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.

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.

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.

An evaluation of the need for shared blood donor deferral registries.

OBJECTIVE: This study examined the frequency with which allogeneic, volunteer blood donors who had been deferred from donation at one blood collection facility donated, or attempted to donate, at a second blood collection facility. METHODS: The blood donor computer files of two local blood collection facilities were-combined and matched donors on the donor deferral registry of each blood collection facility were identified. RESULTS: Of 26,300 donors in the hospital-based blood bank file, 6732 (25.6%) were matched to the community blood center donor file (active donor base approximately 275,000). Matched donors on the donor deferral registry at each blood collection facility numbered 427 (6.3% of total matched donors). A total of 103 evaluable donors (1.5% of total, or 24.1% of deferred, matched donors) had been deferred at one blood collection facility and then later donated, or attempted donation, at the other blood collection facility. Of these 103, 51 were allogeneic donors who had been notified of their deferral status and should not have subsequently attempted blood donation. Thirty-two donors on the donor deferral registry of one blood collection facility made donations at the second blood collection facility which entered the general blood inventory. CONCLUSION: Shared donor deferral registries may be valuable at the local or regional level to prevent deferred blood donors from donating at other blood collection facilities. Whether or not a national donor deferral registry would be efficacious remains to be proven and deserves further study.

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.

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)

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.

An analysis of autologous blood donor motivational factors.

Little is known about the patient's reasons for participating in an autologous blood (AUB) collection program. We surveyed 110 AUB donors in our hospital-based collection program. Although fear of infection from an allogeneic blood transfusion was cited by 20% of AUB donors, 68.2% indicated that their physician's recommendation was a motivating factor. For 18.2% self-initiated motivation was a factor in pursuing AUB. The overwhelming majority (97.1%) stated they would donate AUB even if the risk of getting AIDS from a blood transfusion was zero. Fear of infection does not appear to be the primary reason for patients to donate AUB. Recommendation by their personal physician is an important component in the patients' decision-making process to set aside their own blood for upcoming surgery.

Autologous blood transfusion and intraoperative cell salvage in a patient with homozygous sickle cell disease.

BACKGROUND: Autologous transfusion can eliminate the need for homologous transfusions. In addition, hypotensive anesthesia and devices that salvage red blood cells for return to the patient can reduce operative blood loss. However, blood from patients with sickle cell disease is difficult to store. SUMMARY: A 16-year-old black girl with homozygous sickle cell disease needed surgery for progressive scoliosis. Her family's religious convictions precluded homologous transfusions. During surgery, 400 mL of autologous blood that had been successfully stored was transfused, as was 800 mL of blood salvaged using a cell-saving device, and 3800 mL of nonblood plasma expanders. Intravenous agents were used to maintain hypotension. However, following a rise in the patient's prothrombin and thromboplastin times, four units of homologous packed red cells were transfused with the permission of the patient's parents. CONCLUSIONS: Patients with sickle cell disease can be given hypotensive anesthesia and autologous transfusions of blood donated before surgery and blood salvaged during surgery using a cell-saving device.

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.

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.

Is your staff wise in waste management?

All laboratorians must learn their labs' protocols for hazardous waste disposal, including emergency plans and comprehensive procedures for storage, packing, and hauling.

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.

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.

Computer-assisted audits of blood component transfusion.

Comprehensive review of clinical blood transfusion practice at a tertiary-care medical center is complicated by the extraordinary number of patients that receive such therapy. Computer-assisted review of the key objective data used in making the decisions about transfusion is necessary to evaluate the process. Use of 15,873 units of red blood cells, 3,641 units of plasma, 2,619 pools of platelets or pheresis units, and 259 pools of cryoprecipitate was screened by comparing pre-transfusion and post-transfusion blood counts with the medical staff's evaluation criteria. On this basis, 81.4% of transfusion episodes (TEs) were considered fully justified. Medical records were selected for audit from the cases in which the transfusion decisions could not be justified by on-line information. Abstracted data subsequently justified 82 of 139 audited cases; 68.4% of the comments pertaining to the remaining 57 cases adequately explained the transfusion decision. Thus, nearly 96% of the TEs were justifiable as determined by peer review.

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.

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.

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.