Fresh Frozen Plasma Samples Research Articles

Comparison of Plasma Components between Frozen Plasma and Fresh Frozen Plasma

To compare the plasma components of frozen plasma (FP) and fresh frozen plasma (FFP). Twenty samples of FP and 20 samples of FFP from Beijing Red Cross Blood Center were randomly selected. Immediately after plasma melting, 12 plasma components including coagulation factor, fibrinolytic system and anticoagulation protein were detected, including activated partial thromboplastin time (APTT), prothrombin time (PT), coagulation factor Ⅷ (FⅧ) activity, coagulation factor Ⅴ (FⅤ) activity, fibrinogen(FIB) level, ADAMTS-13 activity, von Willebrand factor(vWF) activity, D-dimer (D-dimer, DD), fibrin degradation products (FDP), antithrombin (AT), protein C (PC), and protein S (PS). All these coagulation components between the two types of plasma were compared and analyzed. Compared with FFP, APTT in FP was significantly prolonged(t=3.428, P<0.01), and PT was also significantly prolonged(z=-2.140, P<0.05), and FⅧ activity was decreased (t=-3.372, P<0.01), but all in the reference range, and PS activity was decreased(t=-2.458，P＜0.05), there was no statistical difference in the other part between two types of plasma(P>0.05). FP can substitute FFP in the treatment of some diseases, although it is lack of some coagulation factors and anticoagulation protein.

Point‐of‐care blood clotting test and its correlation with fibrinogen level: Potential in goal‐directed transfusion in postpartum hemorrhage

To study the correlation of conventional point-of-care clotting test (POCCT) of whole blood with laboratory fibrinogen levels in women with primary major postpartum hemorrhage (PPH) to generate evidence regarding its potential in rationalizing transfusion of blood components for supplementing fibrinogen. A total of 68 samples were studied: 40 from women with primary major PPH, 20 from women without PPH, and eight samples of Fresh Frozen Plasma (FFP). POCCT was performed in the PPH and non-PPH groups and at the same time, sample for laboratory fibrinogen was sent. Values were correlated using Pearson's correlation coefficient (r). Depending upon the laboratory fibrinogen values, POCCT in the PPH group was divided into three subgroups of less than 7, 7-11, and more than >11min as subgroups 1, 2, and 3, respectively. Women in the PPH group (n=40) had a mean fibrinogen level of 346.15±143.37mg% with a significant negative correlation with POCCT (r=-0.69, P<0.001). Mean fibrinogen levels of non-PPH group and FFP samples were 602.1±169.72 and 286.75±103.42mg%, respectively. PPH subgroups 1, 2, and 3 had mean fibrinogen of 452.2±141.8, 332.58±138.51, and 158.71±145mg%, respectively. These values may potentially guide FFP transfusions. POCCT is easy, costs nothing and has a potential role in rationalizing FFP transfusion in low- and middle-income countries.

A Preliminary Study on Coagulation Parameters and Sterility of Thawed Refrozen Fresh Frozen Plasma.

Thawed fresh frozen plasma (FFP) if not used within 6h, may have to be discarded due to the risk of contamination and uncertainty about its quality. The main objective of this study was to evaluate the levels of coagulation Factor II (FII), Factor VIII (FVIII), fibrinogen and bacterial growth in thawed refrozen FFP. Thirty FFP samples were collected from healthy donors. FFP were thawed in water bath at 37°C for 20-25min. Approximately 10mL of plasma from each FFP unit was tested for FII, FVIII, fibrinogen and sterility. The thawed FFP units were then kept at 4°C for 6h before being refrozen and stored at - 20°C. Twoweeks later, the refrozen FFP were thawed again and representative samples were analysed as before. There was a significant decline in the mean FVIII level, from 155.77% to 85.6% at second thaw. The mean FII level increased significantly from 74.9% to 82%, whereas the mean fibrinogen level fell from 3.34g/L to 3.28g/L, but the decline was not statistically significant. There was no bacterial contamination in all samples at both time points. Refrozen plasma may be considered as an alternative to the storage of thawed unused FFP provided they arekept in a controlled environment to reduce wastage. These thawed refrozen FFP can be used later in bleeding cases like other FFP as the levels of FVIII are still within the standard haematology range (0.5-2IU/mL) and above the minimal level of 30%coagulation factors required for adequate haemostasis.

Individual differences of plasma proteins and factors in fresh frozen plasma from Chinese regional blood donors.

Currently, single fresh frozen plasma (FFP) for clinical use is derived from individual blood donors. The objective of this study is to investigate the differences in single FFP units to make related strategies for improving FFP transfusion efficacy. 120 units of single FFP were selected randomly from Chinese PLA Clinical Blood Transfusion Center in Beijing, China. Single FFP samples were thawed in 37°C water bath and were assessed immediately for total protein (TP), albumin (Alb), fibrinogen (Fg), factor V (FV), factor VIII (FVIII), antithrombin-III (AT-III) and protein C (PC). Multiple comparisons were employed to analysis the differences. The quality levels of 120 single FFP units showed wider ranges and presented normal distribution. Fg, FV and PC showed larger fluctuation range but TP, Alb, FVIII and AT-III showed smaller. The mean level of TP was 56.07g/L and FVIII was 0.62IU/mL. There were no significant differences to genders in single FFP units. FFP from younger people with 18-30years old showed a trend towards reduced activity of coagulation factors, especially FV and FVIII. TP and Alb levels in FFP with type O were significantly higher than that in non-O type, but FVIII, AT-III and PC levels were lower in type O than that in non-O type. In a word, the whole quality of single FFP units from Chinese regional donors was acceptable, except FVIII. There were great differences of plasma proteins and factors in single FFP units, age and ABO blood type were main influence factors.

Fibrinogen determination according to Clauss: commutability assessment of International and commercial standards and quality control samples.

Many clinical laboratories use a clotting rate assay according to Clauss for the determination of fibrinogen in citrated plasma. The aim of the present study was to assess the commutability of the current International Standard for fibrinogen (coded 09/264), three commercial fibrinogen standards, and 10 freeze-dried plasma quality control samples from various sources. Clotting rate assays according to Clauss were performed on three automated instruments (Sysmex CA1500, STA-Rack Evolution and ACL-Top 700), using three commercial thrombin reagents (Siemens, Stago, and Instrumentation Laboratory). Relationships between the results obtained with the three instruments were determined with 25 fresh-frozen plasma samples obtained from patients. The deviations of the assay results obtained with the freeze-dried samples were compared with the deviations obtained with the fresh-frozen samples, according to approved CLSI guideline C53A. Freezing and thawing had no influence on the assay results. There were significant differences in the mean assay results (fibrinogen, g/L) for the fresh-frozen plasma samples between the three automated instruments: 2.51 (STA-Rack Evolution), 2.25 (ACL-Top 700) and 2.20 (Sysmex CA1500). Similar differences were observed for several freeze-dried plasma samples. Some freeze-dried plasma samples, including the International Standard, were out of the 95% confidence interval for the relationship between STA-Rack Evolution and Sysmex CA1500. Some freeze-dried plasmas including the international standard for fibrinogen are not commutable among automated instruments for fibrinogen clotting rate assays according to Clauss. Our results have consequences for all interested parties in the traceability chain (WHO, industry, external quality assessment schemes, clinical laboratories).

Paired comparison of methylene blue- and amotosalen-treated plasma and cryoprecipitate.

Cryoprecipitate is used in the treatment of patients with acquired hypofibrinogenaemia. Studies have not directly compared cryoprecipitate produced following pathogen inactivation (PI) of fresh-frozen plasma (FFP) using different systems. The effects of methylene blue (MB) and amotosalen (AS) PI systems on the quality of FFP and cryoprecipitate were investigated in a paired study. Seven group A and 7 group O pools of plasma were prepared and split into individual units and rapidly frozen to produce FFP. Units of FFP were thawed and either PI treated with MB or amotosalen, or left untreated (control). Samples of FFP along with the corresponding cryoprecipitate were tested for a range of coagulation factors, thrombin generation (TGT) and rotational thromboelastometry (ROTEM). AS-FFP showed a smaller decrease following treatment for most coagulation factors analysed than MB-FFP, except fibrinogen (antigen) and factor VII, partly due to lower volume losses. There was no significant difference between treatment methods for fibrinogen content of cryoprecipitate with treated units meeting current UK specification, or TGT and ROTEM parameters studied. MB-cryo contained a significantly higher content of FVIII and lower content of FXIII when compared to AS-cryo, with no difference in fibrinogen activity.

Abstract PD3-8: Circulating tumor DNA and circulating tumor cells in metastatic triple negative breast cancer patients

Abstract Background Preliminary reports suggested that circulating tumor DNA (ctDNA) can be used as a prognostic marker in a way akin to circulating tumor cells (CTC) in metastatic breast cancer patients. However ctDNA detection is often performed on multiple mutations, combining heterogeneous techniques. Here we used the high prevalence of TP53 mutations in triple-negative metastatic breast cancer (TNMBC) to compare CTC and ctDNA detection rates and prognostic value. Methods A cohort of 40 patients treated at the Institut Curie (Paris, France) was enrolled before starting a new line of treatment for TNMBC. CTC were detected by the CellSearch system (in 7.5 mL of blood). Using massively parallel sequencing (NGS), TP53 mutations were first characterized in tumor tissue, then in plasma DNA extracted from fresh frozen plasma samples (from 15-20 mL of blood). ctDNA detection was performed using high depth targeted sequencing using two platforms in parallel (Illumina HiSeq 2500 and Roche 454). Libraries for Illumina were prepared following the TAm-Seq procedure (Forshew et al, Sci Transl Med 2012), with preamplification of all coding TP53 exons and flanking untranslated regions followed by both paired-end 150bp Illumina and 454/Roche sequencing. CTC, ctDNA and usual patient characteristics were correlated with time to progression (TTP) and overall survival (OS). Results Archived tumor (FFPE or frozen) tissue was available for 36 patients, and 31 were successfully sequenced: TP53 mutations were found in 27 patients. As measured on the Illumina platform, ctDNA was detected in 21/27 patients (81%), ranging from 48 to 648,000 copies/mL of plasma (median 1620). Mutant allele fraction in circulating cell-free DNA ranged from 2 to 70% (median 5%). Comparison between ctDNA levels measured by Illumina and 454/Roche platforms in plasma displayed a good correlation (R2 = 0.903), with a single discordance. ≥1 CTC were detected in 19 of these 27 patients (70%). Strikingly, high ctDNA levels had prognostic impact neither on OS, nor on TTP, whatever the dataset used (Illumina or 454) whereas CTC≥5/7.5 mL were correlated with OS (p=0.04) and marginally with TTP (p=0.06). Other known usual factors, such as poor performance status, elevated LDH and number of previous treatment lines had also significant prognostic factors in this cohort. CTC and ctDNA early changes during treatment were available for 12 patients and changes (increase/decrease) of the two biomarkers were globally similar. Conclusion Demonstrating a good sensitivity (81%), ctDNA by the TAm-Seq is more frequently detected than CTCs in the 27 TNMBC with TP53 mutations. The observed correlation between the 2 massively parallel sequencing approaches suggested that ctDNA levels data were quantitative. In contrast to other usual prognostic factors, baseline ctDNA level did not demonstrate a prognostic impact,in this proof-of-principle study, suggesting that mechanisms of ctDNA release in TNMBC rely on biological features that do not dramatically impact patient’s outcome. Citation Format: Francois-Clement Bidard, Jordan Madic, Anna Kiialainen, Fabian Birzele, Guillemette Ramey, Quentin Leroy, Thomas Rio Frio, Virginie Raynal, Virginie Bernard, Alban Lermine, Inga Clausen, Nicolas Giroud, Roland Schmucki, Carsten Horn, Olivia Spleiss, Olivier Lantz, Marc-Henri Stern, Martin Weisser, Ronald Lebofsky, Jean-Yves Pierga. Circulating tumor DNA and circulating tumor cells in metastatic triple negative breast cancer patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD3-8.

Activation of U937 cells in vitro is modified by fresh frozen plasma of healthy donors. Possible role of age and inflammatory profile

Activation of U937 cells in vitro is modified by fresh frozen plasma of healthy donors. Possible role of age and inflammatory profile

A Comparison Study of the Blood Component Quality of Whole Blood Held Overnight at 4°C or Room Temperature

Background. The use of plasma frozen within 24 hrs is likely to increase. Whole blood (WB) and buffy coats (BCs) can be held for a few hrs or overnight before processing. Methods. Twenty-four bags of WB for plasma and 12 bags for platelet (PLT) concentrates were collected. The fresh frozen plasma (FFP) was prepared within 6 hrs. I-FP24 and II-FP24 samples were prepared either from leukodepleted WB that was held overnight or from WB that was held overnight before leukodepletion. The PLT concentrates (PCs) were prepared from BCs within 6 hrs (PC1) and within 18 to 24 hrs (PC2). The typical coagulation factors and some biochemical parameters were determined. Results. Compared to the FFP samples, the levels of FVII and FVIII in the I-FP24 and II-FP24 samples decreased significantly. The pH, Na+, LDH, and FHb levels differed significantly between II-FP24 and FFP. Compared to PC1, PC2 exhibited lower pH, pO2, and Na+ levels, a higher PLT count, and increased pCO2, K+, Lac, and CD62P expression levels. Conclusion. FP24 is best prepared from WB that was stored overnight at 4°C and then leukodepleted and separated within 24 hrs. PCs are best produced from BCs derived from WB that was held overnight at room temperature.

The effect of repeated freezing and thawing on levels of vitamin K-dependent coagulation factors and fibrinogen in fresh frozen plasma

Background:Fresh frozen plasma (FFP) is considered adequate for transfusion immediately after thawing or for up to 24 hours if kept at 1–6°C, and is currently used very often to replace deficient clotting factors. If factor levels in refrozen FFP are within normal limits, then this component can possibly be transfused, thus avoiding wastage of FFP.Aim:To study the fate of vitamin K-dependent coagulation factors (F II, F VII, F IX, F X) and fibrinogen activity levels in repeatedly (twice) frozen and thawed FFP.Materials and Methods:Two hundred FFP units comprising 50 units of each major blood group (A, B, AB, and O) were thawed at 37°C and 10–20 mL of FFP transferred to transfer bags with the help of a sterile connecting device (SCD). The FFP samples were taken into tubes (first sampling), and then the transfer bags were kept for 24 hours at 4°C. After 24 hours, repeat samples were taken in tubes from the transfer bag (second sampling), and then the bags were re-stored at < -18°C. One week later, the above procedure was repeated. Activity of coagulation factors and fibrinogen levels were measured by the automated coagulation analyzer.Results:The levels of F II, F VII, F IX, F X, and fibrinogen of all the 200 FFP units, at all four time points, were above the lower normal value, but well within the normal range.Conclusion:The levels of F II, F VII, F IX, F X, and fibrinogen remain stable and adequate for transfusion in twice-thawed-and-refrozen FFP. This component can be safely used for transfusion as a source of vitamin K-dependent clotting factors and fibrinogen.

Relative concentrations of haemostatic factors and cytokines in solvent/detergent-treated and fresh-frozen plasma

Indications, efficacy, and safety of plasma products are highly debated. We compared the concentrations of haemostatic proteins and cytokines in solvent/detergent-treated plasma (SDP) and fresh-frozen plasma (FFP). Concentrations of the following parameters were measured in 25 SDP and FFP samples: fibrinogen (FBG), factor (F) II, F V, F VII, F VIII, F IX, F X, F XIII, von Willebrand factor (vWF), D-Dimers, ADAMTS-13 protease, tumour necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, IL-8, and IL-10. Mean FBG concentrations in SDP and FFP were similar, but in FFP, the range was larger than in SDP (P<0.01). Mean F II, F VII, F VIII, F IX, and F XIII levels did not differ significantly. Higher concentrations of F V (P<0.01), F X (P<0.05), vWF (P<0.01), and ADAMTS-13 (P<0.01) were found in FFP. With the exception of F VIII and F IX, the range of concentrations for all of these factors was smaller (P<0.05) in SDP than in FFP. Concentrations of TNF-α, IL-8, and IL-10 (all P<0.01) were higher in FFP than in SDP, again with a higher variability and thus larger ranges (P<0.01). Coagulation factor content is similar for SDP and FFP, with notable exceptions of less F V, vWF, and ADAMTS-13 in SDP. Cytokine concentrations (TNFα, IL-8, and IL-10) were significantly higher in FFP. The clinical relevance of these findings needs to be established in outcome studies.

Assessment of ACTH assay variability: a multicenter study

Measurement of plasma ACTH plays a pivotal role in the diagnosis and treatment of several endocrine disorders. Little is known, however, on the variability of ACTH assay results and its impact on clinical practice. The aim of the present study was to assess the performance of routine plasma ACTH measurements. Twenty-five fresh-frozen plasma samples collected from patients with either high, low, or normal ACTH concentrations were measured using seven different assays by 35 different laboratories. Assay precision, method agreement, and result classification were estimated. Inter- and intra-assay coefficient of variation varied considerably with some assays achieving <10%, others consistently achieving >20%. Overall method agreement was good (mean ratio versus target value 1.02) but subject to exceedingly large excursion (lower and upper limits of agreement at 0.13 and 1.91 respectively). Both differences between assays and between laboratories contributed to variability of method agreement. Assays correctly classified most patients with normal and high ACTH concentrations (90% (95% CI 82-97%) and 95% (95% CI 86-100%) respectively), whereas only 60% (95% CI 52-67%) of measurements from patients with low ACTH values were assigned correctly. Field ACTH assays have to be interpreted with caution as they are burdened by high variability and often fail to correctly identify patients with suppressed ACTH secretion. The endocrine community has to include ACTH assays among those requiring standardization.

Evaluation of Clotting Factor Concentrates for Treatment of Thrombotic Thrombocytopenic Purpura.

AbstractAbstract 3678 Introduction:Thrombotic thrombocytopenic purpura (TTP) is characterized by microthrombi, hemolytic anemia as well as thrombocytopenia. These symptoms are caused by a decreased activity of the protease ADAMTS13 which cleaves the von Willebrand Factor (VWF), due to mutation of the ADAMTS13-gene or autoantibodies. At the moment, the only available immediate therapy is plasmapheresis with Fresh Frozen Plasma (FFP) which may induce side effects. Therefore an alternative therapy might be the treatment with clotting factor concentrates. Methods:40 plasma samples were tested, consisting of FFP and solvent/detergent treated plasma, four batches of each blood group; VWF/VIII concentrates (Wilate®; Wilfactin®; Haemate®P; Immunate®; Kogenate®, Beriate®). In all samples ADAMTS13 activity, antigen and autoantibodies against ADAMTS13 were investigated. Additionally, the samples were tested for the presence of ultralarge VWF multimers. The BCS method according to Böhm, two ELISAs (Technozym®ADAMTS13 and Actifluor™ADAMTS13) and the electrophoresis on a SDS gel were used. Results:ADAMTS13 activity was found in all VWF)VIII concentrates, which are produced from human plasma, but only with a very low activity (Wilate® 5.6%; Wilfactin® 2.8%; Haemate®P 13%; Immunate® 3.7%). ADAMTS13 activity was not detectable in the factor VIII concentrates (Kogenate® <2%; Beriate® <2%). Usual FFP and solvent/detergent treated plasma samples, contained much higher ADAMTS13 activity and antigen values than concentrates (FFP 78.6%, solvent/detergent treated plasma 77.6%). However a difference of activity between individual blood groups was evident in FFP samples (blood group A 69.4%; B 64.9%; AB 98.1%; 0 82.0%). Ultralarge VWF multimers could be demonstrated in VWF containing concentrates, less in VIII concentrate from human plasma and not in FFP and solvent/detergent treated plasma samples. As expected recombinant-produced VIII concentrate contained no traces of ultralarge VWF. In all analyzed samples antibodies were negative. Conclusion:For therapy of TTP clotting factor concentrates cannot be used as an alternative to the usual FFP and solvent/detergent treated plasma, because their ADAMTS13 activity values are too low.In addition, the VWF/VIII concentrates contain higher quantities of ultralarge VWF multimers, which are contraindicated for TTP patients. The differences of ADAMTS13 activity in FFP samples varies between the individual blood groups and batches. Solvent/detergent treated plasma shows less variation in ADAMTS13 activity due to the manufacturing process involving plasma pooling. Disclosures:No relevant conflicts of interest to declare.

Flow cytometric assay for the simultaneous determination of residual white blood cells, red blood cells, and platelets in fresh‐frozen plasma: validation and two years' experience

A fully automated single-tube assay with tubes (BD TruCOUNT, BD Biosciences) for absolute counting of residual cells in freshly prepared plasma by flow cytometry was developed (BD Plasma Count). The nucleic acid dye thiazole orange stains white blood cells (WBCs). The monoclonal antibodies anti-CD41a-peridinin chlorophyll protein-Cy5.5 and anti-glycophorin A-fluorescein isothiocyanate label platelets (PLTs) and red blood cells (RBCs), respectively. No fixation, permeabilization, or washing steps were required. Validation was done according to guidelines of the International Conference on Harmonization and the National Committee for Clinical Laboratory Standards. Cell-free plasma was spiked with each cell type for accuracy, reproducibility, and linearity measurements. Results showed no carryover or drift under automated sample acquisition conditions. Nonspecific background was fewer than 0.3 cells per microL for residual WBCs (rWBCs), fewer than 2.7 cells per microL for rRBCs, and fewer than 85 cells per microL for rPLTs. Determinations of rWBC and rPLT counts were linear with a coefficient of variation of less than 12 percent for the imprecision. Owing to cross-linking of the anti-glycophorin A antibody, linearity and precision for rRBCs diverged up to 21 percent at a count of 6000 rRBCs per microL. In a 2-year period, five operators investigated 2666 quality control (QC) samples of fresh-frozen plasma on 108 working days. Maximum cell numbers found were 196 for rWBCs, 3960 for rRBCs, and 28,952 for rPLTs per microL. In 31 cases (1.2%) rWBCs were out of specification. No outlier was observed for rRBCs and rPLTs. Residual RBC cell numbers determined were always within the acceptable concentration range of the assay. These data demonstrate that the single-tube test is suitable for routine QC assessment of the cellular contaminants of therapeutic plasma according to the European recommendations.

Influence of freeze-drying on the clotting properties of fibrinogen in plasma

Freeze-dried plasma standards are often used to calibrate fibrinogen assays. Little is known, however, about the effect of freeze-drying on the clotting properties of fibrinogen. If these properties are altered, the use of freeze-dried calibration standards might influence the results obtained when applying clotting assays to determine fibrinogen concentrations. In order to disclose any discrepancies in fibrinogen concentrations before and after freeze-drying, we determined the fibrinogen concentrations in citrated plasma samples using a total clottable protein method and a clotting-rate assay before and after freeze-drying. When using the clotting-rate assay, significantly higher fibrinogen concentrations were found in fresh-frozen plasma samples compared to freeze-dried samples ( P<.001). In freeze-dried plasma samples, the fibrinogen concentrations were significantly higher using the total clottable protein assay than the clotting-rate assay ( P<.001). When measuring the fibrinogen concentrations in plasma samples with a wide range of fibrinogen concentrations using the routinely employed clotting-rate assay, significantly higher fibrinogen concentrations were found using the freeze-dried calibration plasma, than the fresh-frozen calibration plasma ( P=.02). We conclude that the clotting rate of fibrinogen in citrated plasma is reduced following freeze-drying. When using freeze-dried calibration plasma in a clotting-rate assay, higher fibrinogen concentrations are obtained than by using fresh-frozen plasma. Denaturation of fibrinogen during the freeze-drying process, affecting its polymerization properties, may constitute the main contributor to the reduced clotting rate of freeze-dried plasma.

Hepatitis C infection and viremia in Dutch Hemophilia patients

Serum samples from 316 patients visiting the Dutch National Hemophilia Center were collected from 1979 to 1993 and stored at -30 degrees C. Patients were placed into three different groups: 1) patients ever treated with large pool non-hepatitis C virus (HCV)-safe concentrate (n = 179); 2) patients treated with cryoprecipitate (n = 125); and 3) patients treated exclusively with HCV-save concentrate (n = 12). In order to examine the prevalence of HCV infection in the different treatment groups serum samples were tested retrospectively for anti-HCV antibody using second generation enzyme-linked immunosorbent assay (ELISA) and recombinant immunoblot assay (RIBA-2). Significant differences in the prevalence of HCV infection were found between these 3 groups (group 1: 99%, group 2: 66%, group 3: 0%). The safety of currently administered clotting products is demonstrated in 57 patients who remained without HCV markers between 1989 and 1993. To examine the natural course of HCV infection fresh-frozen plasma samples were obtained recently from a subgroup of 277 hemophilia patients for HCV-RNA detection by a well-validated cDNA-PCR assay. In contrast to other reports, no evidence was found for seronegative HCV carriers. None of 52 patients without anti-HCV had detectable HCV-RNA. Of 225 patients with anti-HCV, 182 (81%) were HCV-RNA positive. None of 39 anti-HCV positive patients with a negative HCV-RNA reaction had serum alanine aminotransferase (ALT) levels above 50 U/l, whereas 44% of HCV-RNA positive patients had persistently elevated ATL levels above 50 U/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Storage conditions of blood samples and primer selection affect the yield of cDNA polymerase chain reaction products of hepatitis C virus.

We have noticed that suboptimal specimen processing and storage conditions may cause false-negative results in the detection of hepatitis C virus (HCV) RNA in plasma or serum. To establish the influence of specimen handling in a serological laboratory on the rate of detection of HCV RNA by the cDNA polymerase chain reaction (cDNA-PCR), we tested routine serum samples and fresh-frozen plasma samples from the same bleeding from confirmed anti-HCV-positive blood donors. When primers from the NS3/NS4 region were used, HCV RNA was detected in fresh-frozen plasma from 67% of the donors, whereas positive results were obtained with only 50% of the serum samples that had been subjected to routine serological procedures. Analysis of the same samples with primers from the highly conserved 5'-terminal region (5'-TR) revealed an HCV RNA detection rate of 92% for both the routine and the fresh-frozen samples. However, the yield of the amplification product in routine samples was strongly reduced compared with that in fresh-frozen plasma. Comparison of both primer sets for cDNA-PCR showed that the 5'-TR primer set was 10- to 100-fold more effective in detecting HCV RNA. We also analyzed the effect of storage of whole EDTA-blood and serum at room temperature and at 4 degrees C on the yield of the amplification product. A rapid decline in detectable HCV RNA of 3 to 4 log units was observed within 14 days when whole blood and serum were stored at room temperature. By contrast, no perceptible reduction in the cDNA-PCR signal was found in freshly prepared serum stored at 4 degrees C.

Automated determination of total plasma cholesterol: a serum calibration technique

The automated (AutoAnalyzer II) determination of cholesterol in nine serum pools in the concentration range 3.465–8.871 mmol/1, gave results that were approximately 10% higher than reference values when the analyses were based on unesterified cholesterol standards containing the same amount of water as the sample extracts (1963 analyses in 12 laboratories during a 12 month period; automated value = 0.032 + 1.10 × (Reference value)). A serum calibration procedure was successful in correcting the observed serum pool values to within +0.2 to −2.1% of reference values, and was equally effective in correcting the values observed for aliquots of 368 fresh-frozen plasma samples analyzed in each of the 12 laboratories during a 38 month period.