Buffy Coat Method Research Articles

Effect of gamma irradiation on cytokines released by platelets during storage

Most published gamma irradiation studies are on the cytokines secreted by leucocytes than platelet cytokines. If cytokines secretion is suppressed by irradiation then it will be an important application of irradiation. To know this we planned to measure cytokines like Regulated upon activation normal T cells expressed and secreted (RANTES), platelet factor 4 (PF-4), Transforming growth factor βeta1 (TGF-β) and βeta Thromboglobulin (β TG) during the storage of irradiated (IR) and non-irradiated (NI) platelets. Ten platelet concentrate (PCs) were prepared using platelet rich plasma and buffy coat methods each and 10 by apheresis on Trima Cell separator. Each PC was transferred in a transfer bag and IR at about 25 Gy and stored at 22 °C in platelet agitator. Samples were taken from NI and IR bag each on 0 day, 3rd day and 5th day from supernatant for cytokine analysis. The samples were stored below −50 °C for cytokines assays using commercial ELISA kits. RANTES levels were in the range of 12–400 ng/ml on 0 day and increased to 108–800 ng/ml on 5th day in NI platelets and 104–800 ng/ml in IR platelet. PF4 increased from 0 day to 5th day showing levels 300–1500 ng/ml in both types of PC. β-TG range was 748–5258 ng/ml in NI and 878–4638 ng/ml in IR platelets on 5th day. TGF-β1 increased up to 780–38431 pg/ml in NI and 461–50,000 pg/ml in IR PC. The study showed that comparison of cytokine levels during the storage in NI and IR platelets was not significant by Mann–Whitney U- test (p > 0.05). Significant increase was observed in these cytokine levels from 0 day to 3rd and 5th day in NI as well as IR samples by Wilcoxon Signed- Rank test (p < 0.05).

Laminin Rich and Immunologically Neutral Epineural Sheath Conduit Supported with Bone Marrow Stromal Cells to Restore Long Nerve Defects: N/A - Not a clinical study

Laminin Rich and Immunologically Neutral Epineural Sheath Conduit Supported with Bone Marrow Stromal Cells to Restore Long Nerve Defects: N/A - Not a clinical study

Clinical efficacy of platelet concentrates prepared by improved buffy-coat method

Objective To explore the clinical efficacy and safety of platelet concentrates prepared by improved buffy-coat method. Methods From October 2012 to February 2014, a total of 89 cases of hospitalized patients in a certain hospital in Zhengzhou City who needed platelet transfusion were collected into this study, they were divided into study group (n=49) and control group (n=40) according to different kinds of platelet preparations they transfused. Patients in study group were transfused with platelet concentrates prepared by improved buffy-coat method, and patients in control group were transfused with apheresis platelets. Platelet counts were detected at pre-transfusion, 1 hour after transfusion and 24 hours after transfusion, then corrected count increment (CCI) was calculated. The improvement of bleeding in patients and transfusion adverse events were observed and recorded before and after transfusion. The platelet counts, CCI, transfusion efficacy and the incidence of adverse reactions in two groups were compared with each other by statistical methods. The study protocol was approved by the Ethical Review Board of Investigation in a certain hospital in Zhengzhou City. Informed consent was obtained from all participants. There were no significant differences of the age, gender ratio, type of diseases and platelet counts before blood transfusion between two groups (P>0.05). Results ①There were no significant differences in platelet content, capacity and the residual of white blood cells and red blood cells between apheresis platelets and platelet concentrates prepared by improved buffy-coat method (P>0.05). ②Platelet counts were significantly increased 1 hour and 24 hours after transfusion compared with the platelet counts before transfusion in two groups, and the differences were statistically different (P 0.05). ③There was no significant difference in rate of adverse reactions after platelet transfusion between two groups (P>0.05). Conclusions Platelet concentrates prepared by improved buffy-coat method are safety and efficacy for patients, whether it is worth of clinical promotion and application, still needs large sample and multicenter randomized controlled trials to confirm. Key words: Platelet concentrates; Improved buffy-coat method; Apheresis platelets; Treatment outcome

Hematocrit alterations and its effects in naturally infected indigenous cattle breeds due to Trypanosoma spp. on the Adamawa Plateau - Cameroon.

Aim:An experimental study was carried out on 148 naturally infected indigenous cattle breeds with either single or mix infections of various species of trypanosomes. The objectives of this study were to determine the species of trypanosomes, observe their hematopathological consequences on host-related risk factors and to determine the packed cell volume (PCV) of the infected group.Materials and Methods:The buffy coat method (BCM) which is a variant of the hematocrit centrifugation method was used for the parasitological and hematological analysis. The May Grünwald-Giemsa method was also used for the identification of different trypanosome species.Results:The infection rate in accordance with the various trypanosomes was as follows: Trypanosoma congolense + Trypanosoma brucei (1.35%), Trypanosoma vivax + T. brucei (1.35%), T. congolense + T. vivax (8.11%), T. congolense + T. vivax + T. brucei (8.78%), T. brucei brucei (11.48%), T. vivax (20.94%), T. congolense (47.97%). The infection rate with respect to breeds showed the following results - Brahman (1.0%), Red Fulani (5.2%), White Fulani (6.5%) and Gudali (16.7%), with no statistical significant difference (p>0.05). The combined mean PCV of single as well as mix infections was not statistically significant (p>0.05). The mean PCV of males (25.64±5.08 standard deviation [SD]) which was lower than that of females (30.82±4.94 SD) was statistically significant (p<0.05). The body condition of infected animals with sex showed that a greater proportion of males with “Poor” and “Medium” conditions showed high prevalence than females with the same conditions, with a significant difference (p<0.05). However, females showed a “Good” condition than males even though it was not statistically significant (p>0.05). The PCV profile of the infected group showed that the highest proportion of infected animals had PCV of ≤31% than PCV >31%. The mean weight of the animals was (265.41±95.36 SD). A scatter-linear plot of infected buffy coat against mean PCV showed a negative parametric correlation.Conclusion:Distinguished Trypanosoma spp. pathogenicity, emaciation and weight loss related anemia, poor body condition, sex and the response of different breeds to various trypanosomes were highly affected and are of vital importance in diagnosis and act as a contribution to future control and treatment plans in this area.

Determining the Effect of Preparation and Storage: An Effort to Streamline Platelet Components as a Source of Growth Factors for Clinical Application

Background: In the present study, different methods for preparation of platelet-rich plasma (PRP) are investigated in order to standardize the component in terms of growth factor content. The effects of concentration technique and storage duration are also analyzed. Methods: PRP was collected from 40 donors by plateletpheresis as well as by the buffy coat and tube method. Concentration of growth factors was performed using double freeze thaw- and CaCl₂-induced degranulation techniques. Growth factor estimation was performed using ELISA. Results: The levels of growth factors were highest in PRP from buffy coat, moderately lower in plasma gained by plateletpheresis and lowest in that obtained by the tube method. Mean levels of platelet-derived growth factors (PDGF) AB and BB are significantly higher when CaCl₂ was used for concentrating the growth factors. The mean levels of transforming growth factor β1 and insulin-like growth factor I were higher when applying the double freeze thaw technique. There was a substantial decline in the levels of growth factors during storage. Conclusion: The buffy coat method is suitable as preparation method for PRP in most settings. The double freeze thaw technique is better suited as concentration technique as it causes lysis of both platelets and white blood cells for releasing growth factors and is easier to perform. Growth factors are not stable in plasma, thus PRP should be frozen immediately after preparation.

Risk Assessment of Single-Donor (Apheresis) Platelet Concentrates and Pooled Whole-Blood-Derived Platelet Concentrates

According to the risk estimates of the Robert-Koch-Institute (RKI) and the Paul Ehrlich-Institute (PEI) an equivalence cannot be assumed to exist between the two different platelet preparations. Differences between single-donor (apheresis) platelet concentrates (ATK) and pooled whole-blood-derived platelet concentrates (PTK) result from donor populations, donation intervals, and preparation techniques. There are no prospective randomized studies with regard to the clinical efficacy, which would unambiguously demonstrate equivalence of the therapeutic efficacy of PTK (buffy coat method) in comparison to ATK. The German Association of Blood Transfusion Services (StKB) points out that, due to the non-equivalence of PTK and ATK, it is incumbent on the transfusion physician to select the platelet concentrate, make the appropriate disclosures, and assume treatment responsibility. Proper compensation for ATK and PTK must be ensured by the health insurance companies, whereby a special indication for the selection of either PTK or ATK is not given. Exceptions are patients with known HLA antibodies in which only selected platelet concentrates may be administered. Otherwise, no indication exists in the selection of the different platelet concentrates (Article is in German).

Influence of Different Methods Preparation on Platelet Activation in Stored Platelet Concentrates

Background: Platelet concentrates are routinely manufactured from whole blood by differential centrifugation. During this storage period under blood bank conditions, biochemical, structural and functional changes occur, a process that is also known as platelet storage lesion. Their quality was assessed using the following parameters: platelets, leukocytes and erythrocytes counts, pH, CD63, lactate dehydrogenase and Annexin V. Materials and Methods: In this experimental study, 25 platelet concentrates prepared with platelet rich plasma-platelet concentrates, 25 units via buffy coat and apheresis-derived platelet methods. The percentages of Annexin V, CD63 expression, lactatedehydrogenase, platelet, leukocytes counts and pH were evaluated. Results: During storage for up to 5 days, no significant pH, difference was observed among all three type of platelet concentrates (p>0.05). The mean leukocytes count buffy coat units, platelet rich plasmaplatelet concentrates and apheresis-derived units were comparable and statistically significant difference was observed (p<0.05). During storage for up to 5days platelet concentrates units displayed significant an increase in lactatedehydrogenase, CD63 and Annexin V expressions, as compared with buffy coat units and apheresisderived units preparation on day 5 (p<0.05). Discussion: The kinetics of CD63 and annexin V levels are influenced by the method used to prepare platelets for storage. The different levels of CD63, annexin V and lactatedehydrogenase in three types of units clearly demonstrating a progressive activation process of platelet concentrates units exceeds that of buffy coat and apheresis-derived units. Further clinical studies will be necessary to determine whether meet the quality criteria or is superior in predicting in vivo viability.

Effects of Prestorage Leukoreduction on the Rate of Febrile Nonhemolytic Transfusion Reactions to Red Blood Cells in a Tertiary Care Hospital.

Background:Febrile nonhemolytic transfusion reactions (FNHTRs) are relatively common complications associated with allogenic transfusion. White blood cells (WBCs or leukocytes) are considered to be an important cause of FNHTRs; the rate of WBC derived pro-inflammatory cytokines increase with storage due to active synthesis of cytokines by these cells. The removal of the WBCs before storage will prevent the accumulation of cytokines during storage that leads to a reduction in the number of FNHTRs.Aim:We have conducted a retrospective analysis comparing the rate of FNHTRs in prestorage leukoreduced (PrSLR) and non leukoreduced RBCs transfusion.Subjects and Methods:A retrospective review of all the transfusion reactions (TRs) reported to the department over a period of 2 years from July 2012 to June 2014 was done. Patients were stratified by the date of reaction and by component received and then divided into two groups: (1) Patients who received allogeneic PrSLR RBCs and (2) nonleukoreduced RBCs. For the PrSLR RBC units, leukoreduction was performed by using buffy coat method of component preparation by quadruple bags and integral bags containing Sepacell® Pure RC filters (Fenwal™ France).Results:37,232 RBCs units were transfused and out of which 14149 (38% i.e. is 14149/37232) were prestorage leukoreduced (PrSLR) and 23083 (62%) were non leukoreduced. A total of 142 (0.38%) TRs were reported during that time period, of which 62 (0.17%) were classified as FNHTRs. In the nonleukoreduced group 124 TRs were reported, of which 55 were classified as FNHTRs to RBCs and the overall rate of FNHTR to RBCs was 0.24%. In pre storage leukoreduced group, 18 TRs were reported, of which 7 were classified as FNHTRs to RBCs and the overall rate of FNHTR to RBCs was 0.05% (P ≤ 0.001). This represents a significant reduction in the rate of FNHTR after institution of prestorage leukoreduction.Conclusion:The rate of FNHTRs to allogenic RBC units after the implementation of prestorage leukoreduction has decreased significantly. Cytokines and chemokines accumulating during storage of cellular blood products are responsible for residual FNHTRs.

Clinical Evaluation of Intralesional Injection of Platelet-Rich Plasma for the Treatment of Proximal Suspensory Ligament Desmitis in Horses

The study clinically evaluated the effectiveness of autologous platelet-rich plasma (PRP), obtained using a double transfusion bag after an ultrasound-guided intralesion injection into an acute lesion of proximal portion of suspensory ligament (SL). Twenty horses with acute proximal suspensory ligament desmitis were included in the study. An ultrasound evaluation of the proximal region of the SL was carried out, and the cross-sectional area (CSA) percentage and fiber score alignment were determined. Four hundred fifty milliliters of blood was collected, and PRP was obtained by means of the buffy coat method, using a double transfusion bag. The PRP was injected into the lesion using an ultrasound guide. A long-term follow-up was carried out by means of a telephonic interview with the owner. The median CSA of the SL lesion at the first examination was 30%. The median platelet count in the PRP was 1,045 × 103 platelets/μL; the final concentration ranged from six to 20 times higher than the platelet blood count. Sixteen horses returned to activity within a period ranging from 12 to 24 weeks from the last PRP injection, and four animals recidivated in different regions of suspensory ligament: midbody desmitis and medial branch. Clinical results confirm that the use of autologous PRP supports healing and reduces recurrence in acute SL lesions.

A Comparison of Different Methods of RBC Leukoreduction and Their Ability to Minimize Neutrophil Priming Activity during Storage

Background:Pre-storage leukoreduction (LR) of whole blood (WB) or red blood cells (RBC) reduces white blood cell (WBC) and platelet contamination and decreases the amount of biologic response modifiers (BRMs) implicated in the development of transfusion-related acute lung injury (TRALI). There are two primary methods of LR used world-wide, however there has not yet been a side-by-side comparison of the buffy coat method to the in-line filtration, in terms of the ability to minimize the neutrophil (PMN) priming activity in the supernatant. We hypothesize that LR by buffy coat is equal to LR by filtration in minimizing PMN priming activity.Method:Five units of WB were drawn from healthy volunteer donors. Plasma was removed by centrifugation and the RBC units were equally divided by weight; 50% were LR by buffy coat removal and 50% were LR by in-line filtration (Haemonetics RC2D) into bags containing AS-3 additive solution (110 ml) and stored at 2-6oC per AABB criteria. Sterile couplers were used to obtain samples weekly during storage. The supernantant was isolated by centrifugation (5,000g for 7 min, followed by 12,000g for 6 min) and stored at -80oC. PMNs were isolated from heparinized WB from healthy volunteer donors by standard techniques. PMNs were incubated with plasma samples for 5 min at 37oC then activated with 1uM formyl-MLF (fMLF) and the maximal rate of O2- was measured. Priming activity is the augmentation of the maximal rate of superoxide production in response to fMLF. Supernatants were immunoblotted for peroxiredoxin-6, an intracellular RBC enzyme.Results:LR by filtration decreased WBC amount by >3 logs and platelets by >2 logs whereas LR by buffy coat reduced both WBC and platelets by only a single log. The supernatants from both buffy coat and filtration LR showed no difference in PMN priming vs. albumin or buffer-treated controls on day (D) 1 and demonstrated an increase in O2- production on D1 vs. D42 of storage (Table 1). However, the supernatants from buffy coat LR RBCs demonstrated significantly greater PMN priming activity as compared to supernatants from filter LR RBCs (2.4-fold). Samples from D14 and D28 also showed a statistically significant greater increase in O2- production in buffy coat LR samples compared to filtered samples. Additionally, there was increased immunoreactivity for peroxiredoxin-6 in buffy coat supernatants vs. filtered RBC supernatants on identical days of storage (Fig.1).Discussion:Filter LR is more effective in reducing WBC and platelet contamination, and significantly decreases the accumulation of PMN priming activity and peroxiredoxin-6 immunoreactivity in the RBC supernatant. LR via filtration appears to produce RBC units with less cellular contamination, decreased BRMs and less release of intracellular enzymes. Further testing is required to determine if these LR RBC units are clinically safer than RBCs which undergo buffy coat LR. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Whole blood treated with riboflavin and ultraviolet light: quality assessment of all blood components produced by the buffy coat method.

Pathogen inactivation (PI) technologies are currently licensed for use with platelet (PLT) and plasma components. Treatment of whole blood (WB) would be of benefit to the blood banking community by saving time and costs compared to individual component treatment. However, no paired, pool-and-split study directly assessing the impact of WB PI on the subsequently produced components has yet been reported. In a "pool-and-split" study, WB either was treated with riboflavin and ultraviolet (UV) light or was kept untreated as control. The buffy coat (BC) method produced plasma, PLT, and red blood cell (RBC) components. PLT units arising from the untreated WB study arm were treated with riboflavin and UV light on day of production and compared to PLT concentrates (PCs) produced from the treated WB units. A panel of common in vitro variables for the three types of components was used to monitor quality throughout their respective storage periods. PCs derived from the WB PI treatment were of significantly better quality than treated PLT components for most variables. RBCs produced from the WB treatment deteriorated earlier during storage than untreated units. Plasma components showed a 3% to 44% loss in activity for several clotting factors. Treatment of WB with riboflavin and UV before production of components by the BC method shows a negative impact on all three blood components. PLT units produced from PI-treated WB exhibited less damage compared to PLT component treatment.

Time Dependent Release of Interleukin-8 and Tumor Necrosis Factor-α in Platelet Concentrate.

Contaminating white blood cells in stored platelet concentrate (PC) are the source of many pro-inflammatory cytokines. These are implicated in transfusion reactions. To study the release of interleukin (IL)-8 and tumor necrosis factor alpha (TNF-α) at different time interval in PC prepared by-platelet rich plasma (PRP) and buffy coat (BC) using different principles. Fifteen PCs were prepared by both the methods. The supernatants of PCs prepared by PRP and BC methods were collected aseptically after 1, 18, 65 and 112h of preparation. pH, platelet and WBC counts were done. The supernatants were frozen in aliquots at -56°C for measurement of IL-8 and TNF-α concentration using ELISA. The Mean±SD value of WBC in PRP-PC was 7.4±3.75×10(7) and in BC-PC 3.9±2.2×10(7). The mean platelet counts were 6.05±1.94×10(10) and 6.54±2.18×10(10) respectively. The highest level of IL-8 in one hour was up to 30pg/ml in both the type of PC. It increased up to 986pg/ml in PRP-PC and 481pg/ml in BC-PC at 112h. IL-8 increased significantly during storage period of 5days in both types of PCs (P0.000 and P0.01). TNF-α level remained low up to 18h. The highest level was 72pg/ml in PRP-PC and 57pg/ml in BC-PC at 65h. IL-8 levels significantly increased after one hour of storage and TNF-α. levels were low up to 18h and then showed increase. The BC-PC had significantly low levels of IL-8 compared to PRP-PC (P0.0001).

Evaluation of Random Donor Platelets Produced from Buffy Coat Stored for 24h at Ambient Temperature: Should This be Implemented in India?

Whole blood derived platelets are made from platelet rich plasma (PRP) method or buffy coat (BC) method. In India majority of random donor platelets (RDPs) are prepared by PRP method. However, BC method offers the advantage of less platelet activation and fewer WBC contamination. Presently in India RDPs are prepared within 8h of whole blood collection, whereas, in Europe this time limit is up to 24h. Our aim was to evaluate the platelet count, WBC contamination, platelet CD62P expression, and biochemical parameters of RDPs prepared from BC within 8h and within 24h of collection. We prepared 40 units of RDP by the BC method from whole blood stored at room temperature within 8h of collection (fresh BC), and another 40 units from BC stored at 22°C for <24h (stored BC). We analyzed the platelet counts, CD62P expression, WBC counts, glucose levels, pH, PO2, PCO2 in both the groups of RDPs, 24h after respective preparation. The platelet counts from stored BC was higher in fresh BC. CD62P expression was low in stored BC compared to fresh BC. There were no differences of pH, pO2, pCO2 and glucose levels in fresh BC and stored BC. WBC contamination was more in fresh BC. Our study stored BC contained higher platelet counts, less WBC contamination and less platelet activation. We conclude that RDP prepared from stored BC is the better method for RDP production.

A quality monitoring program for red blood cell components: in vitro quality indicators before and after implementation of semiautomated processing.

Canadian Blood Services has been conducting quality monitoring of red blood cell (RBC) components since 2005, a period spanning the implementation of semiautomated component production. The aim was to compare the quality of RBC components produced before and after this production method change. Data from 572 RBC units were analyzed, categorized by production method: Method 1, RBC units produced by manual production methods; Method 2, RBC units produced by semiautomated production and the buffy coat method; and Method 3, RBC units produced by semiautomated production and the whole blood filtration method. RBC units were assessed using an extensive panel of in vitro tests, encompassing regulated quality control criteria such as hematocrit (Hct), hemolysis, and hemoglobin (Hb) levels, as well as adenosine triphosphate, 2,3-diphosphoglycerate, extracellular K(+) and Na(+) levels, methemoglobin, p50, RBC indices, and morphology. Throughout the study, all RBC units met mandated Canadian Standards Association guidelines for Hb and Hct, and most (>99%) met hemolysis requirements. However, there were significant differences among RBC units produced using different methods. Hb content was significantly lower in RBC units produced by Method 2 (51.5 ± 5.6 g/unit; p < 0.001). At expiry, hemolysis was lowest in Method 2-produced RBC units (p < 0.05) and extracellular K(+) levels were lowest in units produced by Method 1 (p < 0.001). While overall quality was similar before and after the production method change, the observed differences, although small, indicate a lack of equivalency across RBC products manufactured by different methods.

Extended storage of buffy coat platelet concentrates in plasma or a platelet additive solution

Platelet (PLT) concentrates (PCs) prepared from whole blood in the United States are made using the PLT-rich plasma method. The PCs must be made within 8 hours of blood collection and stored for only 5 days. In Europe and Canada, PCs are made using the buffy coat (BC) method from whole blood held overnight at 22 °C and storage times may be up to 7 days. Our studies were designed to determine how long BC PLTs can be stored in plasma or Plasmalyte while meeting the FDA's poststorage viability criteria. Normal subjects donated whole blood that was stored at 22 °C for 22 ± 2 hours before preparation of BC PLTs. PLTs were stored for 5 to 8 days in either plasma or Plasmalyte concentrations of 65 or 80%. Radiolabeled autologous stored versus fresh PLT recoveries and survivals were assessed as well as poststorage in vitro assays. BC PLTs stored in either plasma or 65% Plasmalyte met FDA poststorage PLT recovery criteria for 7 days but survivals for only 6 days, while storage in 80% Plasmalyte gave very poor results. Both stored PLT recoveries and survivals correlated with the same donor's fresh results, but the correlation was much stronger between recoveries than survivals. In vitro measures of extent of shape change, morphology score, and pH best predicted poststorage PLT recoveries, while annexin V binding best predicted PLT survivals. BC PLTs stored in either plasma or 65% Plasmalyte meet FDA's poststorage viability criteria for 6 days.

Leukodepletion status of blood products and transfusion reactions in thalassemic patients

Background and Objectives: Multi-transfused thalassemic patients are prone to transfusions related complications. Study of these reactions and correlating them with the leukodepletion of the transfused packed red blood cells (PRBCs) reduces transfusion complications due to the transfused leukocytes. Materials and Methods: This is a retrospective study carried out on 1152 transfusions in 161 thalassemic patients at our institute between January 2011 and December 2011. The total transfusions were classified into three categories depending on the leukodepletion status of the PRBC. The clinical records and the reaction workup done to rule out the hemolytic reactions were recorded. Results: Reactions were noted in three (0.2%) out of 161 thalassemic patients were recorded. Two reactions were recorded on transfusions of leukoreduced (buffy-coat method) and one reaction in nonleukoreduced PRBC, respectively. No reactions were recorded on transfusions leucodepleted done by bedside filter. Conclusion: Leukodepletion by using bedside filter is a better method for avoiding transfusion reactions. Though in resource limited settings, leukoreduction using the buffy-coat method is also effective in reducing the transfusion reactions. Leukoreduction of transfused blood cellular components in thalassemic patients is helpful in preventing transfusion reactions.

Evaluation of a Typhoid/Paratyphoid Diagnostic Assay (TPTest) Detecting Anti-Salmonella IgA in Secretions of Peripheral Blood Lymphocytes in Patients in Dhaka, Bangladesh

BackgroundRapid and reliable diagnostic assays for enteric (typhoid and paratyphoid) fever are urgently needed. We report the characterization of novel approach utilizing lymphocyte secretions, for diagnosing patients with enteric fever by the TPTest procedure.MethodologyTPTest detects Salmonella-specific IgA responses in lymphocyte culture supernatant. We utilized TPTest in patients with suspected enteric fever, patients with other illnesses, and healthy controls. We also evaluated simplified modifications of TPTest for adaptation in laboratories with limited facilities and equipment.Principal FindingsTPTest was positive in 39 (27 typhoid and 12 paratyphoid A) patients confirmed by blood culture and was negative in 74 healthy individuals. Among 32 individuals with other illnesses, 29 were negative by TPTest. Of 204 individuals with suspected enteric fever who were negative by blood culture, 44 were positive by TPTest and the patients were clinically indistinguishable from patients with confirmed bacteremia, except they were more likely to be under 5 years of age. We evaluated simplifications in TPTest, including showing that lymphocytes could be recovered using lysis buffer or buffy coat method as opposed to centrifugation, that incubation of cells at 37°C did not require supplemental CO2, and that results were available for majority of samples within 24 hours. Positive results by TPTest are transient and revert to negative during convalescence, supporting use of the test in endemic areas. The results can also be read using immunodot blot approach as opposed to ELISA. Since no true gold standard currently exists, we used a number of definitions of true positives and negatives. TPTest had sensitivity of 100% compared to blood culture, and specificity that ranged from 78–97% (73–100, 95% CI), depending on definition of true negative.ConclusionThe TPTest is useful for identification of patients with enteric fever in an endemic area, and additional development of simplified TPTest is warranted.

Process Improvement by Eliminating Mixing of Whole Blood Units after an Overnight Hold Prior to Component Production Using the Buffy Coat Method

The elimination of a thorough manual mixing of whole blood (WB) which takes place following the overnight hold, but before the first centrifugation step, during buffy coat component production at Canadian Blood Services (CBS) was investigated. WB was pooled after donation and split. Pairs of platelet, red blood cell (RBC), and plasma components were produced, with half using the standard method and half using a method in which the mixing step was eliminated. Quality assessments included yield, pH, CD62P expression and morphology for platelets, hemoglobin, hematocrit, hemolysis, and supernatant K+ for RBCs, and volume and factor VIII activity levels for plasma. All components, produced using either method, met CBS quality control criteria. There were no significant differences in platelet yield between components produced with and without mixing. A significant difference was seen for RBC hemolysis at expiry (P = 0.03), but for both groups, levels met quality control requirements. Noninferiority of components produced without mixing was confirmed for all parameters. Manual mixing is laborious and has a risk of repetitive strain for production staff and its significance is unclear. Elimination of this step will improve process efficiencies without compromising quality.

A COMPARATIVE STUDY OF BLOOD SMEAR, QBC AND ANTIGEN DETECTION FOR DIAGNOSIS OF MALARIA

Background and Objectives: Rapid diagnosis is prerequisite for effective treatment and reducing mortality and morbidity of malaria. Microscopy has been the Gold standard for malaria diagnosis for decades. We made an attempt to compare blood smear, Quantitative Buffy Coat (QBC) and rapid antigen detection methods for the rapid diagnosis of malaria. Methods and Materials: A retrospective study was conducted for 6 months in G.G. Hospital, Jamnagar. A total number of 90 hospitalized clinically suspected malarial cases were collected and confirmed by conventional blood smear, QBC and antigen based Rapid Diagnostic Test (RDT). Blood smears were prepared and stained with Leishman stain. QBC and Rapid Diagnostic tests were done using commercially available kits. Patients were followed-up for signs of clinical recovery. Results: Malaria was diagnosed in 50, 54 and 59 patients by Leishman staining technique, QBC method and Rapid antigen detection test respectively. Sensitivity, specificity, positive and negative predictive values of Antigen detection test were 94%, 70% 79% and 90%, respectively while, those of QBC were 84%, 77%, 82% and 79%, respectively. The sensitivity of rapid diagnostic test is high with grade 3 and 4 parasitemia. Conclusion: In our study, rapid diagnostic tests were found more accurate than PBS and QBC. Microscopy is cost effective but microscopy and QBC requires technical expertise to interpret the results. In places where facilities are not available, rapid, simple and easy to interpret antigen detection test can be used, especially in endemic areas.

Storage of red blood cells affects membrane composition, microvesiculation, and in vitro quality

During storage detrimental biochemical and biomechanical changes occur within a red blood cell (RBC). RBC microparticles (RMPs) produced during storage have been identified as biomarkers of RBC quality, being potentially immunogenic and inhibitory to nitric oxide regulation. In this study, microvesiculation and changes in the composition of the RBC membrane were investigated throughout 49 days of storage and were correlated with in vitro assays examining membrane quality. Leukoreduced RBC units produced using the buffy coat method were collected and stored at 1 to 6°C and were tested weekly for hemolysis, osmotic fragility, deformability, ATP, hematologic indices, and morphology. Microvesiculation was assessed using multicolor flow cytometry. High-performance liquid chromatography and mass spectrometry were used to determine the composition and quantity of phospholipids (PLs) and cholesterol (C) on Days 2 and 43. The assessment of RBCs throughout storage revealed significant increases in percent hemolysis, while significant decreases in ATP concentrations, and the mean corpuscular hemoglobin concentration were observed. Flow cytometry analysis revealed a significant increase in the mean number of microparticles per microliter during storage. Throughout storage, significant decreases were identified in the amount of PLs and total lipids within the RBC membrane. No significant change in the amount of C in the RBC membrane was identified. Significant changes to the RBC membrane occur during storage. The length of storage will influence RMP generation, osmotic fragility, hemolysis, and changes in deformability. These changes in RBC in vitro quality may contribute to transfusion reactions and negative posttransfusion outcomes.