji, prisutnosti poliklonske pozadine niti migracijskom položaju M-proteina. U ovom je radu istražena i opisana mogućnost umjeravanja rezultata imunosuptrakcije. Najusporediviji rezultati s postojećim metodama dobiveni su s rezultatima derivirane varijable D1Ig uz najbolje rezultate procjene preciznosti i točnosti. Dobiveni rezultati upućuju na zaključak da su u računskim modelima za kvantifikaciju M-proteina amplitude krivulja na mjestu vrška M-proteina u imunosuptrakcijskom elferogramu objektivan i vrijedan mjerljivi parametar neovisan o prisutnosti pozadine. Evaluacijom deriviranih varijabli pokazano je da je njihovom primjenom moguće unaprijediti dijagnostiku i praćenje kliničkoga stanja bolesnika s monoklonskom gamapatijom.The presence of M-protein in serum characterizes the state of monoclonal gammopathy. M-protein concentration directly correlates with tumor mass size, except in very rare cases of the nonsecretory disease. Detection, typing, and quantification of M-protein are necessary for diagnosis, assessment of the risk of progression, and monitoring the therapy efficiency. The shortcomings of existing methods for M-protein quantification (densitometric and immunochemical) encourage ongoing research efforts to develop novel procedures that would improve the management of patients with monoclonal gammopathy. This study aimed to develop and evaluate a computational procedure for quantifying serum M-protein using laboratory results of a qualitative immunosubtraction method. The study included 133 samples of patients with monoclonal gammopathy treated at the University Hospital Center Zagreb. Serum protein electrophoresis, immunofixation, and immunosubtraction were performed in all samples, and the concentrations of total protein and of individual immunoglobulin classes G, A, and M were determined. The amplitude of the M-protein spike of the native serum and the globulin curve after immunoprecipitation were recognized as key parameters in the laboratory results of immunosubtraction. Amplitude data, along with the concentrations of total protein, immunoglobulin class involved in M-protein synthesis and globulins represent the basis of explored derived models. A very strong correlation was found between the derived variables AD1nIg, D1Ig, D2Ig, and D1nIg based on data from the immunosubtraction elpherogram with the results of the compared methods. Data on the albumin fraction, generated from immunosubtraction elpherogram, did not improve the correlation. Higher precision of derived variables was demonstrated than in the densitometric method and it was unaffected by concentration, background presence, or migration pattern of M-protein. In this research, the concept of immunosubtraction result normalisation has been described and studied. The most comparable results to both examined methods were obtained with the results of the derived variable D1Ig, with the best precision and accuracy testing results. The findings of this study imply that the change in amplitude of the curves at the M-protein spike position in immunosubtraction elpherogram is an objective, backgroundindependent, measurable, and relevant parameter in M-protein quantification by derived variables. The evaluation of the derived variables showed that their implementation could improve diagnostics and follow-up of patients with monoclonal gammopathy

Quantification of serum M-protein by immunosubstraction method

Prisutnost M-proteina u serumu karakterizira stanje monoklonske gamapatije. Koncentracija M-proteina izravno korelira s veličinom tumorske mase, osim u vrlo rijetkim slučajevima nesekrecijske bolesti. Detekcija, tipizacija i kvantifikacija M-proteina nužne su za postavljanje dijagnoze, procjenu rizika progresije i praćenje uspješnosti terapije. Ograničenja postojećih metoda kvantifikacije serumskoga M-proteina (denzitometrijska i imunokemijska) razlog su kontinuiranih istraživačkih napora u osmišljavanju novih postupaka koji bi unaprijedili dijagnostiku i praćenje kliničkoga stanja bolesnika s monoklonskom gamapatijom. Cilj je ovoga istraživanja bio razviti i evaluirati osmišljeni računski postupak za kvantifikaciju serumskoga M-proteina primjenom laboratorijskih rezultata kvalitativne metode imunosuptrakcije. Istraživanje je obuhvatilo 133 uzorka bolesnika s monoklonskom gamapatijom klinički praćenih u Kliničkom bolničkom centru Zagreb. U svim je uzorcima provedena elektroforeza serumskih proteina, imunofiksacija i imunosuptrakcija te određena koncentracija ukupnih proteina i pojedinih razreda imunoglobulina G, A i M. Amplituda vrška M-proteina nativnoga uzorka i globulinske krivulje nakon imunoprecipitacije prepoznate su kao ključni parametri u laboratorijskim rezultatima imunosuptrakcije. Podatci o amplitudi krivulja su, uz koncentraciju ukupnih proteina, razreda imunoglobulina uključenoga u sintezu M-proteina i globulina, poslužili kao temelj osmišljenih računskih modela. Utvrđena je vrlo dobra povezanost deriviranih varijabli AD1nIg, D1Ig, D2Ig i D1nIg temeljenih na podacima iz imunosuptrakcijskog elferograma s rezultatima uspoređivanih metoda. Podatci iz imunosuptrakcijskog elferograma o albuminskoj frakciji nisu pridonijeli jačini povezanosti rezultata deriviranih varijabli s rezultatima postojećih metoda. Dokazana je veća preciznost određivanja koncentracije M-proteina primjenom deriviranih varijabli u odnosu na denzitometrijsku metodu i nije ovisila o koncentraciji, prisutnosti poliklonske pozadine niti migracijskom položaju M-proteina. U ovom je radu istražena i opisana mogućnost umjeravanja rezultata imunosuptrakcije. Najusporediviji rezultati s postojećim metodama dobiveni su s rezultatima derivirane varijable D1Ig uz najbolje rezultate procjene preciznosti i točnosti. Dobiveni rezultati upućuju na zaključak da su u računskim modelima za kvantifikaciju M-proteina amplitude krivulja na mjestu vrška M-proteina u imunosuptrakcijskom elferogramu objektivan i vrijedan mjerljivi parametar neovisan o prisutnosti pozadine. Evaluacijom deriviranih varijabli pokazano je da je njihovom primjenom moguće unaprijediti dijagnostiku i praćenje kliničkoga stanja bolesnika s monoklonskom gamapatijom.The presence of M-protein in serum characterizes the state of monoclonal gammopathy. M-protein concentration directly correlates with tumor mass size, except in very rare cases of the nonsecretory disease. Detection, typing, and quantification of M-protein are necessary for diagnosis, assessment of the risk of progression, and monitoring the therapy efficiency. The shortcomings of existing methods for M-protein quantification (densitometric and immunochemical) encourage ongoing research efforts to develop novel procedures that would improve the management of patients with monoclonal gammopathy. This study aimed to develop and evaluate a computational procedure for quantifying serum M-protein using laboratory results of a qualitative immunosubtraction method. The study included 133 samples of patients with monoclonal gammopathy treated at the University Hospital Center Zagreb. Serum protein electrophoresis, immunofixation, and immunosubtraction were performed in all samples, and the concentrations of total protein and of individual immunoglobulin classes G, A, and M were determined. The amplitude of the M-protein spike of the native serum and the globulin curve after immunoprecipitation were recognized as key parameters in the laboratory results of immunosubtraction. Amplitude data, along with the concentrations of total protein, immunoglobulin class involved in M-protein synthesis and globulins represent the basis of explored derived models. A very strong correlation was found between the derived variables AD1nIg, D1Ig, D2Ig, and D1nIg based on data from the immunosubtraction elpherogram with the results of the compared methods. Data on the albumin fraction, generated from immunosubtraction elpherogram, did not improve the correlation. Higher precision of derived variables was demonstrated than in the densitometric method and it was unaffected by concentration, background presence, or migration pattern of M-protein. In this research, the concept of immunosubtraction result normalisation has been described and studied. The most comparable results to both examined methods were obtained with the results of the derived variable D1Ig, with the best precision and accuracy testing results. The findings of this study imply that the change in amplitude of the curves at the M-protein spike position in immunosubtraction elpherogram is an objective, backgroundindependent, measurable, and relevant parameter in M-protein quantification by derived variables. The evaluation of the derived variables showed that their implementation could improve diagnostics and follow-up of patients with monoclonal gammopathy

Quantification of serum M-protein by immunosubstraction method

Prisutnost M-proteina u serumu karakterizira stanje monoklonske gamapatije. Koncentracija M-proteina izravno korelira s veličinom tumorske mase, osim u vrlo rijetkim slučajevima nesekrecijske bolesti. Detekcija, tipizacija i kvantifikacija M-proteina nužne su za postavljanje dijagnoze, procjenu rizika progresije i praćenje uspješnosti terapije. Ograničenja postojećih metoda kvantifikacije serumskoga M-proteina (denzitometrijska i imunokemijska) razlog su kontinuiranih istraživačkih napora u osmišljavanju novih postupaka koji bi unaprijedili dijagnostiku i praćenje kliničkoga stanja bolesnika s monoklonskom gamapatijom. Cilj je ovoga istraživanja bio razviti i evaluirati osmišljeni računski postupak za kvantifikaciju serumskoga M-proteina primjenom laboratorijskih rezultata kvalitativne metode imunosuptrakcije. Istraživanje je obuhvatilo 133 uzorka bolesnika s monoklonskom gamapatijom klinički praćenih u Kliničkom bolničkom centru Zagreb. U svim je uzorcima provedena elektroforeza serumskih proteina, imunofiksacija i imunosuptrakcija te određena koncentracija ukupnih proteina i pojedinih razreda imunoglobulina G, A i M. Amplituda vrška M-proteina nativnoga uzorka i globulinske krivulje nakon imunoprecipitacije prepoznate su kao ključni parametri u laboratorijskim rezultatima imunosuptrakcije. Podatci o amplitudi krivulja su, uz koncentraciju ukupnih proteina, razreda imunoglobulina uključenoga u sintezu M-proteina i globulina, poslužili kao temelj osmišljenih računskih modela. Utvrđena je vrlo dobra povezanost deriviranih varijabli AD1nIg, D1Ig, D2Ig i D1nIg temeljenih na podacima iz imunosuptrakcijskog elferograma s rezultatima uspoređivanih metoda. Podatci iz imunosuptrakcijskog elferograma o albuminskoj frakciji nisu pridonijeli jačini povezanosti rezultata deriviranih varijabli s rezultatima postojećih metoda. Dokazana je veća preciznost određivanja koncentracije M-proteina primjenom deriviranih varijabli u odnosu na denzitometrijsku metodu i nije ovisila o koncentraciji, prisutnosti poliklonske pozadine niti migracijskom položaju M-proteina. U ovom je radu istražena i opisana mogućnost umjeravanja rezultata imunosuptrakcije. Najusporediviji rezultati s postojećim metodama dobiveni su s rezultatima derivirane varijable D1Ig uz najbolje rezultate procjene preciznosti i točnosti. Dobiveni rezultati upućuju na zaključak da su u računskim modelima za kvantifikaciju M-proteina amplitude krivulja na mjestu vrška M-proteina u imunosuptrakcijskom elferogramu objektivan i vrijedan mjerljivi parametar neovisan o prisutnosti pozadine. Evaluacijom deriviranih varijabli pokazano je da je njihovom primjenom moguće unaprijediti dijagnostiku i praćenje kliničkoga stanja bolesnika s monoklonskom gamapatijom.The presence of M-protein in serum characterizes the state of monoclonal gammopathy. M-protein concentration directly correlates with tumor mass size, except in very rare cases of the nonsecretory disease. Detection, typing, and quantification of M-protein are necessary for diagnosis, assessment of the risk of progression, and monitoring the therapy efficiency. The shortcomings of existing methods for M-protein quantification (densitometric and immunochemical) encourage ongoing research efforts to develop novel procedures that would improve the management of patients with monoclonal gammopathy. This study aimed to develop and evaluate a computational procedure for quantifying serum M-protein using laboratory results of a qualitative immunosubtraction method. The study included 133 samples of patients with monoclonal gammopathy treated at the University Hospital Center Zagreb. Serum protein electrophoresis, immunofixation, and immunosubtraction were performed in all samples, and the concentrations of total protein and of individual immunoglobulin classes G, A, and M were determined. The amplitude of the M-protein spike of the native serum and the globulin curve after immunoprecipitation were recognized as key parameters in the laboratory results of immunosubtraction. Amplitude data, along with the concentrations of total protein, immunoglobulin class involved in M-protein synthesis and globulins represent the basis of explored derived models. A very strong correlation was found between the derived variables AD1nIg, D1Ig, D2Ig, and D1nIg based on data from the immunosubtraction elpherogram with the results of the compared methods. Data on the albumin fraction, generated from immunosubtraction elpherogram, did not improve the correlation. Higher precision of derived variables was demonstrated than in the densitometric method and it was unaffected by concentration, background presence, or migration pattern of M-protein. In this research, the concept of immunosubtraction result normalisation has been described and studied. The most comparable results to both examined methods were obtained with the results of the derived variable D1Ig, with the best precision and accuracy testing results. The findings of this study imply that the change in amplitude of the curves at the M-protein spike position in immunosubtraction elpherogram is an objective, backgroundindependent, measurable, and relevant parameter in M-protein quantification by derived variables. The evaluation of the derived variables showed that their implementation could improve diagnostics and follow-up of patients with monoclonal gammopathy

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

Humoral immune response in convalescent COVID-19 people with multiple sclerosis treated with high-efficacy disease-modifying therapies: A multicenter, case-control study

Aim: To determine the influence of high-efficacy disease modifying therapy (DMT) on the development of IgG SARS-CoV-2 antibody response in COVID-19 convalescent people with multiple sclerosis (pwMS). ----- Methods: Seventy-four pwMS taking high-efficacy DMTs (specifically natalizumab, fingolimod, alemtuzumab, ocrelizumab, cladribine and ublituximab) and diagnosed with COVID-19 and 44 healthy persons (HC) were enrolled. SARS-CoV2 antibodies were tested with Elecsys® Anti-SARSCoV-2 S assay. ----- Results: pwMS taking high-efficacy DMTs had a significantly higher chance of having negative titer of SARS-CoV2 antibodies compared to healthy controls (33 negative pwMS [44.6%] compared to one negative HC [2.3%], p < 0.001). pwMS taking B-cell depleting therapy (ocrelizumab and ublituximab) had a significantly higher chance of having negative titer of SARS-CoV2 antibodies compared to pwMS on all other DMTs (29 negative pwMS on B-cell therapy [64.4%] compared to four negative pwMS on all other DMTs [13.8%], p < 0.001). Out of other DMTs, two (33.3%) pwMS taking fingolimod and two (16.7%) pwMS taking cladribine failed to develop IgG SARS-COV-2 antibodies. B-cell depleting therapy independently predicted negative titer of IgG SARS-CoV-2 antibody (Exp[B] =0.014, 95%CI 0.002-0.110, p < 0.001). ----- Conclusions: A significant proportion of convalescent COVID-19 pwMS on high-efficacy DMTs will not develop IgG SARS-CoV-2 antibodies. B-cell depleting therapies independently predict negative and low titer of IgG SARS-CoV-2 antibody