Macrophages in Giemsa-stained cerebrospinal fluid specimens predict carcinomatous meningitis.

Carcinomatous meningitis is a condition in which tumor cells spread to the subarachnoid space. Leukocyte counting and typing of cerebrospinal fluid (CSF) cell components are performed manually or using flow cytometry. However, a detailed analysis of these variables using cytological specimens has not yet been reported. The present study analyzed cytological specimens using Giemsa staining and whole slide imaging with computer-assisted image analysis (CAIA) to clarify the characteristics of the leukocyte population in CSF, especially in carcinomatous meningitis. Manual evaluation was performed using 280 Giemsa-stained cytological CSF specimens. For 49 samples, CAIA was used for the whole area of Papanicolaou (Pap) staining, and Giemsa-stained specimens of the same samples were imaged using a virtual slide scanner. The nuclear morphology of the leukocytes was assessed, and the total leukocyte and leukocyte subset (lymphocytes, neutrophils and macrophages) counts were evaluated. Then, the number and percentage of each leukocyte subset population were evaluated. The total leukocyte count was significantly higher in Giemsa-stained specimens compared with in Pap-stained specimens. The percentage of macrophages was significantly higher in samples from patients with non-hematological tumors compared with in samples from patients without tumors, which was confirmed by manual evaluation of the specimens. In addition, the cut-off value of the percentage of macrophages that could discriminate between the tumor history negative cases and cytologically tumor positive cases was determined, revealing that a higher proportion of macrophages reflected the existence of atypical/malignant epithelial tumor cells in CSF samples. Thus, atypical cell screening and analysis of the background characteristics of the leukocyte population should be the focus of cytological specimen screening, especially not to miss carcinomatous meningitis.

High neutrophil incorporation rate of ascitic fluid cytology as an indicator of cancerous ascites.

The cell­in­cell phenomenon (CiCP) involves the incorporation of a viable cell by other cells (host cells) and includes two concepts: Emperipolesis and cell cannibalism. The former involves the incorporation of hematopoietic cells as the incorporated cells, while the latter involves cell incorporation by tumor cells as host cells. A total of 239 peritoneal cavity fluid cytology specimens were evaluated for CiCP and the number of singly detectable nuclei (SDN) were measured by examining virtual slide image files. The rates of CiCP­positive cases (RCPCs) and CiCP emergence rate (CER)/SDN were significantly higher in ascites samples than in peritoneal washing samples (P<0.0001 and P=0.0026, respectively), although the numbers of SDN were not significantly different between the groups (P=0.8063). Both the RCPCs and CER/SDN were significantly higher in tumor­positive specimens than in tumor­negative specimens (P=0.0220 and P=0.0312, respectively), although the numbers of SDN were not significantly different between the samples (P=0.2471). Most of the incorporated cells were lymphocytes and the host cells were macrophages; however, the rate of neutrophil incorporation (NI) by host cells in the total CiCP cells in a sample was significantly higher in tumor­positive specimens than in tumor­negative specimens (P=0.0288). NI was mainly performed via emperipolesis by macrophages, with only six examples not by macrophages observed among all CiCP samples. The threshold NI rate/total CiCP (NI/CiCP) between tumor­positive and tumor­negative groups was 11.1% (P=0.0115). Using this threshold, the peripheral blood leukocyte count was significantly higher in the high­NI/CiCP group than in the low­NI/CiCP group (P=0.0022). The present findings revealed novel aspects of less frequently observed CiCP in ascitic fluid cytology by utilizing combined manual and computer assisted image analysis evaluation of samples. Notably, the present study indicated the importance of increased NI as an indicator of cancerous ascites.

Phosphomolybdic Acid Prevents Nonspecific Nuclear Staining by Picrosirius Red but Is Converted to Molybdenum Blue by Blue Light.

Picrosirius red (PSR) staining is generally used to evaluate liver fibrosis; however, PSR sometimes causes nonspecific nuclear staining. In this study, we evaluated the ability of phosphomolybdic acid (PMA) pretreatment to prevent nonspecific nuclear staining by PSR. In a manual evaluation of 27 non-tumor samples from patients with hepatocellular carcinoma, nonspecific nuclear staining was observed in 3.7% of PMA-treated specimens, compared with 85.2% of untreated specimens. Conversely, computer-assisted image analysis (CAIA) identified nonspecific nuclear staining in 0% of PMA-treated samples, vs 44.4% of untreated samples. Surprisingly, after mounting, PMA-treated specimens exhibited a blue tinge because of molybdenum blue (MB) production following sunlight exposure or virtual slide scanning. Using UV cut film, MB production induced by sunlight exposure was prevented; however, the film did not prevent MB production during virtual slide scanning. Moreover, only blue light-emitting diode exposure resulted in a blue tinge in PMA solution. Our data indicated that PMA pretreatment is effective for evaluating liver fibrosis using CAIA. Meanwhile, improvements in virtual slide scanning protocols would directly improve the quality of PMA-pretreated specimens subjected to CAIA.