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Morphometric evaluation was performed on semi-thin sections of core biopsies of the bone marrow and included 20 cases of each group of diseases besides control specimens. (i) Hyperergic myelitis of rheumatic origin. (ii) Chronic granulocytic leukaemia (CGL). (iii) Polycythaemia vera (P. vera). (iv) Chronic megakaryocytic-granulocytic myelosis (CMGM). (v) Myelofibrosis or osteomyelosclerosis (MF/OMS). The following classification of megakaryopoiesis was applied: normal megakaryocytes; giant forms; microforms; intussusceptions; cytoplasmic fragments; naked nuclei. The density distribution shows an increase of megakaryocyte number in those 5 different marrow disorders, ranging from about 13/mm2 in the normal sample up to 65 cells/mm2 in MF/OMS. Microforms are most frequently encountered in CGL, whereas giant megakaryocytes, intussusceptions and many cytoplasmic fragments characterize P. vera, CMGM and MF/OMS. Our measurements suggests 3 distinct categories of bone marrow lesions with corresponding alterations of the megakaryopoiesis: (i) myelitis and CGL; (ii) P. vera; (iii) CMGM and MF/OMS.

In a retrospective study of 352 patients with primary myelodysplastic syndromes, 61 (17.3%) revealed myelofibrosis in bone marrow biopsies. The fibrosis was observed to occur mostly focally (41/61 cases), and collagen deposits were found very rarely (4/61). The histopathology of bone marrow biopsies revealed hyperplasia and disturbed differentiation in megakaryopoiesis; the frequency and grade of dysplasia in megakaryopoiesis increased with advancing myelofibrosis. Reticulin fibrosis occurred in all subtypes of MDS; however, there was a higher incidence in chronic myelo-monocytic leukaemia (CMMoL). The frequency of cytogenetic aberrations was significantly higher in the MDS cases with myelofibrosis, compared to the cases without fibrosis. Clinical data showed significantly lower values of haemoglobin and lower platelet counts in MDS with myelofibrosis. Life expectancy was reduced to 9.6 months, compared with 17.4 months in MDS without fibrosis. In refractory anaemia, the survival times were 10.0 months in MDS with myelofibrosis, compared to 28.9 months in MDS without myelofibrosis. 36.6% of the patients with MDS and myelofibrosis developed a transformation into ANLL during the course of the disease. Myelofibrosis therefore seems to herald a poor prognosis.

The retrospective evaluation of bone marrow biopsies of 569 patients with primary myelodysplastic syndrome--pMDS--revealed 256 refractory anemias--RA--, 52 refractory anemias with ringed sideroblasts--RARS--, 133 refractory anemias with excess of blasts--RAEB--, 52 refractory anemias with excess of blasts in transformation--RAEB-t--, and 53 chronic myelo-monocytic leukemias--CMMOL--according to FAB-criteria, 23 patients were not otherwise specified (myelodysplastic syndrome: not otherwise specified--MDS.NOS--). RARS-patients had the best prognosis (median survival 41.9 months, incidence of leukemia 3.8%), followed by RA-patients (26.5 months, 16.4%), MDS.NOS-patients (22.4 months, 21.7%), CMMOL-patients (12.5 months, 49.1%). RAEB- and RAEB-t-patients had the worst prognosis (median survival time 8.5 and 4.6 months, incidence of leukemia 42.1% and 57.7%, respectively). But the survival times showed a considerable range in each FAB-subgroup with 0-154 months in RA or 0-52 months in CMMOL. To forecast life expectancy more precisely, a scoring system was developed using nine histopathological parameters, among which the three most important ones were determined: quantity of myeloblasts, myelofibrosis and ALIP's. The scoring system allows a determination of three risk groups with significantly different survival times. It is valid also for patients without increase of myeloblasts (< 5% myeloblasts in the bone marrow) and identifies high-risk MDS patients in this group. By this proposed scoring system, a prognostic approval in primary MDS can be achieved applying histopathology without regarding further methods herewith presenting a system which could be considered independently from hematologic, cytological or laboratory data.

The advanced, fibrotic phase of chronic idiopathic myelofibrosis (CIMF) is preceded by a pre-fibrotic stage. However, the factors which may influence or predict the development of myelofibrosis are not well established. Thus we investigated follow up biopsies of 70 patients with CIMF, diagnosed in stages of no or only scant fiber increase, for the development of myelofibrosis. The influence of histopathological (megakaryocytes, initial fiber content), clinical (age, gender, splenomegaly, chemotherapy) and hematological (Hb, leukocyte- and platelet count) parameters on the development of myelofibrosis was evaluated by using the univariate Log Rank method and the multivariate recursive partition and amalgamation (RECPAM) analysis. Surveying a mean observation period of 47 months we found a development of significant myelofibrosis in 30 of the 70 patients. In the univariate analysis, the development of myelofibrosis was associated with increased megakaryocytes, initial fiber content and age of the patient. In the RECPAM analysis, the patients with a high megakaryocytic count and older age showed the highest risk of developing myelofibrosis (mean 58.3 and 60.1 months). The group with the best prognosis comprised the patients under 60 years which have a low content of megakaryocytes (mean 137 months). We found that the development of myelofibrosis in CIMF was best predicted by an increase of megakaryocytes within the bone marrow, possibly reflecting the release of growth factors by these cells. The next important risk factor was the age of the patients.

In chronic idiopathic myelofibrosis (CIMF) the factors predicting survival in patients who were already in the fibrotic stage have been well documented by numerous studies. Prefibrotic stages were only rarely evaluated so that the prognostic impact of myelofibrosis is currently not well known. Also predictive factors for disease-related events were not included in those studies. Thus, we evaluated the prognostic impact of myelofibrosis and other histopathological (megakaryocytes, blasts) and clinical [age, gender, splenomegaly, chemotherapy, hemoglobin (Hb), leukocyte, and platelet count] parameters in 122 patients in fibrotic and prefibrotic stages of CIMF on event-free survival. The statistical analysis was performed using the univariate log-rank test and the multivariate recursive partition and amalgamation (RECPAM) approach. In 62 patients disease-related events occurred during a mean observation period of 58 months. In univariate analysis they were associated with blast increase in the bone marrow. In RECPAM analysis a shorter event-free survival was found in anemic patients (mean: 9.3 months). In nonanemic patients older than 60 years, advanced myelofibrosis was associated with a shorter event-free mean survival of 23.2 months versus 69.3 months in less advanced cases. A slight or moderate myelofibrosis was not found to have a prognostic impact on event-free survival. The longest event-free survival was found in nonanemic patients who were younger than 60 years (mean: 185 months), regardless of the grade of myelofibrosis. Thus, we found that the most relevant prognostic parameter for event-free survival in CIMF were the Hb value, age, and grade of myelofibrosis.

Zusammenfassung Wegen des erst kurzlich mit Erscheinen der neuen WHO-Klassifikation der chronischen myeloproliferativen Erkrankungen (CMPE) aufgegebenen Festhaltens an einer Knochenmarksfibrose als wesentliches Merkmal der chronischen idiopathischen Myelofibrose (cIMF) sind Verlaufsuntersuchungen zu der Entwicklung einer Myelofibrose (MF) bei dieser Entitat nur von fibrotischen Stadien mit widerspruchlichen Ergebnissen bekannt. Deshalb wurde eine retrospektive Studie an Verlaufsbiopsien von 38 Patienten mit cIMF ohne initiale Fibrose mit der Frage nach der Haufigkeit, der Geschwindigkeit sowie dem Ausmas der Entwicklung einer MF durchgefuhrt. Unabhangig von einer Chemotherapie fanden wir einen Ubergang in eine MF bei 80% der mit Chemotherapie und 75% der symptomatisch behandelten Patienten. Unsere Ergebnisse sprechen fur eine schrittweise Entwicklung der " klassischen " cIMF aus einem " prafibrotischen " Stadium, wobei offenbar eine biologische Heterogenitat in Hinblick auf die Geschwindigkeit bzw. Wahrscheinlichkeit einer MF im Einzelfall besteht, da sich sowohl bei langerer Beobachtungszeit keine Ubergange als auch nach Intervallen von weniger als 2 Jahren eine voll entwickelte MF fanden.

Heterogeneous content of fibres and haematopoesis within the bone marrow may affect diagnosis and staging in chronic myeloproliferative disorders (CMPDs). To evaluate their distribution, we conducted a post mortem histomorphometric study of 22 patients with CMPD in chronic phases. In bone marrow specimens from the anterior and posterior iliac crest (right and left of each), the sternum, the 7th thoracic and the 3rd lumbar vertebra, the argyrophil fibres were counted using the line intersection method and the cellular and fatty bone marrow using the point count method. Statistical analysis was performed by direct comparison of the sites. The distribution of fibres was almost homogeneous in the patients with low fibre content, revealing a random diversity in more advanced stages of marrow fibrosis. 1/22 patient had no fibre increase in one specimen of the iliac crest and overt myelofibrosis in the other sites. 1/22 patient had myelofibrosis in two sites of the iliac crest and no fibre increase in vertebral column and sternum. The bone marrow cellularity was almost homogeneously increased in all patients. Myelofibrosis proved to be a generalised process with heterogeneous grades of severity in different regions of the bone marrow in CMPDs. No topographical bias was found. In contrast to the homogeneous increase of the bone marrow cellularity the topographical heterogeneity of the fibre content may limit the representativity of single bone marrow biopsies in patients with CMPDs.

The present study illustrates characteristic features of histopathology in the 3 non-leukemic, Ph-negative groups of chronic myeloproliferative diseases (CMPD). Attention is paid to the final outcome of CMPD, especially its transformation into acute leukemias and the occurrence of myelofibrosis from bone marrow biopsies (BMB) in a total of 1,716 CMPD patients. Essential thrombocythemia (ET), polycythemia vera (P. vera), and chronic megakaryocytic granulocytic myelosis (CMGM) can readily be distinguished by histopathology from BMB in the great majority of patients without regarding laboratory data, leaving a compartment of about 12% unclassifiable cases. Histologic patterns of staging are the increase in number and pleomorphism of megakaryocytes (MK), increase in number and density of reticulin fibers and collagen fibrosis, and excess of blasts. These 3 criteria are each graded from 0 to 3 in every biopsy. From these, a staging results by means of the histology of BMB in each of the Ph-negative CMPD. This staging provides a classification by defined criteria which permits comparative studies, the possibility of monitoring the individual patients by follow-up histology, and offers a baseline for reliable evaluation of results from therapy protocols.

To compare the prognostic value of scoring systems in primary myelodysplastic syndromes (pMDS), four clinicohematological systems (Rennes, Bournemouth, Düsseldorf, Pavia) and the histopathological Hannover Scoring System were applied to 415 MDS patients from the Bone Marrow Registry of Hannover Medical School. According to the FAB classification, 180 patients (43%) were diagnosed as RA, 33 (8%) as RARS, 99 (24%) as RAEB, 36 (9%) as RAEBt, and 48 (12%) as CMMOL; 19 patients (4%) were not further classified (MDS.UC). All scoring systems revealed three or four groups of patients with significantly different survival times. The ranges and standard deviations in these groups were similar but high in all scoring systems. A good differentiation between short-term survivors (1 year survival time) and intermediate-term survivors (1-4 years) was possible with all tested scoring systems, but the differentiation between intermediate- and long-term survivors (4 years) was not distinctive enough. The problem of risk assessment in the single patient is furthermore elucidated by the values of specificity and sensitivity, which were relatively low in all scoring systems tested. Best results were yielded by the Bournemouth Score for long-term survivors and the Düsseldorf and Hannover Score for intermediate- and short-term survivors. Multivariate analysis of all parameters used in the scoring systems showed the highest negative impact on survival for increase of myeloblasts, anemia, myelofibrosis, high age of the patient, and abnormal localization of immature precursors (ALIPs). Therefore, the histopathological Hannover Scoring System is not only equal to clinicohematological risk assessment in pMDS, but also includes important independent prognostic parameters. Risk assessment for the individual low-risk MDS patient using only initial parameters may be rendered impossible due to the biological nature of pMDS. Therefore, sequential analysis is needed to elucidate random events which alter the prognosis.

The Ph1-negative groups of chronic myeloproliferative diseases (CMPD) are described, and histopathological criteria that distinguish them from each other are given. These are based upon observations in primary biopsies from 2,331 patients with CMPDs among a total of 34,160 patients referred between 1 January 1989 and 30 June 1994 to the Bone Marrow Registry. These cases of CMPD break down into the main groups as follows: CML 23.2%, megakaryocytic myelosis consistent with agnogenic myeloid metaplasia 22.3%, essential thrombocythemia 22.1%, and polycythemia vera 20.4%; 12.0% of cases were unclassifiable. Histological progress in each group is characterized by (1) increasing number and pleomorphy of megakaryocytes, (2) increasing fibrosis, and (3) excess of blasts. These three features can be observed in diagnostic biopsies before any therapy. Therefore, it is recommended that such alterations be reported semiquantitatively. A staging system with four stages from 0 to 3 for each of the three features is introduced. Its application allows staging for the individual patient on the basis of diagnostic biopsies.

Morphometrische Untersuchungen an histologischen Schnitten von Biopsien chronischer myeloproliferativer Erkrankungen (CMPE) konnen die individuelle histologische Diagnose unterstutzen und zur Unterscheidung der 4 Gruppen der CMPE beitragen. Eine Gesamtzahl von 130 Biopsien, unterteilt in 29 Falle von CML ohne Megakaryozytenvermehrung (CML.CT), 26 Falle von CML mit Megakaryozytenvermehrung (CML.MI), 28 Falle von essentieller Thrombozythamie (PTH), 26 Falle von chronischer megakaryozytar-granulozytarer Myelose (CMGM) und 21 Falle von Polycythamia vera (P. vera) sowie eine Anzahl von 30 Kontrollbiopsien gesunder Personen wurden morphometrisch untersucht in Schnitten von nicht entkalkten, in Plastik eingebetteten Knochenmarkbiopsien. Sichere Unterscheidungen der Gruppen sind moglich durch • Grose der Megakaryozyten, • Kernlobulierung und Kerngrose und • die Form der Megakaryozyten. Kern- und Zellflache waren bei der CML signifikant kleiner (Streuung der Mittelwerte der Zellflache: 220–360 μm2) als bei den 3 Ph1-negativen Erkrankungen (Streuung der Mittelwerte der Zellflache: 480–750 μm2). Die Kernlobulierung war bei der PTH ausgepragter als bei der CMGM und der P. vera. Das Clustering von Megakaryozyten war mehr als doppelt so haufig bei der CMGM (8,0–10,5 %), verglichen mit den 3 anderen CMPE (0,1–7,0 %). – Nacktkernige Megakaryozyten waren gegenuber den Kontrollen bei allen CMPE erhoht. Hauptergebnis ist die unterschiedliche Grose der Megakaryozyten zwischen der CML und den 3 Ph1-negativen Gruppen, so das von der kleinmegakaryozytare Ph1-positive CML und den grosmegakaryozytare Ph1-negativen CMPE gesprochen werden kann.

The value of cytogenetics performed simultaneously with histopathology was evaluated in patients with myelodysplastic syndrome (MDS). Clonal karyotype changes of the bone marrow cells supporting the histological diagnosis were found in 38/69 cases (55%). The chromosome aberrations, especially complex changes, were significantly correlated to distinct histopathological findings such as atypias of the haematopoietic cell lines and myelosclerosis. Complex karyotype changes were further associated with short survival of the MDS patients. Our results demonstrate that cytogenetic analyses are helpful in supplementing the histopathological diagnoses. Recent developments in molecular cytogenetics even allow the detection of chromosomal aberrations in non-dividing cells from cytological preparations or tissue sections which may become available for routine diagnosis.

The histologic, hematologic, and morphometric findings of 40 patients positive for the human immunodeficiency virus (HIV) were compared statistically with those of 40 patients with primary myelodysplastic syndromes (MDS) and those of 32 HIV-negative patients with infectious diseases. The severity of anemia and the abnormalities of erythropoiesis in the group of HIV patients were less pronounced than in the group with MDS; megakaryopoiesis showed similarities only with the group of patients with infectious diseases, and characteristics of dysplasia were not observed. Granulopoiesis in MDS showed an increase of blasts in several cases; this was not found in any biopsy specimen from the HIV group. In addition, a statistically significant increase of monocyte-like cells and giant bands could be observed in the bone marrow of the HIV patients. The peripheral blood findings and bone marrow picture in the series of our HIV patients appeared to be related mainly to the influence of opportunistic infections, although a direct effect of the HIV itself could not be excluded.

The distribution and the development of fibrosis were evaluated from bone marrow biopsies of patients with chronic myeloproliferative disorders (CMPD), regarding two groups of patients: (1) 564 with follow-up biopsies over a period of up to twelve years observation time, and (2) 1.787 diagnostic bone marrow biopsies from CMPD patients. Fibrosis was divided into three grades of fiber increase: early myelosclerosis, myelofibrosis, and advanced myelofibrosis. The first group of sequential BMB showed a significant progress to myelofibrosis in so-called "Chronic Megakaryocytic-Granulocytic Myelosis"--CMGM-, which corresponds to Agnogenic Myeloid Metaplasia-AMM-in 72.4% (21/29 patients), as well as in CML with megakaryocytic increase-CML.MI-in 39.2% (20/51). In the second group of diagnostic biopsies, only 30% of CMGM cases showed no fibrosis. In P. vera, 16.2% (18/111) developed myelofibrosis up to twelve years later. This figure was 4.3% (2/46) in Primary Thrombocythemia. Increase of megakaryocytes in CML indicates a high risk for developing fibrosis, combined with reduced life expectancy.

Summary This Diagnostic Seminar intends to announce that CMPDs can be classified from BMB histologically by a rather simple system, which can be applied by interested histopathologists successfully. The rationale of this classification is to stay within the groups of diseases which are outlined by clinical findings including the peripheral blood and bone marrow smears. The concept of traditional classification as given by the WHO and textbooks, however, has to be revised as follows (1) Primary diseases of CMPDs must be distinguished from advanced disorders. Primary diseases are CML, P. vera, Thrombocythemia, CMGM, and unclassifiable CMPD. (2) Idiopathic, primary myelosclerosis of the bone marrow is a reactive feature consecutive to neoplastic transformation of hematopoiesis, i.e. myeloproliferation. (3) Advanced disorders comprise (3.1.) excess of blasts and blast crisis, and (3.2.) early myelosclerosis, myelosclerosis and myelofibrosis, advanced myelofibrosis. Advanced disorders are designated by a composed term classifying them among the groups of primary disease and specifying the advanced stage by a suffix, so that the underlying disease remains coining the term, even in unclassifiable cases in which only CMPDs can be applied. (4) The CML group must be subtyped into CML of common type versus that with increase or predominance of megakaryocytes. By this system of classification, it seems possible to classify and type the spectrum of variations occurring among CMPDs to a satisfying result.

In chronic idiopathic myelofibrosis (CIMF) the factors predicting survival in patients who were already in the fibrotic stage have been well documented by numerous studies. Prefibrotic stages were only rarely evaluated so that the prognostic impact of myelofibrosis is currently not well known. Also predictive factors for disease-related events were not included in those studies. Thus, we evaluated the prognostic impact of myelofibrosis and other histopathological (megakaryocytes, blasts) and clinical [age, gender, splenomegaly, chemotherapy, hemoglobin (Hb), leukocyte, and platelet count] parameters in 122 patients in fibrotic and prefibrotic stages of CIMF on event-free survival. The statistical analysis was performed using the univariate log-rank test and the multivariate recursive partition and amalgamation (RECPAM) approach. In 62 patients disease-related events occurred during a mean observation period of 58 months. In univariate analysis they were associated with blast increase in the bone marrow. In RECPAM analysis a shorter event-free survival was found in anemic patients (mean: 9.3 months). In nonanemic patients older than 60 years, advanced myelofibrosis was associated with a shorter event-free mean survival of 23.2 months versus 69.3 months in less advanced cases. A slight or moderate myelofibrosis was not found to have a prognostic impact on event-free survival. The longest event-free survival was found in nonanemic patients who were younger than 60 years (mean: 185 months), regardless of the grade of myelofibrosis. Thus, we found that the most relevant prognostic parameter for event-free survival in CIMF were the Hb value, age, and grade of myelofibrosis.

The present study, based upon the retrospective evaluation of 352 patients with primary myelodysplastic syndrome (pMDS), revealed hypoplastic MDS in 42 patients (11.9%). Median age is similar in hypo- and normo-/hypercellular MDS (72.6 versus 70.7 versus 72.4 years). Hypoplastic MDS occurred significantly more often in women compared with normo- and hypercellular MDS. Sequential biopsies were performed in 14 patients, showing a persistence of hypoplasia over a period of up to 43 months. The proportion of patients showing mesenchymal reaction, especially an increase of mast cells, was significantly higher in hypoplastic MDS, whereas dysplastic features of hematopoiesis occurred less frequently and were of lower grade in comparison to normo-/hyperplastic MDS. Among the subgroup with hypoplastic bone marrow, the classification according to FAB criteria revealed 28 patients with RA (66.7%), three with RARS (7.1%), and eight with RAEB (19.0%), as well as one patient each with RAEB-T and CMMol (2.4% each), and one case which had to be reckoned among the category of unclassifiable MDS (2.4%). Median survival was 21.8 months for hypoplastic MDS, 26.9 months for normoplastic MDS, and 14.2 months for hyperplastic MDS. During follow-up, 14 patients (33%) with hypoplastic MDS developed acute nonlymphatic leukemia. Although not a constant finding, karyotype abnormalities involving particularly chromosome 7 seem to be associated with hypoplastic MDS. The results confirm the existence of a hypoplastic variant of MDS which seems to more frequently affect female patients, and which requires bone marrow biopsy for its accurate diagnosis.

The distribution and the development of fibrosis were evaluated from bone marrow biopsies of patients with chronic myeloproliferative disorders (CMPD), regarding two groups of patients: (1) 564 with follow-up biopsies over a period of up to twelve years observation time, and (2) 1.787 diagnostic bone marrow biopsies from CMPD patients. Fibrosis was divided into three grades of fiber increase: early myelosclerosis, myelofibrosis, and advanced myelofibrosis. The first group of sequential BMB showed a significant progress to myelofibrosis in so-called "Chronic Megakaryocytic-Granulocytic Myelosis"--CMGM-, which corresponds to Agnogenic Myeloid Metaplasia-AMM-in 72.4% (21/29 patients), as well as in CML with megakaryocytic increase-CML.MI-in 39.2% (20/51). In the second group of diagnostic biopsies, only 30% of CMGM cases showed no fibrosis. In P. vera, 16.2% (18/111) developed myelofibrosis up to twelve years later. This figure was 4.3% (2/46) in Primary Thrombocythemia. Increase of megakaryocytes in CML indicates a high risk for developing fibrosis, combined with reduced life expectancy.

Elliptic Fourier analysis was applied to megakaryocyte nuclei in bone marrow biopsies from 15 patients with chronic myelocytic leukemia with megakaryocyte predominance and from 15 patients with chronic megakaryocytic granulocytic myelosis. To assess the reliability of this procedure, the biopsies were evaluated also by the semiautomatic measurement of nuclear area and form factor, and both methods were compared with respect to the degree of morphologic differences obtained between these two types of chronic myeloproliferative disorders (CMPDs). Discriminant analysis revealed correct reclassification of all cases both for elliptic Fourier analysis and for semiautomatic planimetry, whereas discriminant scores were much higher for Fourier analysis. Thus, simple planimetric features such as nuclear area and form factor, in contrast to Fourier analysis, are not able to detect the full degree of morphologic differences between megakaryocyte nuclei in different CMPDs. Elliptic Fourier analysis therefore seems to be a useful procedure for the accurate description of such complicated structures as megakaryocyte nuclei in CMPD.

In a retrospective study of 352 patients with primary myelodysplastic syndromes, 61 (17.3%) revealed myelofibrosis in bone marrow biopsies. The fibrosis was observed to occur mostly focally (41/61 cases), and collagen deposits were found very rarely (4/61). The histopathology of bone marrow biopsies revealed hyperplasia and disturbed differentiation in megakaryopoiesis; the frequency and grade of dysplasia in megakaryopoiesis increased with advancing myelofibrosis. Reticulin fibrosis occurred in all subtypes of MDS; however, there was a higher incidence in chronic myelo-monocytic leukaemia (CMMoL). The frequency of cytogenetic aberrations was significantly higher in the MDS cases with myelofibrosis, compared to the cases without fibrosis. Clinical data showed significantly lower values of haemoglobin and lower platelet counts in MDS with myelofibrosis. Life expectancy was reduced to 9.6 months, compared with 17.4 months in MDS without fibrosis. In refractory anaemia, the survival times were 10.0 months in MDS with myelofibrosis, compared to 28.9 months in MDS without myelofibrosis. 36.6% of the patients with MDS and myelofibrosis developed a transformation into ANLL during the course of the disease. Myelofibrosis therefore seems to herald a poor prognosis.

A neuronal network, as well as uni- and multivariate statistics and a discriminant analysis were applied to a morphometric database of 58 cases with thyroid neoplasms and normal thyroid tissue. The ability to classify cases correctly according to their diagnoses was compared between the neuronal network and discriminant analysis. For all pairwise comparisons, classification by neuronal network was as least as good as classification by discriminant analysis. For some comparisons, the neuronal network provided more correct diagnoses than discriminant analysis. On the contrary, in a comparison between tumors which are not significantly different according to multivariate statistics, the network reclassifies only half of the cases correctly, whereas discriminant analysis falsely suggests the possibility of classifying cases with either diagnosis. Our results confirm a higher sensitivity of the neuronal network to the diagnostic information contained in the present morphometric database, and we will therefore use this concept for analysis and diagnostic classification in further morphometric studies.

A histological study on sequential bone marrow biopsies in patients with chronic myeloid leukaemia (CML) was performed. We wished to answer the question as to whether a different content of megakaryopoiesis in the bone marrow of CML patients has a prognostic significance for the development of myelofibrosis during the course of disease. In addition, the significance of possible changes in the quantity of megakaryopoiesis in this process was assessed. In 186 patients who had no fibre increase at first diagnosis, the rate of subsequent myelofibrosis varied from 19% for the common or granulocytic subtype (CML.CT) to 40% for patients with features of megakaryocytic increase (CML.MI). No significant differences were found either in the rapidity of progression to fibrosis or in the final rate of osteomyelosclerosis. Whereas in CML.MI most patients (75%) showed an increase of fibres only, this was accompanied by an additional increase of megakaryocytes in CML.CT, changing the histological pattern from CML.CT to .MI or .MP, respectively. The data therefore revealed a correlation between fibre increase and subtyping of CML as suggested by the Hannover classification of chronic myeloproliferative diseases. Subtypes of CML with megakaryocytic increase could be shown to present a "pre-myelofibrotic" stage of disease and may therefore be conceived as a particular pathway of acceleration.

Chromosome analyses of bone marrow and peripheral blood cells were performed in a total of 51 patients with myelodysplastic syndromes (MDS) simultaneously with histopathological examination of resin-embedded bone marrow biopsies. Diagnosis of MDS was established by histopathology according to the French-American-British (FAB) classification, and reassessed by haematological data and clinical course. Clonal karyotypic changes were found in 30 of the 51 patients (59%): in 15 of 19 (79%) patients with refractory anaemia, 7 of 11 (64%) with refractory anaemia and excess of blasts (RAEB), 6 of 10 (60%) with RAEB in transformation, and 2 of 11 (18%) with chronic myelomonocytic leukaemia. The following three features of the histopathology revealed positive correlations with karyotype abnormalities: all cases of myelofibrosis in MDS (7/51) were accompanied by chromosome aberrations, microforms of megakaryocytes with reduced nuclear lobulation were observed in 18 of 30 cases with karyotype changes, and hypocellularity of haematopoiesis was associated with aberrations of chromosome 7 in 2 of 4 cases. No positive correlations were revealed between abnormal karyotypes and the transformation to acute leukaemia. The survival times were significantly decreased in patients with complex (3 and more) karyotype changes, when compared with patients with single (1-2) chromosome aberrations or normal karyotype, independently of the FAB classification.

Planimetry of megakaryocytes (MK) was performed in bone marrow biopsies (BMBs) from patients with chronic myeloproliferative disorders (CMPD) to substantiate cytomorphologic differences in this cell lineage between the four main groups of CMPD. The biopsy specimens were classified histologically prior to morphometry, according to the Hannover Classification of CMPD. Five histological groups were investigated, evaluating between 21 and 30 biopsies in each group. The five groups were as follows: (1) Chronic myelocytic leukemia (CML) of common type (CML.CT), (2) CML with megakaryocytic increase (CML.MI), (3) polycythemia vera (P. vera), (4) primary thrombocythemia (PTH), and (5) chronic megakaryocytic-granulocytic myelosis (CMGM). The results of five variables, i.e. the cellular and nuclear size, the cellular and nuclear form factor, and nuclear segmentation, were determined in at least 50 MK per BMB. The results reveal significant differences in MK nuclear and cellular size, as well as in nuclear segmentation between CML and the three other groups in that the nuclear and cellular size of the MK in CML are smaller than in P. vera, PTH, and CMGM. Moreover, the degree of nuclear segmentation or lobulation differs significantly between the three disorders characterized by large MK. Discriminant analysis permits 78–100% reliability of reclassification by morphometry compared with the histologic classification. A reduced reliability of the morphometric classification to around 80% was found between P. vera and PTH, as well as between P. vera and CMGM. In the design of this study, morphometry of MK lends added weight to the subjective classification of these disorders.

A planimetric study of megakaryopoiesis in various chronic myeloproliferative diseases (CMPD) was performed and the results compared with those from controls and myelitis of rheumatic origin. Morphometric measurements included at least 200 megakaryocytes in each case observed in Giemsa-stained semithin sections of resin-embedded core biopsies. Twenty specimens were evaluated from the controls and inflammatory disorders and from each of the following CMPD: 1, chronic granulocytic leukaemia (CGL); 2, polycythaemia vera (P. vera); 3, chronic megakaryocytic-granulocytic myelosis without or with minimal increase in reticulin fibre content (CMGM); 4, myelofibrosis or osteomyelosclerosis (MF/OMS). Megakaryocytes were classified as follows: 1, normal megakaryocytes at all stages of maturation; 2, giant forms; 3, microforms; 4, intussusceptions; 5, a-nuclear cytoplasmic fragments; 6, naked nuclei or necrotic forms. The results of this study demonstrate obvious abnormalities of megakaryopoiesis in addition to the increase in absolute numbers of megakaryocytes per marrow area and their different sizes as reported earlier (Thiele et al. 1982). Aberrations are particularly conspicuous when pure granulocytic proliferation or neoplasia of CGL is compared with the so-called mixed cellularity of megakaryocytes and granulocytes in CMGM including MF/OMS. Abnormalities of the giant forms of megakaryocytes are especially evident and comprise irregular cellular and nuclear perimeters (as calculated by a modified shape factor) in the two latter entities (CMGM-MF/OMS). This remarkable feature is associated with a disorganization of nuclear development and/or a disproportionate nuclear-cytoplasmic ratio which has never been observed in CGL previously. In combination with this striking cellular anomaly, which is compatible with an extreme amoeboid shape of giant forms in CMGM and MF, intussuceptions and a-nuclear cytoplasmic fragments are frequently encountered. The final stage of megakaryopoiesis, i.e. naked nuclei, are increased in number in all CMPD, probably because of enhanced proliferation and platelet shedding. Naked nuclei are often small in CGL (as remnants of the frequent micromegakaryocytes) and large in P. vera and CMGM/MF (depending on the high incidence of giant megakaryocytes in these latter disorders).

Summary Polyoma virus of the Stuart Eddy strain was tritium-labelled by cultivating the virus in phosphate free Hepes buffered medium in presence of tritium labelled nucleotide triphosphates. The distribution of labelled virus in the organs and in the blood of newborn Wistar rats was examined at graded time intervals from two hours until 40 days after inoculation. The highest amounts of virus per organ was refound in the liver and the highest amount per mg organ weight in the spleen. The virus concentration in most organs exceeded by far the concentration found in the blood, indicating adsorption of virus in these organs. It was found that the distribution of tritium in the target organs for tumor appearance (kidneys and brain) did not differ basically from that in the non-target organs. As a consequence, the distribution of virus in the organs does not explain the fact that the kidneys and the brain are the predilection sites for tumor induction. Tritium labelling did not measurably influence the oncogenicity, infectivity and hemagglutinating activity of the virus.

In a total of 581 patients with the presumptive clinical diagnosis of bone marrow metastases, core biopsies of the iliac crest were performed to evaluate the frequency of infiltrates in routinely taken samples. The frequency of metastases depends greatly on the varying clinical indications to perform an examination. Staging procedures will result in a decline in positive findings whereas overt signs and symptoms of tumor spread naturally increase the rates. Thus, the annual rates have decreased from 45 to 28% during the last 5 years. The mean frequency of metastases in 501 patients with known primaries was 24%; related to particular tumors the rates were 49% in prostate, 41% in breast, 19% in thyroid and 7% in bronchogenic cancer. The patterns of bone marrow involvement displayed an overwhelming focal infiltration of the marrow space in carcinomas of the thyroid gland and bronchus, and a more diffuse extension in tumors of the prostate and breast.

The prognostic value as represented by the predictive implications of the histopathologic bioptic diagnosis in cardiac allografts was studied in a total of 3209 biopsies from 111 patients under triple-drug immunosuppressive therapy after transplantation during a period of more than 2 years (0 to 782 days). The application of the so-called Hannover classification in the histopathologic diagnosis of rejection has revealed that there are certain configurations of histopathologic changes that lead significantly more frequently to the forms of acute rejection (moderate or severe) that require therapy. These are mild acute rejection with retrogressive changes in myocytes and a late-resolving phase of acute rejection with a severe vasculopathy. In cases with the simultaneous presence of the chronic phase of rejection, a slight prolongation of the interval taken by the conversion of these two diagnoses into the therapy-requiring forms of acute rejection could be observed. On average, however, the time interval of conversion of mild acute rejection with retrogressive changes in myocytes and of the late-resolving phase of acute rejection with interstitial vascular reaction into the therapy-requiring forms ranges between 7 and 10 days. Thus a second biopsy should be performed within this time interval.

Morphometry was employed on different entities of chronic myeloproliferative diseases (CMPD) and reactive lesions in addition to normal control specimens. The entities studied included: (1) inflammatory reactions of the bone marrow (so-called myelitis in chronic rheumatoid arthritis), (2) chronic granulocytic leukemia (CGL), (3) agnogenic myeloid metaplasia in an early hypercellular stage (so-called chronic megakaryocytic-granulocytic myelosis, CMGM), (4) agnogenic myeloid metaplasia in an advanced fibrosclerotic stage or osteomyelofibrosis/sclerosis (MF/OMS), (5) polycythemia vera (P. vera), (6) reactive thrombocytosis (TH, as a sequel of miscellaneous conditions) and (7) primary (idiopathic, essential) thrombocythemia (PTH). Evaluation was done on plastic-embedded semithin sections with a constant thickness of 3 micron in approximately 20 cases of each group of CMPD. The following parameters were determined: (1) density distributions of the megakaryocyte and non-megakaryocyte compartments, (2) arrangement of megakaryopoiesis in the bone marrow space (i.e., inhomogeneity or clustering) and (3) the fine structure of megakaryocytes in PTH, with a quantitative analysis of the nuclear morphology by circular deviation and contour factors. The megakaryocyte morphology was closely related to a facultative or obligatory increase of the platelet count in these various entities of CMPD and was separable into two major categories: (1) controls, CGL and myelitis versus (2) CMGM, MF/OMS, P. vera, TH and PTH. These two categories were distinguishable by the prominence of megakaryopoiesis in the bone marrow as well as the elevated platelet counts in the periphery. Moreover, in comparison with CMGM and MF/OMS, PTH was characterized by an apparently normal maturation and a conspicuous polyploidization of megakaryocytes according to the nuclear morphology, which was similar to that of P. vera. Our results suggest that PTH presents a monolinear growth of the megakaryopoiesis in the same way as CGL exhibits a monolinear proliferation of the neutrophilic granulopoiesis. This is in contrast to the mixed cellularity of both the megakaryocyte and granulocyte lineage in CMGM and MF/OMS.

A combined morphometric and ultrastructural study was performed on so called emperipolesis or internal wandering of myeloid cells in the cytoplasm of large mature megakaryocytes. Measurements were made on material from a total of 115 patients comprising a normal control group and 5 groups with subtypes of chronic myeloproliferative diseases, including primary (essential, idiopathic) thrombocythemia (PTH). A significant increase in this peculiar phenomenon was noted in myeloproliferative disorders and especially in PTH where the frequency of emperipolesis showed a positive correlation with the number of anuclear cytoplasmic fragments sectioned, with the circular deviation of the shapes of megakaryocytes and with the extent to which the peripheral thrombocyte count was elevated. Electron microscopy in selected cases displayed the integrity of the plasma membranes of engulfed hematopoietic cells within the dilated cavities of the megakaryocytic demarcation membrane system (DMS) and no evidence of phagocytosis. Moreover there was a close relationship between engulfed myeloid cells and the presumptive sites of platelet shedding which had their openings from the cisternal lumina of the DMS. Our results demonstrate that emperipolesis of hematopoietic cells within megakaryocytes should not be regarded as a special nosological feature, but as an indication of enforced thrombocytogenetic activity which is expressed particularly in PTH.