Search

The microscopic examination of stool for neutrophils can be used as a screen to determine whether bacterial pathogens are present. We reviewed a 1-year experience in a pediatric hospital laboratory to find out how often fecal neutrophil determinations were used, how useful these determinations were, and whether there were potential confounding factors. We found that only 654 (47%) of 1,398 stool cultures were evaluated for fecal neutrophils. If the presence of any number of neutrophils was considered positive, then the sensitivity was 69% and specificity 82%; if greater than five neutrophils per oil immersion field represented a positive determination, sensitivity was 48% and specificity 95%. Sensitivity and specificity differed for each bacterial pathogen species. The test's usefulness therefore varied with definition of a positive test and with the mix of organisms encountered.

In accord with mandates of the College of American Pathologists and Joint Commission of Accreditation of Healthcare Organizations, we have developed a structured function that documents the quality assurance program of the Pathology Department. It is simple and easy to perform, promotes exposure of the department, serves as a database in patient care and laboratory administration, and has been well received by the clinical staff. The program is offered for consideration by other laboratories.

SUMMARY Susceptibility to chemically induced transformation changed as a rat embryo cell culture was passaged. For the first 35 to 60 passages, the cultures were diploid and resistant to transformation by chemical carcinogens. However, cultures infected with a murine leukemia virus were transformed by chemicals. For the next 60 passages, the cultures were heteroploid, but retained contact inhibition and were not tumorigenic. Even without addition of heterotypic viruses, these heteroploid cultures could be transformed by chemicals, but the endogenous rat C-type virus could be demonstrated in the transformed cultures. At higher passages, the rates of spontaneous transformation gradually increased so that the cultures could not be used for transformation studies. Chemically induced transformation of the stable heteroploid cell line (F1706) was manifested by an easy to read focal alteration. Initial observations based on these foci were confirmed by inoculating the morphologically altered cells into isogeneic newborn rats.

Fresh ex vivo vultures of normal human peripheral blood monocytes, which are nonreplicative and known to possess cytochrome P-450 associated mixed- function oxidase activity, were used to assay DNA-excision repair manifested as augmented [3H]thymidine (dThd) incorporation following treatment in culture with diverse mutagenic carcinogens. Untreated monocyte cultures established from pools of 3–6 normal donors incorporated a low level of cytoplasmic [3H]dThd throughout a majority of the cells during an 18-h incubation. This background incorporation into whole cells was 80–90% inhibited by hydroxyurea (HU) at concentrations greater than 5 mM. Dose-related increases in the cumulative 18-h [3H]dThd incorporation in monocytes were observed following treatment with UV, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), mitomycin C (MMC), N-acetoxy-acetylaminofluorene (NA-AAF), aflatoxin B1 (AFB1), benzo[a]pyrene (BaP), and dimethylnitrosamine (DMN). The presence of HU during chemical treatment and throughout this 18 h of incubation with [3H]dThd did not influence the dose—response curves obtained with UV, MMS, NA-AAF and BaP but it increased the input dose of MNNG, MMC, DMN and AFB1 required to give peak repair incorporation. When HU was added to cultures following MNNG damage no interference with repair response was observed. HU apparently influences the extent of DNA damage by direct reactivity with these chemicals or their endogenously generated metabolites rather than inhibiting DNA-repair processes. These results provide evidence that monocytes are enzymatically proficient in base and nucleotide excision pathways and have endogenous capacity to metabolize BaP, AFB1 and DMN to DNA-damaging metabolites. As such, the monocyte is a potentially useful human cell type for detecting genotoxic chemicals and studying individuality in chemical-bioloigical interactions.

The value of histologic evaluation in the analysis of material from first trimester abortions is not completely defined. We prospectively analyzed placenta and decidua from 75 first trimester, spontaneous abortions to ascertain if morphologic features were predictive of karyotype. The histologic features analyzed included hydropic villus change, villus fibrosis, villus scalloping with trophoblastic invaginations, atypical stromal cells, aggregates of lymphocytes in placenta or decidua, and acute inflammation of placenta or decidua. Normal karyotypes were observed in 44 cases and abnormal karyotypes were demonstrated in 31. The presence of villus scalloping with trophoblastic invagination was significantly associated with abnormal karyotypes, particularly triploidy, and the demonstration of acute inflammation was seen significantly more often in cases with normal karyotypes. We conclude that histology can provide only a suggestion as to the likelihood of an abnormal karyotype; the findings are not specific enough to obviate the need for karyotyping in the individual case.Placenta and decidua from 75 1st-trimester spontaneous abortions were prospectively analyzed to determine whether morphologic features were predictive of karyotype. The histologic features analyzed included hydropic villus change, villus fibrosis, villus scalloping with trophoblastic invaginations, atypical stromal cells, aggregates of lymphocytes in placenta or decidua, and acute inflammation of placenta or decidua. Of the 103 cases submitted during the 12-month study period, 75 had successful karyotypes and sufficient histologic material for analysis. Normal karyotypes were found in 44 cases (26 females and 18 males). The 31 cases with abnormal karyotypes included 10 cases of triploidy, 9 cases of trisomy, 6 cases of monosomy, 3 cases of tetraploidy, and 3 cases of unbalanced translocation. The presence of villus scalloping with trophoblastic invaginations was seen significantly more frequently in cases with abnormal karyotypes (p0.05). The positive predictive value of this finding was 59% and the negative predictive value was 75%. Analysis of specific karyotype abnormalities demonstrated that triploid cases contributed the majority of cases with these villus changes. Acute inflammation of the placenta and decidua was significantly associated with a normal karyotype (p0.01). A low frequency of acute inflammation was observed in all the specific karyotype abnormalities. The other histologic features analyzed were found with approximately equal frequency in placentas with normal and abnormal karyotypes. These findings indicate that histology can provide only a suggestion as to the likelihood of an abnormal karyotype; the results are not specific enough to obviate the need for karyotyping in the individual case.

In view of its relatively low virulence for humans Cryptococcus neoformans is considered an opportunistic fungus. Because of its widespread distribution in nature (Emmons, 1951; Muchmore et al, 1963) and the frequent association of cryptococcosis with chronic debilitation (Littman and Zimmerman, 1956), it has been postulated that the virulence of C. neoformans is related chiefly to variations in host resistance. This paper is concerned with studies of the nature of humoral growth-promoting and inhibiting substances affecting this organism and with their significance in relation to natural resistance and tissue predilection in cryptococcosis. METHODS The organism employed in these experiments was isolated at necropsy from a fatal case of human cryptococcosis. Sabouraud's broth (1*^ neopeptone, 2070 glucose) was used as nutrient medium and either Sorensen's phosphate buffer (0.006 M, pH 7.4) or 0.85^o sodium chloride as non-nutrient medium.

Latex particle agglutination for Streptococcus pneumoniae was evaluated in 76 patients. Fifteen of these patients had invasive disease due to S. pneumoniae including 12 with meningitis, 2 with occult bacteremia and 1 with suppurative arthritis. Five of the patients with meningitis also had bacteremia. Pneumococcal antigen was detected in the cerebrospinal fluid of 9 of the 12 patients with meningitis (sensitivity 75%). However, antigen was detected in the serum of only two of the six patients with bacteremia (sensitivity 33%) and was detected in the urine of none of five patients with bacteremia (sensitivity 0%). Consequently latex particle agglutination appears to be useful when cerebrospinal fluid is examined in patients with pneumococcal meningitis but does not appear to be sufficiently sensitive to warrant its use with serum or urine in patients with invasive disease due to S. pneumoniae. The specificity of the system used here appeared satisfactory, since pneumococcal antigen was not detected in any of the body fluids from the 61 patients without evidence of invasive pneumococcal disease (specificity 100%).

CELLS derived from several animal species, including mouse1,2, hamster3 and rat4, have been transformed in vitro from the normal to the malignant state by diverse chemical carcinogens. In similar conditions, however, attempts to transform human cells have usually been unsuccessful and as far as we know, such transformation has been reported only once5. This was a case of two cell lines treated with urethane, obtained from siblings with von Recklinghausen's disease, a familial disorder transmitted by an autosomal dominant gene, and characterised by multiple fibromas with a high predisposition to malignant transformation in vivo6. Although morphologically altered foci of transformed cells were reported, there was the possibility that a few tumour cells in the original population had been selected for by urethane. Therefore we characterised in detail the urethane-treated and untreated cultures. We have found that human cells can indeed be chemically transformed in vitro.

Cultured epithelial cells from human skin generally had 3- to 30-fold more hydrocarbon-metabolizing activity than fibroblasts from skin of the same donor. This activity was constant for up to 55 days in primary culture but was lost rapidly upon physical subdivision of the cultures. Treatment of primary mixed fibroblasts and epithelial cell cultures with methylcholanthrene, but not phenanthrene, led to development of actively growing fibroblastic cultures with many heteroploid cells. Unique marker chromosomes, stable over a number of cell population doublings, were identified in several of the heteroploid cell strains. Pure cultures of fibroblasts from the same donors did not undergo heteroploid conversion in response to methylcholanthrene. Spontaneously occurring heteroploidy in logarithmic phase human fibroblasts is a rare event; thus, heteroploid conversion may be a useful marker for chemical transformation of human cells. Because methylcholanthrene seems to have little transforming effect on human skin fibroblasts, human skin epithelial cells, because of their hydrocarbon-metabolizing activity, may serve to convert methylcholanthrene from a distal to an ultimate carcinogenic form.

In cases of extensive skin burns, there may not be sufficient viable skin to use as autografts. As a solution to this problem, we proposed that skin could be propagated in vitro, and the millions of cells thus grown could be used as autografts. The feasibility of this approach has been studied with rabbit skin. Under tissue culture conditions, small pieces of rabbit skin attach to supporting surfaces and proliferate to form layers of epithelial cells. A satisfactory supporting surface is the dermis of frozen porcine skin. With proper conditions, rabbit epithelial cell surface area can be expanded by a factor of 50 within 7 to 21 days.

In 129 of 140 attempts, human skin cells were successfully cultured on the dermal collagen bed of sterile, dead pigskin. Diploid epithelial cells grew selectively on the collagen bed; fibroblasts grew on the glass surfaces of the culture dishes. The cultures could be subdivided physically up to six times at a 1:2 split ratio, but at least 24 to 48 cell generations were produced over the months the cells could be carried. Much of the cell multiplication resulted in maturation into distinct basal, squamous, granular, and keratinized cell layers. The cultured cells were considered epithelial because of their shape, possession of intercellular bridges, desmosomes and tonofibrils, and because they formed maturating epithelium in vitro and upon transplantation back to the original human donor. As the cells grew they digested the pigskin collagen, thus producing clear zones that could be used to monitor and quantitate cell growth. Multiplication of epilthelial cells, rather than migration, was indicated by mitotic figures in colchicine-treated cultures and by DNA synthesis.

Seventy-five diploid human cell strains were subjected to a number of chemical carcinogens, including urethane and polycyclic hydrocarbons. In most cases, no visible morphological alterations were induced by any treatment. Development of morphologically altered foci was noticed in urethane-treated cultures derived from a patient with von Recklinghausen's disease. This disease is transmitted by an autosomal dominant gene, and has a high rate of spontaneous transformation of neurofibromas to neurofibrosarcomas. Attempts to isolate continuous cell lines from altered foci were successful in only two of several attempts. These continuous cell lines demonstrate altered morphology, loss of contact inhibition, accelerated growth rate, and have attained over 240 generations in a period of 140 weeks. Untreated control cultures became terminal by the 20th generation. Giemsa banding procedures showed that the chromosomal complement consisted of heteroploid human chromosomes. A second diploid cell strain derived from the above patient's sibling, also suffering from von Recklinghausen's disease, likewise was morphologically altered by urethane. Chemical transformation of human cells is difficult to induce; however, selection of genetically predisposed cells and prolonged, intermittent, and repeated chemical treatment may be important factors in achieving transformation.

The test paper here described makes it relatively easy to choose the antibiotic best suited for treatment of a given bacterial infection. Each type of test paper bears both a culture medium and an antibiotic and is supplied, dry and sterile, in a transparent envelope. For a given test, the paper is wetted and inoculated and the envelope with its contents is incubated. It is not necessary to wait for the appearance of visible colonies because the metabolic activity of the bacteria is detected by an indicator (triphenyltetrazolium chloride) which is colorless until reduced to a bright red dye by bacterial enzymes. The reliability of the new test was established by a comparative study with other methods in 100 clinical cases. The test paper is easily stored, and the entire unit can be incinerated after use. It is therefore useful in general office practice and should help to make sure that each patient receives the most effective antibiotic.

The induction of lymphomas in C57BL mice by methylcholanthrene, urethan, or diethylnitrosamine was accompanied by the development of murine leukemia viral antigen in most of the lymphoid tumors. The cell-free transmission of lymphomas induced by methylcholanthrene and the development of antibody to murine leukemia virus prior to the detection of overt lymphoma in these mice suggest that unmasking of a latent leukemia virus is an indigenous actuating cause of the lymphomas.

The coverage of large surface area skin wounds with autograft skin is limited by the amount of available viable donor-site skin. A small amount of donor-site epithelium can be expanded many times over by cultivation in tissue culture on sheets of skin allografts or xenografts. Experiments performed in rabbits indicate that such autograft-allografts or autograft-xenografts can be transplanted to large surface area wounds with essentially complete epithelial coverage of the wounds within two to three weeks, even with wound surface area coverage 50 times the surface area of donor-site skin.