Plasma Centrifuge Research Articles

Effects of Prostaglandin E1on the Preparation of Platelet Concentrates in Dogs

Platelet concentrates (PC) are prepared by centrifugation of platelet-rich plasma (PRP) that is prepared by centrifugation of whole blood. The resuspension of the platelet pellet during PC preparation from dogs is difficult because of platelet activation induced by centrifugation. To investigate the efficacy of adding prostaglandin E(1) (PGE(1) ) to prevent platelet activation during PC preparation from dogs. Fifteen healthy Beagle dogs. Prospective, experimental trial: PGE(1) was added to PRP before the high-speed centrifugation during PC preparation. To estimate the effect of this addition, we assessed the platelet aggregability before transfusion, the survival of the platelets after transfusion, and the platelet reactivity after transfusion, which is estimated by the P-selectin expression of the platelets when stimulated by thrombin. The difficulty associated with platelet resuspension was resolved by PGE(1.) PGE(1) strongly inhibited platelet aggregation induced by collagen and ADP; however, it recovered after the platelets were resuspended in plasma without PGE(1) (mean aggregation ratio; collagen: 10.00-80.80%, ADP: 8.20-53.60%). Survival of the platelets after transfusion was not affected by PGE(1) (mean 8.04 and 7.56 days, without and with PGE(1) ), and thrombin-induced P-selectin expression after transfusion in PGE(1) -treated PC was also well maintained (mean positive ratio 53.7 and 47.9%, before and 24 hours after transfusion). The addition of PGE(1) in PRP before the centrifugation of PRP can improve the preparation efficiency of PC from dogs, while maintaining the therapeutic efficacy of the platelets.

Metrics for comparing plasma mass filters

High-throughput mass separation of nuclear waste may be useful for optimal storage, disposal, or environmental remediation. The most dangerous part of nuclear waste is the fission product, which produces most of the heat and medium-term radiation. Plasmas are well-suited to separating nuclear waste because they can separate many different species in a single step. A number of plasma devices have been designed for such mass separation, but there has been no standardized comparison between these devices. We define a standard metric, the separative power per unit volume, and derive it for three different plasma mass filters: the plasma centrifuge, Ohkawa filter, and the magnetic centrifugal mass filter.

Wave–particle interactions in rotating mirrors

Wave–particle interactions in E×B rotating plasmas feature an unusual effect: particles are diffused by waves in both potential energy and kinetic energy. This wave–particle interaction generalizes the alpha channeling effect, in which radio frequency waves are used to remove alpha particles collisionlessly at low energy. In rotating plasmas, the alpha particles may be removed at low energy through the loss cone, and the energy lost may be transferred to the radial electric field. This eliminates the need for electrodes in the mirror throat, which have presented serious technical issues in past rotating plasma devices. A particularly simple way to achieve this effect is to use a high azimuthal mode number perturbation on the magnetic field. Rotation can also be sustained by waves in plasmas without a kinetic energy source. This type of wave has been considered for plasma centrifuges used for isotope separation. Energy may also be transferred from the electric field to particles or waves, which may be useful for ion heating and energy generation.

Wave-driven countercurrent plasma centrifuge

A method for driving rotation and a countercurrent flow in a fully ionized plasma centrifuge is described. The rotation is produced by radiofrequency waves near the cyclotron resonance. The wave energy is transferred into potential energy in a manner similar to the α channeling effect. The countercurrent flow may also be driven by radiofrequency waves. By driving both the rotation and the flow pattern using waves instead of electrodes, physical and engineering issues may be avoided.

Reply

Sir: The letter from Kakudo et al. indicated the “low yield” (75.1 percent) of platelets in our platelet-rich plasma preparation. In addition, they commented that the double-spin procedure described in the book by Marx and Garg is a standard preparation method for platelet-rich plasma at present1,2 and that the definition of platelet-rich plasma in our article3 is not appropriate because the growth factor concentrations are much lower in our platelet-rich plasma than in the product of the double-spin method. We must explain the difference between the platelet-rich plasma in our article and the platelet-rich plasma that Kukudo et al. describe as the “real” platelet-rich plasma. Platelet-rich plasma is a medical term originated not from orthodontic surgery or from Marx and Garg but from the field of blood transfusion medicine and hematology. Indeed, many scientific articles,4,5 even recently, use the term “platelet-rich plasma” for a product obtained through a single light spin of whole blood, as we used. The American Association of Blood Banks technical manual,6 a typical textbook of blood transfusion medicine, states that “Platelet-rich plasma is separated from whole blood by light-spin centrifugation and the platelets are concentrated by heavy-spin centrifugation with subsequent removal of supernatant plasma” (Fig. 1). Thus, we propose the term “concentrated platelet-rich plasma” or “therapeutic platelet-rich plasma” in scientific articles for the platelet-concentrated plasma product to avoid any confusion or misunderstanding.Fig. 1.: Preparation processes of various serums and plasmas from whole blood. Concentrated/therapeutic platelet-rich plasma can be obtained through heavy-spin centrifugation of platelet-rich plasma; although the concentration of platelets (and platelet-derived cytokines) in concentrated/therapeutic platelet-rich plasma is much higher than that of platelet-rich plasma, the volume of concentration/therapeutic platelet-rich plasma is much smaller than that of platelet-rich plasma, and platelet yield is also smaller in concentration/therapeutic platelet-rich plasma (30 to 60 percent) compared with platelet-rich plasma (70 to 80 percent). White numbers indicate platelet yield of each blood product. The concentration of platelet-derived factors is influenced by both platelet yield and the volume of each product. PRP, platelet-rich plasma; PPP, platelet-poor plasma; SWB, serum from whole blood; SPRP, serum from platelet-rich plasma; SPPP, serum from platelet-poor plasma.In our study, we employed three preparations of human serum: serum from whole blood, serum from platelet-rich plasma, and serum from platelet-poor plasma (Fig. 1). Fetal bovine serum is widely used in research as a standard additive of cell culture, and it is actually bovine serum from whole blood. We attempted to compare human serum from platelet-rich plasma to bovine and human serum from whole blood and human serum from platelet-poor plasma to accurately evaluate the value of serum from platelet-rich plasma for expansion of various types of human cells. For therapeutic use, concentrations of beneficial factors such as platelet-derived cytokines is an important index, and concentrated platelet-rich plasma obtained through the double-spin procedure is more useful than platelet-rich plasma. However, for the purpose of culture additive, either for research or for clinical cell therapy, not only the concentration but also the volume of the product is important; in other words, platelet yield (total amount of platelets) is a critical factor when using human blood with limited availability. Marx reported that the mean platelet yield of the concentrated platelet-rich plasma ranged from 29 to 62 percent that of whole blood, depending on the separation devices used,2 whereas the platelet yield of platelet-rich plasma that we prepared in our study was 75.1 percent that of whole blood. Thus, platelet yield in platelet-rich plasma obtained after a single light spin is much greater than that from concentrated/therapeutic platelet-rich plasma (Fig. 1). Finally, we have to add a note on an error in our article3 that we noticed in the reviewing process for this Reply. We reported that the centrifugation force we used for preparation of platelet-rich plasma and platelet-poor plasma was 93 g and 841 g, respectively; however, 186 g and 1682 g, respectively, are the accurate values. The authors regret the error in calculation. Masakazu Kurita, M.D. Emiko Aiba-Kojima, M.D. Kotaro Yoshimura, M.D. Department of Plastic Surgery University of Tokyo Graduate School of Medicine Tokyo, Japan

Centrifugation improves the detection of HIV-1 p24 antigen in plasma from children born to mothers infected with HIV-1

Detection of HIV proteins and/or nucleic acids is necessary for the diagnosis of perinatal HIV infection. Despite its low sensitivity, detection of p24 antigen in plasma is a simple and economic method for the diagnosis of HIV in exposed children. The aim of this study was to improve the sensitivity of detection of p24 using centrifugation of plasma. Forty-seven selected stored samples from 37 children (23 infected, 14 uninfected, median age of 137 days) were examined. Plasma samples (volume 0.3–1.5 ml) were defrosted, centrifuged at 23,500 × g at 4 °C for 60 min and determination of p24 was carried out in the resuspended pellet (0.12 ml). In 32 plasma samples from infected children, p24 was found originally in 6 (18.7%) and resulted positive in 24 (75%) pellets. When only one sample per child was considered, sensitivity was significantly higher in pellets, 3/23 uncentrifuged plasma samples and 15/23 pellets (McNemar Test, p < 0.001). Specificity was 100%. The absorbance/cut-off ratio was always higher in the pellets from positive children ( p = 0.028). Plasma samples with volumes of 1 ml or more achieved a higher sensitivity (91.7% vs. 36.4%, p = 0.009). Centrifugation of plasma samples prior to determination of p24 in pediatric patients resulted in a significant increase in sensitivity.

A quantitative radiological assessment of outcomes of autogenous bone graft combined with platelet-rich plasma in the alveolar cleft

This longitudinal study evaluated the outcomes of secondary autogenous bone graft combined with platelet-rich plasma (PRP) in the alveolar cleft. Thirty-five alveolar clefts in 30 patients with grafted autogenous bone and PRP (PRP group), and 36 clefts in 30 patients with grafted autogenous bone alone (non-PRP group) were enrolled. PRP was extracted from autogenous blood using a plasma centrifuge system (SmartPReP SMP-1000). The density and resorption of grafted bone were evaluated at 1 week, and 1, 3, 6 and 12 months postoperatively. Bone density was quantitatively assessed as an aluminum-equivalence (Al-Eq) value. Moreover, relationships between bone resorption rate and prognostic factors were discussed. Al-Eq values decreased significantly until 3 months, and then increased up to 12 months in both groups. The Al-Eq rate in the PRP group was significantly smaller than that in the non-PRP group at 3 months. No significant differences were observed in the bone resorption rate between the groups. Regarding prognostic factors, continuous mechanical stress affected bone resorption with or without PRP. The authors suggest that PRP may enhance bone remodeling in the early phase, however, PRP seems to be insufficient as a countermeasure against bone resorption following secondary bone graft in the long term.

Effect of extender, centrifugation and removal of seminal plasma on cooled-preserved Amiata donkey spermatozoa

In the donkey species, the application of cooled semen artificial insemination could aid the survival of endangered breeds. Fifteen ejaculates collected from three Amiata donkeys were used to evaluate the effect of three extenders on spermatozoal motility characteristics after cooling and preservation for up to 72 h. Semen was diluted at a 1:4 semen:extender ratio in INRA96, INRA82 and INRA82 added of 2% centrifuged egg yolk (INRA82-Y) and motility was evaluated by the computerized analyzer CEROS 12.1 at hours 0, 24, 48 and 72. Total motility, and rapid spermatozoa after 24, 48 and 72 h of preservation were higher in INRA82-Y than in INRA96 or INRA82, as was progressive motility after 72 h. INRA82-Y was thus used in a second study, where the effects of centrifugation and of removal of seminal plasma on cooled donkey semen were evaluated on 12 ejaculates from four males. Rapid spermatozoa after 24 and 72 h, and total motility after 72 h were better preserved in the non-centrifuged samples than when seminal plasma was removed, the contrary was true for the proportion of spermatozoa keeping their progressive motility at hour 48. In conclusion, INRA82-Y kept sperm motility characteristics during cooled storage better than INRA82 or INRA96, and removal of seminal plasma during in vitro preservation did not seemed advantageous. Further studies are needed to better understand the changes in motility patterns of donkey spermatozoa caused by seminal plasma and semen extenders, and their relation to fertility.

Determination of amikacin in body fluid by high-performance liquid-chromatography with chemiluminescence detection

A simple and sensitive method was developed for the quantification of amikacin in human plasma and urine samples. The method involves centrifugation of body fluid plasma after dilution with an ethanol/sodium carbonate mixture, and then an aliquot of the supernatant is directly injected into the chromatograph. After separation on a reversed-phase C18 column (runtime 20 min), aminoglycoside is detected on the basis of its complex formation reaction with Cu(II), the catalyst of the luminol/hydrogen peroxide chemiluminescence system. Using a volume of 500 microl biological sample, linearity is established over the concentration range 0.15-2.0 microg/ml and the limit of detection (LOD) is ca. 50 microg/l in plasma or urine. The intra-day and inter-day precision (measured by relative standard deviation, R.S.D.%) are always less than 9%, and relative recoveries are found to be over 92%.

Loss of Factor VIII and von Willebrand Factor Activities during Cold Storage of Whole Blood Is Reversed by Rewarming

Documentation of preanalytical conditions that could result in inaccurate results and misdiagnoses of patients is important. It has recently been reported that a significant loss of factor VIII (FVIII) and von Willebrand factor (vWF) activity occurs when citrated whole blood is stored on ice. We tested the hypothesis that the cold-dependent loss of FVIII and vWF activity is due to the formation of cryoprecipitate and is reversed by rewarming the citrated whole blood before centrifugation and removal of plasma. We collected venous blood from 10 healthy subjects into 3.2% sodium citrate glass tubes. One tube was immediately centrifuged and the plasma was stored at -20 degrees C. Following storage in an ice bath (4 degrees C) for 3.5 hours, 1 tube was immediately centrifuged and processed while the second tube was placed in a 37 degrees C water bath for 5 minutes then centrifuged and processed. Subsequently, plasma samples were quickly thawed at 37 degrees C and the following tests were performed: prothrombin time (PT), partial thrombin time (aPTT), FVIII activity, vWF antigen (vWF:Ag), and vWF activity (vWF:Act). Means for each analyte from the 2 tubes stored at 4 degrees C for 3.5 hours with or without rewarming were compared to baseline tube using the Student t test. Compared to the baseline tube results, PT and aPTT showed no significant changes in either of the tubes stored at 4 degrees C for 3.5 hours. However, FVIII, vWF:Ag, and vWF:Act were significantly lower in the tubes stored at 4 degrees C for 3.5 hours, but no differences were detected between baseline and rewarmed tube results. In conclusion, prolonged storage of citrated whole blood at 4 degrees C causes a clinically significant reduction of FVIII and vWF activities. The losses are completely reversed by rewarming the tube prior to processing. This is consistent with a reversible cryoprecipitation of vWF and FVIII rather than a cold-activated enzymatic degradation.

Apolipoprotein C-III isoforms: kinetics and relative implication in lipid metabolism

Apolipoprotein C-III (apoC-III) production rate (PR) is strongly correlated with plasma triglyceride (TG) levels. ApoC-III exists in three different isoforms, according to the sialylation degree of the protein. We investigated the kinetics and respective role of each apoC-III isoform in modulating intravascular lipid/lipoprotein metabolism. ApoC-III kinetics were measured in a sample of 18 healthy men [mean age (+/-SD) 42.1 +/- 9.5 years, body mass index 29.8 +/- 4.6 kg/m2] using a primed-constant infusion of l-(5,5,5-D3) leucine for 12 h. Mono-sialylated and di-sialylated apoC-III (apo-CIII1 and apoC-III2) exhibited similar PRs (means +/- SD, 1.22 +/- 0.49 mg/kg/day vs. 1.15 +/- 0.59 mg/kg/day, respectively) and similar fractional catabolic rates (FCRs) (0.51 +/- 0.13 pool/day vs. 0.61 +/- 0.24 pool/day, respectively). Nonsialylated apoC-III (apoC-III0) had an 80% lower PR (0.25 +/- 0.12 mg/kg/day) and a 60% lower FCR (0.21 +/- 0.07 pool/day) (P < 0.0001 for comparison with CIII1 and CIII2 isoforms). The PRs of apoC-III1 and apoC-III2 were more strongly correlated with plasma TG levels (r > 0.8, P < 0.0001) than was apoC-III0 PR (r = 0.54, P < 0.05). Finally, the PR of apoC-III2 was strongly correlated with the proportion of LDL <255 A (r = 0.72, P = 0.002). These results suggest that all apoC-III isoforms, especially the predominant CIII1 and CIII2 isoforms, contribute to hypertriglyceridemia and that apoC-III2 may play a significant role in the expression of the small, dense LDL phenotype.

Monitoring magnesium to guide magnesium therapy for heart surgery.

ionized Mg (iMg). Measurement of TMg requires the isolation of plasma (centrifugation of blood sample) or serum (clotting and centrifugation of blood sample). Measurement is made by atomic absorption spectrophotometry or colorimetry. Reference values for plasma and serum TMg typically range from 0.66 to 1.07 mmol·l 1. There is virtually no correlation between plasma/serum TMg and intracellular TMg. Ionized magnesium (iMg Mg2 ) represents the activity of unbound Mg in whole blood plasma, plasma, and/or serum. It is the physiologically active Mg fraction, i.e., the fraction to which tissues respond. Reference values typically range from 0.45 to 0.62mmol·l 1. The fact that it can be measured in a whole blood sample by electrode produces a rapid result ( 150s) on a small sample ( 200μl). A rapid result can be very helpful for patients (1) with arrhythmia, (2) with changes in cardiac output, (3) receiving cardiovascular drugs, (4) sustaining hypoxic damage, and (5) receiving Mg therapy. Blood plasma iMg correlates with intracellular iMg and therefore represents a better indicator of Mg status than TMg. It is typically 70% of the TMg value, but varies with the protein and small ligand concentrations in the blood. The iMg value may be substantially less than 70% of the TMg value in critically ill patients where binding ligand concentrations (heparin, citrate, lactate, phosphate, bicarbonate, etc.) have increased. Comparisons of iMg to TMg were illustrated in nine clinical settings (hypertension, acute myocardial infarction, head trauma, noninsulin-dependent diabetes, stroke, pregnancy, ischemic heart disease, cyclosporin recipients, and asthma) [3]. Ionized magnesium was found to be a better indicator of disease than TMg. In summary, iMg (1) represents the physiologically important Mg measurement, (2) is a better indicator of disease than TMg, and (3) is a more rapid measurement. The literature should be interpreted with caution. Frequently, if not generally, the Mg values reported in

Ultra-fast quantitative bioanalysis of a pharmaceutical compound using liquid chromatography-tandem mass spectrometry

This paper describes the ultra-fast determination of a pharmaceutical compound using TurboIonSpray™ LC-MS-MS on an API 4000 mass spectrometer. Sample preparation consisted of plasma protein precipitation, centrifugation and dilution of the supernatant. The use of small analytical column dimensions ( 2.1 mm×10 mm) and high eluent flow rates (up to 2.2 ml/min) in isocratic mode led to a retention time of 9 s. A sample-to-sample cycle time of only 10 s was achieved by coupling two autosamplers. Partial separation of the drug and its main metabolite could be obtained. The d5-labeled drug used as internal standard compensated for matrix suppression effects. The assay was linear in the concentration range 1–1000 ng/ml, using standards prepared in human plasma. Inter-assay accuracy and precision were 98.5 and 6.2%, respectively. Mean intra-assay accuracy and precision calculated from quality control (QC) samples in human, rat and dog plasma at 3, 30 and 800 ng/ml were 100.8 and 3.8%, respectively. The ultra-fast LC-MS-MS method was successfully cross-validated against a commonly used column-switching LC-MS-MS assay with 2.3 min run time by analyzing real study samples.

Multicenter quality control for the detection of hepatitis C virus RNA in seminal plasma specimens.

The discrepant results available in the literature about the presence of hepatitis C virus (HCV) RNA in seminal plasma of men chronically infected by this agent are related, at least in part, to the molecular techniques used and particularly to the wide range of protocols dedicated to RNA extraction. In order to evaluate these protocols and to standardize the method of detection of HCV RNA in this fluid, a panel of coded specimens was tested blindly in 12 French laboratories; it included 14 seminal plasma specimens and four water controls spiked with HCV RNA ranging from 10 to 20000 IU/ml and two HCV-negative seminal plasma specimens. The extraction step was performed according to methods using either silica beads (NucliSens [Organon Teknika S.A., Fresnes, France]; RNA viral kit [Qiagen, Courtaboeuf, France]) or guanidinium thiocyanate (Amplicor HCV assay; Roche Diagnostics, Meylan, France), preceded or not by a centrifugation of the seminal plasma. For the amplification step, all the laboratories performed the same reverse transcription-PCR technique (Amplicor HCV Cobas assay). The percentage of correct results ranged from 53.3 to 100, the poorest results being obtained when no centrifugation step preceded the Amplicor extraction protocol. The rate of correct results was significantly higher in laboratories using a preliminary centrifugation of the specimen (P = 0.034 by chi-square test). By contrast, the overall number of correct results was not correlated to the initial volume of sample used for the test. These results allowed us to validate standardized techniques adapted to the performance of this test on a routine basis, especially in men infected with HCV and involved in programs of medically assisted reproduction.

Band gap ion mass filter

Ions in a plasma may be radially separated according to mass using a combination of an axial magnetic field and either a radial or azimuthal electric field. The separation is qualitatively different from that obtained by a plasma centrifuge and the characteristics of confined and unconfined ion orbits are analogous to the phenomenon of band gaps in semiconductors.

The effect of time at which plasma separation occurs on biochemical values in small island flying foxes (Pteropus hypomelanus).

Heparinized blood samples from 15 adult small island flying foxes (Pteropus hypomelanus) were stored at 22 degrees C for 0-, 6-, and 24-hr intervals prior to centrifugation and separation of plasma from erythrocytes. Mean plasma biochemical values of 16 analytes were determined from all samples. Mean values of blood urea nitrogen, creatinine, total protein, albumin, globulin, alkaline phosphatase, alanine transaminase, aspartate transaminase, cholesterol, calcium, sodium, and bilirubin did not change significantly over 24 hr at 22 degrees C. Glucose was decreased at 6 and 24 hr. Potassium and phosphorus increased and chloride decreased, respectively, between 6 and 24 hr.

Pre-analytical conditions affecting the determination of the plasma homocysteine concentration.

In the past decade, moderately elevated homocysteine concentration has achieved wide-spread recognition as an independent risk factor for vascular diseases, such as stroke and peripheral vascular disease, as well as for an impaired nutritional status. In general, EDTA plasma is used for the determination of homocysteine. However, from the pre-analytical point of view it is important, that, when plasma is not separated from blood cells within 30 minutes, homocysteine levels increase in samples significantly by about 10% per hour. This 10% increase is very important, because the normal range is between 5 and 15 micromol/l and moderately elevated homocysteine concentrations above 15 micromol/l may signify an increased risk of vascular disease. These preliminary cut-off points show that there is only a small difference between normal and moderately elevated homocysteine concentrations. Most blood samples are obtained outside the hospital, and in these cases homocysteine concentrations will be falsely elevated, if no precautions are taken, such as immediate centrifugation and separation of plasma and cells. This aspect is critical both for clinical studies and in patient care outside the hospital. But even in the hospital it is difficult to separate plasma and cells within 30 minutes. In the past, different approaches were adopted to solve this problem. Potential stabilisers were sodium fluoride (4 g/l) and 3-deazaadenosine (100 micromol/l). Sodium fluoride initially increased the homocysteine concentration, which dropped below the initial values after 72 h. On the other hand, 3-deazaadenosine stabilised homocysteine concentrations for 24 h, but increased it within 72 h by roughly 10%. However, this stabiliser is restricted to HPLC technology but does not work reliably with immunoassays. Lysis of blood stabilised homocysteine, but homocysteine concentrations were systematically lower requiring totally new reference ranges. In addition, acidic citrate (0.5 mol/l) was evaluated, which seems to stabilise plasma homocysteine concentrations at ambient temperatures for several hours. However, small but systematic deviations at baseline are observed. This stabilisation procedure does not interfere with immunoassays. Because immunoassays will be the future method of choice for robust and easy to perform homocysteine measurements, because they easily allow the analyses of high sample numbers, homocysteine stabilisation in whole blood is still an important matter. It must be solved before homocysteine determinations are introduced as a general screening for vascular risk factors in non-specialist laboratories.

Effect of centrifugation and partial removal of seminal plasma on equine spermatozoal motility after cooling and storage

The objective of this study was to determine if centrifugation and partial removal of seminal plasma would improve spermatozoal motility in semen from stallions whose whole ejaculates have poor tolerance to cooling and storage. Stallions were divided into two groups (n=5/group) based on the ability of their extended semen to maintain spermatozoal motility after cooling and storage. Group 1 stallions (“good coolers”) produced semen in which progressive spermatozoal motility after 24 h of cooling and storage was reduced by ≤30% of progressive motility prior to storage. Group 2 stallions (“poor coolers”) produced semen in which progressive spermatozoal motility after 24 h of cooling and storage was reduced by ≥40% of progressive motility prior to storage. The sperm-rich portion of each ejaculate was divided into 4 aliquots. Two aliquots underwent standard processing for cooled transported semen and were examined after 24 and 48 h of cooling and storage in an Equitainer TM. The remaining two aliquots were diluted 1:1 with semen extender, then centrifuged at 400 x g for 12 min at room temperature. After centrifugation, ~90% of the seminal plasma was removed, and the sperm pellet was resuspended in extender to a final concentration of 25 to 50 × 10 6 sperm/mL. These aliquots were then packaged as for the non-centrifuged aliquots and examined after 24 and 48 h of storage.

Enhanced analytical sensitivity of a quantitative PCR for CMV using a modified nucleic-acid extraction procedure

Accurate and rapid diagnosis of CMV disease in immunocompromised individuals remains a challenge. Quantitative polymerase chain reaction (QPCR) methods for detection of CMV in peripheral blood mononuclear cells (PBMC) have improved the positive and negative predictive value of PCR for diagnosis of CMV disease. However, detection of CMV in plasma has demonstrated a lower negative predictive value for plasma as compared with PBMC. To enhance the sensitivity of the QPCR assay for plasma specimens, plasma samples were centrifuged before nucleic-acid extraction and the extracted DNA resolubilized in reduced volume. Optimization of the nucleic-acid extraction focused on decreasing or eliminating the presence of inhibitors in the pelleted plasma. Quantitation was achieved by co-amplifying an internal quantitative standard (IS) with the same primer sequences as CMV. PCR products were detected by hybridization in a 96-well microtiter plate coated with a CMV or IS specific probe. The precision of the QPCR assay for samples prepared from untreated and from pelleted plasma was then assessed. The coefficient of variation for both types of samples was almost identical and the magnitude of the coefficient of variations was reduced by a factor of ten if the data were log transformed. Linearity of the QPCR assay extended over a 3.3-log range for both types of samples but the range of linearity for pelleted plasma was 20 to 40,000 viral copies/ml (vc/ml) in contrast to 300 to 400,000 vc/ml for plasma. Thus, centrifugation of plasma before nucleic-acid extraction and resuspension of extracted CMV DNA in reduced volume enhanced the analytical sensitivity approximately tenfold over the dynamic range of the assay.

Enhanced analytical sensitivity of a quantitative PCR for CMV using a modified nucleic-acid extraction procedure

Accurate and rapid diagnosis of CMV disease in immunocompromised individuals remains a challenge. Quantitative polymerase chain reaction (QPCR) methods for detection of CMV in peripheral blood mononuclear cells (PBMC) have improved the positive and negative predictive value of PCR for diagnosis of CMV disease. However, detection of CMV in plasma has demonstrated a lower negative predictive value for plasma as compared with PBMC. To enhance the sensitivity of the QPCR assay for plasma specimens, plasma samples were centrifuged before nucleic-acid extraction and the extracted DNA resolubilized in reduced volume. Optimization of the nucleic-acid extraction focused on decreasing or eliminating the presence of inhibitors in the pelleted plasma. Quantitation was achieved by co-amplifying an internal quantitative standard (IS) with the same primer sequences as CMV. PCR products were detected by hybridization in a 96-well microtiter plate coated with a CMV or IS specific probe. The precision of the QPCR assay for samples prepared from untreated and from pelleted plasma was then assessed. The coefficient of variation for both types of samples was almost identical and the magnitude of the coefficient of variations was reduced by a factor of ten if the data were log transformed. Linearity of the QPCR assay extended over a 3.3-log range for both types of samples but the range of linearity for pelleted plasma was 20 to 40,000 viral copies/ml (vc/ml) in contrast to 300 to 400,000 vc/ml for plasma. Thus, centrifugation of plasma before nucleic-acid extraction and resuspension of extracted CMV DNA in reduced volume enhanced the analytical sensitivity approximately tenfold over the dynamic range of the assay. J. Clin. Lab. Anal. 14:32–37, 2000. © 2000 Wiley-Liss, Inc.