Detecting bacteria in platelet concentrates by use of reagent strips.

BACKGROUND: Iatrogenic infection of immunosuppressed or immunocompromised hosts secondary to receipt of blood components containing bacteria may result in serious adverse outcomes. Measurement of pH and glucose by use of inexpensive reagent strips has been proposed as a practical means of screening for bacteria in platelet concentrate (PC) units. STUDY DESIGN AND METHODS: Glucose and pH were measured in 3093 PC units by use of reagent strips (Multistix, Bayer Corp.) to screen for bacterial content. Any PC classified by the reagent strip method as containing bacteria was subsequently cultured to confirm the presence and quantity of bacteria present. RESULTS: Thirty of 3093 PC units were classified as containing bacteria by the reagent strip method. Two of the 30 PC units positive by the reagent strip method were also positive by standard bacterial culture technique. Bacillus cereus was isolated from both units. CONCLUSION: Screening PC units by the reagent strip method resulted in 9.7 units per 1000 being wasted, but prevented two patients from receiving a PC unit containing B. cereus.

A java-based application for differential diagnosis of hematopoietic neoplasms using immunophenotyping by flow cytometry.

We describe the implementation of a Java-based application for differential diagnosis of hematopoietic neoplasms using immunophenotyping by flow cytometry. The current version of this Java applet includes the knowledge-base for 33 hematopoietic neoplasms and 43 diagnostic immunophenotyping markers. Java, a new object-oriented computing language, helps facilitate development of this applet, a platform-independent module that can be implemented on the World Wide Web. As the Web rapidly becomes more accessible to users around the world, Web-based software may eventually form the core of decision-support systems in clinical settings. Java-based applications, such as the one described in this paper, are expected to contribute significantly in this area.

A relational database for diagnosis of hematopoietic neoplasms using immunophenotyping by flow cytometry.

A relational database was developed to facilitate the diagnosis of hematopoietic neoplasms using results of immunophenotyping by flow cytometry. This database runs on personal computers and uses backward-chaining search to arrive at conclusions. Results of immunologic marker studies are processed by the database to obtain a set of differential diagnoses. The current version of this database includes diagnostic immunophenotyping pattern for 33 hematopoietic neoplasms. We tested this database using 92 clinical cases from 2 tertiary care medical centers. The database ranked the actual diagnosis as 1 of the top 5 differential diagnoses in 93% of the cases tested. The user can modify the database contents to suit individual needs. This database has been posted on the World Wide Web for direct access. We propose that this user-friendly database is a potential tool for computer-assisted diagnosis of hematopoietic neoplasms.

A rapid and inexpensive method for processing induced sputum for detection of Pneumocystis carinii.

The authors describe a method to process induced sputum specimens for detection of Pneumocystis carinii which is simple, rapid and inexpensive. Induced sputum and bronchoalveolar lavage (BAL) were obtained within a 24-hour period from 41 patients who were HIV-positive and had pulmonary symptoms suspicious for P carinii pneumonia. Induced sputum or BAL fluid was placed into Saccomanno's fixative, blended, and centrifuged. The sediment was stained for P carinii cysts by a modified method with Fungi-Fluor Solution A (Polysciences, Warington, PA) and the Genetic Systems Pneumocystis carinii Immunofluorescence Antibody (Genetic Systems, Seattle, WA). The Genetic Systems stain on the BAL specimen was positive in 35 patients and was the standard for comparison. With a single induced sputum, the Genetic Systems stain detected 31 (89%) positive patients, whereas the Fungi-Fluor stain detected 21 (60%). The sensitivity for detecting P carinii cysts in induced sputum was significantly greater (P < 0.05) for the Genetic Systems stain.

Shear-induced platelet aggregation is inhibited by in vivo infusion of an anti-glycoprotein IIb/IIIa antibody fragment, c7E3 Fab, in patients undergoing coronary angioplasty.

BACKGROUND: Elevated levels of shear stress such as those that occur in stenotic arterial vessels can directly activate and aggregate platelets and thus contribute to the pathogenesis of acute arterial thrombosis. This shear-induced platelet aggregation (SIPA) is mediated by von Willebrand factor binding to platelet membrane glycoprotein (GP) Ib and GPIIb/IIIa. The chimeric Fab fragment of the monoclonal antibody 7E3 (c7E3 Fab) that binds selectively to GPIIb/IIIa is under clinical evaluation in patients undergoing percutaneous transluminal coronary angioplasty (PTCA). This study was undertaken to investigate the effects on ex vivo SIPA of c7E3 Fab administered to patients undergoing PTCA. METHODS AND RESULTS: Six patients received aspirin (325 mg) and boluses of heparin (12,00o U) followed by c7E3 Fab 0.25 mg/kg. Blood collected from each patient before and after heparin treatment and at various time points after c7E3 Fab administration was subjected to laminar shear stress in a cone-and-plate viscometer. Flow cytometry was used to quantify the extents of platelet aggregation and of antibody binding to GPIIb/IIIa. Results indicate that c7E3 Fab injection resulted in a rapid, extensive blockade of GPIIb/IIIa receptors (98.6 +/- 0.2%) and a 50% inhibition of ex vivo platelet aggregation induced by shear stress. c7E3 Fab also completely abolished the formation of large platelet aggregates ("large" refers to particles > 10 microns in equivalent sphere diameter), which are presumably the aggregates of greatest clinical significance. Partial reversibility of the inhibition was noted within 2 days after drug administration, but even after 1 week, platelet function had not been fully restored. CONCLUSIONS: This study demonstrates that c7E3 Fab is a potent inhibitor of SIPA, which may be an important mechanism of its beneficial effect in the treatment of arterial occlusive diseases and in the prevention of thrombotic complications of coronary artery disease after angioplasty.

Flow cytometric studies of platelet responses to shear stress in whole blood.

The objective of this work was to evaluate quantitatively the effects of flow on platelet reactions using a flow cytometric technique. Whole blood was exposed to well defined, laminar shear stress in a cone-and-plate viscometer in the absence of added agonists. Blood specimens were fixed with formaldehyde and incubated with two monoclonal antibodies. Antibody 6D1, specific for platelet membrane glycoprotein Ib (GPIb), was used to identify and enumerate platelets and platelet aggregates on the basis of their characteristic forward scatter and 6D1-FITC fluorescence profiles. Anti-CD62 antibody, specific for the granule membrane protein-140 (GMP-140), was used to measure platelet activation. Results showed platelet aggregation increasing with increasing shear stress with marked increase in this response for a pathophysiological stress level of 140 dyn/cm2 and higher. This stress level also was the apparent threshold for formation of large platelet aggregates ("large" refers to particles larger than 10 microns in equivalent sphere diameter). These platelet responses to shear stress were insensitive to aspirin, but strongly inhibited by agents that elevate platelet cyclic adenosine monophosphate (cAMP) levels. Moreover, pre-incubation of whole blood with monoclonal antibodies that inhibit von Willebrand factor binding to GPIb or von Willebrand factor and fibrinogen binding to GPIIb/IIIa inhibited platelet aggregation. Aggregation induced by shear at 37 degrees C was less in extent than at 23 degrees C. At physiological shear stresses, whole blood was more susceptible to shear-induced platelet aggregation than platelet-rich plasma. This study reaffirms that flow cytometric methods have several important advantages in studies of shear effects on platelets, and extends the methodology to whole blood unaltered by cell separation methods.

Plasma cell leukemia presenting as a pancreatic mass.

A 62-year-old woman presented with a 3-week history of obstructive jaundice. Computed tomography of the abdomen showed marked enlargement of the head of the pancreas and a prominent pancreatic body, suggestive of a neoplasm with associated pancreatitis. The peripheral blood showed an increased number of plasma cells accounting for 50% of the leukocytes. Biopsy specimens of the pancreas, liver, and a peritoneal lymph node showed a diffuse infiltrate of typical and atypical plasma cells (50% of which had cytoplasmic IgG-lambda). Serum and urine protein electrophoresis revealed a monoclonal IgG-lambda spike and Bence Jones-lambda protein, respectively. The bone marrow was diffusely infiltrated by plasma cells. To our knowledge, this is the first reported case of a plasma cell leukemia presenting as a pancreatic mass producing extrahepatic biliary obstruction.

Targeting of the rasT24 oncogene to the proximal convoluted tubules in transgenic mice results in hyperplasia and polycystic kidneys.

Five families of transgenic mice were derived from one-cell-stage embryos injected with gamma GT-rasT24, a fusion gene consisting of the gamma-glutamyl transpeptidase (gamma GT) 5' flanking region containing promoter I linked to a mutated (codon 12) human H-ras oncogene. The transgene was expressed selectively in the kidneys, eyes, and brains of all families as determined by reverse transcription-polymerase chain reaction, nuclease protection assays, and in situ hybridization. In two of five families, kidney lesions consisting of proximal tubular hyperplasia, renal cysts, and microadenomas developed in male animals; males also expressed higher levels of gamma GT/rasT24 RNA. Early lesions consisted of proximal tubular hyperplasia as defined by alkaline phosphatase histochemistry, gamma GT immunohistochemistry, and electron microscopy and could be correlated with the presence of rasT24 RNA within the cystic proximal tubular epithelium by in situ hybridization. Advanced lesions also involved other segments of the nephron and consisted of cysts lined by a flattened unicellular layer of attenuated epithelium. No rasT24 could be identified within cystic lesions of the distal nephron and collecting tubules by in situ hybridization, and they most likely arise by external compression. Animals from the two transgenic strains exhibiting cystic lesions die of renal failure beginning at 8 months of age. No difference in cell-cycle parameters or DNA ploidy between transgenic and control kidneys was identified by flow cytometric analysis. No renal carcinomas developed. The primary renal effects of the H-rasT24 oncogene in this model system consist of proximal tubular hyperplasia and polycystic kidneys. This model appears to provide a useful in vivo system for the study of ras oncogene function and control of renal cell proliferation.

False aneuploidy in benign tumors with a high lymphocyte content: a study of Warthin's tumor and benign thymoma.

The quality of results of flow cytometric DNA content analysis of formalin-fixed, paraffin-embedded tissue may be affected by a number of preanalytical variables. We performed flow cytometric DNA content analysis on two types of benign tumors to investigate the effect of a prominent lymphocytic component: Warthin's tumor (N = 20) and benign thymoma (N = 8). Malignant tumors (N = 23) were included as DNA aneuploid controls. All tissues studied were archival material processed using Hedley's technique either without prolonged rehydration in water (day 0 samples) or with 24- or 48-hour rehydration (day 1 and day 2 samples, respectively). Image cytometric DNA ploidy analysis was also performed on most cases. Eight cases (40%) of Warthin's tumor and five cases (63%) of benign thymoma showed either hyperdiploid peaks or marked asymmetry on the day 0 DNA histograms; nine of the malignant tumors were aneuploid. The DNA histogram abnormalities of the benign tumors could be gated out by excluding the lymphocyte nuclei. None of the DNA indices of the benign tumors corresponded with expected deviations based on published chromosomal studies. All of the DNA histogram abnormalities of the benign tumors disappeared and/or fused with the main peaks on the day 1 or day 2 samples, except for one case of benign thymoma. All the DNA aneuploid peaks on the malignant tumors persisted with prolonged rehydration. Image cytometric DNA analysis showed a diploid pattern in all benign tumors. We conclude that a high lymphocyte content may be a cause of false aneuploidy in these benign tumors. Furthermore, the degree of rehydration appears to be an important factor in achieving optimum fluorochrome staining of DNA.

Hematologic response to acute inflammation: the band neutrophil revisited.

The value of the routine visual (manual) 100-cell differential leukocyte count (DLC) as a screening test in asymptomatic individuals or in patients on admission without suspected distributional or morphologic abnormalities has been challenged in the medical literature. More recently, the usefulness of an increase in the band neutrophils or a "left-shift" in the manual DLC has also been in question. This article discusses the largest sources of error in the routine DLC, namely, cell-identification errors, distributional errors, and statistical sampling errors. The latest flow cytometric, cytochemical, automated leukocyte differential counters offer a viable, reliable alternative to the highly imprecise 100-cell DLC. Studies have shown that these newer instruments are sensitive in detecting the hematologic response to acute inflammation or infection and have the advantage of vastly increased precision and therefore of improved reliability of the differential leukocyte count.

Granulocytic sarcoma manifested as ascites.

Granulocytic sarcoma rarely presents an effusion. We have described an unusual case of granulocytic sarcoma with ascites. Malignant cells may be confused with adenocarcinoma. Eosinophilic myelocytes, peroxidase-positive malignant cells, and the Philadelphia chromosome in the ascitic fluid cells established the definitive diagnosis of granulocytic sarcoma.

An evaluation of the Technicon H-1 automated hematology analyzer in detecting peripheral blood changes in acute inflammation.

The Technicon H-1 (H-1) is an automated hematology analyzer that provides a complete blood cell count, a six-part differential with absolute counts, and morphologic values with a left-shift flag (LS). To determine the sensitivity of the H-1 in detecting peripheral blood changes in acute inflammation, we first correlated the H-1 LS with band counts on the Hematrak 590 (H590), an automated digital image processor differential system. The H-1 sensitivity to H590 band counts above 11% was 76%, specificity was 82%, and efficiency was 80%. Each semiquantitative LS (1+, 2+, 3+), as well as a new factor, lobularity index, was correlated with the actual H590 band count. There was a definite direct proportional relationship between each semiquantitative LS and the mean band count. However, the wide overlaps of band count ranges corresponding to each semiquantitative flag rendered semiquantitation of limited value. Forty cases with the clinical diagnosis of acute appendicitis were similarly studied preoperatively. Thirty-three cases histologically showed acute inflammation. On the H-1, 79% (sensitivity) had LS flags, 88% had absolute neutrophilia (greater than 8 x 10(9)/L), 82% had relative neutrophilia (greater than 75%), and 91% had leukocytosis (greater than 10.5 x 10(9)/L). In comparison, sensitivities on the H590 were 70% for band counts above 11%, 82% for relative neutrophilia, and 85% for absolute neutrophilia. This study shows that the H-1 is at least as sensitive as the H590 to peripheral blood changes that indicate acute inflammation.

A clinical study on the use of a fluorogenic substrate assay of plasminogen.

A fluorogenic substrate assay of plasminogen was compared with acceptable methodologies, i.e., caseinolytic and radial immunodiffusion assays. Reference intervals based on a population of 25 members of the laboratory staff were 2.5-3.8 CTA u/ml (fluorogenic), 2.2-4.0 CTA u/ml (caseinolytic) and 8.7-14.3 mg/dl (RID). Ninety eight determinations were performed on 65 patients. Regression analysis showed linear correlation between the fluorometric and caseinolytic assays (r = 0.8046, p less than 0.03) and between the fluorometric and immunologic assays (r = 0.8572, p less than 0.005). Pre-operative and post-operative determinations were performed on 33 patients undergoing coronary artery bypass surgery and there was a consistent and significant (10%-76%) decrease in the plasminogen levels post-operatively (p less than 0.01). Twenty-five patients with various malignancies were compared with the normal population; no significant difference in the plasminogen levels was observed between the two groups.

Fluorogenic substrate assay of antithrombin III: a clinical study.

The fluorogenic substrate assay of antithrombin III (AT III) was compared with a thrombin inhibition (two-stage) clotting test and a radial immunodiffusion assay. Normal ranges based on a population of 25 individuals were 88-121% (fluorogenic substrate), 72-111% (clotting) and 22.5-35.3 mg/dl (RID). A clinical comparison of the methods showed a linear relationship between the fluorogenic substrate and immunologic methods (r = 0.69, p less than 0.005), as well as between the fluorogenic substrate and clotting methods (r = 0.75, p less than 0.005). The fluorogenic substrate assay reflected the highest incidence of low AT III levels in all three clinical groups included (36 patients with arteriosclerosis, 18 with fractures and 37 with thrombosis/embolism).

Enzyme labeled immunosorbant assay (ELISA) for detection of platelet antibodies.

Although it is widely accepted that patients with immune thrombocytopenia produce platelet antibodies, the demonstration of such antibodies has been difficult and time consuming. A simple and quick enzyme linked immunoassay for platelet antibodies is presented. The platelet associated IgG is coupled with alkaline phosphatase labeled anti-IgG. The resultant complex is determined spectrophotometrically using p-nitrophenyl phosphate as substrate. With this technique, excess of IgG on platelets was detected in 24 out of 33 patients (72 percent) with immune thrombocytopenic purpura and four out of four thrombocytopenic patients with systemic lupus erythematosus. The results of this assay correlate quantitatively with Dixon er al3 complement lysis inhibition assay (r = 0.82).

Laboratory monitoring of heparin therapy--the effect of different salts of heparin on the activated partial thromboplastin time.

Data from the 1978 CAP Hematology Survey were analyzed for the effect of sodium and calcium salts of heparin on the activated partial thromboplastin time (APTT). The results indicate that the sodium salt of heparin prolongs the APTT more than the calcium salt at a heparin concentration of 0.2 units/ml. Furthermore, there was a variable response of the different APTT reagents to the different heparin salts. Data from the 1979 CAP Hematology Survey showed a greater sensitivity of the Automated APTT reagent and the Platelin Plus Activator of General Diagnostics compared with Actin (Dade) and Thrombofax (Ortho) at therapeutic heparin levels of 0.2 units/ml to 0.4 units/ml. The 1979 CAP heparin questionnaire results are presented and analyzed.