Abnormally banded chromosomal regions in doxorubicin-resistant B16-BL6 murine melanoma cells.

B16-BL6 murine melanoma cells were selected for cytogenetic evaluation during the stepwise development of increasing resistance in vitro to the antitumor antibiotic, doxorubicin (DOX). Karyotypic studies demonstrated extensive heteroploidy with both numerical and structural abnormalities which were not present in the parental DOX-sensitive B16-BL6 cells. Trypsin-Giemsa banding revealed the presence of several marker chromosomes containing abnormally banding regions (ABRs) in the 44-fold B16-BL6 DOX-resistant subline. These ABRs appeared to be more homogeneously staining at the higher DOX concentrations. Length measurements (ABR index) in seven banded metaphases indicated a direct correlation with increasing DOX concentration. When the DOX-resistant cells were grown in drug-free medium for 1 yr, the drug-resistant phenotype gradually declined in parallel with the level of resistance and the ABR index. DOX-induced cytogenetic damage examined by sister chromatid exchange methodology in parental B16-BL6 cells indicated a linear sister chromatid exchange:DOX dose-response relationship. However, after continuous treatment of parental B16-BL6 cells with DOX (0.01 microgram/ml) for 30 days, sister chromatid exchange scores were found to return to base-line values. The B16-BL6 resistant cells demonstrated a cross-resistant phenotype with N-trifluoroacetyladriamycin-14-valerate, actinomycin D, and the Vinca alkaloids but not with 1-beta-D-arabinofuranosylcytosine. The results suggest that ABR-containing chromosomes in DOX-resistant sublines may represent cytogenetic alterations of specific amplified genes involved in the expression of DOX resistance. Further studies are required to identify and define the possible gene products and to correlate their relationship to the cytotoxic action of doxorubicin.

Cytogenetic alterations associated with the acquisition of doxorubicin resistance: possible significance of chromosome 7 alterations.

Cytogenetic abnormalities associated with the acquisition of doxorubicin (DOX) resistance (DOXR) were examined in two cell lines (HT1080 fibrosarcoma and LoVo colon adenocarcinoma) which were selected in the presence of increasing concentrations of DOX over a 2-year period. Karyotypes of both tumor lines were initially near-diploid although they differed significantly in their intrinsic sensitivities to DOX (DOX 50% inhibiting concentrations: LoVo, 0.10 micrograms/ml; HT1080, 0.006 microgram/ml). Chromosome banding analysis of DOXR sublines of the LoVo and HT1080 cell lines demonstrated a strikingly different response to DOX selection with regard to both numeric and structural chromosome alterations. DOXR LoVo cells maintained the parental modal chromosomal number of 49 despite a 285-fold increase in the level of resistance, with minimal structural chromosome changes observed. In contrast, the development of DOXR in HT1080 cells was accompanied by marked aneuploidy, including a significant increase in the complexity of the tumor karyotype with increasing levels of DOXR. Cytogenetic evidence suggestive of gene amplification (double minutes and homogeneously staining regions) was also observed in the DOXR HT1080 cell line. Examination of chromosome alterations common to both resistant lines revealed alterations of chromosomes 1, 5, 7, and 11, with chromosome 7q the most frequent site of chromosome change. Reversion of DOXR in both the HT1080 and LoVo cell lines (by continuous in vitro passage once off drug) resulted in an accompanying loss in structurally altered No. 7 chromosomes. Our data suggest that alterations of chromosome 7 are a common and perhaps significant feature of DOXR tumor cells.

Pharmacological and biological evidence for differing mechanisms of doxorubicin resistance in two human tumor cell lines.

The cellular pharmacology of doxorubicin resistance (DOXR) has most commonly been associated with decreased drug uptake, enhanced drug efflux, cross-resistance to multiple anticancer agents, and the overproduction of a Mr 170,000 cell surface glycoprotein (termed P-glycoprotein). In this study, the pharmacological and genetic characteristics of two newly derived human DOXR sublines were examined. These DOXR sublines were established following continuously increasing DOX exposure until a 222-fold resistant fibrosarcoma subline (HT1080/DR4) and a 285-fold resistant colon adenocarcinoma subline (LoVo/DR5) were developed. However, three major lines of evidence suggest that despite the similar selection strategy, the mechanism of DOXR differs significantly between these two cell lines. First, Western blotting using the C219 antibody specific to P-glycoprotein revealed the overexpression of the Mr 170,000 cell surface glycoprotein in LoVo DOXR cells but not in HT1080 DOXR cells. Second, LoVo DOXR cells are cross-resistant to vincristine, actinomycin D, colchicine, etoposide, and gramicidin D, but not to 1-beta-D-arabinofuranosylcytosine. In contrast, HT1080 DOXR cells display cross-resistance to vincristine, actinomycin D, vinblastine, and etoposide; however, they are not cross-resistant to gramicidin D, and show an increased (approximately 18-fold) cross-resistance to 1-beta-D-arabinofuranosylcytosine. Third, intracellular DOX accumulation (as measured by [14C]DOX at 1-h and high-performance liquid chromatography analysis) was decreased approximately 2.7-fold in LoVo DOXR cells and approximately 2.0-fold in HT1080/DR4 cells. However, while net accumulation studies in the presence of 5 micrograms/ml verapamil reversed DOXR to parental values in LoVo colon adenocarcinoma cells, it only minimally decreased DOX resistance (12.6%) in HT1080/DR4 cells. Efflux patterns of [14C]DOX were similar for the DOXR sublines with an approximately 50% decrease in DOX retention after 1 h when compared to their respective parental cell lines. Our results suggest that DOXR in LoVo/DR5 cells may result from overexpression of P-glycoprotein. In contrast, DOXR in HT1080/DR4 appears to be non-P-glycoprotein mediated and may be related to an alternative mechanism capable of altering drug efflux or differential drug binding.

A multicenter investigation with interphase fluorescence in situ hybridization using X- and Y-chromosome probes.

Twenty-six laboratories used X and Y chromosome probes and the same procedures to process and examine 15,600 metaphases and 49,400 interphases from Phaseolus vulgaris-leucoagglutinin (PHA)-stimulated lymphocytes. In Part I, each laboratory scored 50 metaphases and 200 interphases from a normal male and a normal female from its own practice. In Part II, each laboratory scored 50 metaphases and 200 interphases on slides prepared by a central laboratory from a normal male and a normal female and three mixtures of cells from the male and female. In Part III, each laboratory scored 50 metaphases (in samples of 5, 10, 15, and 20) and 100 interphases (in samples of 5, 10, 15, 20, and 50) on new, coded slides of the same specimens used in Part II. Metaphases from male specimens were scored as 98-99% XY with no XX cells, and 97-98% of interphases were scored as XY with 0.04% XX cells. Metaphases from female specimens were scored as 96-97% XX with 0.03% XY cells, and 94-96% of interphases were scored as XX with 0.05% XY cells. Considering the data as a model for any probe used with fluorescence in situ hybridization (FISH), a statistical approach assessing the impact of analytical sensitivity on the numbers of observations required to assay for potential mosaicisms and chimerisms is discussed. The workload associated with processing slides and scoring 50 metaphases and 200 interphases using FISH averaged 27.1 and 28.6 minutes, respectively. This study indicates that multiple laboratories can test/develop guidelines for the rapid, efficacious, and cost-effective integration of FISH into clinical service.

A quality-control system for the general urinalysis laboratory.

Sixteen quality-control products were prepared for general urinalysis testing. Empirically derived formulations simulating the variety found in clinical material monitored the routine analyses for pH, specific gravity, protein, glucose, ketone, and hemoglobin. Formalin-fixed erythrocytes, leukocytes, casts, crystals, and other particulates were added for qualitative identification of the formed sediment. Analytic results outside of the acceptable range were often due to errors in technic or subjective interpretation; re-education of individual personnel was followed by a decrease in the error rate over the first five months of the program.

Alternate chromogens as substitutes for benzidine for myeloperoxidase cytochemistry.

Myeloperoxidase staining methods for classification of leukemias have traditionally employed benzidine dihydrochloride as the chromogen. Recent Occupational Safety and Health Administration regulations have classified benzidine as a carcinogen, which severely restricts its use in the clinical laboratory. Twenty-two specimens from normal control subjects and leukemia patients, previously classified by FAB criteria, were stained with benzidine and two alternate chromogens, diaminobenzidine and Hanker-Yates reagent (p-phenylenediamine and pyrocatechol). In a random, blind fashion, three experienced observers scored the stains from each case according to quality of smear, degree of peroxidase positivity, tinctorial distinction between nucleus and cytoplasm, and overall acceptability of the stain. All three observers rated the substitute chromogens as adequate for routine myeloperoxidase cytochemical staining. It is concluded that either of the methods studied would have clinical utility and can substitute for benzidine as a myeloperoxidase chromogen.

Expression of blood group antigen A by stage I non-small cell lung carcinomas.

To clarify the significance of blood group antigen A (BAA) expression by neoplastic cells, we studied patients who had curative resections of stage I non-small cell lung carcinomas. Immunohistochemical staining using monoclonal antibodies was used to detect BAA expression by paraffin-embedded carcinoma cells. One hundred three patients were studied; mean age was 62.6 years, and 70 (68%) were male. Histologic types were as follows: adenocarcinoma, 52 (50.5%); squamous cell, 25 (24.3%); large cell, 24 (23.3%); and adenosquamous, 2 (1.9%). Histologic grades were as follows: I, 13 (12.6%); II, 26 (25.3%); and III, 64 (62.1%). All patients had American Joint Committee on Cancer stage I tumors: 65 patients (63.1%) had T1 tumors, and 38 (36.9%) had T2 tumors. Recurrences developed in 25 (24.3%) and metachronous malignancies in 4 (3.9%). Survival was 75% +/- 4.8% at 3 years and 66.6% +/- 7.5% at 5 years. Eighty-nine patients (86.4%) were blood group A and 14 (13.6%) were AB. Ninety-five (92.2%) were secretors of BAA and 8 (7.8%) were not. The expression of BAA by neoplastic cells was not detectable in 34 (33%), trace (1% to 5% of neoplastic cells) in 10 (9.7%), 1+ (6% to 25%) in 8 (7.8%), 2+ (26% to 50%) in 12 (11.7%), 3+ (51% to 75%) in 12 (11.7%), and 4+ (76% to 100%) in 27 (26.2%). The pattern of neoplastic cell staining was homogeneous in 14 patients (20.3%) and heterogeneous in 55 (79.7%). Carcinoma recurrence, overall survival, and event-free survival were not related to secretor status, BAA expression, or pattern of staining.(ABSTRACT TRUNCATED AT 250 WORDS)

A multicenter investigation with D-FISH BCR/ABL1 probes.

Twenty-eight laboratories evaluated a new fluorescence in situ hybridization (FISH) strategy for chronic myeloid leukemia. In a three-part study, bcr/abl1 D-FISH probes were used to study bone marrow specimens. First, laboratories familiarized themselves with the strategy by applying it to known normal and abnormal specimens. Then, collectively the laboratories studied 20 normal and 20 abnormal specimens blindly and measured workload. Finally, each laboratory and two experts studied six serial dilutions with 98-0% abnormal nuclei. Using the reported normal cutoff of < 1% abnormal nuclei, participants reported no false-negative cases and 15 false-positive cases (1-6.6% abnormal nuclei). Results provided by participants for serial dilutions approximated the expected percentages of abnormal nuclei, but those from the experts exhibited greater precision. The clinical sensitivity, precision, nomenclature, workload, recommendations for training, and quality assurance in methods using D-FISH in clinical practice are discussed.

Managing hazardous waste in the clinical laboratory.

Clinical laboratories generate wastes that present chemical and biologic hazards. Ignitable, corrosive, reactive, toxic, and infectious potentials must be contained and minimized. A summary of these problems and an overview of the applicable regulations are presented. A checklist of activities to facilitate the annual review of the hazardous waste program is provided.

Documentation of safe work practices in the clinical laboratory.

The safety manual supplemented by explicit safety statements in the laboratory procedure manual should specify the hazards and containment methods for personnel. Control of fire hazards, biohazards, and chemical hazards are specifically addressed along with waste management and radionuclide consideration. Suggestions for incorporation of safety training into the laboratory's continuing education program are provided.

Safety in the medical laboratory.

The clinical laboratory has achieved an enviable record for safety even though the opportunities for accident and misfortune are ever present. This article provides an overview of what constitutes an adequate safety program for a laboratory, as well as a list of sources from which more detailed information can be obtained.

Review of proficiency testing performance of laboratories accredited by the College of American Pathologists.

The Commission on Laboratory Accreditation of the College of American Pathologists (CAP), Skokie, III, monitors the proficiency testing of accredited laboratories by regular review of the Cumulative Survey Management Report. Analytes are targeted for review on the basis of repeated "unacceptable" results in the CAP Survey Program. Approximately 1% of proficiency testing results are repeatedly graded unfavorably. Directors of 271 Great Lakes Region laboratories reported the causes of and the corrective actions taken for 583 targeted results over a two-year period. Targetting of results in 31% was attributed to methodologic or instrumentation problems, 19% to technical performance factors, and 27% to clerical errors. Only 3% were ascribed to the Survey materials or to the criteria used for grading, and 20% of the problems remained unexplained after investigation. There was improvement in 496 (88%) of 583 targeted. During the study period, results from 96% of laboratories showed improved performance for all or a majority of the targeted analytes.

An optimized strategy for choosing the number of platelet concentrates to pool.

CONTEXT: The number of platelet concentrates (PCs) pooled per dose varies widely. The number should be based on quality control statistics. OBJECTIVE: To determine the optimal number of PCs per pool that will achieve a target dose. DESIGN: The population statistics of PC cell counts were obtained from routine quality control records. A target value of 3.00x10(11) cells was chosen. A theoretical model to predict the probability of achieving this target cell count was developed. MAIN OUTCOME MEASURES: The calculated probability was tested in a Monte Carlo simulation. RESULTS: The mean of a sequential series of 100 PC counts (obtained from routine quality control records) was 9.08x10(10), and the standard deviation was 2.70x10(10). Platelet loss during pooling was measured at 8.3%, so that the pooling of 3.27x10(11) cells is required to deliver a dose of 3.00x10(11) cells. The pooling of 4 PCs from this data set predicts an average pool cell count of 3.63+/-2.30x10(11). Using standard tables for normal distribution, the probability of achieving a minimal pool dose of 3.00x10(11) cells is estimated to be greater than 0.76. Similarly, the probability of achieving the target value by pooling 5 PCs is greater than 0.99. In a Monte Carlo simulation selecting 4 PCs, 76.5% of the trials were successful, and for 5 PCs, 99.5% were successful (estimated P values of .77 and .99, respectively). CONCLUSION: Five PCs per pool are necessary and sufficient to achieve the chosen target value given the cell count characteristics of our PC supply.

Proficiency testing in clinical cytogenetics. The 1986 experience of the College of American Pathologists.

In 1986, the College of American Pathologists introduced a proficiency testing program in cytogenetics entitled "survey CY." Approximately 140 laboratories actively participated. The survey consisted of 20 cytogenetic cases sent to the participants in quarterly shipments of five challenges each. Photographic negatives of metaphases were the primary medium studied. The results reported by the participants were highly concordant for the determination of modal chromosome count and sex chromosome designation. More than 90% of the participants correctly identified the following abnormalities: trisomies 8, 13, 18, 21, and X; monosomy X; t(9;22); t(8;21); der(2) t(2;?); and 5p-. Abnormalities A fra(X)(q27), t(5;11), inv(16), and t(15;17) were correctly identified by more than 80%, 70%, 70%, and 60% of the participants, respectively. The participants had the most difficulty with diagnoses that involved photographs of high-resolution chromosome banding. The 1986 survey was not graded, but the 1987 cytogenetics survey will be scored on those challenges where there is at least 80% consensus among the participants.

Accidental fires in clinical laboratories.

The National Fire Protection Association, Quincy, Mass, estimates that 169 fires have occurred annually in health care, medical, and chemical laboratories. On the average, there are 13 civilian injuries and $1.5 million per year in direct property damage. Most fires in which the cause or ignition source can be identified originate in malfunctioning electrical equipment (41.6%) or in the facility's electrical distribution system (14.7%). The prevalence of fire safety deficiencies was measured in the College of American Pathologists Laboratory Accreditation Program. Of the 1732 inspected laboratories, 5.5% lacked records of electrical receptacle polarity and ground checks in the preceding year. Of these inspected laboratories, 4.7% had no or incomplete documentation of electrical safety checks on laboratory instruments. There was no evidence of quarterly fire exit drills in 9% of the laboratories. Deficiencies were also found in precautionary labeling (6.8%), in periodic review of safe work practices (4.2%), in the use of safety cans (3.7%), and in venting of flammable liquid storage areas (2.8%). Fire preparedness would be improved if all clinical laboratories had smoke detectors and automatic fire-extinguishing systems. In-service training courses in fire safety should be targeted to the needs of specific service areas.

Comprehensive Blood Bank Survey.

Approximately 2,400 laboratories participated in the 1982 Comprehensive Blood Bank Survey of the College of American Pathologists. Fourteen Referee Laboratories were utilized to validate the results of participants and to assure lack of deterioration of the specimens during shipment. Results of the participants in ABO grouping, Rh D typing, special antigen typing, and antibody detection and identification are reported and discussed. Special ungraded studies, which provided additional challenges, and supplementary questions with the responses of participants are also included.

Multiple red cell transfusions and alloimmunization. Experience with 6996 antibodies detected in a total of 159,262 patients from 1985 to 1993.

This retrospective study of red cell antibodies covered the period from 1985 to 1993. A three-cell antibody screen, 22% albumin enhancement, and a polyspecific antiglobulin reagent assay were performed. From a total of 159,262 patients in the data set, 6996 antibodies were detected among the sera of 4700 patients (2.9%). Four thousand two hundred thirty-five (60.5%) alloantibodies of potential clinical significance were found. These included anti-K1 (23.0%), -E (17.6%), -D (12.4%), -Le(a) (7.3%), -C (6.3%), -Fya (5.7%), and -c (4.4%). Cold agglutinins were found in 1119 positive antibody screens, 261 had warm autoantibodies, and 554 had high-titered, low-avidity antibodies. Three hundred seven were clinically insignificant (eg, Sda and Bga). Five hundred seventeen were too weak to identify. Most patients' sera demonstrated only one antibody (69.3%), but there was a strong linear correlation between the total number of recorded red cell transfusions and the number of antibodies found (r = .976; P < .0001). There was a higher percentage of females with antibodies than the percentage of females in the total study population (59.2% versus 43.8%, P < .0001). Two hundred nine of 554 (37.7%) high-titered, low-avidity antibodies and 349 (31.2%) of 1119 of the cold agglutinins accompanied or obscured clinically significant antibodies.

Proficiency testing in clinical cytogenetics. A 6-year experience with photographs, fixed cells, and fresh blood.

The College of American Pathologists and the American Society of Human Genetics offer a proficiency testing program in clinical cytogenetics. Two hundred twenty-five laboratories now provide data for this survey, which was begun in 1986. Challenges have consisted of photographed metaphases, fixed lymphoblastoid cell suspensions, fresh peripheral blood, and disarranged karyotypes. The "correct" response was based on 80% or greater consensus among either the referees or the participants. Referee laboratories performed better than participants. More laboratories were able to report accurate recognition of abnormalities by using a coded list than could write the interpretation in standardized nomenclature. Deletions, unbalanced translocations, and inversions were more difficult challenges than balanced translocations or trisomies. Prenatal and lymphocyte challenges were more likely to result in consensus than were bone marrow challenges. Participants performed best on whole-blood challenges. Fixed cell suspensions were less satisfactory. Excellent quality case material is essential for a successful challenge. A grading system has been devised to separate artifacts of the survey process from proficiency variables.

Explaining the risks of blood transfusion to patients.

Many patients express concern about the risk of an infection from blood transfusion. Blood transfusion is one of the safest therapies available, but its risks should never be trivialized when talking with patients. The most common infectious complication is hepatitis C, which occurs in 2% to 4% of transfused patients. Hepatitis B occurs in fewer than 1% of such patients. The risk of HIV infection from a blood transfusion is less than 1 in 100,000 in the United States. Explanation of risks is most effective when comparisons are meaningful and phrased from the patient's point of view.

Platelet transfusion therapy for medical and surgical patients.

Platelet transfusions have become more common as more patients undergo bone marrow transplantation and aggressive chemotherapy for malignant diseases. This paper reviews the indications for platelet transfusions and the factors that can decrease their effectiveness.