An alternative pretreatment procedure in animal transmissible spongiform encephalopathies diagnosis using PrPsc immunohistochemistry.

Because of its sensitivity, immunohistochemistry (IHC) of abnormal prion protein (PrPsc) is used more often in the diagnosis of transmissible spongiform encephalopathies (TSEs), such as scrapie and bovine spongiform encephalopathy (BSE). PrPsc IHC requires a combination of pretreatments (chemical, heating, and enzymatic). The method of application may depend on the anti-prion antibody considered. If these pretreatments are efficient for diagnostic purpose, it may, however, be interesting to use an alternative method to efficiently detect PrPsc IHC immunohistochemically using chemical pretreatments solely. Here we describe such pretreatments reporting the difficulty (section adhesion) but also the potential advantages of such methods (easy, quick, inexpensive, and amplifying effect).     

Amplified immunohistochemical detection of PrPsc in animal transmissible spongiform encephalopathies using streptomycin.

Due to its sensitivity, immunohistochemistry (IHC) of abnormal prion protein (PrPsc) is used to study experimental and natural cases of transmissible spongiform encephalopathies (TSEs) such as Creutzfeldt-Jakob disease in humans or scrapie and bovine spongiform encephalopathy (BSE) in animals. The limits of detection are particularly critical when PrPsc IHC is used for diagnostic purposes. In this article, we describe for the first time the use of streptomycin sulfate in IHC, providing a novel original and easy way to amplify specifically PrPsc immunohistochemical detection in natural cases of BSE and scrapie, as well as in experimental TSEs in mice models using two different PrP antibodies.     

Signal amplification in immunohistochemistry at the light microscopic level using biotinylated tyramide and nanogold-silver staining.

Signal amplification techniques greatly enhance the sensitivity of immunohistochemical (IHC) and in situ hybridization (ISH) methods. In particular, catalyzed signal amplification (CSA) using labeled tyramide or Nanogold-silver staining is an important signal amplification tool. We have applied a combination of both techniques, as has been introduced for ISH, for a further increase in sensitivity of an IHC method to detect cathepsin B. This lysosomal proteinase can also be expressed extracellularly, particularly in relation to cancer metastasis. Higher sensitivity of the IHC method was needed because existing methods failed to demonstrate cathepsin B protein where cathepsin B activity was found with a fluorescence enzyme histochemical method. Combined CSA and Nanogold-silver staining provided the sensitivity that was required. Moreover, this signal amplification method enabled the use of a 10-fold lower concentration of primary antibody (1 microg/ml). Nonspecific background staining was low provided that endogenous biotin, avidin, and peroxidase were completely blocked. The method was reproducible when all steps, and particularly the silver enhancement step, were rigidly controlled. The method resulted in localization patterns of cathepsin B protein that were in agreement with those of cathepsin B activity in serial sections of rat liver containing colon cancer metastases. We concluded that combined application of CSA and Nanogold-silver staining provides high sensitivity for immunohistochemical methods and that activity localization by an enzyme histochemical method is a very attractive alternative to IHC localization of an enzyme because it is at least as sensitive, it is rapid and simple, and it provides direct information on the function of an enzyme.     

三种方法诊断不孕症患者子宫内膜息肉的临床价值研究

目的:探讨单用阴道超声(TVS)、子宫输卵管造影(HSG)、超声子宫水造影(SIS)以及三种方法联合诊断不孕症患者子宫内膜息肉(EP)的临床价值。方法:以206例行宫腔镜联合诊刮或病检的不孕症患者为研究对象,回顾性分析各种检查方法对EP的筛查结果,评价各种检查方法的真实性、可靠性以及预测值。结果:206例不孕症中,共确诊EP患者60例,阳性率29.1%。三种检查方法中,TVS的灵敏度最高(70.0%),特异度最低(73.3%),漏诊率最低(30.0%),误诊率最高(26.7%),正确诊断指数最高(43.3%),阴性似然比最小(0.409),阴性预测值最高(85.6%);SIS检查的灵敏度最低(38.7%),漏诊率最高(61.3%),但是特异性最高(93.3%),误诊率最低(6.7%),阳性似然比最大(4.284),阳性预测值最大(66.6%),正确诊断指数最低(32.0%);HSG检查的上述各项评价指标均介于TVS和SIS之间。TVS和SIS与金标准的符合率低,Kappa值均小于0.4;HSG符合率最高(86.2%),Kappa值0.647。三种检查联合诊断的灵敏度89.3%,漏诊率10.7%,特异度91.4%,误诊率8.6%,正确诊断指数80.7%,阳性似然比10.384,阴性似然比0.117,符合率89.3%,Kappa值0.792,阳性预测值83.3%,阳性预测值94.6%。结论:对于宫腔可能存在内膜息肉的不孕症患者,单一采用阴道超声检查、子宫输卵管造影或超声子宫水造影方法的灵敏度均较低,漏诊率高,与金标准的一致性较差,而三种方法联合用于诊断不孕症患者EP的真实性、可靠性及预测值均较好。     

Fine needle aspiration biopsy with adjunct immunohistochemistry in intraocular tumor management

OBJECTIVE: To assess the effectiveness of fine needle aspiration biopsy (FNAB), with and without immunohistochemistry (IHC), in the management of solid intraocular tumors. STUDY DESIGN: Thirty-three consecutive adults undergoing FNAB of suspected intraocular tumors were studied. Clinical, cytologic and histologic diagnoses were correlated. The positive predictive value, sensitivity and specificity of FNAB for detecting malignancy, the effect of lHC on the final cytologic diagnosis and the number of patients in whom clinical management was altered as a result of cytologic evaluation were determined. RESULTS: The positive predictive value was 96% with and 93% without adjunct IHC. The sensitivity and specificity of FNAB for detecting malignancy were 96% and 83%, respectively, with IHC. Without IHC, the sensitivity was unaltered, but the specificity was 67%. IHC confirmed the morphologic diagnosis in 75% of cases, made a diagnosis in 12.5% and changed a malignant diagnosis from carcinoma to melanoma in 6% of cases. The planned management was changed by the FNAB findings in 24% of patients. In 3 patients (9%), IHC was essential for diagnosis and management. No patients exhibited local tumor dissemination or recurrence associated with the biopsy. CONCLUSION: FNAB is a safe, sensitive and specific method of establishing a tissue diagnosis in a subset of patients with solid intraocular tumors. The routine use of immunohistochemical stain ing increases the diagnostic utility of the technique and may change clinical management.     

Role of Immunohistochemistry in Staging Diffuse Large B-cell Lymphoma (DLBCL)

The use of immunohistochemistry (IHC) in staging bone marrow in non-Hodgkin''s lymphoma (NHL) is largely limited to ambiguous cases, particularly those with lymphoid aggregates. Its role in routine clinical practice remains unestablished. This study aimed to determine whether the routine use of IHC in diffuse large B-cell lymphoma (DLBCL) would improve the detection of lymphomatous involvement in the bone marrow. It also sought to determine the impact of IHC on predicting survival compared with routine histological diagnosis using hematoxylin and eosin (H&E), Giemsa, and reticulin staining. The bone marrow trephines of 156 histologically proven DLBCL cases were assessed on routine histology, and IHC using two T-cell markers (CD45RO and CD3), two B-cell markers (CD20 and CD79a), and κ and λ light chains. IHC detected lymphomatous involvement on an additional 11% cases compared with histology alone. Although both routine histology and IHC were good predictors of survival, IHC was better at predicting survival on stepwise multivariate Cox regression analysis. IHC performed routinely on bone marrow trephines has the ability to improve detection of occult lymphoma in experienced hands. Furthermore, it is a better predictor of survival compared with routine histological examination alone. (J Histochem Cytochem 56:893–900, 2008)     

Correlation between Infectivity and Disease Associated Prion Protein in the Nervous System and Selected Edible Tissues of Naturally Affected Scrapie Sheep

The transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of fatal neurodegenerative disorders characterised by the accumulation of a pathological form of a host protein known as prion protein (PrP). The validation of abnormal PrP detection techniques is fundamental to allow the use of high-throughput laboratory based tests, avoiding the limitations of bioassays. We used scrapie, a prototype TSE, to examine the relationship between infectivity and laboratory based diagnostic tools. The data may help to optimise strategies to prevent exposure of humans to small ruminant TSE material via the food chain. Abnormal PrP distribution/accumulation was assessed by immunohistochemistry (IHC), Western blot (WB) and ELISA in samples from four animals. In addition, infectivity was detected using a sensitive bank vole bioassay with selected samples from two of the four sheep and protein misfolding cyclic amplification using bank vole brain as substrate (vPMCA) was also carried out in selected samples from one animal. Lymph nodes, oculomotor muscles, sciatic nerve and kidney were positive by IHC, WB and ELISA, although at levels 100–1000 fold lower than the brain, and contained detectable infectivity by bioassay. Tissues not infectious by bioassay were also negative by all laboratory tests including PMCA. Although discrepancies were observed in tissues with very low levels of abnormal PrP, there was an overall good correlation between IHC, WB, ELISA and bioassay results. Most importantly, there was a good correlation between the detection of abnormal PrP in tissues using laboratory tests and the levels of infectivity even when the titre was low. These findings provide useful information for risk modellers and represent a first step toward the validation of laboratory tests used to quantify prion infectivity, which would greatly aid TSE risk assessment policies.     

Detection of PrP(sc) in samples presenting a very advanced degree of autolysis (BSE liquid state) by immunocytochemistry.

Although detection of the abnormal isoform of prion protein (PrP(sc)), the specific feature of transmissable spongiform encephalopathies (TSEs), has been previously demonstrated on formalin-fixed autolytic tissue, no samples with autolysis as severe as tested here (i.e., liquid state) have previously been tested. It is inevitable that a small but significant proportion of brains, especially in summer due to delays in postmortem examination, undergo an extremely severe autolysis that makes samples unsuitable for diagnosis by conventional techniques. In this study, 25 bovine samples were diagnosed by applying immunocytochemistry on the corresponding liquid fraction. Four additional portions of brainstem (positive and negative sheep and cattle) were subjected to one of the autolysis regimens at 56C or environmental conditions for up to 80 days and were analyzed with the same methodology. No abnormal protein could be detected in any of the control animals. PrP(sc) accumulation was observed by immunocytochemistry in all cases that were positive by either immunohistochemistry on the corresponding filtrates or by Prionics Western blotting, showing an excellent agreement between the methodology assessed and these routine techniques. The results of this study demonstrate immunocytochemistry as a useful tool for diagnosis in liquid-state samples, solving a most relevant problem in BSE and scrapie epidemiology.     

Transmissible spongiform encephalopathy diagnosis using PrPsc immunohistochemistry on fixed but previously frozen brain samples.

The histological diagnosis of transmissible spongiform encephalopathies (TSEs), such as scrapie and bovine spongiform encephalopathy (BSE), relies on identification in the brain of spongiosis, gliosis, and neuron loss without inflammatory lesions. Because of its sensitivity, immunohistochemistry of abnormal prion protein (PrPsc) is of great help in this diagnosis and can be used on its own or complementary to the biochemical detection of PrPsc. However, in some cases no formalin-fixed material is available, rendering its use as a complementary method impossible. For that purpose, we studied the possibility of detecting PrPsc immunohistochemically in fixed brain samples that had been previously frozen and used for Western blotting analysis. We compared freshly and fixed-frozen brain samples originating from the same sheep, either affected or unaffected with scrapie. We also studied fixed-frozen brain samples from scrapie-affected goats and from cows showing BSE. We showed that in all the species tested, despite damage to the histological structures, PrPsc was still detectable in the fixed-frozen brain sections without unspecific background staining. Notwithstanding the limited number of cases thus far analyzed, we have already demonstrated the possibility of using PrPsc immunohistochemistry on fixed-frozen brain samples with very good efficacy, thus rendering possible its use for diagnostic purposes in TSEs.     

Prevalence of scrapie infection in Great Britain: interpreting the results of the 1997-1998 abattoir survey

An accurate estimate of the prevalence of scrapie infection in the Great Britain (GB) sheep flock is essential when assessing any potential risk to human health through exposure to sheep transmissible spongiform encephalopathies (TSEs). One method for assessing the prevalence is to sample sheep intended for human consumption using a diagnostic test capable of detecting infected animals prior to the onset of clinical signs. An abattoir survey conducted in Great Britain in 1997-1998 tested brain samples from 2809 apparently healthy sheep of which none was found to be positive for scrapie by histopathology or immunohistochemistry (IHC) although 10 were positive for scrapie-associated fibrils (SAF). Subsequently, the tonsils from a subset of the animals sampled were examined using IHC, one of which tested positive. To interpret these results we use a likelihood-based approach, which accounts for the variation in the prevalence of infection with age and test sensitivity and specificity with stage of infection. Combining the results for all of the diagnostic tests yields an estimate of the prevalence of scrapie infection in the GB sheep flock of 0.22% (95% confidence interval: 0.01-0.97%). Moreover, our analysis suggests that all of the diagnostic tests used are very specific (greater than 99%). Indeed, only SAF detection yields a specificity estimate of less than 100%, which helps to account for the high number of samples found to be positive for SAF.     

Ante-mortem detection of chronic wasting disease in recto-anal mucosa-associated lymphoid tissues from elk (Cervus elaphus nelsoni) using real-time quaking-induced conversion (RT-QuIC) assay: A blinded collaborative study

Prion diseases are transmissible spongiform encephalopathies (TSEs) characterized by fatal, progressive neurologic diseases with prolonged incubation periods and an accumulation of infectious misfolded prion proteins. Antemortem diagnosis is often difficult due to a long asymptomatic incubation period, differences in the pathogenesis of different prions, and the presence of very low levels of infectious prion in easily accessible samples. Chronic wasting disease (CWD) is a TSE affecting both wild and captive populations of cervids, including mule deer, white-tailed deer, elk, moose, muntjac, and most recently, wild reindeer. This study represents a well-controlled evaluation of a newly developed real-time quaking-induced conversion (RT-QuIC) assay as a potential CWD diagnostic screening test using rectal biopsy sections from a depopulated elk herd. We evaluated 69 blinded samples of recto-anal mucosa-associated lymphoid tissue (RAMALT) obtained from USDA Veterinary Services. The results were later un-blinded and statistically compared to immunohistochemical (IHC) results from the USDA National Veterinary Services Laboratories (NVSL) for RAMALT, obex, and medial retropharyngeal lymph node (MRPLN). Comparison of RAMALT RT-QuIC assay results with the IHC results of RAMALT revealed 92% relative sensitivity (95% confidence limits: 61.52–99.8%) and 95% relative specificity (95% confidence limits: 85.13–99%). Collectively, our results show a potential utility of the RT-QuIC assay to advance the development of a rapid, sensitive, and specific prion diagnostic assay for CWD prions.     

Charge sensitivity analysis in force-field-atom resolution

Charge sensitivity analysis (CSA) was extended to AMBER force-field resolution. The effective electronegativity and hardness data were found using evolutionary algorithms. Four model hardness matrices based on the classical electrostatic, Mataga–Nishimoto, Ohno, and Louwen–Vogt interpolation formulae were considered. Mulliken population analysis and electrostatically derived charges (CHELPG) were taken into account. It was demonstrated that the Ohno interpolation formula gives the best fit to Mulliken charges. For all molecules from the training set and all model hardness matrices, Mulliken charges were reproduced more accurately than CHELPG charges, indicating their good transferability from system to system. The effective electronegativities and hardnesses obtained were further verified by applying CSA to molecules from a validation set that was different from the training set. The correlation between CSA and Mulliken charges was of the same quality as that obtained for the training set.     

Small cell carcinoma of the uterine cervix--an uncommon variant of cervical cancer with neuroendocrine features

Small cell carcinoma (SCC) of the uterine cervix represents an uncommon variant of cervical cancer with an extremely aggressive biologic behavior, minimum survival chances and rapid and fatal clinical course. This retrospective study included 73 cases of patients treated for invasive squamous carcinoma of the uterine cervix at stages Ib and IIa at the Department of Gynecology in the years 1996-2000. Six patients (8%) with SCC were identified among all cases, sharing the clinical features of young age and early failure of appropriate radical treatment in the presence of apparently low stage disease. Neuroendocrine cellular characteristics were assessed by the biotin-streptavidin-peroxidase (LSAB) method using antibodies against neuron-specific enolase (NSE; DAKO), chromogranin A (CGA; DAKO) and synaptophysin (SYN; DAKO). All tumors examined were positive for NSE and/or CGA and/or SYN. Although the presence of neuroendocrine features appears to correlate with decreased survival, the number of patients is not large enough to determine statistical significance. However, the results confirm that SCC of the uterine cervix is one of the most aggressive tumors of the female genital tract.     

New insights into early sequential PrPsc accumulation in scrapie infected mouse brain evidenced by the use of streptomycin sulfate

To investigate the amplifying potentialities of streptomycin sulfate in the immunohistochemical (IHC) detection of the abnormal prion protein (PrPsc), we used a sequential brain sampling from C506M3 scrapie strain inoculated C57Bl/6 mice. The weekly removed brains, from 7 to 63 days post intra-cranial inoculation were analysed using PrPsc IHC. The introduction of streptomycin sulfate, a technique developed for accurate cellular and regional mapping of PrPsc deposition in several animal TSEs, revealed a substantial amplifying effect and a clear specific PrPsc detection as early as 28 days post inoculation. The location of the first detected PrPsc deposits suggests a possible involvement of the cerebrospinal fluid in the early dissemination of the infectious agent. The meaning of these newly accessible PrPsc deposits is discussed in relation to a possible nascent form of PrPsc molecules detected in situ for the first time. Altogether, these findings argue that this method can be highly useful to study the early stages after infection with prion agents.     

Lipocalin2 Expressions Correlate Significantly With Tumor Differentiation in Epithelial Ovarian Cancer

We recently identified lipocalin2 (LCN2) as being upregulated in ovarian cancer cell lines. The purpose of this study was to validate LCN2 upregulation in ovarian cancers and to investigate its potential as a serum biomarker. We assayed LCN2 expression in ovarian cancers using real-time PCR and IHC. To evaluate the potential of LCN2 as a biomarker, we measured serum LCN2 levels in 54 ovarian cancers, 15 borderline and 53 benign ovarian tumors, and 90 healthy controls. SYBR green PCR and IHC showed LCN2 overexpression in ovarian cancers. LCN2 immunoreactivity was significantly associated with tumor differentiation (p=0.009), as well-differentiated tumors showed the highest LCN2 expression. Serum LCN2 level in ovarian cancer was significantly higher than in the other study groups (p<0.001), and in accordance with IHC results, it also correlated with tumor differentiation, with well-differentiated tumors having the highest value. The sensitivity and specificity of LCN2 in detecting ovarian cancer was 72.2% and 50.4%, respectively. By Cox univariate analysis, LCN2 positivity was an independent prognostic factor for overall survival (hazard ratio = 1.47, p=0.012). In conclusion, LCN2 expressions are upregulated and related to tumor differentiation in ovarian cancers and should be included in future research assessing potential biomarkers for ovarian cancer. (J Histochem Cytochem 57:513–521, 2009)     

Automated selection of DAB-labeled tissue for immunohistochemical quantification.

The increased use of immunohistochemistry (IHC) in both clinical and basic research settings has led to the development of techniques for acquiring quantitative information from immunostains. Staining correlates with absolute protein levels and has been investigated as a clinical tool for patient diagnosis and prognosis. For these reasons, automated imaging methods have been developed in an attempt to standardize IHC analysis. We propose a novel imaging technique in which brightfield images of diaminobenzidene (DAB)-labeled antigens are converted to normalized blue images, allowing automated identification of positively stained tissue. A statistical analysis compared our method with seven previously published imaging techniques by measuring each one's agreement with manual analysis by two observers. Eighteen DAB-stained images showing a range of protein levels were used. Accuracy was assessed by calculating the percentage of pixels misclassified using each technique compared with a manual standard. Bland-Altman analysis was then used to show the extent to which misclassification affected staining quantification. Many of the techniques were inconsistent in classifying DAB staining due to background interference, but our method was statistically the most accurate and consistent across all staining levels.     

Effects of new amphotericin analogues on the scrapie isoform of the prion protein

Prion diseases or Transmissible Spongiform Encephalopathies (TSEs) are a group of neurodegenerative disorders associated with the conversion of a normal host prion protein (PrP(C)) into a pathogenic isoform (PrP(Sc)). Despite years of research, there is still no known cure for TSEs. Amphotericin B (AmB), an anti-fungal antibiotic, has antiprion activity but its usage is limited by its toxicity. This study assessed the antiprion properties of new amphotericin analogues in which the exocyclic carboxyl groups were replaced by methyl groups. These analogues reduced levels of the abnormal PrP(Sc) isoform of the mouse prion protein in cultured cells. 16-descarboxyl-16-methyl-amphotericin B (16B) had antiprion activity equivalent to that of amphotericin B and was significantly less toxic to cells as determined by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide dye reduction assay. A non-anti-fungal analogue, 16-descarboxyl-16-methyl-19-O-(6-deoxyhexosyl)-19-O-desmycosaminyl-amphotericin (16-19B) had higher antiprion activity and significantly lower toxicity than AmB. Some of the new amphotericin analogues may have potential as antiprion drugs.     

Efficacy of immunohistochemical staining in detecting Helicobacter pylori in Saudi patients with minimal and atypical infection

Gastric Helicobacter pylori infection is diagnosed based on histopathological evaluation of gastric mucosal biopsies, urease test, urea breath test, H. pylori culturing, or direct detection using polymerase chain reaction (PCR). This study aimed to evaluate the efficacy of immunohistochemical (IHC) staining in detecting H. pylori in gastric biopsies from patients with chronic gastritis and minimal or atypical infection. Gastric biopsies from 50 patients with chronic gastritis were subjected to routine haematoxylin and eosin (H&E), modified Giemsa, and IHC staining. The results of staining were compared with those of quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis identified 32 (64%) H. pylori-positive cases, whereas IHC, H&E, and modified Giemsa staining identified 29 (58%), 27 (54%), and 21 (42%) positive cases. The sensitivity of IHC staining (87.50%) was higher than that of H&E (59.38%) and modified Giemsa (43.75%) staining. The specificity of H&E, modified Giemsa, and IHC staining was 55.56%, 61.11%, and 94.44%, respectively. IHC staining exhibited the highest diagnostic accuracy (90%), followed by H&E (58%) and modified Giemsa (50%) staining. Active gastritis, intestinal metaplasia, and lymphoid follicles were detected in 32 (64%), 4 (8%), and 22 (44%) cases, respectively, and all of these cases were H. pylori positive. In contrast to routine H&E and modified Giemsa staining, IHC allows for the accurate H. pylori detection in cases with minimal or atypical infection. Moreover, IHC can be an alternative diagnostic method to qRT-PCR for detection of H. pylori in such cases.Key words: Helicobacter pylori, immunohistochemistry, modified Giemsa stain, real-time polymerase chain reaction, chronic gastritis     

Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry

Background

In order to provide reliable tissue material for malignant mesothelioma (MM) studies, we re-evaluated biopsies and autopsy material from 61 patients with a diagnosis of MM from the period of 1980-2002.

Methods

Basic positive (Calretinin, EMA, Podoplanin, Mesothelin) and negative (CEA, Ber-Ep4) immunohistochemical (IHC) marker reactions were determined. If needed, more markers were used. Histological diagnoses were made by three pathologists. Survival data were calculated.

Results

49 cases (80%) were considered being MM by a high degree of likelihood, five more cases possible MM. Of the remaining seven cases, three were diagnosed as adenocarcinoma, three as pleomorphic lung carcinoma, in one peritoneal case a clear entity diagnosis could not be given. One of the possible MM cases and two of the lung carcinoma cases had this already as primary diagnoses, but were registered as MM. With a sensitivity of 100%, Calretinin and CEA were the most reliable single markers. The amount of MM cells with positive immunoreactivity (IR) for Podoplanin and Mesothelin showed most reliable inverse relation to the degree of atypia. In the confirmed MM cases, there had been applied either no IHC or between one and 18 markers. The cases not confirmed by us had either lacked IHC (n = 1), non-specific markers were used (n = 4), IR was different (n = 1), or specific markers had not shown positive IR in the right part of the tumour cells (n = 3). 46 of the 49 confirmed and three of the not confirmed cases had been diagnosed by us as most likely MM before IHC was carried out.

Conclusions

In order to use archival tissue material with an earlier MM diagnosis for studies, histopathological re-evaluation is important. In possible sarcomatous MM cases without any positive IR for positive MM markers, radiology and clinical picture are essential parts of diagnostics. IHC based on a panel of two positive and two negative MM markers has to be adapted to the differential diagnostic needs in each single case. New diagnostic tools and techniques are desirable for cases where IHC and other established methods cannot provide a clear entity diagnosis, and in order to improve MM treatment.