Molecular basis and enzymatic properties of glucose 6-phosphate dehydrogenase volendam, leading to chronic nonspherocytic anemia, granulocyte dysfunction, and increased susceptibility to infections.

We have investigated the blood cells from a woman with a low degree of chronic nonspherocytic hemolytic anemia and frequent bacterial infections accompanied by icterus and anemia. The activity of glucose 6-phosphate dehydrogenase (G6PD) in her red blood cells (RBCs) was below detection level, and in her leukocytes less than 3% of normal. In cultured skin fibroblasts, G6PD activity was approximately 15% of normal, with 4- to 5-fold increased Michaelis constant (Km) for NADP and for glucose 6-phosphate. Activated neutrophils showed a decreased respiratory burst. Family studies showed normal G6PD activity in the RBCs from all family members, including both parents and the 2 daughters of the patient. Sequencing of polymerase chain reaction (PCR)-amplified genomic DNA showed a novel, heterozygous 514C-->T mutation, predicting a Pro172-->Ser replacement. Analysis of G6PD RNA from the patient's leukocytes and fibroblasts showed only transcripts with the 514C-->T mutation. This was explained by the pattern of X-chromosome inactivation, studied by means of the human androgen receptor (HUMARA) assay, which proved to be skewed in the patient, her mother, and one of the patient's daughters. Thus, the patient has inherited a de novo mutation in G6PD from her father and an X-chromosome inactivation determinant from her mother, causing exclusive expression of the mutated G6PD allele. Purified mutant protein from an Escherichia coli expression system showed strongly decreased specific activity, increased Km for NADP and for glucose 6-phosphate, and increased heat lability, which indicates that the defective phenotype is due to 2 synergistic molecular dysfunctions: decreased catalytic efficiency and protein instability.

High DNA content and prognosis in lymph node positive breast cancer. A case control study by the University of Leiden and ECOG. (Eastern Cooperative Oncology Group).

To investigate whether breast cancer cells with unusually high nuclear DNA content are associated with an adverse outcome, Eastern Cooperative Oncology Group investigators selected breast cancer trial patients who suffered an early death (ED) within two years after diagnosis to compare with other trial patients who had a survival of at least 7.5 years. Paraffin blocks of primary breast cancers were obtained from 93 evaluable patients who had been enrolled in two surgical adjuvant trials for lymph node positive (LN+) disease (T1-3N1M0). Single cell monolayer preparations from these blocks were stained with acriflavine-Feulgen and analyzed by image analysis for DNA content with the automated Leiden Television Analysis System (LEY-TAS). Standard prognostic variables (estrogen receptor (ER) status, number of lymph nodes with metastases, and size of the cancer) were compared with three DNA content characteristics: DNA ploidy status, number of nuclei with > 5C DNA content, and percent of nuclei with > 5 C. Estimates of the odds ratio in multivariate comparisons showed that ER negativity was associated with ED (p = 0.0005) and an odds ratio estimate using negative/positive of 4.87. The number of positive lymph nodes associated with ED had a p-value of 0.0005 and an odds ratio estimate of 4.63 when comparing the > 3 nodes group to the 1-3 nodes group. In contrast, the strongest association for any of the DNA content characteristics with ED had a p-value of 0.017 and an odds ratio estimate of 2.76. This power of association disappeared when stratified on ER status.(ABSTRACT TRUNCATED AT 250 WORDS)

Laboratory test of an automated cell analysis system for cervical screening.

An automated cell analysis system (Autoplan-MIAC) for the early detection of precancerous lesions of the cervix was tested under semi-routine conditions in a clinical cytology laboratory. A set of 1500 specimens, highly enriched with abnormal cases, was analysed. Cervical scrapings were collected in suspension and processed by cytocentrifugation for microscopy. Two slides were prepared from each sample: one for staining according to Papanicolaou for the visual reference diagnosis and one for Feulgen staining for automated analysis. The specimens were evaluated in two ways: the first one, which is referred to as the automated machine classification system (AMC), classifies the specimens according to the number and ratio of selected objects (alarms) and is a fully automated system. The second system classifies the specimens after visual evaluation of the stored alarms as they are displayed on a TV monitor, and is designated the interactive machine classification system (IMC). The AMC results showed a false positive rate of 16.5% when the cut-off threshold was selected so that all 117 positively diagnosed specimens were classified 'positive' by the system. In that case 87.4% of the CIN I and 96.9% of the CIN II cases were AMC-positive. The IMC results showed a false positive rate of 2.5%, when 86.3% of the CIN I cases, 96.9% of the CIN II cases and all CIN III and invasive carcinoma cases were positively classified.

The prognostic value of DNA content measured by image cytometry in soft tissue sarcomas.

Nuclear DNA content in soft tissue sarcoma was determined by image cytometry using archival, paraffin embedded material. In a retrospective study 138 specimens of 81 patients have been analysed. The ploidy level was correlated to clinical outcome regarding tumor volume and histological grading, the most important prognostic parameters. Ploidy has a significant prognostic value and correlates well with histological grading (p = 0.01). Tumour volume was found to be an independent prognostic factor (p = greater than 0.1) [chi 2 test]. The DNA content of the primary tumour and of multiple local recurrences remained similar.

Preparation and microscopic visualization of multicolor luminescent immunophosphors.

The preparation of charge-stabilized suspensions of small phosphor particles (0.1-0.3 micron) and their coupling with antibodies to immunoreactive conjugates is described. Phosphor particles consisting of yttriumoxisulfide activated with europium served as a model system in the evaluation of the stabilizing properties of several polycarboxylic acids. The optimal reagents were then applied to other phosphors which differ in spectral characteristics as well as in luminescence lifetime. These phosphors were ground to a size of 0.1-0.3 micron and proteins or other macromolecules were adsorbed to the phosphor particles to prepare conjugates of different physico-chemical properties. A time-resolved microscope, suitable for real time visualization of the time-delayed luminescence of the immunophosphors by the human eye, is described in detail. Since most phosphors require excitation with far UV light, a special fluorescence microscope allowing far UV excitation was developed for conventional visualization of the luminescence emitted by the phosphor. The possibility of multiple color labeling using various phosphor conjugates was demonstrated in a model system consisting of haptenized latex beads.

Remission of monoclonal chronic T-cell expansion associated with severe anemia.

A 49-year-old male with an 8 year history of lowered Hb level, granulocytopenia and fatigue presented in 1986 with progressive fatigue, a dramatically reduced Hb level (45 g/l) and an increased lymphocyte count (6.6 x 10(9)/l). Clinical picture and laboratory studies led to the diagnosis of chronic T lymphocytosis with expansion of CD8+ T cells expressing CD16 IgG Fc receptors (Fc gamma RIII). DNA analyzed with T-cell receptor (TcR) gamma and beta probes revealed extra rearranged bands representing a clonal expansion of T lymphocytes. These T lymphocytes expressed T-cell receptor alpha beta as evaluated by staining with monoclonal antibodies. Because of the severe progressive anemia the patient was transfused with packed red cells. He was then treated with cyclophosphamide. After one month of treatment the transfusions could be discontinued and two months later cyclophosphamide treatment was stopped because of normalized Hb level and lymphocyte counts. The patient remained in a hematologically stable condition, though a minor T-cell population representing the clonal expansion, an inverted CD4/CD8 ratio and low immunoglobulin levels persisted. This is the first report of regression of proven monoclonal CD8+ T gamma-cell expansion and the associated anemia following cyclophosphamide therapy. These observations implicate the expanded monoclonal CD8+ lymphocytes in the pathogenesis of the anemia and granulocytopenia.

Symptomatic cytomegalovirus (CMV) infections identified by image cytometry and other parameters for CMV infection.

Thirty-eight renal transplant recipients were followed during the first 3 months after transplantation. Once weekly, cultures of urine and buffy coat for cytomegalovirus (CMV) were taken and an immunocytochemical assay for immediate early antigens of CMV (IEA assay) was performed. Thirty patients had evidence of a CMV infection and 11 had a symptomatic CMV infection. All symptomatic patients had one or more positive urine cultures or a positive IEA assay. However, 15 patients with positive urine cultures and 12 patients with a positive IEA assay lacked any signs of symptomatic CMV disease. Moreover, 6 out of 15 patients with positive buffy coat cultures for CMV did not have symptomatic CMV disease. Using a computerized system to quantify IEA-positive granulocytes, we show that the absolute number of positive cells per million correlates very well with the occurrence of symptomatic CMV disease.

The present state of the automated micronucleus test for lymphocytes.

This minireview presents the state of the art with respect to automated detection of micronuclei (MN) in binucleated lymphocytes. Emphasis is on an image analysis technique, based on the principles of mathematical morphology (pattern recognition), which combines a personal computer with an image processing board and a board for microscope control. The basic idea behind this procedure is that nuclei plus MN and cytoplasms are analysed separately and sequentially by capturing images from gallocyanin-stained nuclei plus MN and naphthol yellow-S stained cytoplasms from one microscope field by using different filters. Major steps in the identification of nuclei and MN are separation of nuclei and MN from background by determination of periphery of the nuclei and MN, and artefact rejection procedures. After changing the filter, a binary image is constructed from cytoplasms and artefacts. Finally, stored information from selected binucleated objects with/without MN is combined with the cytoplasm image to check whether selected objects belong to the same cytoplasm. The procedure described above allows automated detection of binucleated lymphocytes with or without MN. The current capacity to detect 63% of binucleated cells and 57% of the MN within them is quite acceptable. To avoid false positives, artefact rejection procedures need to be improved before the method can be used routinely.

Trisomy 13 and myelodysplastic syndrome.

The clinical and cytogenetic data of a patient with myelodysplastic syndrome-refractory anemia with excess blasts (MDS-RAEB) and trisomy 13 as the sole abnormality are presented. This appears to be only the second report of such a patient. The presence of trisomy 13 is confirmed by in situ hybridization using an alphoid repeat probe L1.26, which is specific for the centromeres of both chromosomes 13 and 21.

Interobserver variability in the cytological diagnosis of 1500 Papanicolaou stained cervical monolayer specimens.

Cervical specimens from 1500 patients were prepared by means of a centrifugation procedure to obtain monolayer specimens suitable for automated screening using a machine. After staining according to Papanicolaou, each specimen was diagnosed by four independent cytologists from two different institutes. Within each institute, noncorresponding screening results were discussed to arrive at a conclusion diagnosis. After discussion of the discrepancies between the two centers, the conclusion diagnoses were combined to one final cytological diagnosis for each specimen. This final diagnosis is to be used as a reference diagnosis to evaluate machine classification as obtained by the AUTOPLAN/MIAC system. This system is presently being tested both in Leiden and in Frankfurt for its accuracy of detecting abnormal lesions in cervical specimens. The used diagnostic procedure resulted in a negative reference diagnosis for 1217 of the 1500 specimens; 170 specimens were diagnosed CIN I or II (mild or moderate dysplasia) and 113 specimens had a positive reference diagnosis (CIN III or invasive carcinoma). Based on these three diagnostic classes, the agreement between the four independent cytologists and the reference diagnosis varied between 93.60% and 96.60%, whereas 95.33% of all 6000 diagnoses correlated with the reference diagnosis.

Multiple fluorescence in situ hybridization.

A method for multiple fluorescence in situ hybridization is described allowing the simultaneous detection of more than three target sequences with only three fluorescent dyes (FITC, TRITC, AMCA), respectively emitting in the green, red, and blue. This procedure is based on the labeling of (DNA) probes with more than one hapten and visualisation in multiple colors. The possibility to detect multiple targets simultaneously is important for prenatal diagnosis and the detection of numerical and/or structural chromosome aberrations in tumor diagnosis. It may form the basis for an in situ hybridization based chromosome banding technique.

Hb J-Anatolia [alpha 61(E10)Lys----Thr]: structural characterization and gene localization of a new alpha chain variant.

We report the characterization of a new hemoglobin variant having a single amino acid substitution (Lys----Thr) at position 61 of the alpha chain. In addition to the structural analysis, we also describe the strategy used for the identification of the base substitution and the localization of the defect at the gene level using polymerase chain reaction and hybridization with allele-specific oligonucleotides.

Automated image cytometry in cytopathology.

Image cytometry is used more and more for the study of clinical cytology, notably for the determination of morphometrical and densitometrical values, the quantification of monoclonal antibody labelling and the detection of DNA probes after in situ hybridisation. Aspects of automated and interactive image cytometry are discussed, including a brief evaluation of limitations and advantages of the image technique in connection to flow cytometry. Some new technologies such as a sampling technique for paraffin embedded tissue and a new automated microscope, which are of special interest to the pathologist, are described in more detail. Applications in image cytometry include diagnostic and prognostic studies. Examples of diagnostic studies are the automated screening for cervical cancer and the detection of rare remaining cancer cells (minimal residual disease) in the peripheral blood. The use of archival material in image cytometry allows interesting retrospective studies with regard to the relation of the course of the disease with the ploidy characteristics of the tumor.

Automated cell analysis for DNA studies of large cell populations using the LEYTAS image cytometry system.

Image cytometry by means of LEYTAS features analysis of both fresh and archival cellular material. Although not as accurate in ploidy determination as flow cytometry, LEYTAS cytometry incorporates extensive artefact rejection algorithms, thereby allowing detection of low frequency cells. This feature is very useful for the search of rare cells, as e.g. in cervical screening, or for the quantitation of the number of high DNA content cells in the total cell sample. LEYTAS main components are an automated microscope (Autoplan) and a Modular Image Analysis Computer (MIAC), both from Wild Leitz (W-Germany). This paper discusses LEYTAS instrumentation and cell analysis by means of programs especially written for LEYTAS.

Discrepancies in ploidy determination due to specimen sampling errors.

Two techniques are described to enhance the detection of low frequency aneuploid cells in automated cell analysis. One method concerns a cell preparation technique; the other is focused on specific cell selection at the measurement level. The cell preparation method has been designed to select and process the tumour areas in paraffin blocks and can be used for image as well as for flow cytometry. The technique uses incident fluorescence microscopy for visual inspection of the surface of the fluorescently stained tissue block to select the specific tumour parts. Using image cytometry, it is shown that in tissue sections with very small tumour foci and many normal cells, aneuploidy could only be detected after enrichment of the cell sample with the specifically selected areas. The cell selection at the measurement level is directed towards detection of low frequency aneuploid cells on microscope slides using the specific capacities of LEYTAS (Leyden Television Analysis System). With this system, cells of interest can be selected by means of minimum size and intensity thresholds. In addition to measurement of the total cell population, all cells above a minimum DNA value can thus be specifically selected and measured. The advantage of both enrichment techniques is the possibility to detect and measure aneuploid cell lines in cases where normal, diploid cells dominate the paraffin tissue.

Improved detection and quantification of the (immuno) peroxidase product using reflection contrast microscopy.

Reflection contrast microscopy (RCM) is a sensitive tool to detect minor amounts of precipitated diaminobenzidine (DABox) in immunoperoxidase stained specimens. One of the main issues in immunocytochemistry is the ongoing need for more sensitive and quantitative techniques. Therefore we applied RCM, using a new simple model system, to methods previously described for increased sensitivity in immunocytochemistry with bright field microscopy. Addition of imidazole was found the most sensitive method and addition of Nickel and Cobalt ions gave the most enhanced colour intensity. Variation of the enzyme reaction parameters yielded a continuous increase in reflection with time. This was then discussed in view of other model studies of peroxidase kinetics. A quantitative relationship between the amount of peroxidase and the reflection of DABox was observed, indicating that quantitative immunoperoxidase studies with RCM are feasible. In situ hybridization (ISH) was then used as a useful biological model for RCM to test the optimal conditions for DAB staining found in the model system (high concentrations of DAB and peroxidase and 2 h incubation time). There was no background staining in the model system, also after prolonged incubation time. The ISH experiments showed that the contrast (ratio) between specific signal and chromosome background did not increase in time, whereas only the use of high avPO concentrations yielded the highest contrast.

Leptomeningeal plasmacytosis. Case report and considerations on treatment.

We describe a 68-year-old patient with a plasma cell leukaemia in haematological remission presenting with massive intracranial leptomeningeal plasmocytic infiltration (LPI) and hydrocephalus. He was treated with skull irradiation and a combination of intraventricular and lumbar intrathecal therapy with methotrexate. Neurologic improvement and clearance of plasma cells from the cerebrospinal fluid was reached after 2 weeks of treatment but prolonged follow-up was interrupted by a lethal gastro-intestinal haemorrhage, 6 weeks after starting the therapy. From previously reported cases it is known that LPI almost always occurs in either high-grade plasmocytomas or plasma cell leukaemia. These data suggest that therapy of LPI should be the same as in other leukaemias with leptomeningeal infiltration.