bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduced Quality of Life in Veterans at Risk for Drug‐Related Problems
Authors: Okano Gary J. , Malone Daniel C. , Billups Sarah J. , Carter Barry L. , Sintek Charles D. , Covey Douglas , Mason Barbara , Jue Sandra , Carmichael Jannet , Guthrie Kelly , Dombrowski Robert , Geraets Douglas R. , Amato Mary G.
Published: June 2004

The relationships between drug therapy and health‐related quality of life in 1054 patients who received care from Department of Veterans Affairs medical centers (VAMCs) were assessed. Patients at high risk for drug‐related problems were enrolled into a pharmaceutical care study at nine VAMCs. On enrollment, the short form (SF)‐36 was completed and medical records were examined for evidence of coexisting illness. Drug therapy in the year before enrollment was analyzed in relation to SF‐36 scores. Mean ± SD SF‐36 scores ranged from 37.99 ± 41.70 for role physical to 70.78 ± 18.97 for mental health domains, with all domain scores significantly below age‐adjusted national norms (p<0.05). Patients taking a drug that required therapeutic monitoring had significantly lower SF‐36 scores (p=0.0001 to p=0.0033) across all domains except for bodily pain and mental health, compared with patients not taking these agents.
Short‐Term Lorazepam Infusion and Concern for Propylene Glycol Toxicity: Case Report and Review
Authors: Cawley Michael J.
Published: June 2004

A 34‐year‐old woman with a history of renal insufficiency induced by long‐term cocaine use was admitted with acute shortness of breath remarkable for submandibular and anterior throat swelling. She required intubation, mechanical ventilation, and sedation. Sedation was administered with daily infusions of intravenous lorazepam 65, 313, and 305 mg for 3 days, respectively. Forty‐eight hours into the infusion the patient experienced anion gap metabolic acidosis with hyperlactatemia, hyperosmolality, and increased osmolal gap. Propylene glycol (PG), a component of lorazepam intravenous formulation, was considered the potential source of the metabolic abnormality. The patient received greater than 40 times the acceptable recommended amount of PG over 72 hours. Cessation of lorazepam produced major improvements in lactic acid, serum osmolality, and anion and osmolal gaps. The large PG exposure associated with long‐term cocaine‐induced renal insufficiency produced a toxic metabolic state. Agents containing PG should be avoided in patients with compromised renal function (creatinine clearance ≤ 30 ml/min) induced by cocaine use.
Prevalence and Risk of Thrombocytopenia with Valproic Acid: Experience at a Psychiatric Teaching Hospital
Authors: Conley Erin L. , Coley Kim C. , Pollock Bruce G. , DaPos Stacey V. , Maxwell RaeAnn , Branch Robert A.
Published: June 2004

Study Objective. To investigate the prevalence and potential risk factors of thrombocytopenia in hospitalized patients receiving valproic acid (VPA) fo psychiatric indications (excluding epilepsy).

Design. Retrospective study.

Setting. University‐affiliated psychiatric facility.

Patients. Two hundred sixty‐four patients treated with VPA.

Intervention. Data from patients hospitalized between January 1, 1994, and December 31, 1998, who were receiving VPA and had at least one platelet count recorded.

Measurements and Main Results. Of 264 VPA‐treated patients, 31 (12%) met our criteria for thrombocytopenia. Mild thrombocytopenia (platelet count of 101–150 times 103/mm3) occurred in 25 (9%) patients, and moderate thrombocytopenia (platelet count of 40–100 times 103/mm3) occurred in 6 (2%) patients. Age older than 65 years (p=0.02) and VPA dosage greater than 1000 mg/day (p<0.001) were identified as significant risk factors for developing thrombocytopenia.

Conclusion. The estimated prevalence of thrombocytopenia is 12% in the general psychiatric population receiving VPA, with the elderly at greatest risk.
Interaction Between N‐Acetylcysteine and Activated Charcoal: Implications for the Treatment of Acetaminophen Poisoning
Authors: Tenenbein Paul K. , Sitar Daniel S. , Tenenbein Milton
Published: June 2004

Study Objective. To determine if the presence of N‐acetylcysteine reduces the ability of activated charcoal to adsorb acetaminophen both in the absence and presence of a coingestant.

Design. In vitro laboratory study.

Setting. University hospital research laboratory.

Measurements and Main Results. The adsorption of acetaminophen and salicylic acid by activated charcoal in the presence and absence of N‐acetylcysteine was measured in vitro. Acetaminophen and salicylic acid analyses were conducted with high‐performance liquid chromatography. Adsorption data were compared using the appropriate parametric statistical test. The addition of N‐acetylcysteine significantly decreased the binding of acetaminophen by activated charcoal (p<0.005). When salicylic acid was added to simulate a coingestant, N‐acetylcysteine significantly decreased salicylate adsorption by charcoal (p<0.001).

Conclusions. The presence of N‐acetylcysteine reduces the ability of activated charcoal to adsorb acetaminophen and coingestants. In vivo data will be required to determine the clinical relevance of these interactions.
Pharmacoeconomic Evaluation of a Pharmacist‐Managed Hypertension Clinic
Authors: Okamoto Mark P. , Nakahiro Randy K.
Published: June 2004

Study Objective. To measure clinical, economic, and humanistic outcomes associated with a pharmacist‐managed hypertension clinic compared with physician‐managed clinics.

Design. Prospective, randomized, comparative study.

Setting. Managed care organization.

Patients. A total of 330 patients with mild‐to‐moderate essential hypertension.

Intervention. Hypertension care provided by either the pharmacist‐managed hypertension clinic or physician‐managed general medical clinics.

Measurements and Main Results. Baseline and 6‐month evaluations consisted of systolic and diastolic blood pressure measurements, a short‐form health survey, and collection of health care utilization information. After treatment, blood pressure measurements were significantly lower (p<0.001) in the pharmacist‐managed hypertension clinic group than in the physician‐managed clinic group. Patient satisfaction was significantly higher in the hypertension clinic group. Total costs for the hypertension clinic group were not different from those of the physician‐managed clinic group ($242.46 vs $233.20, p=0.71), but cost:effectiveness ratios were lower in the hypertension clinic group ($27 vs $193/mm Hg for systolic blood pressure readings, and $48 vs $151/mm Hg for diastolic blood pressure readings).

Conclusion. In a hypertension clinic, pharmacists can be a cost‐effective alternative to physicians in management of patients, and they can improve clinical outcomes and patient satisfaction.
Recombinant Human Activated Protein C, Drotrecogin Alfa (activated): A Novel Therapy for Severe Sepsis
Authors: Kanji Salmaan , Devlin John W. , Piekos Krista A. , Racine Eric
Published: June 2004

Sepsis remains a major cause of death in hospitalized patients. Despite a massive research effort over the past 2 decades to identify innovative therapies for sepsis, current treatment strategies consist primarily of antiinfective agents and a variety of supportive measures. Activated protein C, an endogenous protein that inhibits thrombosis and inflammation while promoting fibrinolysis, plays an important role in the pathogenesis of sepsis. Recombinant human activated protein C, drotrecogin alfa (activated), when compared with placebo in a randomized, double‐blind study of 1690 patients with severe sepsis (Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis [PROWESS] trial), decreased the relative risk of death at 28 days by 19.4% (95% confidence interval 6.6–30.5%, p=0.005), although there was a trend for more serious bleeding (3.5% vs 2.0%, p=0.06) with its use. Drotrecogin alfa is the first antisepsis drug found to have a mortality benefit. It should be administered only to patients with severe sepsis who meet the PROWESS study inclusion criteria and should be avoided when risk factors for bleeding are present. Ongoing research will help determine the cost‐effectiveness of drotrecogin alfa, as well as its role in critically ill populations not studied in the PROWESS trial.
The Value of Pharmacist Involvement in a Point‐of‐Care Service, Walk‐In Lipid Screening Program
Authors: Jafari Mahtab , Masih Mona , Emerson Jane F.
Published: June 2004

The leading cause of death in the United States is heart disease. Because an elevated serum cholesterol level is an independent risk factor for development of coronary heart disease (CHD), individuals older than 20 years of age are advised to have their cholesterol level checked every 5 years. Walk‐in screening programs are becoming popular as a method of health care delivery. The program at the University of California—Irvine Medical Center administers point‐of‐care, low‐cost lipid profile testing, directly involves patients in their own care, and provides individualized education to patients regarding cardiovascular risk reduction. A total of 301 patients participated in the program between August 1998 and September 2000. Fifty percent of them (150 patients) required intervention; 34% of these (52 patients) were previously undiagnosed. Their mean age was 57 ± 13 years; 35% were women, 53% had two or more cardiac risk factors, and 5% had CHD. Based on the National Cholesterol Education Program guidelines, 29% had low‐density lipoprotein levels above target, 23% had triglyceride levels higher than recommended, and 21% had high‐density lipoprotein levels below target. It is our hope that our successful experience with the program will encourage pharmacists to develop similar programs.
Physician‐Prompting Statin Therapy Intervention Improves Outcomes in Patients with Coronary Heart Disease
Authors: Hilleman Daniel E. , Monaghan Michael S. , Ashby Catherine L. , Mashni Joyce E. , Woolley Kathleen , Amato Cecily M.
Published: June 2004

Study Objective. To evaluate the effectiveness of a posthospital discharge intervention that prompted physicians to increase the use and effectiveness of statins (3‐hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitors) in patients with coronary heart disease (CHD).

Methods. Participants were 612 patients with CHD who were admitted to a coronary care unit. The control group (303 patients admitted from October 1–December 31, 1998) received no follow‐up intervention. The intervention group (309 patients admitted from January 1–March 31, 1999) had follow‐up letters sent or phone calls made to their primary care physicians with patient‐specific recommendations concerning assessment of lipid profiles and statin therapy. Over a 2‐year follow‐up period, assessment of lipid profiles, use of therapy, and adverse clinical outcomes were compared between the control and intervention groups.

Results. At hospital discharge, there was no significant difference in the use of statins between the groups. At each reported follow‐up interval, the percentages of patients having lipid profiles measured, being treated with a statin, receiving titrated dosages of a statin, and achieving low‐density lipid (LDL) cholesterol goals set by the National Cholesterol Education Program (NCEP) were significantly greater in the intervention group compared with the control group (all p<0.05). At the end of the 2‐year follow‐up period, nearly three‐fourths (72%) of the intervention group were receiving a statin, compared with 43% of the control group. In addition, 55% of the intervention group achieved their NCEP LDL goal, compared with only 10% of the control group. Recurrent myocardial infarction, hospitalization for myocardial ischemia, coronary revascularization, and cardiovascular mortality were significantly reduced in the intervention group compared with the control group (all p<0.05).

Conclusion. A relatively simple physician‐prompting intervention significantly increased assessment of lipid status, frequency of statin use, achievement of LDL treatment goals, and titration of lipid drug dosages. In addition, the improved use of statins significantly reduced adverse cardiovascular outcomes. This intervention tool should be more broadly applied in patient populations eligible to receive these agents.
Metformin as a Cause of Late‐Onset Chronic Diarrhea
Authors: Foss Melissa T. , Clement Kathi D.
Published: June 2004

Metformin is an effective and commonly administered drug for controlling plasma glucose concentrations in patients with type 2 diabetes mellitus. Gastrointestinal adverse effects such as abdominal pain, nausea, dyspepsia, anorexia, and diarrhea are common and widely accepted when occurring at the start of metformin therapy. Diarrhea occurring long after the dosage titration period is much less well recognized. Our patient began to experience nausea, abdominal cramping, and explosive watery diarrhea that occasionally caused incontinence after several years of stable metformin therapy. A trial of metformin discontinuation resolved all gastrointestinal symptoms. A review of the literature revealed two reports that suggest diarrhea occurring long after the start of metformin therapy is relatively common, based on surveys of patients with diabetes. Metformin‐induced diarrhea is differentiated from diabetic diarrhea, which is clinically similar, except diabetic diarrhea is rare in patients with type 2 diabetes. Patients with type 2 diabetes who are taking metformin and experience diarrhea deserve a drug‐free interval before undergoing expensive and uncomfortable diagnostic tests, even when the dosage has been stable over a long period.
Possible Interaction Between Intravenous Azithromycin and Oral Cyclosporine
Authors: Lee Page Robert , Ruscin J. Mark , Fish Doug , LaPointe Marc
Published: June 2004

A 42‐year‐old man who had received a cadaveric kidney transplant 9 years earlier was admitted to the hospital with pneumonia. His oral cyclosporine dosage for the past 2 years was stabilized at 100 mg twice/day; his cyclosporine whole blood trough levels 15 days earlier and on the day he was admitted were both 178 ng/ml. The patient was treated with intravenous ceftriaxone and intravenous azithromycin and continued to receive the same dosage of oral cyclosporine. On hospital day 3, his cyclosporine trough level rose to 400 ng/ml and his dosage was reduced by 50%. Trough levels were 181 ng/ml and 175 ng/ml on hospital days 6 and 9, respectively. On hospital day 9, the patient stopped receiving azithromycin. On hospital day 14, his cyclosporine trough level dropped to 76 ng/ml, and his cyclosporine dosage was increased back to 100 mg twice/day. The dosage produced trough levels consistent with those before he had been admitted. The patient was discharged on day 20, and a follow‐up cyclosporine trough level determined 3 weeks later was 175 ng/ml. Administration of azithromycin may have caused the increased cyclosporine concentrations in this patient through p‐glycoprotein inhibition and/or competition for biliary excretion. Azithromycin's interference may be inferred by the increase in cyclosporine levels after administration of this drug and the decrease in cyclosporine levels after its discontinuation—‐both consistent with the pharmacokinetic properties of cyclosporine. Ceftriaxone and acute‐phase reactant activation during infection, however, also may have interfered with the patient's cyclosporine elimination. Azithromycin generally is considered unlikely to interact with cyclosporine. Nonetheless, practitioners should be aware of this possibility and should monitor cyclosporine levels closely, especially in critically ill patients who have other complications.
Olanzapine‐Associated Severe Hyperglycemia, Ketonuria, and Acidosis: Case Report and Review of Literature
Authors: Seaburg Heather L. , McLendon Beth M. , Doraiswamy P. Murali
Published: June 2004

Olanzapine has been associated with insulin resistance and new‐onset diabetes mellitus. A 27‐year‐old African‐American man developed new‐onset severe hyperglycemia—‐glucose 1240 mg/dl, with ketonuria and acidosis, but no weight gain—‐2 years after starting olanzapine. Although his diabetes was stabilized with insulin, his family had difficulty monitoring his therapy, and insulin was discontinued. Subsequent monotherapy with pioglitazone stabilized the patient's glucose levels, allowing him to continue taking olanzapine. Health care professionals should be aware of links between olanzapine and diabetes mellitus and of the potential for delayed recognition of complications associated with diabetes in patients who are psychotic. Insulin poses additional problems because families of patients with schizophrenia have to deal with compliance and risk of accidental or suicidal overdose. This case and others described in the literature illustrate such dilemmas and highlight the need to further study links connecting diabetes, insulin resistance, and olanzapine. Further research to determine proportionality and risk differences among various atypical antipsychotics also is warranted.
Measurement of Functional Properties: Overview of Protein Functionality Testing
Authors: Smith Denise M.
Published: June 2004

This section provides an introduction to food protein functionality testing. Factors that must be considered when devising or selecting a valid testing method are summarized.
Measurement of Protein Hydrophobicity
Authors: Nakai Shuryo
Published: June 2004

Among numerous methods reported in the literature for analysis of protein hydrophobicty, the only methods of potential use for routine food analysis were chosen here. The most popular methods are probe spectrofluorometry using ANS, CPA, DPH, and Prodan. Additional methods selected were SDS binding, hydrophobic interaction chromatography, contact angle, and hydrophobic partition. All methods are relatively easy to conduct when need has arisen to confirm controversial data, such as those obtained by using probe spectrofluorometry. Advantages and disadvantages of different methods are compared. Importance of the relationships with functionality of proteins in food processing is emphasized because the true meaning of surface hydrophobicity of proteins is difficult to define.
Water Retention Properties of Solid Foods
Authors: Regenstein Joe M.
Published: June 2004

Water holding capacity (WHC) has been traditionally measured using a wide variety of techniques, some of which measure very different aspects of water retention. Water uptake ability and expressible moisture measure two such different properties in a practical way that can be used in both industry and research. A third measurement, drip loss, can be applied to raw, cooked, frozen, and otherwise processed samples.
Detecting Enzyme Activity: A Case Study of Polygalacturonase
Authors: Kongruang Sasithorn , Penner Michael H.
Published: June 2004

Chemical and physical approaches to monitoring enzyme activity are illustrated using polygalacturonase as the focus enzyme. Polygalacturonase is a depolymerase that catalyzes the hydrolysis of 1,4‐glycosidic linkages in linear homogalacturonan regions of pectic polymers. Activity measurements of this enzyme may be based on the generation of new product, such as the generation of reducing sugars, or changes in the rheological properties of the polymer that result as a consequence of catalysis. Two basic assay protocols, reducing sugar‐ and viscosity‐based assays, are presented here. Discussions of approaches to enzyme extraction and critical parameters for maintaining assay specificity are included.
Gas Chromatography/Olfactometry
Authors: Acree Terry E. , van Ruth Saskia
Published: June 2004

Volatile odorants can be analyzed by gas chromatography/olfactometry (GC/O), a collection of techniques that range from simple modifications of GC equipment to elaborate commercial GC‐olfactometers and protocols. This unit describes the direct column sniffing, dilution analysis, and time intensity protocols most commonly used in the food industry.
Creep and Stress Relaxation: Step‐Change Experiments
Authors: Whittingstall Peter
Published: June 2004

This article discusses the use of step‐change tests in rheometry of foods. A step change test allows for a material to be probed in its linear viscoelastic region, and beyond, where deformation is no longer reversible. This irreversible deformation behavior is of interest as it marks the onset of flow and can therefore shed light on the yield or failure of materials.
Analysis of Isoflavones in Soy Foods
Authors: Zhang Yu Chu , Schwartz Steven J.
Published: June 2004

Increasing evidence suggests that consumption of soybean products may have a significant impact upon health. The biological activity has been associated, in part, with the presence of isoflavones in soy. Analysis of these bioactive compounds in soybean products is an essential part of any research involving soy isoflavones. Various processing conditions produce soy products with a wide range of isoflavone content and composition. Recent studies demonstrated that the chemical forms and abundance of isoflavones in soy foods have a significant impact on their bioavailability and biological effects. It is thus very important to avoid altering the natural forms and abundance of the twelve soy isoflavones during extraction, identification, and quantification. In this unit, an attempt has been made to provide reliable protocols with the most commonly used analytical techniques for this purpose.
Fluidics
Authors: Stovel Richard
Published: June 2004

The primary function of the fluidics of a flow cytometer is to deliver cells to the sensing area in a single file well aligned with the detectors. In a sorting instrument, the fluidics must also be capable of physically isolating those cells selected on the basis of measurements made by the instrument. The unit discusses primary and secondary fluidic functions, types of sensing area, types of sample delivery, potential problems affecting sample flow, and sorting.
Optical Filter Sets for Multiparameter Flow Cytometry
Authors: Waggoner Alan
Published: June 2004

One of the most important components of a fluorescence detection system, the optical filter set selects wavelengths of light for fluorophore excitation and fluorescent signal discrimination. Of the variety of optical devices available for wavelength selection, optical filters have gained wide acceptance because of their wavelength selectivity, ease of installation, and relative inexpensiveness. This unit discusses the properties, compositions, and uses of optical filters in flow cytometry. The information should allow investigators to select quality filters for modifying systems for new dyes and multicolor analyses.
High‐Speed Cell Sorting
Authors: Leary James F.
Published: June 2004

A flow cytometer has the ability to analyze several thousand cells per second. This is particularly valuable for the detection of rare events. For many biological and clinical applications, cells must be isolated on the basis of multiple quantitative properties. This need is ideally met by the analytical power of flow cytometry, which provides the front‐end selection mechanism for cell sorting. In essence, cell sorting represents real‐time data classification coupled with actual physical isolation of cells with the properties of interest. This unit discusses the principles and practices of operating a cell sorter to maximize the sorting rate, including the trade‐offs between speed and resolution.
Principles of Gating
Authors: Wood James C. S.
Published: June 2004

Few aspects of flow cytometry are common to every application. Gating principles is one. This unit provides an excellent overview as well as detailed conceptual material on how gating works, when certain types of gating should or should not be applied, and the advantages and disadvantages of various techniques. This unit should be required reading for every technician using a flow cytometer and every graduate student starting out in the area, and is an excellent refresher for those more advanced in the technology. It brings out the historical development of our understanding of gating applications as well as providing a high level of technical accuracy.
Techniques for Flow Cytometer Alignment
Authors: Jett James H. , Martin John C. , Habbersett Robert C.
Published: June 2004

This unit provides essential knowledge for correctly using any flow cytometer to ensure that data collected are accurate and reliable. Two levels of system alignment are described: routine alignment checks and complete alignment, both of which can be applied to a variety of instrument configurations.
Light Scatter: Detection and Usage
Authors: Salzman Gary C.
Published: June 2004

Light scatter is one of the most basic of measurements made in flow cytometry. This unit presents a short commentary on what light scatter is and how it is measured. First, the author describes the basic principles of the scattering of light from small particles such as cells. This is followed by a discussion of methods for detecting scattered light in a flow cytometer. The unit ends with a brief survey showing how light scattering is used in a broad range of flow cytometry applications.
Time‐Resolved Fluorescence Measurements
Authors: Steinkamp John A.
Published: June 2004

This unit provides a detailed review and technical discussion of the principles of time‐resolved fluorescence. The unique feature of this particular unit is that the principles are directly tied to the use of flow cytometry. There are a number of advantages in being able to discriminate between fluorochromes based upon their lifetimes as opposed to their emission wavelengths. One particularly good application is the determination of bound versus free molecules. The author covers both the theoretical and practical components of measuring fluorescence in the time domain. In addition, the unit provides a listing of the most common fluorescent dyes with comparative data of lifetime and excitation conditions.
Plug Flow Cytometry
Authors: Edwards Bruce S. , Sklar Larry A.
Published: June 2004

Although flow cytometry has the powerful ability to rapidly screen large collections of cells, the technology has yet to be efficiently applied to large‐scale screening operations involving multiple discrete suspensions. High‐throughput flow cytometry would be beneficial to many areas of biological investigation, such as modern drug discovery, which involves testing of cellular targets against millions of potentially valuable compounds. The authors have developed a flow injection analysis approach to automated sample handling in which individual sample suspensions are sequentially inserted as plugs of precisely defined volumes into a flowing fluid which delivers them to the laser beam. The unit describes the basic elements and concepts of this plug flow system and discusses representative applications.
Dynamic Thermoregulation of the Sample in Flow Cytometry
Authors: Graves Steven W. , Habbersett Robert C. , Nolan John P.
Published: June 2004

Fine control of temperature is an important capability for any analytical platform. A circulating water bath has been the traditional means of maintaining constant temperature in the sample chamber of a flow cytometer, but this approach does not permit rapid changes in sample temperature. This unit explains the use of Peltier modules for regulation of sample temperature. The heat pumping generated by the passage of current through properly matched semiconductors, known as the Peltier effect, makes it possible for these thermoelectric modules to both heat and cool. The authors describe the construction of a Peltier module based thermoregulation unit in step‐by‐step detail and present a demonstration of flow cytometry measurements as a function of temperature.
Excitation and Emission Spectra of Common Dyes
Authors: Shapiro Howard M.
Published: June 2004

This unit tabulates fluorescence excitation and emission spectra of a number of fluorescent probes and labels commonly used in cytometry. This information facilitates selection of probes, light sources, and filters to be used in multicolor experiments.
Contrast Enhancement in Light Microscopy
Authors: Keller H. Ernst
Published: June 2004

The optical microscope is a fundamental component of an image cytometry system. Electronic imaging has both greatly enhanced the capabilities of light microscopes and placed ever‐increasing demands on their optical performance. This unit covers the basic concepts of light microscopy, including Köhler illumination, resolution, contrast, and numerical aperture, and reviews the many types of instruments and techniques for contrast enhancement.
Cameras
Authors: Castleman Kenneth R.
Published: June 2004

Another critical component of an image cytometer is the camera, which converts the optical image to an electrical form to view on a monitor, record, or digitize for subsequent analysis. Any image degradation introduced at this stage affects the quality and accuracy of the system output. This unit discusses the fundamentals of camera phenomena and presents extensive data on available CCD chips and cameras.
Optical Filtering Systems for Wavelength Selection in Light Microscopy
Authors: Waggoner Alan S. , Wachman Elliot S. , Farkas Daniel L.
Published: June 2004

Because of its sensitivity and specificity, fluorescence‐based detection is one of the foremost methods for microscopic imaging of biological tissues and cells. Dramatic improvements in filter system design and implementation coupled with development of an ever‐widening range of sensitive fluorescent dyes have made multicolor imaging a powerful tool for structural and functional analysis. This revised and expanded unit reviews some of the main principles and developments of optical filtering.
Calibration: Sampling Density and Spatial Resolution
Authors: Young Ian T.
Published: June 2004

This unit presents a discussion of procedures for measuring the sampling density and spatial resolution of a quantitative microscope system. These two independent quantities are fundamental characteristics of a system that must be known for proper processing and interpretation of digitized microscope images and of measurements extracted from such images.
Microscope Alignment
Authors: Friedman Marc M. , Abramowitz Mortimer
Published: June 2004

In order to obtain good images using any microscopic imaging modalities, it is necessary to understand the microscope thoroughly and set up properly. This unit should be required reading for all students and staff using a light microscope. It clearly defines the types of illumination used in microscopes and details the correct set up and operation.

Keywords: light microscopes; Köhler illumination; microscope alignment.
Multiphoton Imaging
Authors: Amos Brad
Published: June 2004

This unit presents practical details and summary for biologists of state‐of‐the‐art multiphoton imaging in cytology. The usefulness of multiphoton imaging is critically discussed and compared with confocal. Details of laboratory setup are also included.
Method for Visualizing Aerosol Contamination in Flow Sorters
Authors: Oberyszyn Andrew S.
Published: June 2004

The routine operation of a flow cytometer generates aerosols, and most commercially available instrument are not designed to be 100% effective in their elimination. Aerosols are a concern for operators as well as for others in the facility because of the potential for exposure to contamination, especially during sorting of biohazardous samples. This unit describes a visual method for examining aerosol containment using a commercial resin that fluoresces under UV or black light illumination. The method is rapid, inexpensive, and simple. Results are immediate and potentially quantitative
Titering Antibodies
Authors: Stewart Carleton C. , Stewart Sigrid J.
Published: June 2004

Flow cytometry using fluorochrome‐conjugated antibodies has emerged as a major approach to automated cellular identification. One of the most important issues in immunophenotyping is using the correct amount of antibody. This unit presents techniques for ascertaining the optimal titer for individual, dual, and multiple antibodies used for simultaneous phenotyping, stressing the importance of quality control in making batches of antibody for routine use. Keywords: flow cytometry; monoclonal antibodies; antibody titration; immunophenotyping.
Conjugation of Fluorochromes to Monoclonal Antibodies
Authors: Holmes Kevin L. , Lantz Larry M. , Russ Wesley
Published: June 2004

Detection of cell surface molecules labeled by monoclonal or polyclonal antibodies conjugated to a fluorochrome is probably the most widely used application of flow cytometry. This unit contains protocols for tagging monoclonal antibodies with fluorescein, biotin, Texas Red, and phycobiliproteins. In addition, it provides a procedure for preparing a PE‐Texas Red tandem conjugate dye that can then be used for antibody conjugation. These protocols enable investigators to label antibodies of their choice with multiple fluorochromes and permit more combinations of antibodies for multicolor flow applications.

Keywords: flow cytometry; monoclonal antibodies; fluorochromes; antibody labeling.
Nucleic Acid Probes
Authors: Poot Martin
Published: June 2004

This unit describes the physicochemical structures of commonly used nucleic acid probes, classified into four groups based on their mode of nucleic acid binding. The unit provides an excellent background for the many protocols employing these dyes and should be a prerequisite for any studies involving nuclear probes.
Cellular Function Probes
Authors: Johnson Iain D.
Published: June 2004

This unit covers many of the fluorescent probes used in cell biology, such as green fluorescent protein, esterase and peptidase substrates, intracellular thiols, lipid probes, and probes for intracellular ions. It brings to light the chemical relationships between certain probes and relates these properties to their functional application. Correct use of these probes requires an understanding of how they are taken up and distributed within the cell and where appropriate, what factors will affect the fluorescence emission from the probe. This is particularly important for probes of pH and calcium flux.
Handling, Storage, and Preparation of Human Blood Cells
Authors: McCoy, Jr. J. Philip
Published: June 2004

Human peripheral blood samples are probably the most common specimens submitted for flow cytometric analysis. Handling and preparation will vary depending on the cell lineage to be examined and the type of assay. This unit presents protocols for separating white cells by lysing erythrocytes, isolating mononuclear cells by trilineage separation, and assorted methods for isolating or enriching specific cell populations, including monocytes, neutrophils, T lymphocytes, B lymphocytes, NK cells, stem cells, and platelets. Keywords: flow cytometry; human blood; leukocyte preparation; mononuclear blood cells; cell population enrichment; cell population isolation; cell population purification
Handling, Storage, and Preparation of Human Tissues
Authors: Dressler Lynn G. , Visscher Dan
Published: June 2004

Human tissue for flow cytometry must be prepared as an adequate single‐cell suspension. The appropriate methods for tissue collection, transport, storage, and dissociation depend on the cell parameters being measured and the localization of the markers. This unit includes a general method for collecting and transporting human tissue and preparing a tissue imprint. Protocols are supplied for tissue disaggregation by either mechanical or enzymatic means and for preparation of single‐cell suspensions of whole cells from fine‐needle aspirates, pleural effusions, abdominal fluids, or other body fluids. Other protocols detail preparation of intact nuclei from fresh, frozen, or paraffin‐embedded tissue. Support protocols cover fixation, cryospin preparation, cryopreservation, and removal of debris. Keywords: flow cytometry; human tissue; tissue imprint; single‐cell suspension preparation; tissue disaggregation; paraffin‐embedded tissue
Flow Analysis and Sorting of Plant Chromosomes
Authors: Doležel Jaroslav , Macas Jiří , Lucretti Sergio
Published: June 2004

The use of flow cytometry for evaluation of plant chromosomes requires some specialized attention to preparation and instrumentation. This unit deals exclusively with plant cytogenetics and presents an outline of this area as well as methods for accumulation of cells in metaphase, preparation of chromosome suspensions, flow analysis and sorting of chromosomes, and processing of the sorted chromosomes. Each method is described in tremendous detail because in many aspects dealing with plant cells is quite different from dealing with mammalian cells. Supporting histograms are presented as well as a range of special hints on dealing with plant material and a discussion of the utility of sorted chromosomes for plant genome mapping.. Keywords: flow cytometry; plant chromosomes; cell cycle synchronization; chromosome isolation; chromosome sorting; physical gene mapping; chromosome‐specific DNA libraries
Immunophenotyping
Authors: Stewart C.C. , Stewart S.J.
Published: June 2004

This unit presents basic techniques for immunophenotyping by flow cytometry, direct using a conjugated monoclonal antibody and indirect using an unconjugated primary antibody followed by a conjugated secondary antibody. Combinations of these methods are described for two‐, three‐, and four‐color staining. Analysis of data acquired from cells stained by these procedures is detailed. A procedure is given for the detection of the location of nonviable cells so that they can be gated out of the analysis.

Keywords: flow cytometry; immunophenotyping; indirect staining; direct staining; multicolor staining; immunophenotypic analysis.
Enumeration of CD34+ Hematopoietic Stem and Progenitor Cells
Authors: Sutherland D. Robert , Keeney Michael , Gratama Jan W.
Published: June 2004

CD34 is the first documented cell surface antigen whose expression within the hematopoetic system is restricted to stem and progenitor cells of all lineages. Transplantation of CD34+ cells has created a need for accurate and rapid assessment of CD34+ cell concentrations in peripheral blood as well as for the optimal timing of apheresis sessions to insure the harvest of sufficient CD34+ cells. However, accurate enumeration of these cells represents a rare‐event analysis, a demanding and somewhat complicated procedure. This comprehensive unit presents the clearest and most definitive methods yet available for CD34+ enumeration by flow cytometry, evaluates a number of commercially available procedures, and provides invaluable supporting information and detailed analysis which we believe is found in no other publication. All the possible problems are discussed and examples provided, allowing the clinical hematology lab to be certain that analysis of CD34+ cells is performed in an appropriate manner.
Immunophenotypic Analysis of Peripheral Blood Lymphocytes
Authors: Ruiz‐Argüelles Alejandro , Pérez‐Romano Beatriz
Published: June 2004

This unit on basic phenotyping describes two basic and two alternate protocols for the immunophenotypic identification and classification of human peripheral blood lymphocytes. The presented protocols comply with consensus recommendations from professional organizations that regulate the clinical use of such assays. The authors discuss whole blood assay systems as well as enriched systems from several perspectives, including absolute number determination.

Keywords: flow cytometry; peripheral blood; lymphocytes; immunophenotype; lymphocyte subsets
Immunophenotypic Analysis of Human Mast Cells by Flow Cytometry
Authors: Escribano Luis , Navalón Raquel , Núñez Rosa , Díaz Agustín Beatriz , Bravo Pilar
Published: June 2004

The immunophenotypic identification and enumeration of human bone marrow mast cells represents a clear demonstration of the utility of flow cytometry for rare‐event analysis. The basic approach can be applied to a variety of specimens, including bone marrow, peripheral blood, ascitic fluid, and lymphoid tissue such as adenoids. Special emphasis is placed on markers with potential utility for distinguishing between normal, reactive, and pathological mast cells. From the clinical aspect, immunophenotypic analysis of mast cells may have great utility in supporting the diagnosis of tissue involvement in mastocytosis.
Immunophenotypic Identification, Enumeration, and Characterization of Human Peripheral Blood Dendritic Cells and Dendritic‐Cell Precursors
Authors: Almeida Julia , Bueno Clara
Published: June 2004

Study of dendritic cells is complicated by their low frequency and the lack of specific markers for sensitive and specific identification. Furthermore, these cells are not a homogeneous population, but contain a variety of subsets. Flow cytometry has well‐established applications for the analysis of cells present at low frequencies; the use of a two‐step acquisition procedure increases the number of cells for analysis. This unit presents protocols for the identification and enumeration of dendritic cells and cell subsets in erythrocyte‐lysed whole peripheral blood. Special attention is paid to the combinations of reagents used for the identification of all dendritic cells as well as each subset, and on the multiparametric gating strategies for specific analysis.
Immunophenotypic Analysis of Platelets
Authors: Krueger Lori A. , Barnard Marc R. , Frelinger A.L. , Furman Mark I. , Michelson Alan D.
Published: June 2004

Platelets are the smallest cellular component in the peripheral circulation. Their primary role is maintenance of hemostasis. The evaluation of platelets by flow cytometry has proven beneficial in the investigation of many disease states, including inherited defects, cardiovascular disease, stroke, and many other inflammatory processes. In the absence of an added exogenous platelet agonist, the activation state of in vivo circulating platelets can be measured. Inclusion of an exogenous agonist in the assay enables analysis of the reactivity of circulating platelets in vitro. This unit describes protocols to evaluate platelet surface glycoproteins, platelet activation status, and platelet responsiveness to agonist. Detailed discussion of appropriate sample handling, reagent preparation, flow cytometric set‐up, and data interpretation are provided for three independent assays.
Immunophenotypic Analysis of PNH Cells
Authors: Richards Stephen J. , Hillmen Peter
Published: June 2004

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired hematopoietic stem‐cell disorder in which a gene mutation results in cellular inability to synthesize the glycosylphosphatidylinositol (GPI) anchors needed to attach various proteins to the cell membrane. Traditional clinical laboratory PNH screening and diagnosis are based upon demonstration of increased erythrocyte susceptibility to lysis by activated complement. These methods are neither very specific nor very sensitive, and have largely been superseded by the rapid, sensitive, and specific flow cytometric analysis of GPI‐linked antigen expression. The protocols in this unit cover immunophenotypic analysis of red blood cells, peripheral blood granulocytes, and peripheral blood monocytes, as well as a four‐color modification for simultaneous analysis of peripheral blood granulocytes and monocytes.
Quantitative Flow Cytometric Analysis of Membrane Antigen Expression
Authors: D'hautcourt Jean‐Luc
Published: June 2004

Immunological analysis for cell antigens has been performed by flow cytometry in a qualitative fashion for over thirty years. During that time it has become increasingly apparent that quantitative measurements such as number of antigens per cell provide unique and useful information. This unit on quantitative flow cytometry (QFCM) describes the most commonly used protocols, both direct and indirect, and the major methods of analysis for the number of antibody binding sites on a cell or particle. Practical applications include detection of antigen under‐ or overexpression in hematological malignancies, distinguishing between B cell lymphoproliferative disorders, and precise diagnosis of certain rare diseases.
Immunophenotyping Using a Laser Scanning Cytometer
Authors: Tárnok Attila , Gerstner Andreas O.H.
Published: June 2004

Three‐ and four‐color immunophenotyping is routine in traditional flow cytometry, as is measurement of cell proliferation, but there are drawbacks. The techniques cannot analyze cell morphology or permit restaining of cells of interest. This unit describes a slide‐based method of immunophenotyping using a laser scanning cytometer. In general, many assays originally developed for flow can be adapted to LSC. Although the speed of analysis is comparatively slow, LSC has the advantage that cells are not lost. Considerable additional information can be obtained by morphological examination and/or by further staining because specimens can be repeatedly analyzed and archived. The method has potential to become a powerful tool in clinical diagnosis.
Enzymatic Amplification Staining for Cell Surface Antigens
Authors: Kaplan David
Published: June 2004

Many molecules of biological significance function at very low levels and are difficult to detect with the standard staining procedures for flow cytometric analysis. This unit describes a technology for cell staining that enhances the specific fluorescence signal by as much as 100‐fold. The system is based on the enzyme‐catalyzed deposition of a tagged molecule. Enzymatic amplification staining is qualitatively different from the inclusion of additional layers of binding molecules because background fluorescence levels are not increased along with the specific signal. The technique is compatible with multicolor staining. An alternate protocol explains the performance of multiple amplifications on the same cell population by adding a peroxide incubation.
Flow Cytometric Assessment ofHLAAlloantibodies
Authors: Bray Robert A. , Gebel Howard M. , Ellis Thomas M.
Published: June 2004

Antibodies against HLA molecules are formed in response to exposure to foreign HLA molecules, which can occur as a result of blood transfusion, pregnancy, or transplant. Blood components, particularly those containing cellular elements, are the most common cause of HLA antibodies. This unit describes technical aspects of the flow cytometric crossmatch (FCXM), flow cytometric microparticle assays, and cell‐based flow cytometric screening assays. The collective goal for these assays is to clearly identify the presence of HLA antibody, determine the titer of antibody, and elucidate the specificities (i.e., HLA antigens) to which they will react. Knowledge of this information is critical for organ allocation and accurate assessment of the immunological risk for a patient at the time of transplantation. In addition, the identification of HLA antibodies in blood components may be useful in planning appropriate transfusion support strategies for selected patients.
Differential Staining of DNA and RNA
Authors: Darzynkiewicz Zbigniew , Juan Gloria
Published: June 2004

Cell cycle analysis by means of differential staining of RNA and DNA permits determination of RNA content, which in turn allows one to discriminate G0 versus G1 cells and to detect cell differentiation. This unit presents two protocols for differential staining, one using the metachromatic dye acridine orange (AO) and the other a combination of pyronin Y (PY) and Hoechst 33342. Each method has its advantages and limitations, and the Hoechst‐PY method is not applicable to single‐laser instruments.
DNA Content Measurement for DNA Ploidy and Cell Cycle Analysis
Authors: Darzynkiewicz Zbigniew , Juan Gloria
Published: June 2004

This unit presents the most common methods of DNA content analysis. The techniques differ primarily in the mode of cell permeabilization (detergents versus prefixation with alcohol) and composition of the stain solution. Protocols are given for analysis of fixed cells, tissue samples, live cells (for subsequent sorting and culture), archival paraffin‐embedded tissue, and apoptotic cells.