Preparation and Pathogen Inactivation of Double Dose Buffy Coat Platelet Products using the INTERCEPT Blood System

Blood centers are faced with many challenges including maximizing production yield from the blood product donations they receive as well as ensuring the highest possible level of safety for transfusion patients, including protection from transfusion transmitted diseases. This must be accomplished in a fiscally responsible manner which minimizes operating expenses including consumables, equipment, waste, and personnel costs, among others.Several methods are available to produce platelet concentrates for transfusion. One of the most common is the buffy coat method in which a single therapeutic platelet unit (≥ 2.0 x10¹¹ platelets per unit or per local regulations) is prepared by pooling the buffy coat layer from up to six whole blood donations. A procedure for producing "double dose" whole blood derived platelets has only recently been developed.Presented here is a novel method for preparing double dose whole blood derived platelet concentrates from pools of 7 buffy coats and subsequently treating the double dose units with the INTERCEPT Blood System for pathogen inactivation. INTERCEPT was developed to inactivate viruses, bacteria, parasites, and contaminating donor white cells which may be present in donated blood. Pairing INTERCEPT with the double dose buffy coat method by utilizing the INTERCEPT Processing Set with Dual Storage Containers (the "DS set"), allows blood centers to treat each of their double dose units in a single pathogen inactivation processing set, thereby maximizing patient safety while minimizing costs. The double dose buffy coat method requires fewer buffy coats and reduces the use of consumables by up to 50% (e.g. pooling sets, filter sets, platelet additive solution, and sterile connection wafers) compared to preparation and treatment of single dose buffy coat platelet units. Other cost savings include less waste, less equipment maintenance, lower power requirements, reduced personnel time, and lower collection cost compared to the apheresis technique.     

Subacute bacterial endocarditis; a report on 57 patients treated with massive doses of penicillin

Fifty-seven patients with subacute bacterial endocarditis were treated with doses of penicillin varying from 500,000 to 20,000,000 units per day. Diagnosis was confirmed in some cases by growths on blood culture, in others by postmortem examination. In those cases in which the diagnosis was established by blood culture, the in vitro sensitivity of the organism to penicillin was determined and penicillin then was administered by continuous intramuscular infusion in a dosage calculated to produce blood levels of penicillin four to five times that required for in vitro inhibition. Penicillin was given for a period of 21 days, and blood cultures were made periodically during and after treatment. Of the 57 patients, 38 were cured (66.7 per cent), and 19 died (33.3 per cent). Of the 19 who died, three did so within 48 hours of hospitalization and seven died despite adequate treatment. Of these seven, three died of cerebral emboli, two because of resistance to penicillin and streptomycin, one because of congestive heart failure, and one of undetermined cause. The remaining nine who died were considered to have been inadequately treated in that there was (1) failure to obtain sensitivity, (2) inadequate dossage of penicillin, (3) delay in starting treatment, or (4) failure to recognize mixed infections. There were five patients with repeatedly sterile blood cultures during life. In all of these cases, streptococcus viridans was recovered at postmortem examination. In an attempt to determine how long therapy should justly be withheld in patients with repeatedly sterile blood cultures, 140 cases of subacute bacterial endocarditis in which positive blood cultures had been obtained were reviewed. From the review it was determined that if blood cultures taken during the first two days are reported sterile, the chance of subsequent cultures proving positive is minimal. Therefore, for patients in whom the diagnosis seems otherwise obvious, delaying treatment for more than two days is not justified even though the blood culture be sterile. In cases in which blood cultures are repeatedly sterile, a dosage of 6,000,000 to 10,000,000 units of penicillin daily for 21 days is advisable.High bacterial resistance to penicillin and streptomycin was found in four fatal cases. In one of these, the infecting organism was streptococcus viridans, and in three it was staphylococcus albus. There was one patient with penumococcal meningitis complicated by unrecognized streptococcal viridans bacterial endocarditis.     

SUBACUTE BACTERIAL ENDOCARDITIS—A Report on 57 Patients Treated with Massive Doses of Penicillin

Fifty-seven patients with subacute bacterial endocarditis were treated with doses of penicillin varying from 500,000 to 20,000,000 units per day. Diagnosis was confirmed in some cases by growths on blood culture, in others by postmortem examination. In those cases in which the diagnosis was established by blood culture, the in vitro sensitivity of the organism to penicillin was determined and penicillin then was administered by continuous intramuscular infusion in a dosage calculated to produce blood levels of penicillin four to five times that required for in vitro inhibition. Penicillin was given for a period of 21 days, and blood cultures were made periodically during and after treatment.Of the 57 patients, 38 were cured (66.7 per cent), and 19 died (33.3 per cent).Of the 19 who died, three did so within 48 hours of hospitalization and seven died despite adequate treatment. Of these seven, three died of cerebral emboli, two because of resistance to penicillin and streptomycin, one because of congestive heart failure, and one of undetermined cause. The remaining nine who died were considered to have been inadequately treated in that there was (1) failure to obtain sensitivity, (2) inadequate dossage of penicillin, (3) delay in starting treatment, or (4) failure to recognize mixed infections.There were five patients with repeatedly sterile blood cultures during life. In all of these cases, streptococcus viridans was recovered at postmortem examination. In an attempt to determine how long therapy should justly be withheld in patients with repeatedly sterile blood cultures, 140 cases of subacute bacterial endocarditis in which positive blood cultures had been obtained were reviewed. From the review it was determined that if blood cultures taken during the first two days are reported sterile, the chance of subsequent cultures proving positive is minimal. Therefore, for patients in whom the diagnosis seems otherwise obvious, delaying treatment for more than two days is not justified even though the blood culture be sterile. In cases in which blood cultures are repeatedly sterile, a dosage of 6,000,000 to 10,000,000 units of penicillin daily for 21 days is advisable.High bacterial resistance to penicillin and streptomycin was found in four fatal cases. In one of these, the infecting organism was streptococcus viridans, and in three it was staphylococcus albus. There was one patient with penumococcal meningitis complicated by unrecognized streptococcal viridans bacterial endocarditis.     

High correlation between Chagas' disease serology and PCR-based detection of Trypanosoma cruzi kinetoplast DNA in Bolivian children living in an endemic area

Abstract The detection of Trypanosoma cruzi kinetoplast DNA by polymerase chain reaction (PCR) amplification is a potentially powerful tool for the parasitological diagnosis of Chagas' disease. We have applied this technique in a field situation in Bolivia, where 45 children from a primary school were subjected to serological testing, buffy coat analysis and PCR diagnosis. 26 of the 28 serology-positive individuals were also positive by PCR. In addition, two serology-negative children gave a positive result by PCR, including one who was positive in the buffy coat test. These results suggest that PCR detection of T. cruzi DNA in blood can be a very useful complement to serology in Chagas' disease diagnosis in Bolivia.     

Cell separation in the buffy coat

One of the most rapid methods to determine cell counts in whole blood is by way of layer thickness measurements of the buffy coat. The purpose of this study was to determine the separation and purity of blood cells in the different layers of the buffy coat. Blood samples were centrifuged at 10,000 g in microhematocrit tubes with an inserted float to expand the buffy coat region. Whole blood from normal laboratory individuals separates by density into four regions: platelets, a layer of lymphocyte and monocytes, granulocytes and erythrocytes. A thin band of highly swollen red cells was discovered between the buffy coat layers of many normal volunteers and patients. Stereological analysis of electron micrographs showed that mixing of formed elements within the layers is less than 2% with the exception of some erythrocytes, which can make up a higher volume fraction in the lymphocyte/monocyte and granulocyte layers. The red cell column contains about 95.7% erythrocytes and is depleted of platelets and leukocytes. In approximately 5% of hospital blood samples, the granulocyte-erythrocyte interface was feathered and undetectable, and a significantly higher volume fraction of red cells was found among the granulocytes. Cell mass density determinations indicate that the erythrocytes in these abnormal granulocyte layers have a lowered mass density, overlapping with that of the granulocytes.     

Sensitive and specific detection of Trypanosoma cruzi DNA in clinical specimens using a multi-target real-time PCR approach

Background

The laboratory diagnosis of Chagas disease is challenging because the usefulness of different diagnostic tests will depend on the stage of the disease. Serology is the preferred method for patients in the chronic phase, whereas PCR can be successfully used to diagnose acute and congenital cases. Here we present data using a combination of three TaqMan PCR assays to detect T. cruzi DNA in clinical specimens.

Methods/Principal Findings

Included in the analysis were DNA extracted from 320 EDTA blood specimens, 18 heart tissue specimens, 6 umbilical cord blood specimens, 2 skin tissue specimens and 3 CSF specimens. For the blood specimens both whole blood and buffy coat fraction were analyzed. The specimens were from patients living in the USA, with suspected exposure to T. cruzi through organ transplantation, contact with triatomine bugs or laboratory accidents, and from immunosuppressed patients with suspected Chagas disease reactivation. Real-time PCR was successfully used to diagnose acute and Chagas disease reactivation in 20 patients, including one case of organ-transmitted infection and one congenital case. Analysis of buffy coat fractions of EDTA blood led to faster diagnosis in six of these patients compared to whole blood analysis. The three real-time PCR assays produced identical results for 94% of the specimens. The major reason for discrepant results was variable sensitivity among the assays, but two of the real-time PCR assays also produced four false positive results.

Conclusions/Significance

These data strongly indicate that at least two PCR assays with different performances should be combined to increase the accuracy. This evaluation also highlights the benefit of extracting DNA from the blood specimen''s buffy coat to increase the sensitivity of PCR analysis.     

Determining Toxoplasma high-risk autologous and allogeneic hematopoietic stem cell transplantation patients by systematic pre-transplant PCR screening of stem cell originated buffy coat

The diagnosis of Toxoplasma infection or disease in hematopoietic stem cell transplantation (HSCT) patients is achieved mainly by PCR screening; however screening did not find wide field of use in practice due to costly expenditures of PCR. This study aimed to determine patients at high risk of Toxoplasma infection or disease before transplantation by stem cell originated buffy coat PCR and subsequently to screen them. Buffy coats collected from 12 autologous and 18 allogeneic HSCT patients' donors were investigated by PCR before transplantation. After transplantation, blood and sera collected at fixed time intervals were screened by two PCR methods and serological assays. Screening results first time assessed a toxoplasmosis incidence level as 25% in autologous HSCT patients and increased incidence level in allogeneic HSCT patients to 22%. Importantly, buffy coat PCR was first time performed before transplantation, to determine the risk of toxoplasmosis. Buffy coat PCR results showed that four patients were at high risk of toxoplasmosis before transplantation. After transplantation, these patients experienced toxoplasmosis. In conclusion, for the determination of patients at risk of toxoplasmosis, clinicians should consider buffy coat PCR in combination with serology before transplantation. After transplantation, PCR screening can be initiated in high risk patients upon clinical suspicion.     

Direct fluorescent and indirect immunofluorescent assay in buffy coat for candidemia diagnosis in pediatric patients: a comparative study

The diagnosis of candidemia by blood culture has poor sensitivity; therefore, immunossupresed patients and those with risk factors may receive empirical antifungal therapy, wich is potentially toxic. Fluorescent tests have been developed to obtain an early and more sensitive diagnosis than blood culture. The aim of this study was to compare indirect immunofluorescence vs direct calcofluor white fluorescence in buffy coat for candidemia diagnosis. Sensitivity, specificity, predictive values, of positive and negative samples were 60%, 86%, 33%, 95% and 90%, 80%, 35%, 99%, for indirect immunofluorescence and calcofluor white, respectively.     

Rapid diagnosis of cytomegalovirus in immunocompromised patients using monoclonal antibodies that detect early viral antigens

A technique was applied to detect early fluorescent antigens (DEFA) of cytomegalovirus (CMV) using the E13 monoclonal antibodies in 52 immuno-compromised patients hospitalized in the Nephrology Institute of Havana. Of the 75 urine or blood (buffy coat) samples taken, 15 were found positive to CMV. Using classical diploid human fibroblast isolation technique, 12 CMV strains were isolation of previously detected positive samples by DEFA. In addition, CMV was isolated from one sample reported to be negative by DEFA. A coincidence of 80% was found between both techniques. With the ELISA test, all the sample studied have IgG antibodies to CMV.     

Predictors of Bacteraemia in Patients with Suspected Community-Acquired Pneumonia

Introduction

The diagnostic yield of blood cultures is limited in patients with community-acquired pneumonia (CAP). Yet, positive blood culture results provide important information for antibiotic treatment and for monitoring epidemiologic trends. We investigated the potential of clinical predictors to improve the cost-benefit ratio of obtaining blood cultures.

Methods

Data from two prospective cohort studies of adults with suspected CAP, admitted to non-ICU wards, were combined. Two models were created, one using readily available parameters and one additionally including laboratory parameters.

Results

3,786 patients were included (2,626 (69%) with X-ray confirmed CAP). Blood cultures were obtained from 2,977 (79%) patients (and from 2,107 (80%) with X-ray confirmed CAP). 266 (8.9%) of the patients with a blood culture had bacteraemia. Clinical predictors of bacteraemia were absence of pre-admission antibiotic treatment, pleuritic pain, gastro-intestinal symptoms, tachycardia, tachypnea, hypotension and absence of hypoxia. After including laboratory results in the model, younger age, C-reactive protein, leukocytosis or leukopenia, low thrombocyte count, low sodium level, elevated urea and elevated arterial pH were added, while gastro-intestinal symptoms and hypotension were no longer significant. The area under the receiver operating characteristics curve was 0.66 (95% confidence interval 0.63–0.70) for the first model and 0.76 (95% confidence interval 0.73–0.79) for the second model.

Conclusion

In conclusion, in patients hospitalized with CAP, bacteraemia was moderately predictable using clinical parameters only. We recommend against the use of a risk prediction model for the decision to obtain blood cultures.     

Bacteraemia caused by Leptotrichia trevisanii in a neutropenic patient

We describe an episode of Leptotrichia trevisanii bacteraemia in a neutropenic hemato-oncology patient receiving chemotherapy for Refractory Anemia with Excess Blasts-2 (RAEB-2). Although Leptotrichia spp. colonize the oral cavity and genitourinary tract, serious episodes of bacteraemia might occur in immunocompromised patients, particularly in those with severe neutropenia. Therefore, microbiologists should consider the possibility of Leptotrichia spp. septicemia in patients with blood cultures positive for gram negative bacilli, when routine microbiology tests fail to reveal a correct identification of the organism.     

Correlation of tuberculin-induced lymphocyte transformation with skin test reactivity and with clinical manifestations of sarcoidosis

In vitro lymphocyte reactivity to tuberculin (PPD) was studied in buffy coat cultures from 87 patients with sarcoidosis and from 64 controls. A strong correlation was found between PPD-induced lymphocyte transformation and skin reactivity. No significant differences were found in the in vitro response of lymphocytes from skin test positive patients with sarcoidosis and from controls with the same degree of skin test reactivity. In patients with sarcoidosis negative to 100 TU, tuberculin sensitivity could be demonstrated in vitro significantly more often than in comparison subjects. Both in vivo and in vitro tuberculin sensitivity and “spontaneous” transformation were significantly more frequent in patients with erythema nodosum.     

Towards developing a diagnostic regimen for the treatment follow-up of Trypanosoma brucei gambiense

BALB/c mice infected with a high virulent strain of Trypanosoma brucei gambiense IL3707 were treated intraperitoneally (i.p.) with either Melarsoprol (Mel-B) or PSG(+) buffer as controls. The mice were subsequently monitored regularly for parasites by direct microscopic examination of their tail blood or buffy coat and by polymerase chain reaction (PCR). Mel-B was found to be an effective drug for treatment against T.b. gambiense because at the end of the first treatment schedule, all treated mice were negative for parasites even by PCR, while all the control animals were positive. Three of the five Mel-B treated mice, while parasitologically negative, were PCR positive between 53 and 80 days post infection (DPI), indicating that they still harbored an infection. All treated mice were subsequently negative for parasites even by PCR at 88 DPI. A combination of conventional microscopic examination and PCR offers a good prediction of cure following treatment of trypanosomosis.     

CFU-F circulating in cord blood

CFU-F (colony forming units-fibroblast) were studied from cord blood and, as controls, from normal bone marrow of older children and adults. Numbers of CFU-F in cord blood buffy coat cells are lower by a factor of 10 in comparison to bone marrow CFU-F. Cytomorphology and staining with monoclonal antibody identify the progeny cells of CFU-F as fibroblasts. Cord blood CFU-F derived fibroblasts have properties supporting hematopoiesis: They produce CSF (colony stimulating factor) to which fresh cord blood CFU-GM (colony forming units-granulocytic, monocytic) react by colony formation in a dose-response manner. In addition, fibroblast colonies discharge clonogenic round cells into the medium forming CFU-GM and CFU-F colonies in secondary methyl cellulose cultures. We conclude that fetal blood contains clonogenic stromal cells (CFU-F) that give rise to fibroblasts with properties of hematopoietic support.     

A New Tube for Preparing the Buffy Coat for Electron Microscopy

Anderson (1965) has described a simple method for preparing relatively pure fields of leukocytes from peripheral blood for electron microscopy using an angular spatula to detach and remove the buffy coat from the wall of a standard conical centrifuge tube. the buffy coat is, however, fragile, especially after gentle centrifugation (1500 × g for 7-9 min) and brief (5-10 min) fixation. the spatula often breaks the buffy coat and mixes it with the red cells below. the conical centrifuge tube is also disadvantageous. Its large cross-section and capacity require relatively large amounts of blood and it gives a buffy coat which, for normal blood, is too thin to permit adequate representation in individual transverse sections of those leukocytes which occur in low percentages. the use of special tubes to circumvent these problems (Wright and Douglas 1902, Endres 1927, Bessis 1940, Butler and Cushmann 1941, Chevillard and Hamon 1943, Ottesen 1954, Bessis 1956) has been very useful for cell separation but the design of the tubes proposed is not suitable for routine electron microscopy.     

Survival in vivo of platelets stored for 48 hours in the buffycoat at 4 degrees C compared to platelet rich plasma stored at 22 degrees C

High speed centrifugation allows separation of whole blood into cell free plasma, a buffy coat and leukocyte poor red cells. The buffy coat can be used for the preparation of platelet concentrates. High lactate production at 22 degrees C requires storage of the buffy coat at 4 degrees C. Survival in vivo of platelet concentrates prepared from buffy coats stored at 4 degrees C for 48 h (BC-PC) was compared with the survival in vivo of platelet concentrates from platelet rich plasma stored at 22 degrees C for 48 h (PRP-PC). Both methods were studied in the same healthy volunteers (n = 8) using 51Cr labeled autologous platelets. The mean +/- SD recovery 15 min after reinfusion of the BC-PC was 30.5% +/- 13.3% and for PRP-PC 41.4% +/- 7.9% (p less than 0.0001). The survival in vivo for BC-PC was 2.4 days +/- 0.4 days and for PRP-PC 7.0 days +/- 1.4 days (p less than 0.0001). Since the survival in vivo is significantly less for platelets derived from the buffy coat stored at 4 degrees C, we advocate storage of platelets at 22 degrees C.     

Decrease in anaerobe-related bacteraemias and increase in Bacteroides species isolation rate from 1998 to 2007: A retrospective study

Conflicting data have accumulated in recent years regarding the incidence of anaerobic bacteraemias. The aim of this study was to determine the prevalence of bacteraemias due to anaerobic bacteria and evaluate the importance of anaerobic blood cultures in a university hospital in Israel. A retrospective survey which focused on anaerobic blood culture bottles was performed on blood cultures received in our laboratory during the decade from January 1998 to December 2007. Anaerobic-related bacteraemias decreased during that period, whereas a significant increase was observed in Bacteroides species isolated from the blood cultures (from 18% during 1998–2002 to 43% during 2003–2007). Comparison of the medical records of 54 patients with Bacteroides-related bacteraemia during the two end periods (1998–1999 and 2006–2007) revealed a marked increase in complex underlying diseases. Hypertension and diabetes mellitus type II were found in 29% of the patients in 1998–1999 and increased to 43–45% of the patients in 2006–2007. Ischemic heart disease also increased from 14% of the patients in 1998–1999 to 43% in 2006–2007. We conclude that although positive anaerobic blood cultures account for a small percentage of positive blood samples, the growing involvement of Bacteroides species-related bacteraemias together with an increase in complex underlying diseases in these patients emphasize the importance of anaerobic blood cultures, particularly in patients with co-morbidities.     

Cord blood buffy coat DNA methylation is comparable to whole cord blood methylation

Cord blood DNA methylation is associated with numerous health outcomes and environmental exposures. Whole cord blood DNA reflects all nucleated blood cell types, while centrifuging whole blood separates red blood cells, generating a white blood cell buffy coat. Both sample types are used in DNA methylation studies. Cell types have unique methylation patterns and processing can impact cell distributions, which may influence comparability. We evaluated differences in cell composition and DNA methylation between cord blood buffy coat and whole cord blood samples. Cord blood DNA methylation was measured with the Infinium EPIC BeadChip (Illumina) in eight individuals, each contributing buffy coat and whole blood samples. We analyzed principal components (PC) of methylation, performed hierarchical clustering, and computed correlations of mean-centered methylation between pairs. We conducted moderated t-tests on single sites and estimated cell composition. DNA methylation PCs were associated with individual (P_PC1 = 1.4 × 10^?9; P_PC2 = 2.9 × 10^?5; P_PC3 = 3.8 × 10^-5; P_PC4 = 4.2 × 10^-6; P_PC5 = 9.9 × 10^-13, P_PC6 = 1.3 × 10^?11) and not with sample type (P_PC1-6>0.7). Samples hierarchically clustered by individual. Pearson correlations of mean-centered methylation between paired samples ranged from r = 0.66 to r = 0.87. No individual site significantly differed between buffy coat and whole cord blood when adjusting for multiple comparisons (five sites had unadjusted P<10^?5). Estimated cell type proportions did not differ by sample type (P = 0.46), and estimated proportions were highly correlated between paired samples (r = 0.99). Differences in methylation and cell composition between buffy coat and whole cord blood are much lower than inter-individual variation, demonstrating that both sample preparation types can be analytically combined and compared.     

Adenine nucleotide content and energy charge of Azotobacter vinelandii during batch growth in dialysed-soil medium

Abstract In human peripheral blood, a factor which induces gonococcal resistance to complement-mediated killing by fresh human serum is more concentrated in the white blood cells of buffy coat than in red blood cells. Futhermore, the resistance-inducing factor is present in both polymorphonuclear phagocytes and mononuclear cells (monocytes and lymphocytes) separated from the buffy coat by centrifugation on Ficoll-Hypaque gradients. These results imply that inflammatory cells mobilised from the blood to sites of infection carry a host factor which, if it is available to the gonococci, would materially increase their ability to resist a major defence mechanism and hence enhance their capacity to maintain and increase infection.     

Influence of fungal isolates on germination and growth of perennial ryegrass

Summary The influence of fungi isolated from perennial ryegrass roots on the germination and development of seedlings of perennial ryegrass was investigated. The basic procedure employed was to sterilise the seed surface and then inoculate with fungi and plant in non-sterile soil. It was realised that the fungal isolate inoculated on to the sterile seed surface would not remain dominant in the root region of the host and would have an influence on the host which would decline with time from when the seed germinated. This was because it would have to face antagonism from the normal components of the root microflora present in the non-sterile soil.Trichoderma viride delayed the emergence of the seedlings and reduced the production of herbage, an observation consistent with results of some other investigators. A sterile hyaline fungus stimulated the emergence of the seedlings, but subsequent tests showed that the presence of the microflora of the seed coat, or the soil microflora, or the sterile hyaline fungus was effective in promoting the rate of germination of seed that had been surface sterilised. Leaching seed in water brought about an increase in the rate of seed germination, and it is suggested that there might be a germination inhibitor soluble in water present in the seed coat, which might be inactivated by saprophytic micro-organisms.