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PubMed2000

Diagnosis of venom allergy by flow cytometry. Correlation with clinical history, skin tests, specific IgE, histamine and leukotriene C4 release.

BACKGROUND: Potent allergens such as hymenoptera venoms are capable of inducing severe and life threatening clinical reactions. Percentage of false negative results obtained by the usual diagnostical methods is comprised between 10 and 25%. OBJECTIVE: Evaluation of the sensitivity and the specificity of cellular tests and particularly evaluation of a new flow cytometric method. METHODS: Forty-five allergic patients having experienced a local, a systemic reaction or an anaphylactic shock and 10 controls having undergone hymenoptera stings without clinical reactions were selected on the basis of the clinical history, skin tests and specific IgE. Three cellular tests were performed on the same cell suspensions and in the presence of 2 ng/mL of rIL3: histamine release (RIA), leukotriene C4 release (ELISA) and basophil activation test (flow cytometry after double anti-IgE FITC, anti-CD63 PE labelling). RESULTS: As compared to the clinical history, sensitivities of skin tests, specific IgE, flow cytometry, histamine release and leukotriene release were, respectively; 85%, 88%, 100%, 89% and 100%. Flow cytometric analysis of basophil activation showed a significant decrease of the mean fluorescence density and number of IgE positive cells and a significant increase of the number of CD63 positive cells. The 10 controls tested by flow cytometry were negative. CONCLUSION: As compared to the clinical history and to the other parameters tested here, flow cytometry showed a high sensitivity and a high specificity. The excellent correlation observed between this method and the other cellular tests such as histamine and leukotriene release are in favour of the specificity of flow cytomery and in favour of the use of this method for venom allergy diagnosis.

Adolescent↗

PubMed1988

Future clinical role for flow cytometry.

It appears that there is an essential, not just supportive, role for flow cytometry in the clinical context, particularly in providing early information in clinical oncology. High flow rate enumeration and sorting of rare cells, combined with microscopy, offer immediate benefits in the clinical processing of at least some cancers. These benefits would be in diagnosis (perhaps very early detection of metastatic cells in the present prediagnostic phase of solid tumor growth), monitoring, prognosis, and therapy. Importantly, flow cytometric measures can be implemented immediately, and measurement times are short. The value of high flow rate operation of existing facilities in clinical, "supportive" flow cytometry should be better appreciated, if only because shorter measurement times and on-line analysis would make the existing facilities more cost effective: higher throughput for the same overheads. Finally, the wisdom of employing nonsorting cytometers for clinical use should be strongly questioned. Thus, what future impact will the application of flow sorting have in clinical fields old and new, e.g., in bacterial infection measurements in peripheral blood? In particular, nonsorting machines will be unable to adopt the "essential" clinical role I have proposed here.

Animals↗

PubMed2002

External quality assurance schemes in flow cytometry.

Quality assurance schemes were devised to monitor the performance of laboratory tests commonly utilized in routine clinical practice. In the area of Flow Cytometry, external quality assurance schemes are very much in their infancy. There are 3 major applications of Flow Cytometry in the clinical laboratory: lymphocyte subset enumeration, CD34 enumeration and leukemia immuno-phenotyping. Pilot schemes for CD34 enumeration and leukemia immuno-phenotyping have revealed a considerable lack of consensus with a wide divergence in practices. The coefficient of variation in reported results from these assessments have been very large, ranging from 20-200%. Results for lymphocyte subset enumeration surveys are sometimes not much better despite having been around for a longer period of time. Quality assurance programs in Flow Cytometry are difficult to conduct and 3 or 4 concurrent factors are often cited as reasons for the large variability. The need for viable, functionally intact cells as suitable assay material is probably the biggest contributor to the large variability. This difficulty in providing stable cells for inter-laboratory comparison is compounded by the lack of consensus in testing methodology. Participation in a distant external quality assurance scheme is consequently not very helpful to improving the practices in the laboratory. Currently, there are more problems than solutions. An awareness of these issues will hopefully contribute to the development of future consensus and offer up solutions to some of these problems.

Flow Cytometry↗

PubMed1993

Validation of flow cytometry analysis in the objective assessment of spermatogenesis: comparison to the quantitative testicular biopsy.

Objective determination of spermatogenesis has been accomplished by quantitative testicular biopsy, which, although laborious, has served as the standard for spermatogenic assessment. Aspiration deoxyribonucleic acid (DNA) flow cytometry of the testis, however, has simplified this determination, and has correlated with indirect hormonal parameters of spermatogenesis and qualitative observations of the seminiferous epithelium. Nevertheless, this important modality has yet to be validated against quantitative micrometry of the testis. To determine this correlation we submitted 29 incisional testicular biopsies for simultaneous quantitative analysis and DNA flow cytometry. Micrometric parameters included the mean tubular wall thickness, and the mean tubular concentration of late spermatids and Sertoli cells. The percentage of haploid, diploid and tetraploid cells was determined for each patient. For the entire patient population a statistically significant correlation was observed between the percentage of haploid cells and the tubular concentration of late spermatids (r = 0.784, p < 0.0005) as well as the mean tubular spermatid-to-Sertoli cell ratio (r = 0.824, p < 0.0005). A similar correlation was noted for various etiological subsets of patients: spinal cord injury (r = 0.809, p < 0.002), genital tract obstruction (r = 0.705, p < 0.02) and miscellaneous diagnoses (r = 0.828, p < 0.02). For the group with testicular failure quantitative micrometry and flow cytometry demonstrated severe impairment in all patients although a statistically significant correlation could not be shown because of the small range of values. DNA flow cytometry analysis correlates strongly with the current standard of quantitative spermatogenic assessment and, therefore, it may be validated as a simplified and highly objective method of determining spermatogenesis.

Biopsy↗

PubMedDate not supplied

Clinical applications of flow cytometry.

OBJECTIVE: To describe the basics of three clinical applications of the flow cytometer. DATA SOURCES: Recent articles and books on flow cytometry and laboratory diagnosis. STUDY SELECTION: Not applicable. DATA EXTRACTION: Performed by the author. DATA SYNTHESIS: Immunophenotyping is the classification of cells based on antigens present on their surfaces. These antigens can be detected and quantified by a flow cytometer using monoclonal antibodies conjugated to fluorescent dyes. Reticulocytes are immature red blood cells that contain varying amounts of RNA and DNA. They can be stained with a fluorescent dye and enumerated by a flow cytometer. DNA ploidy analysis of solid tumors involves staining the nuclei of cells with a fluorescent dye. The amount of DNA in each cell is determined, and the percentage of cells in the S phase is calculated. CONCLUSION: Flow cytometry is a relatively new technology in the clinical laboratory. There are many clinically useful applications for which it is suited. As the technology continues to grow, so will the use of the flow cytometer.

Blood Cell Count↗

PubMed2005

Detection of Anisakis simplex-induced basophil activation by flow cytometry.

BACKGROUND: Laboratory diagnosis of anisakidosis is based on specific serum IgE detection. Recently, detection of allergen-induced basophil activation by flow cytometry has been proposed as a valuable tool for allergy diagnosis. OBJECTIVE: To evaluate if detection of Anisakis-induced basophil activation by flow cytometry is a useful tool in the diagnosis of Anisakis allergy. METHODS: Patients with Anisakis allergy (A.s.+, n = 37), patients reporting chronic urticaria or abdominal pain unrelated to fish ingestion (A.s.-, n = 51), and healthy controls (n = 12) were studied. Specific IgE to Anisakis simplex (A. simplex) was quantified with CAP-FEIA method, and basophil activation test was performed with three different concentrations of an Anisakis crude extract. Basophil gating was performed with CD123 and HLA-DR, and cellular activation was measured with CD63. RESULTS: A.s.+ patients showed significantly higher age and total IgE levels than did the A.s.- patients. Specific IgE to A. simplex correlated with the activated basophil percentages obtained with 15 microg/mL (r = 0.80; P < 0.001), 1.5 microg/mL (r = 0.84; P < 0.001), and 0.15 microg/mL (r = 0.82; P < 0.001) of A. simplex crude extract. Nine individuals (3 in the A.s.+ group and 6 in the A.s.- group) were nonresponders to basophil stimulation with anti-IgE. Five A.s.- patients showed positive IgE values to A. simplex while the basophil activation test was negative. According to the receiver operating characteristics curves performed between A.s.+ vs. A.s.- and A.s.+ vs. healthy controls, the cutoff for a positive basophil activation test was >or=21% (specificity = 96%, sensitivity = 100%), and 16% (sensitivity and specificity of 100%) respectively. When nonresponders were included in the A.s.+ vs. A.s.- analysis, sensitivity decreased to 95%. Multivariate logistic analysis showed that the specific basophil activation was a factor independently associated with clinical symptoms of A. simplex allergy. CONCLUSIONS: Detection of A. simplex-induced basophil activation by flow cytometry is a useful laboratory technique for the diagnosis of anisakidosis, supplementing specific IgE determinations.

Adult↗

PubMed1985

DNA image cytometry on machine-selected breast cancer cells and a comparison between flow cytometry and scanning cytophotometry.

A DNA image cytometry method, implemented on the LEYTAS image processing system, has been applied to acriflavine-Feulgen-stained breast cancer cytology specimens. An essential feature of the LEYTAS image cytometry method (LCM) is the automated selection of single nuclei according to predetermined specifications. Visual interaction has been used to reject remaining artefacts like overlapping nuclei. DNA profiles obtained with LCM have been compared with DNA profiles obtained by scanning cytophotometry (SCM) or flow cytometry (FCM). The resolution of DNA profiles obtained with LCM is similar to that from SCM but lower than that from FCM. However, a high correlation is found for the DNA indices measured with LCM and FCM (r = 0.97). The LCM profiles of aneuploid tumours generally showed lower accessory diploid fractions than FCM profiles due to the automated rejection of leukocyte nuclei. Also, LCM profiles frequently showed the presence of minor subpopulations of highly aneuploid/polyploid tumour cells that could not be identified by FCM. Therefore, LCM appears to be supplementary to FCM for studying tumour cell stemline heterogeneity.

Aneuploidy↗

PubMed1999

The use of flow cytometry to measure neutrophil function.

Neutrophils are important professional phagocytic cells that provide the host with a first line of defense against acute bacterial and fungal diseases and recurrent, severe or unusual infections are associated with inherited defects of neutrophil function. Furthermore, abundant evidence links inappropriate neutrophil-mediated tissue damage to the pathogenesis of conditions such as acute respiratory distress syndrome, septicemia with multiorgan failure, ischemia-reperfusion injury and rheumatoid arthritis. Flow cytometry has been increasingly used to evaluate the functional capabilities of neutrophils. In this review, we discuss the use of flow cytometry to assess neutrophil functional responses including calcium mobilization, F-actin assembly, adhesion, aggregation, degranulation, phagocytosis and reactive oxygen species (ROS) production. The use of flow cytometry to identify neutrophil priming is also discussed. The advantage of flow cytometry is that the majority of neutrophil functions can be measured using a small volume of whole blood that reduces artifactual changes in function caused by purification procedures. The advent of numerous new fluorochromes and multiparametric analysis allows the simultaneous measurement of several neutrophil functions in the same population of cells. Flow cytometric analysis provides a rapid screen for abnormalities of neutrophil function and reflects more accurately their behavior in vivo.

Animals↗

PubMed2005

Resolving and classifying haematopoietic bone-marrow cell populations by multi-dimensional analysis of flow-cytometry data.

The study of normal or malignant haematopoiesis requires the analysis of heterogeneous cell populations using multiple morphological and molecular criteria. Flow cytometry has the capacity to acquire multi-parameter information of large haematopoietic cell populations, utilizing various combinations of >200 molecular markers (clusters of differentiation, CD). However, current flow cytometry analyses are based on serial gating of two-parametric scatter plots--a process that is inherently incapable to discriminate all subgroups of cells in the data. Here we studied the cellular diversity of normal bone marrows (BM) using multi-dimensional cluster analysis of six-parametric flow cytometry data (four CD, forward scatter and side scatter), focusing mainly on the myeloid lineage. Twenty-three subclasses of cells were resolved, many of them inseparable even when examined in all possible two-parametric scatter plots. The multi-dimensional analysis could distinguish the haematopoietic progenitors according to International Society of Haematotherapy and Graft Engineering criteria from other types of immature cells. Based on the defined clusters, we designed a classifier that assigns BM cells in samples to subclasses based on robust six-dimensional position and extended shape. The analysis presented here can manage successfully both the increasing numbers of haematopoietic cellular markers and sample heterogeneity. This should enhance the ability to study normal haematopoiesis, and to identify and monitor haematopoietic disorders.

Antigens, CD↗

PubMed2001

[Whole blood flow cytometry for detection of activated platelets. I. Platelet identification, blood collection and storage].

In order to assess platelet activation by flow cytometry and activation-dependent monoclonal antibodies, a minute volume(2.5 microliters) of citrated whole blood was mixed for 15 minutes with a cocktail of monoclonal antibodies(MoAb), including PAC1, (a MoAb specific for fibrinogen receptors), and a MoAb against CD62P, (an alpha-granule membrane protein which associates with platelet surface membranes when platelets are activated). After fixation with 1% formaldehyde, the percentage of platelets positive for PAC1 and/or MoAb-CD62P was measured by flow cytometry. Even in resting platelets without stimulation, about 15% of platelets were found to be PAC1-positive and 0.6% of platelets were CD62P-positive. If there was a one-minute delay after needle puncture and before blood collection, the number of PAC1-positive platelets was increased compared to that of blood obtained immediately after puncture. If blood was allowed to stand at room temperature, there was a gradual increase in the number of PAC1-positive platelets, and after 60 minutes, this increase became statistically significant. The addition of Iloprost, (a prostacyclin analogue), immediately after venipuncture did not completely prevent the increase in PAC1-positive platelets after 60 minutes. If the inhibitor was added after first incubating for 60 minutes, however, the percentage of PAC1-positive platelets was reduced to preincubation value. Although flow cytometry is a simple and powerful tool to assess platelet activation, there remain several methodological problems to be resolved before this method may be employed in routine clinical use.

Adult↗

PubMed1995

DNA ploidy status in 84 ocular melanomas: a study of DNA quantitation in ocular melanomas by flow cytometry and automatic and interactive static image analysis.

Deoxyribonucleic acid (DNA) ploidy was quantified in 84 ocular melanomas (median follow-up, 11-years) by flow cytometry, CAS 200 interactive image analysis, and Pathology Image Processing Environment (PIPE; Department of Quantitative Pathology, Free University, Amsterdam, The Netherlands) automatic image analysis (75). Overall, 32.1% of the melanomas were aneuploid, 2.3% were tetraploid, and 66.6% were diploid. Pathology Image Processing Environment analysis estimated DNA ploidy in 12 tumors that were unprocessable by flow cytometry. Of 10 tumors that were diploid by flow cytometry, PIPE detected stemline aneuploidy in five and some aneuploid cells in five more. Seven tumors were aneuploid by PIPE but diploid by CAS 200, five of which contained occasional DNA aneuploid cells on the CAS 200 histograms. Pathology Image Processing Environment analysis quantified tumor samples with an average of 500 (250 to 1,050) cells in less than 10 minutes. All cells classified as spindle A according to the Callender system (more than 10,000) were diploid. Spindle B and epithelioid cells occupied both diploid and aneuploid peaks on the DNA histograms. Dioxyribonucleic acid variables did not correlate with established prognosticators, such as Callender cell type, largest tumor dimension, or glaucoma, nor did they reach significance by univariate and multivariate analyses. The value of these findings as a diagnostic support in uveal melanoma, particularly in combination with fine needle aspiration biopsy, is discussed.

DNA, Neoplasm↗

PubMed1990

Flow cytometry detection and evaluation of bladder tumors.

The diagnosis and classification of bladder cancer are based primarily on histologic and cytologic light microscopy. The significant cytologic alternations are abnormal (increased) DNA content and structural changes in chromatin. Measurements of DNA content carried out by flow cytometry on suspensions of tumor cells and bladder irrigation specimens correlate well with clinical and biopsy findings. Badalament et al (Badalament RA, Kimmel M, Gay H, et al. Cancer 1987;59:2078-2085) reported that a single bladder wash flow cytometry correctly detected 83% of bladder cancers. The technique is most sensitive in detecting early, in situ, and superficially invasive carcinoma. In 100 urologic patients with non-neoplastic disease of the bladder, Klein et al (Klein FA, Herr HW, Sogani PC, et al. J Urol. 1982;127:946-948) reported only two false-positive examinations. Flow cytometry appears to be at least as valuable as conventional urinary cytology, without the need of an experienced cytopathologist. However, as specimens must be collected by bladder irrigation via catheter or cytoscope, the technique is most suitable not for population screening, but in monitoring high-risk populations: industrial workers or others exposed to carcinogens, persons with a history of urothelial tumors, and adult patients referred for urologic examinations because of hematuria, unexplained, recurrent cystitis, or other urologic symptoms. DNA flow cytometry also has been valuable in monitoring the conservative treatment of bladder cancer and of predictive value in the intravesical bacille Calmette-Guérin treatment of superficial carcinomas of the bladder. Dual parameter measurements of DNA content and antigen expression are now under evaluation, as are measurements of chromatin structure alteration, metabolism, and proliferative rate. These promise to discriminate subsets of bladder cancer that may be predictive of clinical behavior.

Antibodies, Monoclonal↗

PubMed1998

Deoxyribonucleic acid flow cytometry and traditional pathologic variables in invasive penile carcinoma: assessment of prognostic significance.

OBJECTIVES: The identification of reliable prognostic factors to guide the selection of patients at high risk of harboring subclinical metastases in penile cancer is important. We evaluated traditional pathologic variables and deoxyribonucleic acid (DNA) flow cytometry to determine the prognostic significance of these variables for the subsequent development of lymph node metastases. METHODS: Clinical data and pathologic specimens were retrospectively reviewed from patients treated surgically at university-affiliated hospitals from 1958 to 1987. Pathologic analysis (grade, depth of invasion, and pathologic stage) and DNA flow cytometry were performed on specimens from 46 patients with invasive penile carcinoma and complete medical records. Pathologic variables were compared with DNA flow cytometry results in patients who never developed lymph node metastasis (32 patients, median follow-up 121 months) and in those who presented with or developed proved lymph node metastases (14 patients, median follow-up 18 months). RESULTS: The distributions of diploid and nondiploid tumors were similar in patients with or without lymph node metastasis. In addition, there was no significant difference in the grade distributions of tumors with respect to lymph node status. Patients with positive nodes more commonly had tumors that invaded greater than 0.5 cm or that exhibited pathologic Stage T2 or greater (deep invasion). All 14 patients who presented with or subsequently developed metastasis had deep primary tumors. Thirteen of 36 patients with clinically negative nodes had superficially invasive tumors (pathologic Stage T1 and depth of invasion 0.5 cm or less), and none developed metastasis (median follow-up 124 months [range 58 to 240]). Tumor grade was significantly related to the likelihood of deep invasion but was not an independent prognostic factor for metastasis. CONCLUSIONS: DNA flow cytometry does not add prognostic information to that obtained by pathologic assessment in patients with invasive penile carcinoma. The presence of pathologic Stage T2 or greater or depth of invasion greater than 0.5 cm defines a group of patients at high risk of inguinal node metastasis. A novel finding was that patients with minimally invasive lesions (0.5 cm or less) and no evidence of corporal invasion (pathologic Stage T1) have little risk of inguinal node metastasis. Close observation of reliable patients meeting these criteria may be a safe alternative to prophylactic lymphadenectomy.

DNA, Neoplasm↗

PubMed1996

T and B cell crossmatching using three-colour flow cytometry.

We have developed a highly sensitive method for the simultaneous crossmatching of T and B cells using three-colour flow cytometry. This method identifies T and B cell populations in a lymphocyte sample and analyses each for the presence of bound patient IgG alloantibodies. A fluorescein isothiocyanate conjugated rabbit anti-human IgG antibody was used to detect bound IgG following incubation of donor lymphocytes with patient serum. R-phycoerythrin conjugated anti-human CD19, and Quantum Red conjugated anti-human CD3 monoclonal antibodies were used to detect T and B cell populations, respectively. A comparison of results between conventional and three-colour flow cytometry crossmatches showed that there was no difference in detection of bound IgG between the two methods for ten test sera, doubling dilutions of a positive control serum and 72 donor-recipient pairs. Three-colour flow cytometry has proved particularly helpful in determining whether an antibody is directed against donor T and/or B cells and was shown to be more sensitive than complement-dependent cytotoxicity assay in eight out of 72 crossmatches.

B-Lymphocytes↗

PubMed2006

Identification of acute myeloid leukemia in dogs using flow cytometry with myeloperoxidase, MAC387, and a canine neutrophil-specific antibody.

BACKGROUND: Flow cytometry may be used to determine immunophenotype or lineage of leukemic cells, but few antibodies are available that are specific for cells of monocytic and granulocytic lineage. OBJECTIVE: The purpose of this study was to evaluate the flow cytometric staining patterns of 3 commercial monoclonal antibodies for monocytes and granulocytes in clinically healthy dogs and in dogs with acute myeloid leukemia (AML). METHODS: Mouse antihuman macrophage antibody (MAC387), mouse anti-human myeloperoxidase (MPO), and a canine neutrophil-specific antibody (NSA) were evaluated using flow cytometry on blood from 6 clinically healthy control dogs, and on blood (n = 7) and/or bone marrow (n = 2) from 8 dogs with AML. A diagnosis of acute leukemia was confirmed by >30% blasts in bone marrow or >30% blasts in peripheral blood, together with bi- or pancytopenia, circulating CD34-positive blast cells, and clinical signs of disease. Leukemic samples also were evaluated using a wide panel of monoclonal antibodies. RESULTS: MAC387 stained neutrophils and monocytes from control dogs, although the staining profiles for the 2 cell types differed. MPO and NSA resulted in strong positive staining of neutrophils; MPO also stained monocytes weakly. Lymphocytes did not stain with any of the antibodies. One case was classified as AML of granulocytic lineage (AML-M1), 6 cases were classified as acute monocytic leukemia (AML-M5), and 1 case was classified as acute myelomonocytic leukemia (AML-M4). Neoplastic myeloblasts in the dog with granulocytic AML were positive for MPO, NSA, MAC387, and CD4. All monoblasts from the dogs with AML-M5 were positive for CD14, 5 of 6 were positive for MAC387, and 2 were positive for MPO. NSA staining was negative in the 2 dogs with AML-M5 in which it was evaluated. In the dog with AML-M4 variable percentages of blast cells were positive for CD14, MPO, MAC387, CD4, and NSA. CONCLUSIONS: Antigens identified by antibodies to MAC387, MPO, and NSA were expressed not just by normal mature neutrophils and monocytes, but also by neoplastic myeloblasts and monoblasts. These 3 antibodies may be useful as part of a wider panel for immunophenotyping AML in dogs.

Animals↗

PubMed1997

Comparison of a semi-automated new Coulter methylene blue method with fluorescence flow cytometry in reticulocyte counting.

A semi-automated new methylene blue method for reticulocyte counting was compared to the manual method and fluorescence flow cytometry. Over a wide range of reticulocyte counts (0.3-12.7%) the new methylene blue method was found to be comparable to that of fluorescence flow cytometry, with high precision. Between-run precision studies showed coefficients of variation (CVs) of 4.8, 6.9 and 14.5% for reticulocyte counts of 9.5, 2.4 and 0.7%, respectively. Within-run precision studies showed CVs of 8.6, 8.1 and 6.1% for reticulocyte counts of 0.9, 5.3, and 18.4%, respectively. The correlation coefficient between the Coulter method and the manual method was 0.83, and 0.85 between the Coulter method and flow cytometry. The possibility of extending the incubation time to at least 4 h and the insignificant changes observed with samples stored for up to at least 72 h at 4-8 degrees C make this new method most convenient and allow any laboratory having Coulter STKS or MAXM cell counters to perform reticulocyte counts in an efficient, reliable manner.

Cell Separation↗

PubMed1990

A comparison of flow cytometry and other techniques used for diagnosing Mycoplasma infections.

Mycoplasma isolates from chickens are usually identified by a fluorescent antibody (FA) technique that requires 7 to 10 days from sampling to the completion of identification. The FA procedure uses Mycoplasma-positive antibodies conjugated with fluoroisothiocyanate (FITC) to identify the colonies of the organism on agar. This paper describes a flow cytometric procedure that uses the same antibody-FITC conjugate and can be completed in 3 or 4 days. Broilers and egg-type hens inoculated with Mycoplasma gallisepticum and M. synoviae were swabbed from the choanal cleft and cultures were grown in broth. Broth aliquots were briefly incubated with polyclonal antisera to M. gallisepticum and M. synoviae conjugated with FITC, the same reagents routinely used in the FA technique. The sample was then analyzed by flow cytometry for the association of the fluorescent label and the organism. Samples from inoculated chickens incubated with the homologous antibody-FITC had greater fluorescent intensity than did those incubated with the heterologous antibody-FITC. Diagnosis by flow cytometry was consistent with that by the FA technique except that in one trial one sample was incorrectly identified by flow cytometry as positive for M. gallisepticum, although it was not inoculated with M. gallisepticum. Also, flow cytometry did not correctly identify M. synoviae organisms as readily when broilers were inoculated with both M. gallisepticum and M. synoviae.

Agglutination Tests↗

PubMed1986

Rat spermatogenesis in vitro traced by quantitative flow cytometry.

In vitro differentiation of germ cells in rat seminiferous tubule segments at stages II-III of the epithelial cycle was studied. DNA flow cytometry was used for quantitation of absolute cell numbers from the cultured tubule segments that were compared to freshly isolated stages of the cycle, as identified by transillumination stereomicroscopy of the seminiferous tubules and phase-contrast microscopy of live cell squashes. Spermatogonia and spermatocytes from stages II-III showed normal morphological differentiation during 7 days in vitro. Round spermatids differentiated to Step 7 of spermiogenesis but Step 16 spermatids failed to develop. Acid phosphatase activity in the spermatogenic cells changed normally during the culture. As compared with freshly isolated control tubule segments, 35% of round spermatids and 42% of pachytene spermatocytes were present in culture after 7 days. The cell numbers recovered from defined stages by DNA flow cytometry were close to those found in morphometric studies. Flow cytometry is an efficient quantitation method for cells liberated from seminiferous epithelium. Spermatogonia, spermatocytes, and early spermatids are able to differentiate in vitro, but spermatids approaching the elongation (acrosome) phase, and particularly the maturation phase, fail to differentiate under present culture conditions.

Acid Phosphatase↗