Flow cytochemistry in clinical hematology

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<ul><li><p>10. Cytochemische Leukocytenbefunde ~} '~@S~e~ </p><p>Peroxidase und Naphthol-AS-D-Chloracetatesterase gelingt auch die Abgrenzung atypischer Reifungsstufen der Neutrophi- lenreihe (Para-Neutrophile) sowie mit einigen anderen Techni- ken die Erkennung yon atypischen eosinophilen Granulocyten. Peroxidase als Bestandteil der PrimS.rgranula neutrophiler Vor- stufen ist der wichtigste Marker der granulocyt/iren, unspezifi- schen Esterase der monocyt~iren Zellreihen. Morphologisch nicht abgrenzbare Vorstufen der Megakariocyten sind bisher nur fiber eine spezielle P1/ittchen-Peroxidase, zum Tell fiber immnno- logische Marker abgrenzbar, ffir atypische Vorstufen der Erythropoese ist bisher der Membranbestandteil Glykophorin A das sicherste Nachweiskriterium. </p><p>Die Gruppe der akuten lymphatischen/undifferenzierten Leuk/imien wird aufgrund der Membraneigenschaften charakte- risiert. Dies erfolgt entweder mit Hilfe von traditionell hergestell- ten Antiseren oder dutch monoklonale Antik6rper. Zus/itzlich hat das Enzym TdT Bedeutung ffir die Routinediagnostik erlangt, das mittels Immunfluorescenz oder Immunperoxidase- technik im Ausstrich nachgewiesen werden kann. Mit Ausnahme der B-ALL zeigen im allgemeinen alle Formen der ALL Aktivi- tilt, Bedeutung hat die Reaktion auch zum Nachweis des sogenannten lymphatischen Blastenschubes der chronischen myeloischen Leuk/imie. </p><p>Da trotz langjfihriger intensiver Forschung keine leukS.mie- spezifischen Marker gefunden wurden, ist der Anwender enzy- matischer oder immunologischer Untersuchungsmethoden auf die Abgrenzung normaler von pathologischen Zellpopulationen mit konventioneller Technik angewiesen. Im Prinzip arbeiten auch moderne Zellsorter oder Differenzierungsapparate nach dem Prinzip der Mustererkennung und benutzen dieselben Techniken, die in der Cytochemie fiblich sind. </p><p>Bei speziellen wissenschaftlichen Fragestellungen k6nnen mikrophotometrische Verfahren, autoradiographische Metho- den oder ultrastrukturelle Cytochemie sowie die Durchflul3cyto- photometrie als ErgS.nzung Verwendung linden, in der Routine sind sie bisher nicht eingeffihrt. </p><p>Differenzierungsautomaten nutzen neben den qualitativen Merkmalen auch die M6glichkeit zu einer gewissen Quantifizie- rung aus, atypische Zellen miissen jedoch nach wie vor vom erfahrenen Untersucher mikroskopisch identifiziert werden. </p><p>Die modernen Methoden zur Analyse atypischer Zellen liefern eine Ffille von Informationen, die jedoch nur sinnvoll integriert werden k6nnen, wenn man die konventionelle Mor- phologie beherrscht. </p><p>Fresenius Z Anal Chem (1984) 317 : 628 - 629 9 Springer-Verlag 1984 </p><p>Flow Cytochemistry in Clinical Hematology </p><p>S. A. Bentley </p><p>Dept. of Pathology, School of Medicine, Chapel Hill, NC 27514, USA </p><p>Durchflug-Cytochemie in der klinischen H~imatologie </p><p>The differential leucocyte count (DLC) is the most frequently requested of all clinical cytological investigations. This, in combination with the relatively small number and clear de- finition of the various leucocyte subclasses, makes the DLC an excellent candidate for an automated cytological procedure. Another important objective in automating the DLC would be an improvement in the quality of the data generated. </p><p>Differential leucocyte analysis has not changed significantly since it was first described by Paul Ehrlich in /879. When compared with modern analytical techniques, the DLC is both imprecise and inaccurate. Errors result from maldistribution of cells on the slide, from observer misclassification of cells and from unfavorable counting statistics. The latter is by far the major source of error in the visual DLC. </p><p>Commercially successful automated DLC systems employ two types of technology, namely digital image processing and flow cytochemistry. Systems based on digital image processing use fixed, Romanowsky-stained blood films which are scanned microscopically by an optical sensing device. Analog signals corresponding to leucocytes are digitized and the digital data processed to yield descriptive parameters upon which cell classification is based. </p><p>Image processing systems closely parallel the visual DLC and are therefore subject to the same sources of error. Film scanning is a relatively slow process and it is not possible to examine large numbers of leucocytes. Image processing, al- though a valid approach to DLC automation, does not therefore offer any improvement in the quality of DLC data when compared to the visual method. </p><p>Flow cytochemistry is employed in only one automated DLC system, namely the Technicon H 6000. This instrument, besides </p><p>generating a conventional complete blood count, also performs a DLC. Leucocyte classification is based primarily on simul- taneous measurement of cellular peroxidase activity and small angle forward light scatter, measured in continuous flow. Basophils are identified by Alcian Blue uptake, measured in a separate flow channel. The H 6000 DLC is based on an analysis of approximately/0,000 cells and the instrument has a through- put capacity of 90 specimens per hour. </p><p>Evaluations of the H 6000 stress its reliability as a system for routine DLC analysis. In addition, flow cytochemistry can provide further diagnostically valuable data that cannot be obtained in any other way. This relates to certain unique properties of the flow cytochemical technology. </p><p>The extreme speed of the system enables the examination of very large numbers of cells, even in patients with low total leucocyte counts. It is thus possible, for the first time in the history of hematological cytology, to obtain differential leu- cocyte counts that are statistically significant. This is particularly valuable in the enumeration of minor cell populations, both physiological and pathological. It also enables precise moni- toring of changes in the DLC in response to various therapeutic manipulations. Flow cytochemistry is especially valuable in the treatment of patients with malignant diseases - both hema- tological and non-hematological - who frequently suffer peripheral cytopenias during therapy and in whom small changes in the DLC can be of great significance. </p><p>Another novel property of this technology derives from its substantial capacity for data reduction. In conventional differen- tial leucocyte analysis, data are expressed in terms of the relative numbers of a defined set of leucocyte classes. Flow systems provide scattergrams (light scatter vs. peroxidase activity) that effectively reduce data obtained from 10,000 leucocytes to a single image, offering valuable diagnostic information. Those experienced with the H6000 system often examine the scat- tergram before even looking at the numerical DLC data. </p><p>Flow cytochemical technology has thus expanded the dia- gnostic capacity of analytic hematology. The use of this type of system in conjunction with peroxidase-linked antibodies offers virtually limitless potential for examination of the leucocyte population. </p><p>Fresenius Z Anal Chem (1984) 317 : 629 9 Springer-Verlag 1984 </p><p>629 </p></li></ul>