Use of CD157 in FLAER-based assays for high-sensitivity PNH granulocyte and PNH monocyte detection.

BACKGROUND: Recent Flow Cytometric guidelines to detect Paroxysmal Nocturnal Hemoglobinuria (PNH) in white blood cells recommend using FLAER-based assays to detect granulocytes and monocytes lacking expression of GPI-linked structures. However national proficiency testing results continue to suggest a need for improved testing algorithms, including the need to optimize diagnostic analytes in PNH. METHODS: CD157 is another GPI-linked structure expressed on both granulocytes and monocytes and here we assess its ability to replace CD24 and CD14 in predicate 4-color granulocyte and monocyte assays respectively. We also assess a single tube, 5-color combination of FLAER, CD157, CD64, CD15, and CD45 to simultaneously detect PNH clones in granulocyte and monocyte lineages. RESULTS: Delineation of PNH from normal phenotypes with 4- or 5-color CD157-based assays compared favorably with 4-color predicate methods and PNH clone size data were similar and highly correlated (R(2) >0.99) with predicate values over a range (0.06%-99.8%) of samples. Both CD157-based assays exhibited similar high levels of sensitivity and low background levels in normal samples. CONCLUSIONS: While CD157-based 4- and 5-color assays generated closely similar results to the predicate assays on a range of PNH and normal samples, the 5-color assay has significant advantages. Only a single 5-color WBC reagent cocktail is required to detect both PNH granulocytes and monocytes. Additionally, sample preparation and analysis time is reduced yielding significant efficiencies in technical resources and reagent costs. All 4- and 5-color reagent sets stained stabilized whole blood PNH preparations, used in external quality assurance programs.

Use of CD157 in FLAER-based assays for high-sensitivity PNH granulocyte and PNH monocyte detection.

Background: Recent Flow Cytometric guidelines to detect Paroxysmal Nocturnal Hemoglobinuria (PNH) in white blood cells recommend using FLAER-based assays to detect granulocytes and monocytes lacking expression of GPI-linked structures. However national proficiency testing results continue to suggest a need for improved testing algorithms, including the need to optimize diagnostic analytes in PNH. Methods: CD157 is another GPI-linked structure expressed on both granulocytes and monocytes and here we assess its ability to replace CD24 and CD14 in predicate 4-color granulocyte and monocyte assays respectively. We also assess a single tube, 5-color combination of FLAER, CD157, CD64, CD15 and CD45 to simultaneously detect PNH clones in granulocyte and monocyte lineages. Results: Delineation of PNH from normal phenotypes with 4- or 5-color CD157-based assays compared favorably with 4-color predicate methods and PNH clone size data were similar and highly correlated (R2 >0.99) with predicate values over a range (0.06% - 99.8%) of samples. Both CD157-based assays exhibited similar high levels of sensitivity and low background levels in normal samples. Conclusion: While CD157-based 4- and 5-color assays generated closely similar results to the predicate assays on a range of PNH and normal samples, the 5-color assay has significant advantages. Only a single 5-color WBC reagent cocktail is required to detect both PNH granulocytes and monocytes. Additionally, sample preparation and analysis time is reduced yielding significant efficiencies in technical resources and reagent costs. All 4- and 5-color reagent sets stained stabilized whole blood PNH preparations, used in external quality assurance programs. © 2013 Clinical Cytometry Society.

Immunofluorescent staining of metastatic carcinoma cells in serious fluid with carcinoembryonic antibody, epithelial membrane antibody, AUA-1 and Ber-EP4.

Using an indirect immunofluorescence technique, we assessed the accuracy and clinical usefulness of a panel of monoclonal and polyclonal antibodies. The panel consisted of carcinoembryonic antibody (CEA) and epithelial membrane antibody (EMA), AUA-1, and Ber-EP4 conjugated with fluorescein isothiocyanate. Twenty-six specimens from pleural, peritoneal or pericardial effusions known to contain carcinoma cells (adenocarcinoma or large cell anaplastic carcinoma) and 16 specimens without carcinoma were first examined. The sensitivity and specificity for each of the antibodies were as follows: CEA, 71% and 75%; EMA, 96% and 81%; AUA-1, 80% and 100%; and Ber-EP4, 85% and 100%, respectively. The panel of antibodies was then applied to a group of 14 'problematic' fluids. These had been identified as causing dilemmas in interpretation, either because the cells in the fluids were of equivocal appearance on light microscopy, or the cytological diagnosis was different from that expected in the light of the clinical condition of the patient. Insufficient cellular material was present in one specimen. In five (39%) of the cases the immunochemical staining supported the light microscopic diagnosis. In four (30%) cases, however, the results indicated that the original light microscopic report was incorrect. Two of these were examples of large cell carcinoma of the lung, in which false negative reports had been issued on pleural fluids. The other two were cases of benign ovarian tumours in which a false positive report had been issued. The immunostaining also clarified the final diagnosis in the three patients (23%) on whom 'suspicious' cytological reports had previously been issued. The remaining case, fluid from a patient with a high grade mixed Mullerian tumour of the ovary, was unresolved. We conclude that immunofluorescent staining by AUA-1, EMA and Ber-EP4 is an aid in the cytological interpretation of serous fluids. CEA is much less helpful.