Effect of cryopreservation on a rare McLeod donor red blood cell concentrate.

Units of red blood cell (RBC) concentrates with rare phenotypes are typically not included in method validation studies for cryopreservation processes; rather, they are reserved for patients with rare blood needs. Some rare RBC phenotypes may demonstrate membrane abnormalities, like acanthocytosis as observed for RBCs with the McLeod phenotype, and are specifically banked for these rare attributes; however, the impact that rare RBC phenotypes have on post-thaw quality has not been well studied. To evaluate how a rare RBC phenotype is affected by the cryopreservation process, 4 RBC units, cryopreserved in 1993 using manual methods, were selected for evaluation. These RBCs included one with the McLeod phenotype and three with phenotypes not known to cause significant membrane changes. Post-thaw, an altered deglycerolization protocol, implemented to reduce supernatant glycerol after cryopreservation, was used before processing RBCs on an automated closed system (ACP 215; Haemonetics, Boston, MA) to accommodate the use of a closed system cell processor not available when the RBC units were previously cryopreserved. RBC quality was tested at 24 hours, 7 days, and 14 days post-deglycerolization. Before deglycerolization, an extracted sample from the thawed glycerolized RBC unit was used to obtain genetic material for phenotype confirmation. Genotyping confirmed the McLeod phenotype. When comparing McLeod with non-McLeod units, RBCs from the McLeod donor exhibited acanthocytosis, higher rigidity, and lower morphology scores than RBCs from the non-McLeod units post-deglycerolization. Hemolysis, however, was comparable across all 4 units, meeting regulatory standards. Therefore, McLeod RBCs can withstand cryopreservation, suggesting that units from these donors, glycerolized using older methods, can be deglycerolized using the ACP 215 and stored hypothermically for 14 days. It was also determined that genotyping can be performed on non-leukocyte-reduced cryopreserved RBCs, allowing for confirmation of genetic profiles of donor units banked before the implementation of molecular methods.Units of red blood cell (RBC) concentrates with rare phenotypes are typically not included in method validation studies for cryopreservation processes; rather, they are reserved for patients with rare blood needs. Some rare RBC phenotypes may demonstrate membrane abnormalities, like acanthocytosis as observed for RBCs with the McLeod phenotype, and are specifically banked for these rare attributes; however, the impact that rare RBC phenotypes have on post-thaw quality has not been well studied. To evaluate how a rare RBC phenotype is affected by the cryopreservation process, 4 RBC units, cryopreserved in 1993 using manual methods, were selected for evaluation. These RBCs included one with the McLeod phenotype and three with phenotypes not known to cause significant membrane changes. Post-thaw, an altered deglycerolization protocol, implemented to reduce supernatant glycerol after cryopreservation, was used before processing RBCs on an automated closed system (ACP 215; Haemonetics, Boston, MA) to accommodate the use of a closed system cell processor not available when the RBC units were previously cryopreserved. RBC quality was tested at 24 hours, 7 days, and 14 days post-deglycerolization. Before deglycerolization, an extracted sample from the thawed glycerolized RBC unit was used to obtain genetic material for phenotype confirmation. Genotyping confirmed the McLeod phenotype. When comparing McLeod with non-McLeod units, RBCs from the McLeod donor exhibited acanthocytosis, higher rigidity, and lower morphology scores than RBCs from the non-McLeod units post-deglycerolization. Hemolysis, however, was comparable across all 4 units, meeting regulatory standards. Therefore, McLeod RBCs can withstand cryopreservation, suggesting that units from these donors, glycerolized using older methods, can be deglycerolized using the ACP 215 and stored hypothermically for 14 days. It was also determined that genotyping can be performed on non-leukocyte­reduced cryopreserved RBCs, allowing for confirmation of genetic profiles of donor units banked before the implementation of molecular methods.

An investigation of red blood cell concentrate quality during storage in paediatric-sized polyvinylchloride bags plasticized with alternatives to di-2-ethylhexyl phthalate (DEHP).

BACKGROUND AND OBJECTIVES: Di-2-ethylhexyl phthalate (DEHP) is a blood bag plasticizer. It is also a toxin, raising concerns for vulnerable populations, for example, neonates and infants. Here, the in vitro quality of red cell concentrates (RCC) stored in paediatric bags formulated with alternative plasticizers to DEHP was compared. MATERIALS AND METHODS: RCC were pooled and split into polyvinylchloride (PVC)/DEHP, PVC/1,2-cyclohexanedicarboxylic acid diisononyl ester (DINCH) or PVC/butyryl trihexyl citrate (BTHC) bags. Quality was assessed on storage days 5, 21, 35 and 43. RESULTS: Metabolism differed among the bags: pCO2 levels were lowest and pO2 were highest in BTHC bags. Glucose consumption and lactate production suggested higher metabolic rates in BTHC bags. ATP levels were best maintained in DINCH bags (day 43 mean level: 2·86 ± 0·29 µmol/g Hb). RCC in BTHC bags had the greatest potassium release (54·6 ± 3·0 mm on day 43). From day 21, haemolysis was higher in BTHC bags (P < 0·01) and by day 43 had exceeded 0·8% (0·85 ± 0·10%). RCC in BTHC bags showed more microparticle formation than RCC in DEHP or DINCH bags. CONCLUSION: The results suggest that the BTHC formulation used was detrimental to RBC quality. DINCH bags could be a viable alternative to DEHP: they outperformed DEHP bags energetically, with better maintenance of ATP levels.

The effect of processing method on the in vitro characteristics of red blood cell products.

BACKGROUND AND OBJECTIVES: While the clinical impact of differences in red blood cell (RBC) component processing methods is unknown, there are concerns they may be confounding variables in studies such as the ongoing 'age of blood' investigations. Here, we compare the in vitro characteristics of red cell concentrates (RCCs) produced by several different processing methods. MATERIALS AND METHODS: Nine processing methods were examined: three apheresis methods (Alyx, MCS+ and Trima), as well as leucoreduced whole blood-derived RCCs produced by buffy coat and whole blood filtration and non-leucoreduced RCCs. RCCs were stored in saline-adenine-glucose-mannitol or additive solutions (AS) 1 or 3 for 42 days, with quality tested on day 5 and day 42. RESULTS: Many significant product differences were observed both early in and at the end of storage. Mean haemoglobin (Hb) ranged from 52 to 71 g/unit and mean Hct from 59·5 to 64·8%. Most RCC passed regulated quality control criteria according to Canadian Standards Association guidelines, although there were some failures relating to Hb content and residual WBC counts. CONCLUSION: Processing method impacts RCC characteristics throughout storage; better understanding of these differences and reporting of processing method details is critical.

Adult age confounds estimates of static allometric slopes in a vertebrate.

In many animal groups, the size of male genitalia scales shallowly with individual body size. This widespread pattern appears to admit some exceptions. For instance, steep allometries have been reported for vertebrate genitalia. This exception, however, may be due to a confounding effect arising from the continued growth of some structures during adulthood in vertebrates. Consider the possibility that genitalia continue to grow in adults while body size does not. If so, taking measurements from adults of different ages could yield steeper allometries than would be obtained from measurements of adults of the same age. We used vervet monkeys to test this hypothesis. We found that all body parts continued to grow in adult vervet monkeys, with sexual traits (including genitalia) showing faster growth rates. Traits with faster growth rates over adult ages had steeper allometries. And accounting for variation in adult age yielded shallower allometries, bringing vervet monkey genitalia in line with the predominant pattern observed in other animal groups. These results suggest that steep allometric slope estimates reported for other vertebrates may be due in part to mixing of adult ages, and reinforces one of the most consistent patterns yet detected in the study of static allometry.

The effect of timing of gamma-irradiation on hemolysis and potassium release in leukoreduced red cell concentrates stored in SAGM.

While irradiation of red cell concentrates (RCC) prevents graft-versus-host disease in susceptible transfusion recipients, it also damages red blood cells (RBC). To understand the ability of irradiation regulations to prevent transfusion of inferior units, we irradiated 980 RCC in saline-adenine-glucose-mannitol (SAGM) using various combinations of pre-irradiation age and post-irradiation storage times, and measured hemolysis and extracellular potassium levels. We observed unacceptably high hemolysis (>0·8%) in some RCC and elevated extracellular potassium levels in all gamma-irradiated RCC. This suggests that more restrictive storage times should be considered for RCC in SAGM.

Identification of a novel HLA-A*02 allele, HLA-A*02:01:01:32, in a deceased Caucasoid donor.

HLA-A*02:01:01:32 differs from HLA-A*02:01:01:01 by a single nucleotide substitution (G→A) at position 2456.

Identification of the novel allele, HLA-B*14:56, in a Brazilian individual.

Novel allele, HLA-B*14:56, generated by a gene conversion event was identified in a Brazilian individual.

Single molecule real-time DNA sequencing of HLA genes at ultra-high resolution from 126 International HLA and Immunogenetics Workshop cell lines.

The hyperpolymorphic HLA genes play important roles in disease and transplantation and act as genetic markers of migration and evolution. A panel of 107 B-lymphoblastoid cell lines (B-LCLs) was established in 1987 at the 10th International Histocompatibility Workshop as a resource for the immunogenetics community. These B-LCLs are well characterised and represent diverse ethnicities and HLA haplotypes. Here we have applied Pacific Biosciences' Single Molecule Real-Time (SMRT) DNA sequencing to HLA type 126 B-LCL, including the 107 International HLA and Immunogenetics Workshop (IHIW) cells, to ultra-high resolution. Amplicon sequencing of full-length HLA class I genes (HLA-A, -B and -C) and partial length HLA class II genes (HLA-DRB1, -DQB1 and -DPB1) was performed. We typed a total of 931 HLA alleles, 895 (96%) of which were consistent with the typing in the IPD-IMGT/HLA Database (Release 3.27.0, January 20, 2017), with 595 (64%) typed at a higher resolution. Discrepant types, including novel alleles (n = 10) and changes in zygosity (n = 13), as well as previously unreported types (n = 34) were observed. In addition, patterns of linkage disequilibrium were distinguished by four-field resolution typing of HLA-B and HLA-C. By improving and standardising the HLA typing of these B-LCLs, we have ensured their continued usefulness as a resource for the immunogenetics community in the age of next generation DNA sequencing.

A novel allele, HLA-B*51:220, identified in an individual from south of Brazil.

Novel allele, HLA-B*51:220 generated by a gene conversion event was identified in a Brazilian individual.

Mutagenicity of 1,2-dichloroethane and 1,2-dibromoethane in two human lymphoblastoid cell lines.

1,2-Dichloroethane (DCE) and 1,2-dibromoethane (DBE) were tested for the ability to induce gene mutations in two human lymphoblastoid cell lines, designated AHH-1 and TK6. Both chemicals were 'direct-acting' mutagens in both cell lines. DBE was essentially equally mutagenic in TK6 cells and AHH-1 cells. In contrast, DCE was 25-fold more mutagenic in the AHH-1 cell line than in the TK6 cell line. This differential sensitivity between AHH-1 cells and TK6 cells was related to the levels of glutathione S-transferase activity in these two cell lines.

Statistical discriminant analysis of arrhythmias using intracardial electrograms.

The problem of classifying ventricular arrhythmias from intracardial electrograms is considered. Standard statistical discrimination procedures are applied using a simple parametric model for the shape of the pulse near its peak. This approach makes simultaneous use of the model parameters, has well known statistical properties, and involves computations that can be carried out efficiently. Preliminary analyses of real data sets, using both linear and quadratic discrimination functions, yield promising results.

Pregnancy outcome in free-ranging vervet monkeys (Cercopithecus aethiops).

A preliminary assessment of individual female differences in conception rate and fetal wastage has been determined for a population of wild vervet monkeys (Cercopithecus aethiops). One of three troops of vervet monkeys, the subjects of a long-term behavioral study, was trapped and blood was obtained for electrophoretic analysis. Pregnant females exhibited a distinctive serum aminopeptidase phenotype allowing a conclusive determination of pregnancy. Of the seven females diagnosed as pregnant, three later gave birth. Of the females that aborted, two were nulliparous and one was very old. Studies of captive animals have indicated that age and rank may affect a female's ability to carry a fetus to term. These factors, rather than the trapping procedure, may have been responsible for most of the fetal loss in the trapped troop. A comparison of all three troops for a 3-year period indicated that there were fluctuations in yearly birth success of individual females, as well as a relatively high miscarriage rate. The results of this study indicate the advantages of obtaining joint behavioral and biological data.

Dietary fiber and laxation in postop orthopedic patients.

The addition of wheat fiber in the diet of post-surgical orthopedic patients as a means of preventing constipation was studied using a quasi-experimental design. It was hypothesized that a 20 gm supplement of All Bran and natural bran would promote spontaneous bowel movements, reduce the incidence of constipation, and thus decrease the need for elimination interventions. The results show that the study group had more spontaneous bowel movements and required fewer elimination interventions than did the control group.

Evaluation of adverse events in cats receiving long-term piroxicam therapy for various neoplasms.

The role of cyclo-oxygenase 2 (COX-2) and prostaglandins (PG) in carcinogenesis has been documented in many species. Piroxicam has shown efficacy against several neoplasms and is frequently prescribed for chronic use. There are no studies investigating chronic piroxicam administration in cats and the chronic use of non-steroidal anti-inflammatory agents in this species has long been cautioned against. This retrospective study aimed to evaluate adverse effects in cats receiving long-term daily piroxicam. Seventy-three cats received daily piroxicam at doses of 0.13-0.41mg/kg. Treatment duration ranged from 1 to 38 months. Treatment with piroxicam was found to significantly increase frequency of vomiting during the first month of therapy, though this was most significant for cats receiving concurrent chemotherapy. Piroxicam administration was not significantly associated with hematologic, renal or hepatic toxicities. Adverse events were not correlated with dosage. Adverse events were reported in 29% of cats, and were generally mild and transient. Eight percent discontinued piroxicam due to adverse reaction, and 4% due to difficult administration. This study indicates that long-term daily piroxicam is generally well tolerated in cats at conventional doses.

Comparative postcranial body shape and locomotion in Chlorocebus aethiops and Cercopithecus mitis.

Body weight and length, chest girth, and seven postcranial limb segment lengths are compared between two guenon species, Chlorocebus (Cercopithecus) aethiops (vervets) and Cercopithecus mitis (blue monkeys), exhibiting different habitual locomotor preferences. The subjects, all adults, were wild caught for a non-related research project (Turner et al. [1986] Genetic and morphological studies on two species of Kenyan monkeys, C. aethiops and C. mitis. In: Else JG, Lee PC, editors. Primate evolution, proceedings of the Xth International Congress of Primatology, Cambridge. London). The morphological results are interpreted within the context of previously published observations of primate locomotion and social organization. The sample is unique in that the body weight of each individual is known, allowing the effects of body-size scaling to be assessed in interspecific and intersexual comparisons. C. mitis has a significantly (P < 0.05) greater body weight and trunk length than C. aethiops. A shorter trunk may function to reduce spinal flexibility for ground-running in the latter. Proximal limb segments (arm and thigh) are significantly greater in C. mitis, reflecting known adaptations to committed arboreal quadrupedal locomotion. By contrast, relative distal limb segments (forearm, crus, and foot) are significantly longer in C. aethiops, concordant with a locomotor repertoire that includes substantial terrestrial quadrupedalism, in addition to arboreal agility, and also the requisite transition between ground and canopy. Although normally associated with arboreal monkeys, greater relative tail length occurs in the more terrestrial vervets. However, because vervets exploit both arboreal and terrestrial habitats, a longer tail may compensate for diminished balance during arboreal quadrupedalism resulting from the greater "brachial" and "crural" indices that enhance their ground quadrupedalism. Most interspecific differences in body proportions are explicable by differences in locomotor modalities. Some results, however, contradict commonly held "tenets" that relate body size and morphology exclusively to locomotion. Generally associated with terrestriality, sexual dimorphism (male/female) is greater in the more arboreal blue monkeys. A more intense, seasonal mating competition may account for this incongruity.

Brief communication: the 1996 American Association of Physical Anthropology membership survey.

The 1996 AAPA membership survey included 1,033 participants. This number represents 72.6% of the membership of the association. Data were collected on gender, academic rank, highest degree, decade highest degree was awarded, discipline, employment, and rank order of subdiscipline. There are statistically significant differences (Chi-square test, P < 0.05) in subdiscipline membership including, among others, human and primate evolution, primatology, and skeletal biology. There are also gender differences in number of students and academic rank.

Blood protein variation in a population of Ethiopian vervet monkeys (Cercopithecus aethiops aethiops).

Seven small groups of vervet monkeys (Cercopithecus aethiops aethiops), totaling 124 individuals, from Awash National Park, Ethiopia, were trapped and blood samples were obtained. Twenty-three loci were examined by starch gel electrophoresis and four loci, Tf, E, ADA, and PMG2 were found to be polymorphic. The average heterozygosity (H) of the population was calculated to be 5.6%. No significant deviations from Hardy-Weinberg equilibrium proportions occurred and a chi-square test for group homogeneity was also not significant. Average Fst for all polymorphic loci was calculated to be 0.062. This suggests that the entire group functions as a single Mendelian population.

Variation in erythrocyte enzyme activity among primate species.

Significant differences between species in the mean level of enzyme activity, ranging from 50 to 400%, were detected during a study of 10 enzymes from erythrocytes of humans and five non-human primates. The activity differences were consistent with variation, between species, at the structural loci for specific enzymes rather than differences in mean cell age, hemoglobin content or other pleiotropic effects. For seven of the eight loci at which direct comparisons among the six species could be made, the number of species-specific activity differences was greater than the number of variants observed by electrophoresis. Thus not only do activity measurements detect previously "hidden" variation among species but they also provide information regarding genetic differences which may be of potential physiological and/or selective relevance.

Thermostability characteristics of glucosephosphate and triosephosphate isomerase in erythrocytes from several species.

Significant differences in the thermostability of both glucosephosphate and triosephosphate isomerase were noted among a series of six primate and five nonprimate species. The enzyme structural differences among species, as assessed by thermostability profiling, was greater than expected from electrophoretic mobility patterns. Microheterogeneity of GPI, i.e. differences in thermostability within a species that are not detectable by electrophoresis, was detected in two primate species. Major differences in the levels of erythrocyte enzyme activity were observed with human and cow differing by 18-fold for TPI and baboon and cow differing by seven-fold in GPI activity.

Growth, development, and sexual dimorphism in vervet monkeys (Cercopithecus aethiops) at four sites in Kenya.

Body weight and ten body segment measurements were collected from 367 wild-trapped vervet monkeys (Cercopithecus aethiops) in central and southern Kenya. The animals represent between 70 and 95% of the animals in each of 30 troops at four geographical locations separated by 80 to 380 km. The capture sites differed in altitude, mean annual rainfall and temperature. Two questions are addressed: (1) what are the differences in male and female growth patterns, and (2) what is the relationship between size, climate, and availability of food? Each animal was assigned to an age class based on dental examination. Means for all variables do not diverge for males and females from birth to age class 4 (15-18 months). After this, male and female growth rates diverge. This sexual dimorphism in growth pattern may reflect timing of entry into the reproductive community. A nested analysis of variance (ANOVA) was employed to compare sites, groups within sites and individuals within groups. Statistically significant differences between sites in body weight and body segment measurements are found for adult females. Except for tail length, these differences do not follow Bergmann's or Allen's Rules correlating size differences and temperature, but rather may reflect proximity to cultivated areas or tourist lodges with greater access to human food.