Norbert Böhm

Fluorescence cytophotometry: a valuable method for the quantitative determination of nuclear feulgen-DNA

SummaryA cytofluorometric apparatus with incident illumination for fluorescence excitation is described here.Cytophotometric fluorescence measurements (UV- and blue excitation) of acriflavine-acridine yellow-, coriphosphine- and pararosaniline-Schiff stained di-, tetra- and octoploid liver nuclei, leucocytes and sperms (Feulgen reaction) were found to agree with the absorbance data obtained from the same slide by means of the integrating microdensitometer.The stoichiometry of the fluorescence emission is discussed in detail. It is emphasized that not a linear, but an exponential relationship exists between the emitted fluorescence intensity and the concentration of the fluorescent substance.Cytofluorometry of Feulgen-stained nuclei has proved to be as reliable and fast as the absorbance scanning measurements at the intergrating microdensitometer.

Proportionalitätsfehler bei der Feulgen-Hydrolyse

SummaryComparing the Feulgen dye-content of different nuclei (liver cells, lymphocytes, granulocytes and sperms) after alcohol-fixation deviations were found between the measured Feulgen values and the DNA-content to be expected from the DNA-constancy law. The Feulgen values of lymphocytes and granulocytes proved to be lower (up to minus 20%) than those of diploid liver nuclei at all hydrolysis times, while in the postmaximal range of hydrolysis the values of the haploid sperms were too high (even higher than those of the diploid nuclei). Such differences did not appear when nuclei of the same cell type in different DNA- ploidy classes (liver nuclei) were compared.Those deviations of leucocytes and sperms were no longer found after fixation in methanol-formalin-glacial acetic acid and, in addition, could not be confirmed by UV-absorption measurements. For that reason we suppose them to be due to proportionality errors caused by variations in the hydrolytic behaviour of the different nucleoproteins.Thus fixation in methanol-formalin-glacial acetic acid seems to be more suitable for quantitative Feulgen measurements than alcohol-fixation, which may easily give rise to proportionality errors during Feulgen hydrolysis.Moreover, to avoid any false interpretation of Feulgen data we should suggest controll measurements using another independent method (f. e. UV-absorption), or — if this is impossible — to check the Feulgen values after different fixations and variant hydrolysis times (premaximal, maximal, postmaximal).ZusammenfassungBeim Vergleich des Feulgen-Farbgehaltes verschiedener Zellkerne (Leberzellen, Lymphozyten, Granulozyten und Spermien) traten nach Alkoholfixierung Abweichungen der gemessenen Feulgen-Werte von den nach dem Gesetz von der DNS- Konstanz zu erwartenden DNS-Gehalt dieser Kerne auf. Verglichen mit den Feulgen-Werten der diploiden Leberzellkerne ergaben Lympho- und Granulozyten bei allen Hydrolysezeiten zu niedrige (bis zu minus 20%), haploide Spermien im postmaximalen Hydrolysebereich zu hohe Feulgen-Werte (z. T. sogar höhere Werte als die diploiden Zellkerne). Innerhalb desselben Zelltypes wurden dagegen, auch beim Vergleich der verschiedenen Ploidiestufen der Leberkerne, keine Differenzen festgestellt.Da die an Leukozyten und Spermien beobachteten Abweichungen nach Methanol-Formalin-Eisessig-Fixierung nicht mehr auftraten und auch durch UV-absorptionscytophotometrische Messungen nicht bestätigt werden konnten, muß man annehmen, daß es sich um Proportionalitätsfehler handelt, die auf Hydrolyseunterschieden beruhen.Für die quantitative Feulgen-Cytophotometrie scheint daher die Methanol-Formalin-Eisessig-Fixierung besser geeignet zu sein als die Alkoholfixierung, bei deren Verwendung es leicht zu Proportionalitätsfehlern während der Feulgen-Hydrolyse kommen kann.

Durchflußfluorescenzcytophotometrie für ultraschnelle DNS-Messungen an großen Zellpopulationen

SummaryA flow-through cytophotometer based upon the Leitz-microscope photometer (MPV) is described. The light source is a high pressure Xenon arc HBO 100 in an arrangement for epiillumination.The flow system is designed in such a way that each cell, after previous Feulgenstaining in aqueous suspension, has to pass through the focal plane of the objective. This is achieved by a self-constructed flow channel with a vertical fluid stram carrying the stained cells along the optical axis of the microscope at a flow rate of 100 to 1000 cells per sec. After passage through the focal plane with simultanneous registration of the highest fluorescence reading the cells are flushed off by a horizontal water current.The fluorescence light impulses emitted by the cells are classified and stored by an impulse height discriminator, from which they may be recalled either analogically as a histogram (DNA distribution pattern) or digitally as a series of numbers.In addition, the fluorescence impulses can be visualized at the screen of an oscillograph. The flow rate of the fluorochromized cells is determined by means of a counter unit.The steps of preparation of the cell suspension as well as fixation, hydrolysis and Feulgen-staining with acriflavine-Schiff of the suspended tissue cells is described in detail.The possibilities of application of the instrument as a cyto-morphometric approach to cytology and cytochemistry automation are discussed.ZusammenfassungAusgehend vom Leitz-Mikroskopphotometer MPV mit Auflicht-Anregung wird ein Durchflußfluorescenzcytophotometer beschrieben, das es gestattet, die relativen Feulgen-DNS-Mengen von bis zu 1000 Zellen pro Sekunde zu bestimmen. Die Meßwerte werden in einem Impulshöhenanalysator klassifiziert und gespeichert. Auf Abruf wird der Speicherinhalt analog als Histogramm ausgeschrieben oder digital als Zahlenfolge ausgedruckt.Das Vorgehen bei der Zellsuspensionsherstellung, Fixierung, Hydrolyse und Färbung mit dem Feulgen-Farbstoff Akriflavin wird dargelegt.Anwendungsbeispiele werden besprochen.

Signet ring cell carcinoma of the eccrine sweat glands in the eyelid.

BACKGROUND Signet ring cell carcinoma of the eyelid is a rare variant of eccrine sweat gland carcinoma and has been reported previously in only five patients. METHODS The authors report the clinical findings of a 55-year-old man with a signet ring cell carcinoma in the left eyelid as well as a clinical follow-up of 4.5 years. Several biopsies and the exenteration specimen were analyzed by routine light microscopy, electron microscopy, and comprehensive immunohistochemical stains on paraffin sections. RESULTS Histologically, the tumor was shown to be a rare type of eccrine sweat gland carcinoma with signet ring cells and Indian file growth pattern reminiscent of invasive lobular carcinoma of the breast. Estrogen and progesterone receptors were identified immunohistochemically. On electron microscopy, intracytoplasmic pseudolumina with microvilli were positive for anti-human milk fat globulin and the lectin peanut agglutinin. Clinically, the tumor followed a malignant course with orbital invasion and lymph node metastases. CONCLUSIONS Histologic recognition of this variant of eccrine sweat gland carcinoma is important because of its aggressive and malignant behavior and the wide range of differential diagnoses. Primarily, metastatic mammary carcinoma must be excluded. The treatment is primary excision with histologic control of the excision margins. In more advanced stages, radiation therapy, neck dissection, and anti-estrogen therapy should be considered.

Adrenal, cutaneous and myocardial lesions in fulminating endotoxinemia (Waterhouse-Friderichsen syndrome).

Summary Waterhouse-Friderichsen syndrome (WFS) was diagnosed in ten autopsy cases (6 to 16 years of age) according to the following criteria: 1. fulminating meningococcic septicemia, 2. massive cutaneous hemorrhages, and 3. bilateral hemorrhagic necrosis of the adrenals. Only one case showed disseminated intravascular coagulation (DIC) and hemorrhagic diathesis. Skin histology revealed small numbers of microthrombi to be present in all cases. The vital organs, however, did not all disclose such microthrombi: only 3 adrenal, 4 kidney, and 2 lung specimens presented microthrombi. The hearts showed no evidence of DIC, but did reveal histological signs of myocarditis accompanied by endothelial damage, interstitial edema, fibrinous transudation, granulocytic infiltration, hemorrhages, and foci of necrotic heart muscle fibers. These findings suggest a toxic (endotoxinogenic) damage of the endothelial cells, vascular walls and adjacent interstitial and parenchymal tissues. The hemorrhagic necroses observed in the adrenal and the hemorrhagic skin lesions, likewise, appear to be the result of direct endotoxinic damage, rather than microcirculatory ischemia resulting from microthrombi. Thus, we believe that overwhelming toxemia plays the immediate and dicisive role in the fatal outcome of WFS. Adrenal failure and systemic shock with DIC merely present additional complications which may or may not be present. In all ten cases investigated, interstitial myocarditis was found; this and other supportive clinical and morphological findings point heavily to cardiac arrest as the immediate cause of death in fatal WFS.

Graft-versus-Host Disease in two Newborns After Repeated Blood Transfusions Because of Rhesus Incompatibility

Summary Fatal GVHD developed in two male newborn babies, who had been treated by repeated intrauterine (only case 2) and exchange blood transfusions because of severe Rhesus incompatibility. The clinical manifestations of the disease were fever, enlargement of liver and spleen, diarrhea, exanthema, anemia, and blood eosinophilia. Both babies died at the age of three weeks. In case 2 identical HL-A antigens were found in the blood of the last donor and in the lymphocytes of the baby obtained shortly after death. Autopsy and histologic examinations disclosed a marked atrophy of the lymphatic organs with depletion of lymphocytes, together with an hypoplastic bone marrow. Around blood vessels in the systemic connective tissue and in many organs infiltrates of eosinophilic granulocytes, histiocytes, lymphocytes and lympho-monocytoid blasts were found. The gastro-intestinal tract, the liver and the skin were predominantly affected. In addition we observed hemorrhagic necroses of lymph nodes with extreme dilatation of lymph vessels, which were occupied by mature and immature erythroid and monocytoid cells. Ringshaped fibrinoid and hemorrhagic necroses were also found in the spleen around the Malpighian corpuscles. These inflammatory and necrotizing tissue damages are attributed to local immune reactions between proliferating T-lymphocytes of the donor and tissue antigens of the host. No primary defect of the immune system of the babies could be verified. It is therefore postulated that intrauterine transfusions (or an accidental materno-fetal transfusion via the placenta) induced a state of nonspecific immune tolerance by exhaustion of the immature cellular immune defence mechanisms of the fetus , thus allowing subsequent implants of immune competent cells not to be rejected but to proliferate and inhabit the lymphatic organs of the host. This hypothesis is supported by two facts: 1. Intrauterine and subsequent exchange transfusions are usually required to induce GVHD in primary immunologically normal babies (with Hassals corpuscles and immune globulines shown to be present). 2. Only lymphocytes of the exchange transfusion donors and non of the intrauterine donors were found in the blood of the GVHD babies. Both these requirements were also met in the cases of Naiman et al. (1969) and Parkman et al. (1974). Our case 1 may have been caused by a slightly different mechanism in that instead of intrauterine transfusions, maternal blood cells had crossed the placenta and had induced a state of nonspecific fetal immune tolerance. This, however, could not be directly proven because no immunological and cytogenetic studies were performed in this case.

Reversible Hyperplasie und Hypertrophie der Mäuseleber unter funktioneller Belastung mit Phenobarbital

Abstract Introduction: Administration of phenobarbital to rats and mice is well known to cause enlargement of the liver, where the drug is metabolized by hydroxylation and oxydation. The increase of the liver weight is thought to be due to an enlargement of the individual hepatocytes (hypertrophy) caused by an augmentation of the smooth endoplasmic reticulum, as well as to cell multiplication (hyperplasia). The present investigation deals with the nuclear DNA content of mouse hepatocytes during and after administration of different doses of phenobarbital. The data are related to liver weight with due consideration of mitotic activity and cell loss by necrobiosis. Material and Methods: 172 five to six weeks old male albino NMRI mice with a body weight of 23 to 33 gms were randomly divided into four groups, one of which served as the controls. The three test groups received 75 mg, 100 mg and 150 mg phenobarbital per 1 kg body weight intraperitoneally once every day for a total of 10 days. Thereafter the administration of the drug was discontinued. Beginning with the third day of the experiment 3 animals of each group were sacrificed by exsanguination every secound day after their body weight had been carefully determined. Then the liver weights were measured. The nuclear DNA content of the hepatocytes was determined from liver smears by means of acriflavine-Feulgen fluorescence cytophotometry. The number of mitotic figures and of necrobiotic liver cells was counted in histologic sections. Results: With animals receiving 150 mg and 100 mg phenobarbital per 1 kg body weight a rapid increase of the relative liver weight (up to 74% above the controls) was observed, which was reduced back to normal levels within 10 days after discontinuation of the drug. Parallel with the increase of the liver weight a striking DNA-polyploidisation of the liver nuclei occurred which proved to be reversible during the reduction phase. Mitotic figures were found only in the initial phase of the experiment (third to fifth day), while the number of necrobiotic hepatocytes was increased after the drug was discontinued. Similar but markedly less pronounced effects were encountered with animals of the 75 mg group. Discussion: It is concluded that the increase of the liver weight of mice after phenobarbital administration is partly due to cell multiplication (hyperplasia) — as is shown by a high number of mitotic figures in the initial phase of the experiment-, partly due to the enlargement of hepatocytes with concommitant polyploidisation of the nuclei (hypertrophy). When the drug administration is discontinued the liver weights return to normal levels within 10 days. Since at the same time the number of high-ploidy nuclei is reduced with no evidence of an increased mitotic activity, the reduction of the liver weight should be partly caused by an elimination of high-ploidy hepatocytes, which are no longer required after the hyperfunctional stimulus has ceased. These cells break down necrobiotically, — thus reducing both cell number and DNA-ploidy to the normal age-dependent level.

Staphylococcal scalded skin syndrome (SSSS) and consecutive septicaemia in a preterm infant.

Staphylococcal scaled-skin syndrome (SSSS) is a toxin-related epidermolytic disease that usually affects infants and children under 5 years. We report herein a case of a premature infant who had developed SSSS after an infection of the pharynx with staphylococci and who died of septicaemia due to pseudomonas aeruginosa. The primary mechanism of action of epidermolysin still remains unknown. We demonstrate that acantholysis is due to an early edema of the intercellular space with separation of ultrastructurally unaltered desmosomes.

Results after surgery in undifferentiated large cell carcinoma of the lung: the role of neuroendocrine expression

OBJECTIVE The objective of this study was to define the incidence of light microscopically undifferentiated large cell carcinomas, to analyze tumor stages, types of resections necessary and postsurgical survival. Additionally we tried to evaluate whether or not neuroendocrine expression influences the biological behavior of these tumors. METHODS Light microscopic specimens of 105 patients having undergone surgery for undifferentiated large cell carcinoma were reviewed following the 1981 WHO criteria. Fifty eight cases were excluded because elements of adeno- or squamous cell carcinoma, neuroendocrine or combined patterns of histological differentiation were observed. The remaining 47 cases of pure undifferentiated large cell carcinoma were evaluated immunohistochemically for neuroendocrine differentiation using a combination of the markers neuron specific enolase, synaptophysin and chromogranin A. The hospital charts of the patients were analyzed retrospectively recording tumor stage, operative procedure, postoperative complications, postoperative adjuvant treatment procedures, actual tumor state and survival time. RESULTS Thirteen patients (27.7%) had postsurgical tumor stage I, 5 (10.6%) stage II, 15 (31.9%) stage IIIA, 9 (19.1%) stage IIIB, and 5 (10.6%) stage IV. In 46 of 47 patients resections of lung parenchyma were performed (wedge resection n = 5, segmental resection n = 1, lobectomy n = 27, bilobectomy n = 3, pneumonectomy n = 10), in 6 patients combined with broncho- and/or angioplastic procedures. At the time of chart review 20 (42.5%) patients were still alive. The cause of death in the remaining patients was recurrent lung cancer in the majority of cases (24 or 92.30%). The overall mean survival of the 46 patients undergoing parenchymal resections was 19 months, the 3-year survival rate 31.7%. The immunohistochemical examination demonstrated expression of neuron specific enolase in 15 cases. Synaptophysin and chromogranin A were not detected in any case. For these 15 patients the mean survival was 25.6 months (+/- 4.3) and the 1-year survival rate 67% (confidence interval 43-91%) compared to 13.8 (+/- 2.1) months and 33.5% (confidence interval 15.3-51.7%) in the remainder. The difference was not significant (P = 0.06). CONCLUSIONS The light microscopic diagnosis of undifferentiated large cell carcinoma revealed to be subject to considerable interobserver variability. Undifferentiated large cell carcinoma takes a more unfavorable clinical course than other non-small cell carcinomas. Despite lack of statistical significance, expression of neuron specific enolase appeared to be associated with less aggressive biological behavior of the respective neoplasms. Immunohistochemical evaluation of undifferentiated large cell carcinomas using a combination of neuron specific enolase, chromogranin A, and synaptophysin did not provide more therapeutically relevant information than that obtained by light microscopic assessment.

EBV‐associated lymphoproliferative syndrome with a distinct 69 base‐pair deletion in the LMP‐1 oncogene

Summary. We describe an immunocompetent 12‐year‐old boy with chronic EBV infection and lymphoid interstitial pneumonitis. Lymph node biopsies showed effacement of the architecture with polymorphic cellular infiltrates, consisting predominantly of T cells and natural killer cells. No clonal rearrangement of TCR or immunoglobulin genes was seen. DNA was extracted from hilar lymph nodes; sequencing of the carboxy terminal region of the latent membrane protein 1 (LMP‐1) oncogene revealed a 69 base‐pair deletion and four point mutations. Immunosuppressive treatment with prednisone and cyclosporine reversed the lymphadenopathy.