Stability of lymphocytes and Epstein-Barr virus during red blood cell storage.

BACKGROUND: The Epstein-Barr virus (EBV) establishes and maintains latent infection in B lymphocytes of the healthy adults. Lymphocytes remain viable during red blood cell (RBC) storage. The effect of RBC storage on the stability of EBV-infected B lymphocytes and EBV genome is not known. STUDY DESIGN AND METHODS: Eight randomly selected non-leukoreduced AS-5 RBC units were stored for 42 days under standard blood bank refrigerated at 1-6 degrees C. Cell count and EBV genomes in CD19+ B lymphocytes were measured in fresh products and weekly for 6 weeks. Total white blood cells (CD45+), T lymphocyte (CD3+), and B lymphocyte (CD19+) were quantified by a single platform flow cytometric assay. EBV genomes were quantified by real-time polymerase chain reaction using DNA purified from CD19+ B cells. RESULTS: Viable white blood cell, T and B lymphocytes followed a biphasic decline curve during RBC storage consisting of a steep steady decline during the first 3 weeks followed by a plateau for the remainder of the storage. At the end of the RBC shelf-life, 19% of the original T and B cells remained viable. EBV genomes per 10(5) CD19+ B lymphocytes remained constant during RBC storage. However, the total EBV genomes in the RBC units decline by more than 80% of their original value at the end of RBC storage due to loss of viable B lymphocytes. CONCLUSIONS: The results indicate that lymphocytes and EBV latently infected B cells can survive the normal storage conditions for RBC.

The prevalence of feline A/B blood types in the Sydney region.

OBJECTIVE: To determine the distribution of A/B blood types in pedigree and crossbred cats in the Sydney region, and to estimate the associated risk of administering incompatible blood in an unmatched random transfusion. DESIGN: A prospective/retrospective study of blood specimens collected from both sick and healthy cats. MATERIALS AND METHODS: Blood was collected from 355 cats from the Sydney region over a 12-year period from 1992 to 2003. Specimens were obtained from 187 domestic crossbred cats (short and long-haired) and 168 pedigree cats. The blood type of each cat was determined by one of three different laboratories using standard methods that varied over the duration of the survey. RESULTS: The distributions of blood types obtained by the three laboratories were not significantly different. The prevalence of type-A, type-B and type-AB blood types in crossbred cats was 62%, 36% and 1.6%, respectively. This is the highest percentage of type-B cats so far reported for an outbred population of domestic cats, and is significantly higher than the 26% reported previously for cats in the Brisbane region. The calculated frequency for the type-B allele assuming Hardy-Weinberg equilibrium for this feline population is 0.60; the corresponding frequency of the type-A allele is thus approximately 0.40. The calculated proportion of random transfusions from this population giving rise to an incompatible blood transfusion is 46%, with half of these being life-threatening events. The calculated proportion of random matings from this population at risk for developing neonatal isoerythrolysis is 23%. The distribution of A and B blood types for pedigree cats was in general agreement with data reported previously for cats in North America and Europe, suggesting that the distribution of blood types in these purebred populations is relatively consistent throughout the world. CONCLUSIONS: The prevalence of type B cats in the owned domestic and pedigree cat population is so high that blood typing or cross matching prior to transfusion should be mandatory, except in Siamese/Oriental cats.

Viability of cryopreserved BM progenitor cells stored for more than a decade.

BACKGROUND: PBPC or BM is increasingly being harvested in remission for possible use in the event of relapse. Although the value of this approach has not been demonstrated, the long-term storage of progenitor cell components has become commonplace in many facilities. METHODS: We used multi-parameter flow cytometry to determine the viability of 11 long-term cryopreserved BM components (mean = 11.8 years) in liquid phase nitrogen. The components, prepared for autotransplantation but deaccessioned after confirming patient death, were carefully thawed, washed, and assayed immediately. The flow cytometry assay was performed according to the ISHAGE protocol, modified by the addition of 7AAD for analysis of progenitor viability (CD45+ CD34+ 7AAD-) and total leukocyte viability (CD45+ 7AAD-). In addition, total viability was assessed by fluorescence microscopy using acridine orange dye exclusion; granulocyte-monocyte colony-forming units (CFU-GM) were measured after 14 days culture. RESULTS: Leukocyte viability by flow cytometry and fluorescence microscopy agreed well (r2 = 0.55, slope = 0.83, P < 0.0005 by linear regression). CFU-GM did not correlate with CD34% or any of the viability parameters. Compared with short-term stored (mean = 33 days) PBPC assayed at infusion, long-term stored BM had a comparable percentage of CD34+ cells, comparable CFU-GM activity, increased CD34 viability, but decreased total cell viability, the latter most likely due to an increased proportion of differentiated myeloid cells. DISCUSSION: The results indicate that BM products can be cryopreserved for more than a decade without apparent loss of progenitor activity, as measured by these laboratory surrogates. This agrees with clinical anecdotes describing successful engraftment with long-term stored BM, and argues that expiration dates cannot be set for cryopreserved hematopoietic stem-cell components stored in liquid phase nitrogen.

Solid phase extraction gas chromatography/electron capture detector method for the determination of organochlorine pesticides in wildlife whole blood.

A gas chromatographic method for the analysis of 10 organochlorine pesticides in 0.5 mL of whole blood is described. Sample preparation involved an ethyl ether and hexane extraction, followed by a silica solid phase extraction cleanup. The pesticides are quantified by gas chromatography/electron capture detection. Method limits of detection ranged from 1.1 to 5.2 microg/L. The mean and standard deviation for the recovery of 10 pesticides was 97.9 +/- 5.5%. Recoveries from whole blood were comparable to recoveries from plasma. This indicates that the preparation of plasma is unnecessary for the quantification of organochlorine pesticides in blood. This approach is particularly useful as a nonlethal approach for monitoring pesticide contamination in small animals for which the volume of blood is limiting.

Reversed-phase ion-pair liquid chromatographic determination of chlorophacinone and diphacinone in steam-rolled oat baits and steam-rolled oat/wax baits.

A reversed-phase ion-pair liquid chromatographic (LC) method was developed for analysis of steam-rolled oat (SRO) baits fortified with either chlorophacinone or diphacinone. Baits were prepared with and without paraffin wax. Chlorophacinone or diphacinone was extracted from wax-free SRO baits with 5 mM tetrabutylammonium phosphate methanolic ion-pairing solution. Wax baits were initially extracted with petroleum ether and then cleaned up by liquid extraction into methanolic ion-pairing solution containing 20% water. SRO extracts were analyzed with reversed-phase ion-pair LC. Chlorophacinone and diphacinone were quantified by UV absorption at 325 nm. Recoveries from SRO fortified with chlorophacinone at 25 and 150 micrograms/g were 90.7 and 90.8%, respectively, whereas for diphacinone at the same levels, recoveries were 93.5 and 92.3%, respectively. Recoveries from wax baits fortified at 25 and 75 micrograms/g chlorophacinone were 98.5 and 100%, respectively, whereas for diphacinone at the same levels, recoveries were 93.6 and 98.0%, respectively. Method limits of detection for chlorophacinone and diphacinone in SRO baits were estimated to be 1.0 and 0.76 micrograms/g, respectively. Method limits of detection for chlorophacinone and diphacinone in wax baits were estimated to be 4.2 and 2.8 micrograms/g, respectively.

Evaluation of monoclonal antibody-mediated anti-acute myeloid leukemia immunotherapy in a SCID/hu model.

The therapeutic potential of the IgM complement-fixing murine monoclonal antibody (mAb) PM-81 (anti-CD15) against acute myeloid leukemia (AML) was assessed in a SCID/hu leukemia model. Intraperitoneal (i.p.) injection of NB4 leukemia cells resulted in aggressive growth of leukemia cells in the peritoneal cavity of irradiated SCID/CB-17 mice. Flow cytometric analysis of human CD15, 33 and 45 expression, as well as cytologic examination, revealed that leukemia cells disseminated into the peripheral blood and multiple tissues of the mice. The approximately linear relationship between the injected leukemia cells and the subsequent leukemia cell proliferation provided a reliable model for monitoring the therapeutic effects of immunotherapy. Intraperitoneal injection of the mAb PM-81 markedly suppressed leukemia cell growth in this SCID/leukemia model. Most of the untreated mice died within 35-50 days of leukemia cell inoculation. Four weeks after inoculation of NB4 cells, five of nine mAb PM-81 treated mice had no solid tumor growth and six of nine had no detectable peritoneal exudate leukemia cells as determined by flow cytometry. In contrast, 100% of the mice in the untreated or control mAb groups were found to have both solid and peritoneal leukemia growth. In further experiments designed to evaluate the effects of therapy on survival, 50% (4/8) of PM-81 treated mice survived to 150 days, and had no detectable solid or suspension leukemia cells detectable at necropsy. In contrast, the median survival of untreated or negative control antibody-treated mice was 40 days (comparison to PM-81 treated; p = 0.006 and p = 0.03, respectively). The mechanism of leukemia cell suppression is not likely due to complement fixation since we could not demonstrate in vitro any cytotoxicity mediated by SCID mouse plasma. Further study is required to understand the mechanism of the antileukemia effect of PM-81 in this model.

Effect of ingesta and of tablets of different strengths on the systemic availability of digoxin in normal dogs.

The plasma concentrations of digoxin were measured in eight normal dogs given digoxin on four occasions, using three different feeding regimens and tablets of two strengths. Although ingesta tended to slow the absorption of digoxin, the systemic availability of the drug, based on measurements of Cmax, tmax and AUC did not differ when digoxin tablets were given with canned food, with dry food, or without food. However, some of the pharmacokinetic characteristics and smaller individual variations with the dry food regimen would be considered advantageous for maintenance therapy. Tablets containing 62.5 micrograms or 250 micrograms of digoxin had a similar relative bioavailability. The peak plasma digoxin concentrations were higher in female dogs, and the trends in other data also suggested that the systemic availability of digoxin was better in female dogs.

An evaluation of 4 commercially available ELISA kits for the diagnosis of Dirofilaria immitis infection in dogs.

Serum samples from 100 pound dogs were used to evaluate 4 commercial ELISA kits available for the diagnosis of Dirofilaria immitis. The kits were assessed on sensitivity (the ability to identify infected dogs), specificity (the ability to identify uninfected dogs) and accuracy (sensitivity plus specificity). The kits varied in sensitivity from 36% to 86%, in specificity from 44% to 70%, and in accuracy from 53% to 65%. The sensitivity was not affected by the age of the dogs, nor by the number of circulating microfilariae. The kits were most specific when testing the youngest dogs (less than = 3 years). The problems associated with the serological diagnosis of D. immitis infection in practice are discussed.

Home health care: drug-related problems detected by consultant pharmacist participation.

Consultant pharmacists conducted a drug history and regimen review on one-fourth of the patient population served by a community hospital-based home health department. About one-third of study patients had inappropriately stopped taking their medications; over one-fourth of the study patients had changed their schedule or dose without health professional consultation. Fifteen percent of all medications were duplications, and only 8% of prescription medications were correctly identified by name. Consultant pharmacist activities that could reduce drug-related problems were identified as drug therapy consultation with patients and staff, drug regimen review, drug information, and patient medication education.

Resistance to benzimidazole anthelmintics in equine strongyles. 1. Frequency, geographical distribution and relationship between occurrence, animal husbandry procedures and anthelmintic usage.

A survey was conducted to determine whether benzimidazole resistant populations of equine strongyles are present in New South Wales and north central Victoria; what is their frequency and geographical distribution; which species are involved; and whether different methods of parasite control could be related to the occurrence and frequency of anthelmintic resistant populations. Resistant populations of strongyles were found over wide areas of New South Wales and in north central Victoria. There was no relationship between geographical location and the occurrence of benzimidazole resistance. The species involved were small strongyles of the sub-family Cyathostominae. There was a direct correlation between the occurrence of resistance (including the level at which it is present) and the frequency of use of benzimidazole anthelmintics. Examination of management practices showed that resistance is not an important problem on farms where different chemical classes of anthelmintics were used in a slow rotation programme; combination anthelmintic therapy (for example, benzimidazole/piperazine/organophosphates) was used and anthelmintic treatment was given at intervals of not less than 16 weeks. Tentative suggestions are made for the control of small strongyles in the light of an emerging resistance problem.

Trichostrongylus colubriformis and Ostertagia circumcincta resistant to levamisole, morantel tartrate and thiabendazole: occurrence of field strains.

Field strains of Trichostrongylus colubriformis and Ostertagia circumcincta, designated PF4 and PF5 respectively, were recovered from a farm on which the sole use of levamisole over a preceding 12 year period led to the development of anthelmintic resistance. The results of field observations and preliminary critical trials in both Merino and crossbred sheep showed that both species have varying degrees of resistance to three major anthelmintics; levamisole, morantel tartrate and thiabendazole. Mean worm count reductions for adult T colubriformis (PF4) for therapeutic doses of morantel tartrate, thiabendazole and levamisole in crossbreds were 45.7 per cent, 97.3 per cent and zero respectively, and for Merinos 80.7 per cent, 88.3 per cent and 92.0 per cent respectively. Against O circumcincta the corresponding reductions for crossbreds were 51.4 percent, 95.4 per cent and 20.3 per cent and for Merinos, 52.5 per cent, 73.1 per cent and 29.8 per cent. There was no statistically significant difference in the responses of both parasite species to either levamisole or morantel. This result suggests that resistance to the two chemically unrelated drugs may be co-inherited.