Hans W. von Heyden

Hairy Cell Leukaemia: Surface Markers and Functional Capacities of the Leukaemic Cells Analysed in Eight Patients

Summary. In eight patients with hairy cell leukaemia (HCL) peripheral blood cells and in two patients also spleen cells were analysed for surface markers and functional capacities. Only cells containing the tartrate resistant isoenzyme 5 of the acid phosphatase were considered. Hairy cells (HC) of all patients were found to adhere spontaneously to glass and plastic surfaces and to spread after adherence like monocytes. They ingested latex particles of more than 1 μm diameter, but, in contrast to monocytes, did not phagocytose erythrocytes sensitized either by IgM or by IgG antibodies. HC of all patients bore Fc‐receptors with a high binding affinity for aggregated IgG. Using 125I‐labelled F(ab′)2‐fragments of monospecific antibodies in autoradiography, only one light chain type was detected on HC of individual patients. In four patients μ‐ and δ‐chains were simultaneously expressed on HC, whereas in two patients only γ‐chains and in one case only μ‐chains were observed on HC. One patient showed a combination of γ‐ and δ‐chains on his HC. A great variation in density of surface immunoglobulins of HC was observed within individual patients. After removal by capping, surface immunoglobulin reappeared on HC during cell culture, but more slowly than on normal B‐lymphocytes. As shown in two patients by internal labelling, HC secreted immunoglobulin light chains, but no heavy chains. On the basis of these findings the classification of HC as belonging to the B‐cell lineage, rather than to the monocytic lineage, seems to be justified.

Isoenzym-Muster saurer Phosphatasen aus normalen und leukämischen weißen Blutzellen

SummaryThe activities of acid phosphatases (AP) were measured in leukocytes from patients with chronic myelocytic leukemia (CML), macrophages, granulocytes, in the fractionated mononuclear cells of patients with CML and with hairy-cell-leukemia (HCL) and in the cells from patients with acute leukemia (AL). The lowest activities were found in lymphocytes of normal subjects and of patients with chronic lymphatic leukemia (CLL) and in thrombocytes.Isoenzyme (IsE) l was characteristic for thymocytes, IsE 2 for granulocytes, IsE 3 for pathologic blast-cells, lymphocytes and thrombocytes, IsE 4 for macrophages, IsE 5 with components a and b for the mononuclear fraction of patients with HCL. In addition IsE5 was detected in lymphocytes, macrophages and CLL-cells.In 4 patients with HCL the relative percentage of IsE-5-fraction was slightly greater than the percentage of tartrate resistant cells. In two patients with questionable HCL well marked IsE-5-fractions were recognized but no tartrate resistant cells. In one patient with HCL a relatively high percentage of tartrate resistant hairy-cells and in comparison an inadäquate low IsE-5-fraction was found. These different relations were explained with the more sensitive method of gelelectrophoresis and different affinity of substrates to AP.ZusammenfassungDie durchschnittlichen Aktivitäten (IE/106 Zellen) der sauren Phosphatasen und die prozentuale Verteilung der Isoenzyme der sauren Phosphatasen wurden aus Lysaten von normalen und leukämischen weißen Blutzellen bestimmt.Bei 4 Patienten mit Haarzell-Leukämie lag der Anteil des Isoenzyms 5 geringfügig über dem der tartratresistenten Zellen. Bei 2 Patienten mit einer fraglichen Haarzell-Leukämie waren deutliche Isoenzym-5-Fraktionen, aber keine tartratresistenten Zellen zu erkennen. Bei einem Patienten mit Haarzell-Leukämie wurde bei einem hohem Anteil tartratresistenter Haarzellen eine auffallend geringere Isoenzym-5-Fraktion gefunden. Diese unterschiedlichen Beziehungen wurden mit der empfindlichen Nachweismethode der Gelelektrophorese und unterschiedlicher Substrataffinität erklärt.The activities of acid phosphatases (AP) were measured in leukocytes from patients with chronic myelocytic leukemia (CML), macrophages, granulocytes, in the fractionated mononuclear cells of patients with CML and with hairy-cell-leukemia (HCL) and in the cells from patients with acute leukemia (AL). The lowest activities were found in lymphocytes of normal subjects and of patients with chronic lymphatic leukemia (CLL) and in thrombocytes. Isoenzyme (IsE) 1 was characteristic for thymocytes, IsE 2 for granulocytes, IsE 3 for pathologic blast-cells, lymphocytes and thrombocytes, IsE 4 for macrophages, IsE 5 with components a and b for the mononuclear fraction of patients with HCL. In addition IsE 5 was detected in lymphocytes, macrophages and CLL-cells. In 4 patients with HCL the relative percentage of IsE-5-fraction was slightly greater than the percentage of tartrate resistant cells. In two patients with questionable HCL well marked IsE-5-fractions were recognized but no tartrate resistant cells. In one patient with HCL a relatively high percentage of tartrate resistant hairy-cells and in comparison an inadaquate low IsE-5-fraction was found. These different relations were explained with the more sensitive method of gelelectrophoresis and different affinity of substrates to AP.

[Culture of leukocytes of patients with hairy cell leukaemia in the diffusion chamber (author's transl)].

ZusammenfassungPeriphere Blutzellen von Patienten mit Haarzell-Leukämie wurden in intraperitonealen Diffusionskammern kultiviert. Bei allen Patienten konnte eine Proliferation von Haarzellen beobachtet werden. Granulo-, Erythro- und Monozytopoese zeigten im Vergleich zu Normalpersonen keine änderung der Proliferation und Differenzierung. Damit blieb die Gegenwart von Haarzellen ohne Einfluß auf das Kulturverhalten der restlichen Hämatopoese. Gleichzeitig darf nach diesem Befund ein normaler Gehalt an hämatopoetischen Stammzellen im Blut der Patienten vermutet werden. Dagegen wiesen das Verhalten der Lymphozyten und die Anzahl entstehender Plasmazellen deutliche Unterschiede zu Normalpersonen auf, deren Ursachen bisher nicht geklärt sind.SummaryPeripheral blood cells from patients with hairy cell leukaemia were cultured in diffusion chambers implanted intraperitoneally. Proliferation of hairy cells could be observed in all patients. Granulo-, erythro- and monocytopoiesis showed no difference of proliferation and differentiation as compared to normal persons. This demonstrates that the presence of hairy cells in the culture did not influence the growth pattern of the residual haemopoiesis. From this result it can be additionally supposed that patients have a regular content of haeniopoietic stem cells in peripheral blood. However the growth of lymphocytes and plasmacells showed a significant difference compared with normal persons. The cause is unknown as yet.

Characteristics of Macrophages in vitro derived from Peripheral Blood Cells

SummaryCharacteristics of macrophages in vitro derived from the peripheral blood of healthy donors are described. Macrophages generated as clusters in the liquid culture system are morphologically similar to those in the agar system. Peroxidase positive granules are found in macrophages and in fibroblastoid cells. Few macrophages are formazan positive. In the liquid culture system macrophages fixed as monolayer cells are in equilibrium with those in the supernatant.ZusammenfassungCharakteristika von Makrophagen in vitro werden beschrieben, die vom peripheren Blut gesunder Spender stammen. Makrophagen, die als cluster im Flüssigkultursystem entstanden, ähneln morphologisch jenen im Agarsystem. Peroxydase-positive Granula wurden in Makrophagen und in fibroblastoiden Zellen gefunden. Einige Makrophagen sind Formazan-positiv. Im Flüssigkultursystem sind die Makrophagen, die als Monolayer fixiert sind, im Gleichgewicht mit jenen im Überstand.

Isoenzym-Muster der sauren Phosphatasen von Epstein-Barr-Virus-DNS enthaltenden, permanent wachsenden lymphoiden Zell-Linien

SummaryThe expression of acid phosphatases is cytochemically one of the most important features in permanent growing B-cell-lines. In few cell lines acid phosphatase is resistant against tartrate.Tartrate resistant isoenzyme 5 with components a und b can be demonstrated in monocytes, lymphocytes, chronic lymphatic leucemic cells and especially in hairy cells as well as in cell lines derived from a healthy donor.Fractionation of acid phosphatase by gelelectrophoresis in separated lymphocytes demonstrates especially isoenzyme 3, in separated macrophages isoenzyme 4. Isoenzyme 4 could not be detected in several cell lines. It is therefore concluded that these cell lines are probably derived from lymphocytic precursors. Cell lines with isoenzyme 4 may be the result of a facultative hybridisation between lymphocytes and monocytes.Profiles of acid phosphatases in virus-negative cell lines (Ramos, BJAB) were not significantly altered by conversion with EBV.ZusammenfassungDie Expression der sauren Phosphatasen sind das stärkste zytochemische Merkmal lymphoider Zellinien. In einigen Zellen erscheint die saure Phosphatase zytochemisch resistent gegen Tartrat.Die tartratresistenten Isoenzyme 5 mit den Komponenten a und b lassen sich sowohl bei Fraktionierung der sauren Phosphatasen aus Monozyten, Lymphozyten, chronisch lymphatischen Leukämiezellen und besonders aus Haarzellen als auch in unterschiedlicher Ausprägung in Zellinien von einem gesunden Spender darstellen.Die gelelektrophoretische Fraktionierung der sauren Phosphatasen in angereicherten Lymphozyten zeigt vorwiegend das Isoenzym 3, in angereicherten Makrophagen das Isoenzym 4. In einigen Zellinien ist das Isoenzym 4 nicht nachzuweisen. Es wird daraus geschlossen, daß diese Zellinien möglicherweise ausschließlich lymphozytären Ursprungs sind, während diejenigen mit Nachweis des Isoenzyms 4 vielleicht Produkte einer fakultativen Hybridisierung aus Lymphozyten und Monozyten sind.Profile der sauren Phosphatasen in Virus-negativen Zellinien (Ramos, BJAB) werden durch Konversion mit EBV nicht eindeutig verändert.

CYTOLOGICAL CHARACTERISTICS OF NORMAL HUMAN PERIPHERAL BLOOD CELLS IN CULTURE BEFORE ESTABLISHMENT INTO LYMPHOCYTOID CELL LINES

Human peripheral blood cells were observed in culture. Attached cells and those floating in the supernatant fluid were classified as macrophages, lymphoid and fibroblastoid cells. Peroxidase positive granules were demonstrated in macrophages and fibroblastoid cells. It is concluded that (a) appearance of macrophages is dependent on the formation of a monolayer, (b) a gradient relationship between the attached and supernatant cells exists, (c) some fibroblastoid cells are derived from macrophages, and (d) lymphocytes alone are not able to form a monolayer.

Criteria for the differentiation of lymphoid cell lines

SummaryEleven lymphoid cell lines were initiated for culture simultaneously and sequentially from one healthy male donor and three from one female donor. There was no difference in respect to morphology (light- and electronmicroscopy) and growth characteristics. Chromosome pattern demonstrated tendency to aneuploidy except for one heteroploid cell line. Subterminal constrictions were found as B- and C-marker chromosomes in cell line II 1 and III 2. No other aberrations were typical for one cell line.Epstein-Barr Virus particles (EBV) were only seen in the oldest cell line. Seven cell lines were EBV-antigen positive, detected by immunofluorescent cell staining. The immunoglobulin pattern was constant. IgM was found in seven, IgG in three, and IgA in four cell lines. One cell line produced simultaneously IgM and IgG. It is concluded that cell lines are derived from B-type precursor cells and that they demonstrate clonal growth.ZusammenfassungElf lymphoide Zellinien wurden gleichzeitig und nacheinander von einem gesunden männlichen und drei von einem weiblichen Spender zur Kultur angesetzt. Hinsichtlich der Morphologie (Licht- und Elektronenmikroskopie) und Wachstumseigenschaften gab es keinen Unterschied. Die chromosomalen Verteilungen zeigten eine Neigung zur Aneuploidie mit Ausnahme einer Zellinie, die heteroploid war. Subterminale Einschnürungen wurden als B und C gekennzeichnete Chromosomen in Zellinie II 1 und III 2 gefunden. Andere Abweichungen waren für keine Zellinie typisch.Epstein-Barr-Viruspartikel (EBV) wurden nur in der ältesten Zellinie gesehen. Mit der Immunofluoreszenz-Methode waren sieben Zellinien EBV-Antigen positiv. Das Immunglobulinmuster war konstant. IgM wurde in sieben, IgG in drei und IgA in vier Zellinien gefunden. Eine Zellinie produzierte gleichzeitig IgM und IgG. Es wird angenommen, daß die Zellinien von Vorläuferzellen des B-Typs abstammen und daß sie ein klonales Wachstum aufweisen.