Extra-prostatic extension grading system: correlation with MRI features and integration of capsular enhancement sign for "enhanced" detection of T3a lesions.

PURPOSE: This study aims to confirm the diagnostic accuracy of extra-prostatic extension (EPE) grading system and to explore the predictive capabilities of the prostate MRI while considering various MRI features such as lesion location, apparent diffusion coefficient (ADC) values and capsular enhancement sign (CES). METHODS: Our monocentric study is based on a retrospective analysis of 99 patients who underwent radical prostatectomy from January 2021 to January 2023. The observers reviewed for each lesion, including location (transitional or peripheral zone, anterior or posterior location), capsular contact length, irregular bulging of the capsule, asymmetry of the neurovascular bundle, obliteration of the recto-prostatic angle, macroscopic EPE, ADC value, and CES. RESULTS: Among 99 patients, 31 patients had EPE. Lesions with EPE have broadercapsule contact (24 mm vs 12 mm) with contact ≥14 mm being the optimal cut-off for EPE discrimination. Among the morphological MRI criteria used to determine the EPE, the one with major sensitivity was shown to be bulging (sen 81%), while macroscopic extension had highest specificity (100%). Univariate analysis showed as significative risk factors for EPE: capsular contact ≥14 mm (P < .001), International Society of Urological Pathology score ≥3 (P = .005), CES (P < .001), bulging (P = .001), neurovascular bundle asymmetry (P < .001) and EPE score ≥2 (P < .001), and in multivariate analysis CES (P = .001) and EPE score ≥2 (P = .004) were significant. The AUC of the EPE score was 0.76, raised to 0.83 when combining it with CES (P = .11). CONCLUSION: CES in the setting of multiparametric MRI can increase diagnostic accuracy for the prediction of extracapsular disease. ADVANCES IN KNOWLEDGE: This study highlights the potential of contrast media in prostate cancer local staging.

The synergism of SMC1A cohesin gene silencing and bevacizumab against colorectal cancer.

BACKGROUND: SMC1A is a subunit of the cohesin complex that participates in many DNA- and chromosome-related biological processes. Previous studies have established that SMC1A is involved in cancer development and in particular, is overexpressed in chromosomally unstable human colorectal cancer (CRC). This study aimed to investigate whether SMC1A could serve as a therapeutic target for CRC. METHODS: At first, we studied the effects of either SMC1A overexpression or knockdown in vitro. Next, the outcome of SMC1A knocking down (alone or in combination with bevacizumab, a monoclonal antibody against vascular endothelial growth factor) was analyzed in vivo. RESULTS: We found that SMC1A knockdown affects cell proliferation and reduces the ability to grow in anchorage-independent manner. Next, we demonstrated that the silencing of SMC1A and the combo treatment were effective in increasing overall survival in a xenograft mouse model. Functional analyses indicated that both treatments lead to atypical mitotic figures and gene expression dysregulation. Differentially expressed genes were implicated in several pathways including gene transcription regulation, cellular proliferation, and other transformation-associated processes. CONCLUSIONS: These results indicate that SMC1A silencing, in combination with bevacizumab, can represent a promising therapeutic strategy for human CRC.

BRAFV600E immunohistochemistry can reliably substitute BRAF molecular testing in the Lynch syndrome screening algorithm in colorectal cancer.

AIMS: The Lynch syndrome (LS) screening algorithm requires BRAF testing as a fundamental step to distinguish sporadic from LS-associated colorectal carcinomas (CRC). BRAF testing by immunohistochemistry (IHC) has shown variable results in the literature. Our aim was to analyse concordance between BRAFV600E IHC and BRAF molecular analysis in a large, mono-institutional CRC whole-slide, case series with laboratory validation. METHODS AND RESULTS: MisMatch repair (MMR) protein (hMLH1, hPMS2, hMSH2, and hMSH6) and BRAFV600E IHC were performed on all unselected cases of surgically resected CRCs (2018-2023). An in-house validation study for BRAFV600E IHC was performed in order to obtain optimal IHC stains. BRAFVV600E IHC was considered negative (score 0), positive (scores 2-3), and equivocal (score 1). Interobserver differences in BRAFV600E IHC scoring were noted in the first 150 cases prospectively collected. Nine-hundred and ninety CRCs cases (830 proficient (p)MMR/160 deficient (d)MMR) were included and all cases performed BRAFV600E IHC (BRAFV600E IHC-positive 13.5% of all series; 66.3% dMMR cases; 3.4% pMMR cases), while 333 also went to BRAF mutation analysis. Optimal agreement in IHC scoring between pathologists (P < 0.0001) was seen; concordance between BRAFV600E IHC and BRAF molecular analysis was extremely high (sensitivity 99.1%, specificity 99.5%; PPV 99.1%, and NPV 99.5%). Discordant cases were reevaluated; 1 score 3 + IHC/wildtype case was an interpretation error and one score 0 IHC/mutated case was related to heterogenous BRAFV600E IHC expression. Among the 12 IHC-equivocal score 1+ cases (which require BRAF molecular analysis), three were BRAF-mutated and nine BRAF-wildtype. CONCLUSION: BRAFV600E IHC can be used as a reliable surrogate of molecular testing after stringent in-house validation.

Synchronous and metachronous primary colorectal cancers with concordant and discordant mismatch repair status.

Multiple primary colorectal carcinomas (CRCs) synchronous, when detected approximately at the same time, or metachronous, when a significant amount of time has elapsed between diagnoses can show both mismatch repair (MMR) status concordance and discordance between primary tumors. The aim was to evaluate the MMR status of a monoinstitutional, retrospective cohort of synchronous and metachronous CRCs, with a focus on the frequency of cases with discordant MMR status, and explore the MMR status of metastatic nodal deposits. All synchronous and metachronous CRCs diagnosed in our institution between 2011 and 2023 were collected. Clinicopathologic characteristics were evaluated, including MMR status of all CRCs, BRAF mutation, and MLH1 promoter methylation analyses. MMR status discordant cases were further analyzed, and MMR testing was performed on nodal metastases. Of 3671 patients, 107 (2.9%) had multiple CRCs (94 synchronous and 13 metachronous; total number of CRCs 220). Sixty CRCs were MMR deficient (dMMR) (27.3%), and most were right-sided and high-grade and showed special histologic features (P < .00001). Ninety-three patients showed intertumoral MMR concordance: 70 (65.4%) with MMR-proficient (pMMR) CRCs, and 23 (21.5%) were dMMR. Fourteen patients (13.1%) showed intertumoral MMR discordance (at least one dMMR and one pMMR), and in 5 patients, nodal metastases were present: 2 patients harbored metastases only from their pMMR cancer, 2 only from their dMMR cancer, and in 1 patient both pMMR and dMMR metastases were present. In conclusion, all multiple primary CRCs should be analyzed for MMR status as discordant MMR is possible as well as discordant metastatic nodal deposits, and this may be important for patient management.

Impact of formalin fixation on mismatch repair protein evaluation by immunohistochemistry.

Mismatch repair/microsatellite instability (MMR/MSI) status in colorectal cancer (CRC) has become fundamental as a diagnostic, prognostic, and predictive factor. MMR immunohistochemistry (IHC) is considered a simple and reliable approach; however, its effectiveness depends on pre-analytic factors. Aim of this study was to investigate the impact of different fixation times/protocols on MMR protein IHC quality. Left over tissue from surgically resected CRC samples (cold ischemia time < 30 min) where fixed as follows: standard formalin fixation (24-48 h); hypo-fixation (<20 h); hyper-fixation (>90 h); cold (4°C) fixation (24-48 h); standard fixation for small sample size (0.5×0.5 cm). Samples for each group were collected from 30 resected CRC and the following parameters were evaluated on 600 immunohistochemical stains: intensity of expression; patchiness of staining; presence of central artefact. Forty-six immunoreactions were inadequate (score 0 intensity), the majority regarding MLH1 or PMS2 in the hypo-fixation group (47.8%), followed by the hyper-fixation group (28.1%); cold formalin fixation showed the least inadequate cases. Patchiness and central artefact were more frequent in hypo-fixation and standard fixation group compared to the others. MLH1 (closely followed by PMS2) performed worse with regard to immunostaining intensity (p=0.0002) in the standard and in the hypo-fixation group (p< 0.00001). Using a small sample size improved patchiness/central artefacts. This is the first study specifically created to evaluate the impact of fixation on MMR protein IHC, showing that both formalin hypo- and hyper-fixation can cause problems; 24-h formalin fixation as well as cold (4°C) formalin fixation are recommended for successful IHC MMR evaluation.

A High Percentage of CD16+ Monocytes Correlates with the Extent of Bone Erosion in Chronic Lymphocytic Leukemia Patients: The Impact of Leukemic B Cells in Monocyte Differentiation and Osteoclast Maturation.

Significant skeletal alterations are present in Chronic Lymphocytic Leukemia (CLL) patients; bone erosion, particularly evident in the long bone shaft, appeared increased in the progressive disease stage. Moreover, the partial colonization of the bone with reactive bone marrow we documented via PET-FDG imaging suggests that neoplastic cell overgrowth contributes to bone derangement. Indeed, cytokines released by leukemic B cells impair osteoblast differentiation and enhance osteoclast formation in vitro. CD16, Fcγ-RIIIa, has been previously indicated as a marker of osteoclast precursors. We demonstrate, here, that the percentage of circulating monocytes, CD16+, is significantly higher in CLL patients than in normal controls and directly correlated with the extent of bone erosion. When we assessed if healthy monocytes, treated with a CLL-conditioned medium, modulated RANK, RANKL and CD16, we observed that all these molecules were up-regulated and CD16 to a greater extent. Altogether, these findings suggest that leukemic cells facilitate osteoclast differentiation. Interestingly, the evidence that monocytes, polarized toward the M2 phenotype, were characterized by high CD16 expression and showed a striking propensity to differentiate toward osteoclasts may provide further explanations for the enhanced levels of bone erosion detected, in agreement with the high number of immunosuppressive-M2 cells present in these patients.

HLA-G as a prognostic marker in stage II/III colorectal cancer: not quite there yet.

Identifying innovative molecules involved in the tumor immune escape process could help refine the survival stratification of colorectal cancer (CRC) patients. HLA-G, a non-classical HLA molecule, physiologically involved in tolerogenic mechanisms, has recently emerged as a relevant prognostic marker in other tumor types, but ambiguous data are reported in the CRC setting. This study aims to evaluate the HLA-G expression and prognostic potential in a series of stage II/III CRCs. HLA-G expression was evaluated in 100 pT3 CRC cases by means of immunohistochemistry using the 4H84 and MEM-G/2 monoclonal antibodies. We observed heterogeneous expression of HLA-G showing different ranges: 4H84 expression ranged from > 1 to 40%-median 7%; MEM-G/2 expression ranged from 20 to 90%-median 50%. HLA-G positivity (any intensity > 1%) varied according to the antibody employed, identifying: 8 4H84 positive, 34 MEM-G/2 positive, 6 double-positive and 52 negative cases. Correlation with clinico-pathologic data showed a significant association with a poor tumor differentiation in stage III right-sided CRC subgroup (p = 0.043), while no other pathologic variable was significantly associated. Survival analysis revealed a reduced disease-free survival rate (HR 4.304613; p = 0.031) in the subgroup of CRC-related death cases, while no correlations were observed considering the whole series and the overall survival. In conclusion, HLA-G is a promising CRC prognostic marker however much work is still required regarding technical aspects and evaluation of expression.

MiR-146b-5p regulates IL-23 receptor complex expression in chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (CLL) cells express the interleukin-23 receptor (IL-23R) chain, but the expression of the complementary IL-12Rß1 chain requires cell stimulation via surface CD40 molecules (and not via the B-cell receptor [BCR]). This stimulation induces the expression of a heterodimeric functional IL-23R complex and the secretion of IL-23, initiating an autocrine loop that drives leukemic cell expansion. Based on the observation in 224 untreated Binet stage A patients that the cases with the lowest miR-146b-5p concentrations had the shortest time to first treatment (TTFT), we hypothesized that miR-146b-5p could negatively regulate IL-12Rß1 side chain expression and clonal expansion. Indeed, miR-146b-5p significantly bound to the 3'-UTR region of the IL-12Rß1 mRNA in an in vitro luciferase assay. Downregulation of miR-146b-5p with specific miRNA inhibitors in vitro led to the upregulation of the IL-12Rß1 side chain and expression of a functional IL-23R complex similar to that observed after stimulation of the CLL cell through the surface CD40 molecules. Expression of miR-146b-5p with miRNA mimics in vitro inhibited the expression of the IL-23R complex after stimulation with CD40L. Administration of a miR-146b-5p mimic to NSG mice, successfully engrafted with CLL cells, caused tumor shrinkage, with a reduction of leukemic nodules and of IL-12Rß1-positive CLL cells in the spleen. Our findings indicate that IL-12Rß1 expression, a crucial checkpoint for the functioning of the IL-23 and IL-23R complex loop, is under the control of miR-146b-5p, which may represent a potential target for therapy since it contributes to the CLL pathogenesis. This trial is registered at www.clinicaltrials.gov as NCT00917540.

Ultra-hyper-fractionated radiotherapy for high-grade gliomas.

High-grade gliomas (HGGs; WHO grades III and IV) are invariably lethal brain tumors. Low-dose hyper-radiosensitivity (HRS) of HGG is a well-established phenomenon in vitro. However, possibly linked to the unavailability of accurate animal models of the diseases, this therapeutic effect could not be consistently translated to the animal setting, thus impairing its subsequent clinical development. The purpose of this study was to develop radiotherapeutic (RT) schedules permitting to significantly improve the overall survival of faithful animal models of HGG that have been recently made available. We used primary glioma initiating cell (GIC)-driven orthotopic animal models that accurately recapitulate the heterogeneity and growth patterns of the patients' tumors, to investigate the therapeutic effects of low radiation doses toward HGG. With the same total dose, RT fractions ≤0.5 Gy twice per week [ultra-hyper-fractionation (ultra-hyper-FRT)] started at early stages of tumor progression (a condition that in the clinical setting often occurs at the end of the guidelines treatment) improved the effectiveness of RT and the animal survival in comparison to standard fractions. For the same cumulative dose, the use of fractions ≤0.5 Gy may permit to escape one or more tumor resistance mechanisms thus increasing the effectiveness of RT and the overall animal survival. These findings suggest investigating in the clinical setting the therapeutic effect of an ultra-hyper-FRT schedule promptly extending the conventional RT component of the current guideline ("Stupp") therapeutic protocol.

The Seeds of Doubt: Finding Seeds in Intriguing Places.

Introduction: Seeds may be found in gastrointestinal tissue samples, and their multifaceted appearance may be challenging. The aim is to report a rough incidence of pathology samples which show seeds, specify the most frequent sample types and show an iconography of the most commonly identified seeds. Materials and Methods: Between 2017 and 2020, all gastrointestinal pathology cases in which seeds/seed parts were found, were collected and seed type described by referencing a seed image library. Results: Fifty cases with complete seeds/seed parts were collected: 16 colonic resections for colorectal cancer and diverticulosis, 13 appendiceal resections for appendicitis, 1 gastric resection. Fifteen cases were found in polypectomy specimens and 5 cases in colorectal endoscopic biopsies. Most frequent seed types were tomato, kiwi, blueberry, and blackberry seeds. Conclusion: Seeds may be found in up to 4% of specimens; their recognition may be useful to exclude parasitic infections as well as in forensic sciences.

Methods for restoration of ki67 antigenicity in aged paraffin tissue blocks.

Pathology archives are a treasure trove of paraffin embedded tissue spanning many years and covering a wide variety of tissues and diseases. The possibility of using old archival formalin fixed paraffin embedded (FFPE) tissues for diagnostic updates and research projects is a widespread need and it requires archives of stable, well-preserved samples. Immunohistochemistry performed on old archival paraffin blocks may give unreliable results, in particular for some antigens, such as Ki67. In consideration of this phenomenon, our aim is to comprehensively test and identify methods which may be used to obtain Ki67 immunohistochemical reactions of good quality from old archival FFPE blocks. Various methods were tested in order to evaluate their possible efficacy in increasing Ki67 immunointensity in a collection of 40-year-old, archival blocks including re-embedding, with deeper sectioning of tissue from the block and increasing heat-based pretreatment times (20 cases) and re-processing (20 cases). All reactions were performed using an automated immunostainer and Ki67 stained immunosections compared using a visual colour-based scale (the first immunostained section was considered as baseline). The combination of deep sectioning (1000 µM) and prolonged heat-based pretreatment (64 min) markedly increased immunoreactivity for Ki67. Re-embedding and reprocessing did not have a significant effect. Large tissue samples showed heterogeneity of Ki67 immunoexpression between the periphery of the sample and the central area. In conclusion, the study defines a useful protocol to increase antigen retrieval applicable to dated archival tissues.

Chronic lymphocytic leukemia cells impair osteoblastogenesis and promote osteoclastogenesis: role of TNFα, IL-6 and IL-11 cytokines.

Bone skeletal alterations are no longer considered a rare event in chronic lymphocytic leukemia (CLL), especially at more advanced stages of the disease. This study is aimed at elucidating the mechanisms underlying this phenomenon. Bone marrow stromal cells, induced to differentiate toward osteoblasts in osteogenic medium, appeared unable to complete their maturation upon co-culture with CLL cells, CLL-cell-derived conditioned media (CLL-cm) or CLL-sera (CLL-sr). Inhibition of osteoblast differentiation was documented by decreased levels of RUNX2 and osteocalcin mRNA expression, by increased osteopontin and DKK-1 mRNA levels, and by a marked reduction of mineralized matrix deposition. The addition of neutralizing TNFα, IL-11 or anti-IL-6R monoclonal antibodies to these cocultures resulted in restoration of bone mineralization, indicating the involvement of these cytokines. These findings were further supported by silencing TNFα, IL-11 and IL-6 in leukemic cells. We also demonstrated that the addition of CLL-cm to monocytes, previously stimulated with MCSF and RANKL, significantly amplified the formation of large, mature osteoclasts as well as their bone resorption activity. Moreover, enhanced osteoclastogenesis, induced by CLL-cm, was significantly reduced by treating cultures with the anti-TNFα monoclonal antibody infliximab. An analogous effect was observed with the use of the BTK inhibitor, ibrutinib. Interestingly, CLL cells co-cultured with mature osteoclasts were protected from apoptosis and upregulated Ki-67. These experimental results parallel the direct correlation between amounts of TNFα in CLL-sr and the degree of compact bone erosion that we previously described, further strengthening the indication of a reciprocal influence between leukemic cell expansion and bone structure derangement.

Increased Arginase1 expression in tumor microenvironment promotes mammary carcinogenesis via multiple mechanisms.

Arginine metabolism plays a significant role in regulating cell function, affecting tumor growth and metastatization. To study the effect of the arginine-catabolizing enzyme Arginase1 (ARG1) on tumor microenvironment, we generated a mouse model of mammary carcinogenesis by crossbreeding a transgenic mouse line overexpressing ARG1 in macrophages (FVBArg+/+) with the MMTV-Neu mouse line (FVBNeu+/+). This double transgenic line (FVBArg+/-;Neu+/+) showed a significant shortening in mammary tumor latency, and an increase in the number of mammary nodules. Transfer of tumor cells from FVBNeu+/+ into either FVB wild type or FVBArg+/+ mice resulted in increase regulatory T cells in the tumor infiltrate, suggestive of an impaired antitumor immune response. However, we also found increased frequency of tumor stem cells in tumors from FVBArg+/-;Neu+/+ transgenic compared with FVBNeu+/+ mice, suggesting that increased arginine metabolism in mammary tumor microenvironment may supports the cancer stem cells niche. We provide in vivo evidence of a novel, yet unexploited, mechanism through which ARG1 may contribute to tumor development.

Coping with formalin banning in pathology: under vacuum long-term tissue storage with no added formalin.

Formalin is toxic and has recently been classified as carcinogenic leading to a proposed European formalin ban. But, the pathology use of formalin has however been completely overlooked, and this is proving to be a relevant issue, as no alternative, reliable, tissue fixative is available. Various systems have been proposed to reduce formalin use and exposure; long-term storage and disposal of formalin is also a problem. With this in mind, under vacuum sealing (UVS) systems have been proposed for transportation/storage, however, for how long tissue retains its characteristics (morphological and molecular) is unknown. This study aims to compare histology specimens stored by formalin immersion (FI) and specimens stored after fixation with UVS technique with no additional formalin, at different time periods. Twenty tissue samples (10FI; 10UVS) were stored for different time periods (15 days, 1-2-3-6-12 months) for a total of 120 samples, compared with regard to their morphology, histochemistry, immunoreactivity (24 specific antibodies) and DNA status. All samples showed well-preserved morphology and overlapping staining quality. A significant reduction in immunoreactivity was however identified in the various time periods, particularly for heat pre-treated nuclear antigens, and this commenced earlier (1 month) for FI. UVS storage showed higher DNA content than FI but slightly poorer DNA integrity. These results add important knowledge to the use of UVS in daily practice, as long-term storage of pre-fixed tissue in UVS is not detrimental to the quality of tissue while having the boon of using very little formalin with less operator exposure and lower disposal costs.

HLA-G expression in gastric carcinoma: clinicopathological correlations and prognostic impact.

To analyze expression of human leukocyte antigen-G (HLA-G) in gastric adenocarcinoma and correlate its expression with histological and clinical variables. A continuous series of 94 unselected patients with gastric adenocarcinoma (stage I to III) were selected. All histological and clinical variables were collected including the intensity of intra- and peri-tumor lymphocytic infiltration. HLA-G expression was investigated using immunohistochemistry. All histological samples analyzed for HLA-G expression were taken from the primary gastric lesion and included non-neoplastic mucosa. Evaluation of HLA-G expression was performed on the transition zone between tumor and non-neoplastic mucosa, and the invasive front of the tumor and assessment was performed as follows: percentage of positive (strong expression vs weak) cells. A variable amount of HLA-G-positive tumor cells was found in 24 out of 94 cases (25.5%). No significant correlation was found between HLA-G expression and other clinicopathological variables (sex, age, stage, grade, histotype). The overall median survival was worse in patients with HLA-G-positive adenocarcinoma (24.3 months, CI95% 7.7-41.0) compared to those with HLA-G-negative tumors (66.3 months, CI95% 53.0-79.7; p < 0.0001). Two- and 5-year survival rates of HLA-G-negative patients were 88 and 44%, respectively, while were 42 and 11% in those HLA-G-positive. This trend was observed in all stages but was more marked in stage III. HLA-G expression is associated with poor survival in stage III gastric cancer patients and represents a possible immunoescape mechanism of cancer cells.

Immunohistochemistry on old archival paraffin blocks: is there an expiry date?

Few studies have focused on antigen preservation in formalin-fixed and paraffin-embedded (FFPE) tissue in old archival material and additional studies are required, especially considering that these samples are an irreplaceable resource for scientific and clinical research. The purpose of this study is to verify antigen preservation in FFPE tissue samples stored for several decades. From the pathology archives, FFPE blocks were selected dating back to the 1960s, 1970s, 1980s, 1990s, 2000s and 2010. A panel of 12 antibodies was applied and immunoreactivities were compared. While cytoplasmic antigens showed no reduction in immunostaining intensity over time, membrane and nuclear antigens presented reduced staining intensity in older blocks. In particular, the nuclear antigen, Ki67 and CD31 showed the most pronounced antigen decay in the oldest archival blocks. In order to test possible antigen recovery, deep sectioning and lengthening of heat pretreatment were applied. Both strategies partially recover antigenicity, but their simultaneous application shows the best results.

Section detachment in immunohistochemistry: causes, troubleshooting, and problem-solving.

Section detachment in immunohistochemistry (IHC) is a common phenomenon, increasing times and costs of diagnosis and research. However, it has poorly been investigated. The aim of this study was to identify the causes of section detachment, with the purpose of defining a quality assured laboratory procedure to minimize detachment frequency. We screened 3349 IHC sections from formalin-fixed paraffin-embedded tissue, identifying 177 cases with section detachment (5.3% of the sample). Detachment regarded mainly samples of surgical breast tissue and IHC procedures in which heat pretreatment was used. Focusing on pre-analytical factors, we investigated seven main critical issues: (1) section aging; (2) section thickness; (3) slide contamination; (4) slide aging; (5) slide brand; (6) "human" influence; and (7) sample size and fixation. Each of these issues was individually investigated to establish their influence on detachment. Targeted experiments were performed by varying section age, thickness, cleanliness, slide brand and age, and sample size and fixation. Finally, to investigate operator-dependent causes, sections were cut by different operators blinded to aim. The most important factors influencing section detachment were demonstrated to be: section thickness, slide aging, slide brand, "human" influence, and size and fixation of samples. The pre-analytical phase, including all the aforementioned issues, should be standardized within a quality assurance program. By adopting these recommendations, we obtained a 34% drop in section detachment. Although section detachment remains difficult to eradicate completely, many other influences can be addressed and corrected in any laboratory leading to an increase in efficiency and cost saving.