胃癌前哨淋巴结检测的临床研究

目的:探讨胃癌术中前哨淋巴结(sentinel lymph node,SLN)定位检测的可行性及其临床意义。方法:使用亚甲蓝对40例胃癌患者行前哨淋巴结术中标识活检,随后行D2或D2以上手术。结果:40例胃癌患者中,38例找到前哨淋巴结,检出率为38/40(95%),有32例存在SLN转移,8例SLN为唯一转移部位,且均为T1、T2期。由SLN的病理学状态来预测胃周围淋巴结转移情况的敏感性为32/34(94.12%),特异性为4/4(100%),假阴性率为2/34(5.88%),准确率为34/38(89.47%),其中假阴性的2例,肿瘤都处于T4期。结论:胃癌SLN定位及活检技术能较准确反映早期胃癌的淋巴结转移状况,但对进展期胃癌而言假阴性率较高,对胃癌整个区域淋巴结状态预测的可靠性和可行性尚需进一步验证。     

胃癌前哨淋巴结检测的临床研究

目的：探讨胃癌术中前哨淋巴结（sentinel lymph node,SLN）定位检测的可行性及其临床意义。方法：44t用亚甲蓝对40例胃癌患者行前哨淋巴结术中标识活检,随后行D2或D2以上手术。结果：40例胃癌患者中,38例找到前哨淋巴结,检出率为38／40（95％）,有32例存在SLN转移,8例SLN为唯一转移部位,且均为T1、T2期。由SLN的病理学状态来预测胃周围淋巴结转移情况的敏感性为32／34（94．12％）,特异性为4／4（100％）,假阴性率为2／34（5．88％）,准确率为34／38（89．47％）,其中假阴性的2例,肿瘤都处于T4期。结论：胃癌SLN定位及活检技术能较准确反映早期胃癌的淋巴结转移状况,但对进展期胃癌而言假阴性率较高,对胃癌整个区域淋巴结状态预测的可靠性和可行性尚需进一步验证。     

检测pT_(1-4a)N_(1-3)M_0期胃癌淋巴结微转移检测的临床意义

目的:探讨胃癌淋巴结微转移及临床病理因素对p T1-4aN1-3M0期胃癌患者术后5年无瘤生存率的影响。方法:选取我院2009年1月至12月期间胃肠外科单一手术组行D2胃癌根治术p T1-4aN1-3M0期患者63例1427枚HE染色阴性淋巴结,应用免疫组化法检测这些淋巴结中CK19表达,观察微转移的情况并分析发生微转移的胃癌患者临床病理特征及对患者5年无瘤生存率的影响。结果:临床病理分期p T1-4aN1-3M0胃癌患者中,经免疫组化染色,1427枚HE常规染色阴性淋巴结中CK19阳性表达率为15.49%(221/1427);63例胃癌患者中CK19表达阳性率39.68%(25/63);术后随访时间5.6~68.5月(平均时间43.88月),淋巴结中CK19阴性表达、阳性表达患者的总5年生存率分别为52.63%、28.00%;两者无瘤生存率差异有统计学意义(x2=8.677,P=0.003)。淋巴结CK19阳性表达与胃癌患者的肿瘤直径(P0.05)、浸润胃壁深度(P0.05)有关。COX生存回归分析显示淋巴结微转移为独立预后因素。25例患者发现淋巴结微转移并推荐再分期,再分期率39.68%(25/63)。结论:p T1-4aN1-3M0期胃癌病人,CK-19免疫组化法染色能检出常规HE染色阴性淋巴结中的微转移,有助于细化分期、判断预后及指导治疗。     

Use of Indocyanine Green for Detecting the Sentinel Lymph Node in Breast Cancer Patients: From Preclinical Evaluation to Clinical Validation

Assessment of the sentinel lymph node (SLN) in patients with early stage breast cancer is vital in selecting the appropriate surgical approach. However, the existing methods, including methylene blue and nuclides, possess low efficiency and effectiveness in mapping SLNs, and to a certain extent exert side effects during application. Indocyanine green (ICG), as a fluorescent dye, has been proved reliable usage in SLN detection by several other groups. In this paper, we introduce a novel surgical navigation system to detect SLN with ICG. This system contains two charge-coupled devices (CCD) to simultaneously capture real-time color and fluorescent video images through two different bands. During surgery, surgeons only need to follow the fluorescence display. In addition, the system saves data automatically during surgery enabling surgeons to find the registration point easily according to image recognition algorithms. To test our system, 5 mice and 10 rabbits were used for the preclinical setting and 22 breast cancer patients were utilized for the clinical evaluation in our experiments. The detection rate was 100% and an average of 2.7 SLNs was found in 22 patients. Our results show that the usage of our surgical navigation system with ICG to detect SLNs in breast cancer patients is technically feasible.     

吲哚菁绿联合亚甲蓝在乳腺癌前哨淋巴结活检中的临床应用

目的:探讨吲哚菁绿(indocyanine green,ICG)联合亚甲蓝在乳腺癌前哨淋巴结活检(sentinel lymph node biopsy,SLNB)中的临床应用价值。方法:共入组39例乳腺癌患者,在光学分子影像手术导航系统(surgical navigation system,SNS)的引导下,使用ICG联合亚甲蓝实施SLNB。快速冰冻病理证实前哨淋巴结(sentinel lymph node,SLN)转移者,即刻行腋窝淋巴结清扫(axillary lymph node dissection,ALND);SLN阴性者免于ALND。结果:联合法实施SLNB检出率95%,成功检出SLN125个,平均检出3.2个SLN,准确率95.0%,灵敏度100%,假阴性率0%;蓝染法检出率87.2%,成功检出SLN71个,平均检出1.8个SLN,准确率89.7%,灵敏度93.7%,假阴性率为6.3%;统计结果采用x2检验,检验结果具有统计学意义。结论:ICG联合亚甲蓝实施乳腺癌SLNB成功率高,假阴性率低,临床效果不亚于亚甲蓝,是实施SLNB的新方法。     

Sentinel Lymph Node Detection Using Carbon Nanoparticles in Patients with Early Breast Cancer

Purpose

Carbon nanoparticles have a strong affinity for the lymphatic system. The purpose of this study was to evaluate the feasibility of sentinel lymph node biopsy using carbon nanoparticles in early breast cancer and to optimize the application procedure.

Methods

Firstly, we performed a pilot study to demonstrate the optimized condition using carbon nanoparticles for sentinel lymph nodes (SLNs) detection by investigating 36 clinically node negative breast cancer patients. In subsequent prospective study, 83 patients with clinically node negative breast cancer were included to evaluate SLNs using carbon nanoparticles. Another 83 SLNs were detected by using blue dye. SLNs detection parameters were compared between the methods. All patients irrespective of the SLNs status underwent axillary lymph node dissection for verification of axillary node status after the SLN biopsy.

Results

In pilot study, a 1 ml carbon nanoparticles suspension used 10–15min before surgery was associated with the best detection rate. In subsequent prospective study, with carbon nanoparticles, the identification rate, accuracy, false negative rate was 100%, 96.4%, 11.1%, respectively. The identification rate and accuracy were 88% and 95.5% with 15.8% of false negative rate using blue dye technique. The use of carbon nanoparticles suspension showed significantly superior results in identification rate (p = 0.001) and reduced false-negative results compared with blue dye technique.

Conclusion

Our study demonstrated feasibility and accuracy of using carbon nanoparticles for SLNs mapping in breast cancer patients. Carbon nanoparticles are useful in SLNs detection in institutions without access to radioisotope.     

早期子宫颈恶性肿瘤腹腔镜手术中两种示踪剂前哨淋巴结活检结果的对比分析

目的:通过在早期子宫颈恶性肿瘤患者中应用吲哚箐绿(ICG)及纳米炭混悬液为示踪剂行腹腔镜下前哨淋巴结(sentinel lymph node,SLN)切除术,对比两种示踪剂的示踪效果,寻找临床更适宜普遍使用的示踪剂。方法:选取仁济医院妇瘤科2016.8～2019.10期间诊断明确的122例早期子宫颈恶性肿瘤患者为研究对象。随机采用ICG或纳米炭为前哨示踪剂。对两种示踪剂的显影情况和SLN的示踪效果进行分析。结果:在122例子宫颈恶性肿瘤病例中,宫颈注射ICG64例,检出SLN385枚,平均每个患者检出6.02枚SLN,检出率100%(64/64),特异度96.77%,敏感度75%。宫颈注射纳米炭混悬液58例,检出SLN265枚,平均每个患者检出4.57枚SLN,检出率96.9%(56/58),特异度96.36%,敏感度66.67%。两种示踪剂都有较好的示踪效果(P=0.9356)。结论:早期子宫颈恶性肿瘤行宫颈注射ICG或纳米炭混悬液,腹腔镜下显影的SLN均具有较高的检出率与准确率,是一种较为可行的方法,可普遍开展,值得推广。     

Clinical evaluation of a novel intraoperative handheld gamma camera for sentinel lymph node biopsy

ObjectivePreoperative lymphoscintigraphy (PLS) combined with intraoperative gamma probe (GP) localization is standard procedure for localizing the sentinel lymph nodes (SLN) in melanoma and breast cancer. In this study, we evaluated the ability of a novel intraoperative handheld gamma camera (IHGC) to image SLNs during surgery.MethodsThe IHGC is a small-field-of-view camera optimized for real-time imaging of lymphatic drainage patterns. Unlike conventional cameras, the IHGC can acquire useful images in a few seconds in a free-running fashion and be moved manually around the patient to find a suitable view of the node. Thirty-nine melanoma and eleven breast cancer patients underwent a modified SLN biopsy protocol in which nodes localized with the GP were imaged with the IHGC. The IHGC was also used to localize additional nodes that could not be found with the GP.ResultsThe removal of 104 radioactive SLNs was confirmed ex vivo by GP counting. In vivo, the relative node detection sensitivity was 88.5 (82.3, 94.6)% for the IHGC (used in conjunction with the GP) and 94.2 (89.7, 98.7)% for the GP alone, a difference not found to be statistically significant (McNemar test, p = 0.24).ConclusionSmall radioactive SLNs can be visualized intraoperatively using the IHGC with exposure time of 20 s or less, with no significant difference in node detection sensitivity compared to a GP. The IHGC is a useful complement to the GP, especially for SLNs that are difficult to locate with the GP alone.     

结直肠癌淋巴结微转移灶的临床病理研究

摘要目的：探讨结直肠癌淋巴结微转移灶的特点及其临床病理意义。方法：对我院2001年1月-2006年10月收治的56例无组织学淋巴结转移的结直肠癌患者的650个淋巴结进行微转移灶的免疫组织化学和组织学检测,并分析微转移灶与患者临床病理特征之间的关系。结果-通过对41,zm厚淋巴结连续切片进行观察,连续切片五片的微转移灶检出率较一片和两片的的微转移灶检出率显著升高（P〈0．05）。按解剖部位分组的患者微转移灶阳性率1组有66．67％（16／24）,2组有60％（9／15）,3组有41．2％（7／17）,无显著性差别（P〉0．05）。微转移灶与无组织学淋巴结转移的结直肠癌患者的肿瘤浸润深度（P=0．013）和肿块大小（P=0．037）存在显著性相关,但有微转移灶和无微转移灶的无组织学淋巴结转移的结直肠癌患者总体生存状况比较无显著差异（P〉0．05）。结论：微转移灶在无组织学淋巴结转移的结直肠癌淋巴结中较常见,与肿瘤大小和浸润深度有关,但与患者的预后无显著相关性。     

Tumor-derived TGFbeta-1 induces dendritic cell apoptosis in the sentinel lymph node

Lymphatic flux from a primary tumor initially flows into a tumor-draining lymph node (LN), the so-called sentinel LN (SLN). Carried by the lymph fluid are a variety of mediators produced by the tumor that can influence immune responses within the SLN, making it a good model with which to investigate tumor-related immunology. For instance, dendritic cell (DC) numbers are reduced in SLNs from melanoma and breast cancer patients. In the present study, we investigated the mechanism by which DC numbers were reduced within SLNs from patients with non-small cell lung cancer. We found that the incidence of apoptosis among DCs was higher in SLNs than in non-SLNs, as were levels of TGFbeta-1. In contrast, levels of TGFbeta-1 mRNA did not differ between SLNs and non-SLNs, but were 30 times higher in tumors than in either LN type. In vitro, incubation for 2 days with TGFbeta-1 induced apoptosis among both cultured DCs and DCs acutely isolated from normal thoracic LNs, effects that were blocked by the TGFbeta-1 inhibitor DAN/Fc chimera. Taken together, these results suggest that tumor-derived TGFbeta-1 induces immunosuppression within SLNs before the movement of tumor cells into the SLNs, thereby facilitating metastasis within those nodes.     

The reliability and accuracy of intraoperative imprint cytology of sentinel lymph nodes in breast cancer

OBJECTIVE: Sentinel lymph node (SLN) biopsy is a new component of the surgical treatment of breast cancer that accurately predicts axillary status. In this study the authors evaluated the accuracy of intraoperative imprint cytology (IC) in comparison with definitive histologic evaluation of SLN in breast cancer patients. METHODS: A total 413 women with breast carcinoma and clinically negative axillary nodes underwent breast surgery and SLN biopsy. Mapping of SLN involved injection of (99m)Technecium labelled human albumin nanocolloid particles and Patent Blue dye. At the Department of Pathology, SLNs were bisected along its major axis. Both halves were imprinted 2-4 times on the slides and immediate staining with Hemacolor (Merck Germany) was performed for intraoperative examination. Imprint node negative women underwent no further surgery, while node positive women proceeded to full axillary clearance. Histological analysis of the SLN involved serial sectioning of the whole node with H&E and immunostaining for cytokeratin. RESULTS: Definitive histology revealed metastases (pN+) in 159/413 patients (38.5%): 69 (16.7%) macro metastases, 57 (13.8%) micro metastases, and 33 (8%) women with only isolated IHC positive cells or positive cell groups smaller than 0.2 mm (pNO sn+). The other 254 women had negative SLN biopsy. Imprint cytology detected 54/69 macro metastases, and 4/57 micro metastases. In the group with negative SLN (254), 2 cases were 'false positives'. CONCLUSIONS: Imprint of SLN biopsy can identify a negative axilla with high accuracy (specificity 99.2%). Overall sensitivity is only 36.5%, but macrometastases are detected in 77% which is important for performing ALDN in one session with operation of primary tumour.     

初诊腋窝淋巴结阳性乳腺癌新辅助化疗后对腋窝淋巴结转阴性前哨活检的影响分析

摘要 目的：探究对初诊腋窝淋巴结阳性乳腺癌行新辅助化疗患者开展腋窝前哨淋巴结活检的临床意义。方法：选择2017年1月至2020年10月于我院接受改良根治术或保乳术治疗的100例初诊腋窝淋巴结阳性乳腺癌患者,将其中50例病理检测II B、III期行4~8个疗程新辅助化疗后实施前哨淋巴结活检患者设为研究组,将50例I、II A期直接行前哨淋巴结活检患者设为对照组,对比两组患者前哨淋巴结检出率、准确率、假阴性率和灵敏度,同时就患者病理特征与前哨淋巴结检出率的相关性开展分析。结果：（1）比较显示研究组患者与对照组患者在前哨淋巴结检出数、前哨淋巴结检出率以及前哨淋巴结假阴性率方面组间差异不大（P>0.05）;（2）病理学特征分析显示肿瘤直径以及临床N分期同新辅助化疗后患者前哨淋巴结检出阳性率密切相关（P<0.05）。结论：对初诊腋窝淋巴结阳性行新辅助化疗乳腺癌患者实施前哨淋巴结活检具有较显示的临床意义,能够较好的预测患者腋窝淋巴结状况,同时化疗前肿瘤直径、临床N分期是影响前哨淋巴结检出率的重要影响因素。     

Tissue rinse liquid‐based cytology: a feasible tool for the intraoperative pathological evaluation of sentinel lymph nodes in breast cancer patients

K. Yamashiro, K. Taira, M. Nakajima, D. Okuyama, M. Azuma, H. Takeda, H. Suzuki, H. Jotoku, K. Watanabe, M. Takahashi, K. Taguchi and M. Tamura
Tissue rinse liquid‐based cytology: a feasible tool for the intraoperative pathological evaluation of sentinel lymph nodes in breast cancer patients Objectives: A unique diagnostic method was designed for the intraoperative pathological evaluation of sentinel lymph nodes (SLNs) in breast cancer patients, and the results were verified with 2 years of experience. Methods: Excised lymph nodes were cut into 2‐mm‐thick slices and rinsed thoroughly in CytoRich Red^®. The sliced tissues were embedded in a paraffin block. Three cytological glass slides of the cells exfoliated in CytoRich Red^® were prepared by the SurePath^® liquid‐based cytology (LBC) technique. Two slides were stained by the Papanicolaou method, and the remaining slide was immunostained with an anti‐keratin antibody. This process is called tissue rinse liquid‐based cytology (TRLBC). The results of TRLBC were compared with those of the final pathological diagnoses, including immunostaining with an anti‐keratin antibody on paraffin blocks (PB). Results: This study analysed 444 SLNs from 247 consecutive breast cancer patients. It required 35 minutes to complete the intraoperative diagnosis on a single node, and it took an additional 5 minutes per node if more than one node was submitted. When the results of PB were assumed to be the gold standard, the sensitivity and specificity of TRLBC were 81.9% and 96.1%, respectively. TRLBC detected all nodes with macrometastasis and 23 of 24 nodes with micrometastasis. Fifteen false‐negative TRLBC results were ‘isolated tumour cell clusters’ on PB, but there was one with micrometastasis histologically. Four of 14 false‐positive TRLBC results were proven to be true positive by supplementary examination using step sectioning of the paraffin blocks of the nodes. Conclusion: TRLBC is a feasible and promising intraoperative cytopathological tool showing a comparable efficacy to PB while still allowing the conventional postoperative histological examination.     

Sentinel lymph node detection by combining nonradioactive techniques with contrast agents: State of the art and prospects

The status of sentinel lymph nodes (SLNs) has a substantial prognostic value because these nodes are the first place where cancer cells accumulate along their spreading route. Routine SLN biopsy (“gold standard”) involves peritumoral injections of radiopharmaceuticals, such as technetium-99m, which has obvious disadvantages. This review examines the methods used as “gold standard” analogs to diagnose SLNs. Nonradioactive preoperative and intraoperative methods of SLN detection are analyzed. Promising photonic tools for SLNs detection are reviewed, including NIR-I/NIR-II fluorescence imaging, photoswitching dyes for SLN detection, in vivo photoacoustic detection, imaging and biopsy of SLNs. Also are discussed methods of SLN detection by magnetic resonance imaging, ultrasonic imaging systems including as combined with photoacoustic imaging, and methods based on the magnetometer-aided detection of superparamagnetic nanoparticles. The advantages and disadvantages of nonradioactive SLN-detection methods are shown. The review concludes with prospects for the use of conservative diagnostic methods in combination with photonic tools.     

Clinical relevance of novel imaging technologies for sentinel lymph node identification and staging

The sentinel lymph node (SLN) concept has become a standard of care for patients with breast cancer and melanoma, yet its clinical application to other cancer types has been somewhat limited. This is mainly due to the reduced accuracy of conventional SLN mapping techniques (using blue dye and/or radiocolloids as lymphatic tracers) in cancer types where lymphatic drainage is more complex, and SLNs are within close proximity to other nodes or the tumour site. In recent years, many novel techniques for SLN mapping have been developed including fluorescence, x-ray, and magnetic resonant detection. Whilst each technique has its own advantages/disadvantages, the role of targeted contrast agents (for enhanced retention in the SLN, or for immunostaging) is increasing, and may represent the new standard for mapping the SLN in many solid organ tumours. This review article discusses current limitations of conventional techniques, limiting factors of nanoparticulate based contrast agents, and efforts to circumvent these limitations with modern tracer architecture.     

Étude de faisabilité du ganglion sentinelle dans le cancer de la prostate par cœlioscopie : premiers résultats

Objectives

The standard lymphadenectomy is currently a challenge in the management of prostate cancer. The aim of this prospective study was to evaluate the performance of the sentinel lymph node (SLN) by laparoscopy in patients with localized prostate cancer, candidates for local treatment.

Patients and methods

Patients were injected transrectal ultrasound-guided with 0.3 mL/100MBq 99^mTc-Sulfur rhenium colloid in each prostatic lobe, the day before surgery. Lymphoscintigraphy was performed after 2 hours. The detection was realized intraoperatively with a laparoscopic probe (Gamma Sup Clerad^®) followed by extensive dissection. Counts of SLN were performed in vivo and confirmed ex vivo. The histological analysis was performed by HPS staining and followed by immunochemistry if SLN was free.

Results

Seventy patients with carcinoma of the prostate at intermediate or high risk of lymph node metastases (D’Amico), PSA median 9.5 ng/mL [6–130], were included in the study. The lymphoscintigraphic detection rate was 94.2% (66/70) and intraoperative of 97.0% (68/70). Fourteen patients had lymph node metastases, six only in SLN. The false negative rate was 2/14 (14.0%). The internal iliac region is the first metastatic site (40.9%). Limited or standard lymph node dissection would have ignored respectively 72.7% and 59.0% of lymph node metastases.

Conclusion

The laparoscopy is adapted to a broad identification of SLN and targeted dissection of these lymph nodes significantly limits the risk of surgical extended dissection while maintaining the accuracy of the information.     

Primary Tumor-Secreted Lymphangiogenic Factors Induce Pre-Metastatic Lymphvascular Niche Formation at Sentinel Lymph Nodes in Oral Squamous Cell Carcinoma

Objectives

The objectives of this study were to evaluate the formation of lymphvascular niches in lymph nodes of patients with oral squamous cell carcinoma (OSCC), and investigate the roles of lymphangiogenic and angiogenic factors, such as vascular endothelial growth factor (VEGF)-A, VEGF-C, and VEGF-D, expressed in the primary tumors.

Materials and Methods

Forty-four patients with previously untreated clinically late T2 or T3 OSCC of cN0 were evaluated for primary tumors and 166 sentinel lymph nodes (SLNs). Primary tumors were immunohistochemically analyzed for expressions of VEGFs. Densities of lymphatic vessels (LVD_podoplanin) and high endothelial venules (HEVD) in the SLNs were also calculated using antibodies for each marker, podoplanin and MECA-79, respectively.

Results

In 25 patients, all lymph nodes were metastasis-negative, whereas, in 19 patients, metastasis was positive for at least one lymph node (either at SLN, non-SLN, or nodal recurrence). From the analyses of 140 SLNs without metastasis, LVD_podoplanin in 50 SLNs of metastasis-positive cases was significantly higher than that in 90 SLNs of metastasis-negative cases (p = 0.0025). HEVD was not associated with lymph node metastasis. The patients with VEGF-A-High or VEGF-D-High tumors had significantly higher LVD_podoplanin than patients with their Low counterparts (p = 0.0233 and p = 0.0209, respectively). In cases with lymph node metastasis, the VEGF-D-expression score was significantly higher than in those without lymph node metastasis (p = 0.0006).

Conclusions

These results suggest that lymph node lymphangiogenesis occurs before metastasis in OSCC. VEGF-A and VEGF-D play critical roles in this process. VEGF-D is a potential predictive marker of positive lymph node metastasis in cN0 patients.     

Optimization of an immunostaining protocol for the rapid intraoperative evaluation of melanoma sentinel lymph node imprint smears with the 'MCW melanoma cocktail'

BACKGROUND: In the management of cutaneous melanoma, it is desirable to complete the regional lymphadenectomy during the initial surgical procedure for wide excision of biopsy site and sentinel lymph node (SLN) biopsy. In this study, we optimized and evaluated a rapid 17 minutes immunostaining protocol. The discriminatory immunostaining pattern associated with the 'MCW Melanoma Cocktail' (mixture of Melan- A, MART- 1, and tyrosinase) facilitated the feasibility of intraoperative evaluation of imprint smears of SLNs for melanoma metastases. METHODS: Imprint smears of 51 lymph nodes from 25 cases (48 SLNs and 3 non-SLNs, 1 to 4 SLNs/case) of cutaneous melanoma were evaluated. RESULTS: Sixteen percent, 8/51 lymph nodes (28%, 7/25 cases) were positive for melanoma metastases in immunostained permanent sections with the 'MCW melanoma cocktail'. All of these melanoma metastases, except 1 SLN from 1 case, were also detected in rapidly immunostained wet-fixed and air-dried smears (rehydrated in saline and postfixed in alcoholic formalin). The cytomorphology was superior in air-dried smears, which were rehydrated in saline and postfixed in alcoholic formalin. Wet-fixed smears frequently showed air-drying artifacts, which lead to the focal loss of immunostaining. None of the 5 SLNs from 5 cases exhibiting capsular nevi showed a false positive result with immunostained imprint smears. CONCLUSIONS: Melanoma metastases can be detected intraoperatively in both air-dried smears and wet-fixed smears immunostained with the MCW Melanoma cocktail. Air-dried smears rehydrated in saline and postfixed in alcoholic formalin provide superior results and many practical benefits.