Susana Castañón

Flow Cytometric Analysis of Cerebrospinal Fluid Samples and Its Usefulness in Routine Clinical Practice

Low volume and few cells have hampered the use of flow cytometry for studying cerebrospinal fluid (CSF) in routine clinical practice, although information about the cellular phenotypes present in this type of sample is of great value in many diseases. We developed a novel flow cytometric strategy capable of identifying total CSF T lymphocytes and the CD4+ subset, even in CSF samples with as few as 1 leukocyte per 3 microL of sample. We also showed that identification of CD8+ T cells could be achieved in most samples, while B lymphocytes are detectable only in samples with more than 5 cells per microliter. These findings demonstrate the reliability of this method to improve the diagnostic accuracy of classic cytologic studies in many neurologic disorders.

Features of the CD4+ T-cell response in liver and peripheral blood of hepatitis C virus-infected patients with persistently normal and abnormal alanine aminotransferase levels.

BACKGROUND/AIMS The liver is the primary site of hepatitis C virus (HCV) replication; intrahepatic T-cell responses may influence liver disease severity. METHODS HCV-specific CD4(+) T-cell reactivity was investigated ex vivo in paired liver tissue and peripheral blood from 42 chronic HCV patients. RESULTS The frequencies with which HCV-specific HLA class-II-restricted CD4(+) T-cell proliferation were observed were 29% in liver and 36% in peripheral blood. Among responses, non-structural-3 protein (NS3)-specific T-cell proliferation was dominant but non-exclusive and did rarely occur concurrently in liver infiltrate and peripheral blood suggesting liver compartmentalization of a CD4(+) T-cells population. Compared with 24 patients with abnormal ALT levels, 18 HCV carriers with persistently normal ALT levels had similar serum and liver viral loads but showed: (i) a low-activity grade and stage chronic hepatitis (P<0.001); (ii) less intrahepatic CD4(+) T-lymphocytes (P<0.01); (iii) less frequent intrahepatic (17 vs. 33%) and peripheral (17 vs. 38%) NS3-specific CD4(+) T-cell proliferation; (iv) less often in vitro T-helper (Th)1 (interferon-gamma) cytokine production (2 vs. 18%; P<0.001). CONCLUSIONS Our data show a low frequency of intrahepatic HCV-specific HLA class-II-restricted CD4(+) Th1 responses in patients with chronic HCV. However, these Th1 responses are detected more often in those patients with overt clinical and histological disease.

Immunophenotype in chronic myelomonocytic leukemia: is it closer to myelodysplastic syndromes or to myeloproliferative disorders?

Chronic myelomonocytic leukemia (CMML) is a heterogeneous disease balanced between myelodysplastic syndromes (MDS) and myeloproliferative disorders (MPD). We used flow cytometry to describe and compare the immunophenotypic profile of 20 patients with CMML, 38 patients with MDS, and 20 patients with MPD. CMML and MDS only showed statistically significant differences (P<0.05) in CD56 monocyte expression. CMML and MPD showed significant differences in CD45 myeloid distribution, myeloid antigenic profile, CD56 and CD2 monocyte expression, and B-cell development. These data support the classic concept of CMML as part of MDS diseases and encourage including immunophenotyping among the studies to be performed in these diseases.

Role of flow cytometry immunophenotyping in the diagnosis of leptomeningeal carcinomatosis

PURPOSE To explore the contribution of flow cytometry immunophenotyping (FCI) in detecting leptomeningeal disease in patients with solid tumors. EXPERIMENTAL DESIGN Cerebrospinal fluid (CSF) samples from 78 patients who received a diagnosis of epithelial-cell solid tumors and had clinical data suggestive of leptomeningeal carcinomatosis (LC) were studied. A novel FCI protocol was used to identify cells expressing the epithelial cell antigen EpCAM and their DNA content. Accompanying inflammatory cells were also described. FCI results (positive or negative for malignancy) were compared with those from CSF cytology and with the diagnosis established by the clinicians: patients with LC (n = 49), without LC (n = 26), and undetermined (n = 3). RESULTS FCI described a wide range of EpCAM-positive cells with a hyperdiploid DNA content in the CSF of patients with LC. Compared with cytology, FCI showed higher sensitivity (75.5 vs 65.3) and negative predictive value (67.6 vs 60.5), and similar specificity (96.1 vs 100) and positive predictive value (97.4 vs 100). Concordance between cytology and FCI was high (Kp = 0.83), although misdiagnosis of LC did not show differences between evaluating the CSF with 1 or 2 techniques (P = .06). Receiver-operator characteristic curve analyses showed that lymphocytes and monocytes had a different distribution between patients with and without LC. CONCLUSION FCI seems to be a promising new tool for improving the diagnostic examination of patients with suspicion of LC. Detection of epithelial cells with a higher DNA content is highly specific of LC, but evaluation of the nonepithelial cell compartment of the CSF might also be useful for supporting this diagnosis.

Circulating immune complexes from HIV‐1+ patients induces apoptosis on‘qc normal lymphocytes

Isolated immune complexes from sera of 49 out of 67 human immunodeficiency virus‐1‐positive (HIV‐1+) patients (CIC–HIV+), composed of anti‐HIV–HIV‐Ag, could induce apoptosis on normal phytohaemagglutinin (PHA)‐activated lymphocytes. DNA degradation was detected by propidium iodide staining. This activity is directed against CD4+ lymphocytes as demonstrated by double binding of CIC–HIV+ and anti‐CD4 on apoptotic cells. Expression of Fas antigen is prior to apoptotic phenomena. CIC–HIV+ apoptosis inducers belong mainly to asymptomatic HIV‐infected patients, indicating that immune complexes from these patients can destroy CD4+ lymphocytes.

Differences in cerebrospinal fluid inflammatory cell reaction of patients with leptomeningeal involvement by lymphoma and carcinoma

Dissemination of neoplastic cells into the cerebrospinal fluid (CSF) and leptomeninges is a devastating complication in patients with epithelial cell neoplasia (leptomeningeal carcinomatosis [LC]) and lymphomas (lymphomatous meningitis [LyM]). Information about the surrounding inflammatory cell populations is scarce. In this study, flow cytometry immunophenotyping was used to describe the distribution of the main leukocyte populations in the CSF of 83 patients diagnosed with neoplastic meningitis (LC, n = 65; LyM, n = 18). These data were compared with those obtained in the CSF from 55 patients diagnosed with the same groups of neoplasia without meningeal involvement (solid tumors, n = 36; high-grade lymphoma, n = 19). Median (interquartile) rates of lymphocytes, monocytes, and polymorphonuclear (PMN) cells were 59.7% (range, 35-76.6%), 24% (range, 16-53%), and 1.5% (range, 0-7.6%) in LC, respectively, and 98.5% (range, 70.8-100%), 1.5% (range, 0-29.3%), and 0% in LyM, respectively (P < 0.001). No difference was observed between patients with breast adenocarcinoma (n = 30) and lung adenocarcinoma (n = 21), nor with different rates of malignant CSF involvement. Patients with lymphoma (with or without LyM) had a similar CSF leukocyte distribution, but cancer patients with LC and without LC had a distinctive PMN cell rate (P = 0.002). These data show that CSF samples from patients with LC have a greater number of inflammatory cells and a different leukocyte distribution than seen in the CSF from patients with LyM. Description of PMN cells is a distinctive parameter of patients with LC, compared with the CSF from patients with LyM and patients with cancer but without LC.

Role of antiretroviral regimes in HIV‐1 patients in reducing immune activation

We assessed whether antiretroviral regimes are able to diminish apoptosis and markers of lymphocyte activation and restore lymphocyte proliferation. T‐cell subset, spontaneous and induced apoptosis, CD95 and soluble Fas antigen and cell proliferation were analysed in 41 human immunodeficiency virus type 1‐positive patients. Twenty‐five were in asymptomatic stage A and 16 were in stage B/C. Thirty‐five received antiretroviral treatment: 18 received two inhibitors of reverse transcriptase and one protease inhibitor and 17 received three inhibitors of reverse transcriptase. Six patients did not receive treatment, for different reasons, but continued to participate in the study. Studies were performed at baseline, 3, 6 and 12 months. Levels of CD4 increased slightly until 6 months of antiretroviral treatment, as a whole, in all the patients treated. Naïve CD4 lymphocytes, as well as memory CD4 lymphocytes, remained constant. Spontaneous apoptosis of lymphocytes, after 72 hr of culture, decreased in all patients treated, but to a much smaller extent than phytohaemagglutinin‐induced apoptosis. In both groups treated, levels of soluble Fas decreased until 6 months of treatment and then increased again. Lymphocyte proliferation reached normal levels after 1 year of treatment. In patients without treatment CD4 cells decreased slowly and no modification in activation markers was found. Antiretroviral regimes decrease immune activation as well as viral load and this deactivation restores lymphocyte proliferation.