F. Schmalzl

The Application of Cytochemical Methods to the Study of Acute Leukemia

The comparison of cytochemical criteria with conventional morphologic and cytologic criteria as well as with electron microscopic and biochemical investigations all confirm the value of cytochemistry

Cytochemistry and ultrastructure of pathologic granulation in myelogenous leukemia

SummaryThe production of characteristic granules is one of the most specialized functions concerned with the development of granulocytes and monocytes. Leukemia-associated defects in the production of these highly specialized cell organelles reflect the perturbation of the RNA metabolism within the leukemic cells. Pathologic granulation can be of some clinical importance as a diagnostic aid in identifying various cytological types of acute leukemia as well as giving an indication for the profound perturbation in the intrinsic leukocyte regulation, as it occurs in preleukemic states or following treatment with some cytotoxic agents. The defects in granule production may lead either to a partial or complete reduction of a single or of several granular enzymes as well as in the partial or complete failure of the production of a given type of granules. On the other hand unusual increases of specific enzyme activities can be found as well as combination of characteristic granules of different cell types within one homogenous leukemic cell population. Ultrastructural evidence has been obtained that the production of granules may be disturbed at any morphologically identifiable stage of granulogenesis. Pathological agglomeration or fusion of granules with atypical cytosomes may lead to further abnormal subcellular structures exhibiting granular enzyme activities.ZusammenfassungDie Vorstufen der Granulozyten und der Monozyten sind während ihrer Reifung durch die Bildung charakteristischer Granula gekennzeichnet. Mit der Leukämie in Zusammenhang stehende Defekte in der Bildung dieser hochspezialisierten Zellorganellen spiegeln eine Störung des RNA-Stoffwechsels der leukämischen Zelle wieder. Eine pathologische Granulierung kann klinisch von Bedeutung sein: 1. kann hierdurch die Klassifizierung der akuten Leukämie nach verschiedenen zytologischen Typen erleichtert werden; 2. kann hierdurch eine Störung des Leukozyten-Stoffwechsels sichtbar gemacht werden, wie sie bei präleukämischen Zuständen oder als Folge der Behandlung mit Zytostatika auftreten kann. Der Defekt in der Bildung kann sich äußern entweder in einer teilweisen oder vollständigen Einschränkung einzelner oder mehrerer Enzyme der Granula, oder aber sogar in dem teilweisen oder vollständigen Versagen in der Bildung eines bestimmten Granulatyps. Weiterhin kann in einer homogenen leukämischen Zellpopulation eine außergewöhnliche Vermehrung spezifischer Enzymaktivitäten gefunden werden, sowie das Nebeneinander von für verschiedene Zelltypen charakteristischen Granula. Elektronenmikroskopisch lassen sich Hinweise dafür gewinnen, daß die Bildung der Granula in jeder morphologisch zu unterscheidenden Reifestufe der Granula-Bildung gestört sein kann. Eine Zusammenballung oder ein Verschmelzen von Granula und atypischen Zytosomen kann weitere pathologische subzelluläre Strukturen mit Enzym-Aktivitäten der Granula verursachen.

Hairy Cell Leukemia (‘Leukemic Reticuloendotheliosis’), Reticulosarcoma, and Monocytic Leukemia

Cytochemical and electron-microscopic studies have been carried out on leukemic monocytes and ‘hairy cells’ (HC), ‘reticulosarcoma’ (RS) cells and cells of cases of ‘reticulosis’ and ‘reticulosarcoma cell leukemia’. Additional investigations included quantitative determinations of the urinary lysozyme excretion, skin window studies, testing of the phagocytosis of ferritin by HC, and labelling of the Fc receptors on HC at the ultrastructural level. Clear evidences against any cytological relationship among leukemic HC and monocytes have been provided. Further results argued also against the frequently stressed relationship among leukemic monocytes and RS cells. Cases of ‘RS cell leukemia’ and ‘reticulosis’ had to be reclassified as lymphosarcoma cell leukemia, acute lymphatic, and myeloblastic leukemias. Besides distinct ultrastructural differences among HC, RS cells, and lymphocytes, mainly gradual differences have been noted using cytochemical methods and by evaluating the phagocytosis of ferritin particles. A further common trait of HC, RS cells, and B lymphocytes seems to be the presence of surface Fc receptors. A more precise classification instead of the diagnosis ‘reticulosarcoma’ and ‘reticulosarcoma cell leukemia’ is required, and the use of the term ‘hairy cell’ leukemia is suggested instead of the misleading term ‘leukemic reticuloendotheliosis’.

Atypical myeloblastic leukemia with differentiation into “Paraneutrophils”

SummaryA case of a patient is presented, who suffered from acute leukemia, showing predominantly atypical myeloblasts in the bone marrow. In contrast, a high amount of atypical “neutrophils” were present in the peripheral blood, as well as there was a conspicuous number of monocyte-like intermediate forms between the myeloblasts and the atypical segmented “neutrophils.” The cytochemical pattern of the “Neutrophils” was highly atypical, the main part being devoid of sudanblack B-positive lipids, as well as of peroxidase and chloroacylesterase activity, which characteristically are present in large amounts in the neutrophil granulocytes. Moreover, atypical distribution of alkaline phosphatase and naphthylamidase activities were encountered in these cells. The intermediate or transitional cells exhibited a cytochemical pattern different from that of normal blood monocytes, but corresponding to that of the atypical neutrophils. Thus, the assumption of a direct maturation way from myeloblasts to “paraneutrophils” with mononuclear, monocyte-like intermediate stages was supported by the results of cytochemical investigations. In contrast to the elevated number of neutrophils in the peripheral blood (10 350/mm3) very rare normal neutrophil precursors with typical cytochemical pattern were encountered in the haematopoietic organs.ZusammenfassungEs wird über eine Patientin mit einer akuten myeloischen Leukämie berichtet, die im Knochenmark überwiegend atypische Myeloblasten aufwies. Im Gegensatz dazu fanden sich im peripheren Blut eine große Anzahl ebenfalls atypischer “Neutrophiler” sowie eine beträchtliche Zahl monozytenähnlicher Zellen, die wirals Zwischenformen zwischen den atypischen Myeloblasten und den pathologischen Neutrophilen auffassen. Die pathologischen “Neutrophilen” zeigen ein ungewöhnliches zytochemisches Muster, in dem der größte Teil dieser Zellen keine Sudanschwarz-B-positiven Lipide sowie kaum Peroxydase-Naphthol-AS-D-Cloroacetat-Esterase-Aktivität aufweist. Diese drei zytochemischen Reaktionen geben normalerweise eine starke positive Reaktion in den neutrophilen Granulozyten. Außerdem wurde eine atypische Werteinteilung sowohl der alkalischen Phosphatase als auch der Naphthylamidase festgestellt. Die monozytenähnlichen Zwischenformen unterscheiden sich deutlich in ihrem zytochemischen Muster von normalen Monozyten und gleichen den atypischen neutrophilen Zellen. Diese zytochemischen Befunde stützen die Ansicht, daß es bei der Patientin zu einer Ausreifung der Myeloblasten über ein monozytenähnliches Zwischenstadium zu atypischen “neutrophilen” Granulozyten gekommen ist. Trotz der hohen Zahl “neutrophiler” Granulozyten im peripheren Blut (10 350/mm3) wurden in den blutbildenden Organen nur wenig typische neutrophile Vorstufen mit einem normalen Enzymmuster festgestellt.

Hydrolysis of a chymotrypsin substrate and of naphthol AS-D chloroacetate by human leukocyte granules

SummaryThe subcellular distribution has been investigated of two esterases from human neutrophil granulocytes obtained from patients with chronic myelocytic leukemia. One esterase hydrolyzed the typical chymotrypsin substrate N-acetyl-L-tyrosine ethyl ester, the other hydrolyzed naphthol AS-D chloroacetate, a substance which is used in cytochemistry for the demonstration of esterase activity. Both enzymes could be recovered almost exclusively in the granule fraction and were optimally active at about pH 7.4. After lysis of the granules by hypoosmotic shock 73% of the naphthol AS-D chloroacetate esterase activity could be dissolved in buffered 0.15M sodium chloride (pH 7.0) in contrast to only 17% of the N-acetyl-L-tyrosine ethyl esterase activity. The soluble lysate contained 59% of the total granule protein. It was subjected to acrylamide-gel electrophoresis at acid pH and multiple bands of naphthol AS-D chloroacetate esterase activity were demonstrated on the gels. Acrylamide-gel electrophoresis of the insoluble proteins and the soluble lysate of the granule fraction was carried out after dissolution of the proteins in phenol-acetic acid-urea and the resulting electrophoretic patterns were compared. Hydrolysis of naphthol AS-D chloroacetate by the granule fraction was compared to hydrolysis of the same substrate effected by the pure proteases chymotrypsin, trypsin, and pancreatopeptidase E.ZusammenfassungEs wurde die subzelluläre Verteilung zweier Esterasen in neutrophilen Granulozyten von Patienten mit chronischer Myelose bestimmt. Eine der beiden Esterasen hydrolysierte den N-Acetyl-L-Tyrosinäthylester, der ein typisches Substrat für Chymotrypsin ist, die andere hydrolysierte Naphthol-AS-D-chloracetat, das in der Zytochemie zur Darstellung von Esteraseaktivitäten verwendet wird. Beide Esteraseaktivitäten konnten in der Granulafraktion lokalisiert werden, das pH-Optimum ihrer Aktivität liegt bei pH 7,4. 59% der gesamten Granulaproteine konnten in 0,15M NaCl (pH 7,0) gelöst werden. Die löslichen Granulaproteine enthielten 73% der Naphthol-AS-D-chloracetat-Esteraseaktivität, jedoch nur 17% der Esteraseaktivität gegenüber dem N-Acetyl-L-Tyrosinäthylester. Die Acrylamid-Gelelektrophorese bei pH 4,3 zeigte mehrere Banden mit Esteraseaktivität gegenüber Naphthol-AS-D-chloracetat. Zum Vergleich des Proteinmusters des löslichen Lysats mit dem des unlöslichen Rückstands der Granulafraktion wurde nach dem Lösen der Proteine in einer Mischung aus Phenol, Eisessig und Harnstoff eine weitere Acrylamid-Gelelektrophorese durchgeführt. Die Hydrolyse des Naphthol-AS-D-chloracetats durch die Granulafraktion wurde mit der Spaltung des gleichen Substrats durch die Proteasen Chymotrypsin, Trypsin und Pankreatopeptidase E (Elastase) verglichen.

Naphthol AS-D chloroacetate esterases in granule extracts from human neutrophil leukocytes

SummarySoluble proteins of a granule fraction derived from human neutrophils were subjected to electrophoresis on 15% acrylamide gels at pH 4.3 Several of the protein bands were shown to possess hydrolytic activity on naphthol AS-D chloroacetate at pH 7.4. Electrophoretic movement of NASDCA esterases is similar to those of trypsin, chymotrypsin and elastase.ZusammenfassungLösliche Proteine einer Granulafraktion aus menschlichen Neutrophilen wurden einer Acrylamidgel-Elektrophorese (15% Acrylamidgel bei pH 4,3) unterwerfen. Verschiedene Proteinbanden zeigten eine hydrolytische Aktivität gegenü ber Naphthol-AS-D-Chloracetat bei pH 7,4. Die elektrophoretische Wanderung der Naphthol-AS-D-Chloracetat spaltenden Esterase ist ähnlich der von Trypsin, Chymotrypsin und Elastase.

Abnormal megakaryopoiesis in patients with myelodysplastic syndromes: analysis of cellular and humoral defects.

Summary. In 13 patients with myelodysplastic syndrome (MDS) mature and immature erythropoietic (CFU‐E, BFU‐E), granulopoietic (CFU‐GM) and megakaryopoietic (CFU‐Meg) colony formation from human bone marrow mononuclear cells was evaluated in a microagar culture system. All but three patients exhibited abnormal CFU‐Meg. The defect of CFU‐Meg paralleled the reduction of BFU‐E, whereas CFU‐GM number declined to a lesser extent. Not only the CFU‐Meg number, but also the number of megakaryocytes (Mk) per colony was reduced suggesting an additional functional CFU‐Meg defect.

Megakaryoblastic micromegakaryocytic crisis in chronic myeloid leukemia.

SummaryAtypical megakaryoblasts (MKB) or megakaryocytes (MK) are occasionally present in the peripheral blood during the terminal development of chronic myeloid leukemia (CML). We report on a 49-year-old female suffering from Ph1 chromosome-positive CML with typical megakaryoblastic transformation in the peripheral blood and in the bone marrow. The small “blasts” were at the most only slightly larger and were occasionally even smaller than lymphocytes but showed megakaryoblastic or atypical megakaryocytic differentiation. The cytoplasmic cytochemical pattern of the atypical megakaryocytic cells was identical to that of large atypical thrombocytes. Platelet peroxidase was detected upon electron-microscopic (EM) examination. Immunologic characterization disclosed the presence of MK-specific antigens. When cultured in vitro on agar, the blasts transformed spontaneously into large mature MK, exhibiting characteristic cytochemical and immunological patterns. Cytogenetic examination of peripheral blood showed severe abnormalities. The patient did not respond to therapy and died 3 months after manifestation of the blast crisis.

Abnormal expansions of granular lymphocytes: Reactive lymphocytosis or chronic leukemia? case report and literature review

SummaryA case of chronic lymphoproliferative disorder is presented, wherein a morphologically homogeneous population of lymphoid cells displayed properties similar to those described for large granular lymphocytes (LGL). Besides their LGL-like phenotype (VEP 13+, OKM 1+, OKT 10+ Fc-IgG-receptor+, OKT 3−), the proliferating cells were cytotoxic to NK targets as well as to antibody-coated target cells. Clinically, our patient presented low-grade lymphocytosis, splenomegaly, neutropenia, hyperimmunoglobulinemia and recurrent infections. Based upon this and 32 similar cases reported in the literature, we conclude that lymphoproliferative disorders involving GL encompass a variety of clinical entities, ranging from reactive GL lymphocytoses to overt lymphocytic malignancies.

Large granular lymphocytes: Morphological and functional properties I. Results in normals

SummaryLarge granular lymphocytes (LGL) represent a morphologically distinct mononuclear cell type. In healthy individuals an average of 12.5±5.2% of peripheral mononuclear cells exhibit this morphology. Structurally they are characterized by azurophilic granules, light cell density, low surface adherence and expression of Fc-IgG receptors. Functional analyses on LGL suspensions enriched by means of density gradient fractionation and adherence procedures included testing of T-cell and monocyte-specific in vitro reactions as well as evaluation of their natural cytotoxicity (NC). Results indicated a close correlation between the percentages of LGL and the capacity to bind and to lyse NC-susceptible tumor targets. In agreement with others we would thus conclude that LGL embrace the majority of NK-effector cells. The morphological definition of LGL provides an additional tool for the clinical evaluation of this cell mediated immune reactivity.