Stabilization media increases recovery in paucicellular cerebrospinal fluid specimens submitted for flow cytometry testing.

BACKGROUND: Flow cytometric immunophenotpying (FCI) of cerebrospinal fluid (CSF) and other paucicellular fluids has been demonstrated to have increased sensitivity in detection of lymphoma and leukemia when compared to cytomorphology [(1) de Graaf et al., Cytometry Part B 2011, 80B:271-281; (2) Szamosi et al., CLSI Document H56-A-Body Fluid Analysis for Cellular Composition; Approved Guideline, Wayne, PA: Clinical and Laboratory Standards Institute, 2006; (3) Kraan et al., Flow Cytometric Immunophenotyping of Cerebrospinal Fluid. Current Protocols in Cytometry, Hoboken, NJ: Wiley, 2008]. However, low cellularity has been an historical problem with these samples. Several studies indicate that immediate addition of a stabilization media (e.g., RPMI with fetal calf serum (FCS)) to CSF improves the cell yield for FCI [(1) de Graaf et al.]. Such stabilization medias can, however, significantly increase cost. METHODS: We compared FCI results in CSF stabilized with RPMI 1640 (without additional additives) to results obtained using non-stabilized CSF. Samples were processed according to published CLSI guidelines [(2) Szamosi et al.]. RESULTS: About 98/105 (93%) CSF specimens stabilized with RPMI had adequate numbers of viable cells (>100) for performing FCI. About 65/217 (30%) CSF specimens without stabilization had adequate numbers of viable cells for analysis (70% either quantity not sufficient (QNS) or specimen viability below analytical limits). CONCLUSIONS: Utilizing RMPI without FCS as a stabilization media results in increased cell yield and improved FCI results. We have found FCS is not required to achieve high quality results in FCI of paucicellular CSF specimens.

Ehrlichia equi infection of horses from Minnesota and Wisconsin: detection of seroconversion and acute disease investigation.

Equine granulocytic ehrlichiosis (EGE) is caused by infection with Ehrlichia equi. EGE has been reported primarily in northern California, where E equi is transmitted by the tick Ixodes pacificus. Reports of EGE and the emergence of human granulocytic ehrlichia in Minnesota prompted a seroprevalence study of E equi in horses of Minnesota and Wisconsin. Tick (Ixodes scapularis) endemic areas of Minnesota and Wisconsin were compared to nonendemic regions of Minnesota. Indirect fluorescent antibody was used to detect the presence of serum antibodies to E equi. Serum samples from healthy horses, 375 samples from I scapularis endemic counties, and 366 samples from nonendemic counties were screened at a 1:40 dilution. Results demonstrated a seroprevalence of 17.6% in endemic areas versus 3.8% in nonendemic areas. Ehrlichial DNA from 2 samples was successfully amplified by polymerase chain reaction and 919 base pairs were sequenced. The DNA sequence of 1 Minnesota/Wisconsin strain differed from the GenBank strain (M73223) of E equi at positions 84 and 886 and from the MRK strain of E equi at position 84, and was identical to the human granulocytic ehrlichiosis (HGE) agent. The 2nd Minnesota/Wisconsin strain was identical to the 1st with the exception of a substitution of "A" at position 453 that is not present in E phagocytophila, E equi, or HGE agent strain sequences. Based on the results of this study, we concluded that E equi is present and causes infection in horses in Minnesota and Wisconsin. The occurrence of infection is higher in tick endemic regions.

Invasion and intracellular development of the human granulocytic ehrlichiosis agent in tick cell culture.

Human granulocytotropic ehrlichias are tick-borne bacterial pathogens that cause an acute, life-threatening illness, human granulocytic ehrlichiosis (HGE). Ehrlichias within neutrophil granulocytes that invade tick bite sites are likely ingested by the vector, to be transmitted to another mammalian host during the tick's next blood meal. Thus, the cycle of replication and development in the vector is prerequisite to mammalian infection, and yet these events have not been described. We report tick cell culture isolation of two strains of the HGE agent directly from an infected horse and a dog and have also established a human isolate from HL60 culture in tick cells, proving that the blood stages of the HGE agent are infectious for tick cells, as are those replicating in the human cell line HL60. This required changes to the culture system, including a new tick cell line. In tick cell layers, the HGE agent induced foci of infection that caused necrotic plaques and eventual destruction of the culture. Using the human isolate and electron microscopy, we monitored adhesion, internalization, and replication in vector tick cells. Both electron-lucent and -dense forms adhered to and entered cells by a mechanism reminiscent of phagocytosis. Ehrlichial cell division was initiated soon after, resulting in endosomes filled with numerous ehrlichias. During early development, pale ehrlichias with a tight cell wall dominated, but by day 2, individual bacteria condensed into dark forms with a rippled membrane. These may become compacted into clumps where individual organisms are barely discernible. Whether these are part of an ehrlichia life cycle or are degenerating is unknown.

Career development for nurses in today's health care environment and the value of nontraditional roles.

Roles that were essential in the health care setting of the past are rapidly disappearing in today's era of redesign. Career development and advancement are tumultuous processes in the current health care environment. The article illustrates what nontraditional roles can offer nurses through a case study on the contract negotiation process. It is hoped that the article will encourage nurses to take risk in choosing career experiences. Such choices will facilitate the learning that is necessary to lead the complex health care organizations of today.

Bridging town and gown: building research partnerships between community-based professional providers and academia.

Strategic research partnerships between community-based professionals and academic researchers are increasingly recognized as a means of evolving evidence-based practice and policy directions in health services delivery. Many barriers impede collaboration, however, and information on how to successfully implement collaborative partnerships is scattered, leaving much to trial and error. This article describes the process of achieving a successful research partnership among community-based home care professionals and academic researchers during a 6-year period of collaboration. The case illustration portrays how to identify and motivate the right research partners, how to mobilize resources, how to sustain an effective partnership process during the lengthy time commitments required to complete research, and how to ensure the experience of return on investment of time, energy, and resources in the partnership process. As measured by the outcomes of their collaborative research experience, the authors conclude that the benefits of bridging town and gown greatly outweigh the challenges of research partnerships.

Natural infection of small mammal species in Minnesota with the agent of human granulocytic ehrlichiosis.

The natural reservoirs for the agent of human granulocytic ehrlichiosis (HGE) are suspected to be the small mammals that host immature stages of Ixodes scapularis ticks. To determine if such small mammals are naturally infected, we collected blood and serum samples from small mammal species in rural and suburban areas of Minneapolis and St. Paul, Minn. Samples were collected from white-footed mice (Peromyscus leucopus), eastern chipmunks (Tamias striatus), southern red-backed voles (Clethrionomys gapperi), and insectivorous shrews (Blarina brevicauda and Sorex cinereus). Blood samples were tested by PCR for active infection with the HGE agent, and sera from P. leucopus mice were tested for serologic evidence of infection by indirect immunofluorescence. PCR analyses revealed the presence of HGE agent DNA in 20 of the 190 samples (10.5%) tested. Of the 119 P. leucopus mouse serum samples that were analyzed, 12 (10.1%) contained Ehrlichia equi antibodies. In 3 of 119 (2.5%) P. leucopus mice from which both blood and serum were collected. HGE agent DNA and antibodies against E. equi were present. Animals with evidence of infection with the HGE agent are widely distributed around the Minneapolis-St. Paul area in regions with known I. scapularis tick activity. Small mammals that are frequent hosts for larval I. scapularis ticks and that are found in areas where HGE occurs are likely to be a major reservoir from which infected ticks that bite humans are derived.

Geographic, clinical, serologic, and molecular evidence of granulocytic ehrlichiosis, a likely zoonotic disease, in Minnesota and Wisconsin dogs.

Seventeen Minnesota and Wisconsin dogs with granulocytic ehrlichosis were studied. The diagnoses were made by finding ehrlichia morulae in peripheral blood neutrophils. Eight dogs were studied retrospectively, and nine dogs were studied prospectively. The medical records of all dogs were reviewed. Eighty-eight percent of the dogs were purebred and 76% were spayed females. The median age was 8 years. Sixty-five percent of the cases were diagnosed in October and November. Fever and lethargy were the most common clinical signs. The most frequent laboratory findings were lymphopenia, thrombocytopenia, elevated activities of serum alkaline phosphatase and amylase, and hypoalbuminemia. No dogs seroreacted to Ehrlichia canis or Ehrlichia chaffeensis antigens, which are cross-reactive. Seventy-five percent of the dogs tested during the acute phase of disease and 100% of the dogs tested during convalescence were seropositive for E. equi antigens. Granulocytic ehrlichial 16S rRNA gene DNAs from six dogs were amplified by PCR. Sequence analysis of a 919-bp sequence of the ehrlichial 16S rRNA gene amplified by PCR from the blood of two dogs revealed the agent to be identical to the agent of human granulocytic ehrlichiosis in Minnesota and Wisconsin and to be very similar to E. equi and Ehrlichia phagocytophila and less similar to E. canis, Ehrlichia ewingii, and E. chaffeensis. The geographic, clinical, serologic, and molecular evidence indicates that granulocytic ehrlichiosis in Minnesota and Wisconsin dogs is not caused by E. ewingii, but suggests that it is a zoonotic disease caused by an agent closely related to E. equi and that dogs likely contribute to the enzootic cycle and human infection.

Neospora caninum pneumonia in an adult dog.

Neospora caninum was identified in a lung aspirate specimen from an adult dog with severe pneumonia. Neosporosis was tentatively diagnosed by cytologic examination of a Wright-Giemsa-stained smear of the aspirate specimen, using light microscopy. The infection was confirmed by immunohistochemical examination of a section of lung tissue obtained at necropsy. Neosporosis is usually a fatal ascending neurologic disease of dogs less than 1 year of age. Neospora caninum infections are uncommon in adult dogs and do not have a consistent clinical course. The disease in the dog of this report was unique because the dog had clinical respiratory tract disease and because preliminary diagnosis was made by cytologic examination.

Use of hexadimethrine bromide as a heparin-neutralizing agent in canine plasma.

Hexadimethrine bromide was evaluated as a heparin-neutralizing agent in a simple modification of the activated partial thromboplastin time test in canine plasma. Addition of various amounts of heparin in vitro to canine plasma indicated that heparin could be neutralized by adding 0.5 micrograms of hexadimethrine bromide 15 s before CaCl2 was added to the reaction mixture of the activated partial thromboplastin time test. In 8 dogs given (subcutaneous injection) 500 USP units of sodium heparin/kg, marked individual variations in clotting time prolongations were observed over the 12-hour period of study. The hexadimethrine bromide modification effectively neutralized the heparin-related clotting time prolongations to values that were not significantly different from base-line (preheparin) activated partial thromboplastin time values. The modification seems to be useful in confirming the presence of heparin and in monitoring heparin therapy in dogs.