Development of plasma quality control material for non-invasive prenatal detection of fetal aneuploidy / 中华检验医学杂志

Objective:To develop a self-made plasma quality control material for non-invasive prenatal testing (NIPT) and evaluate its performance.Methods:139 NIPT-negative maternal plasmas stored in the genetic department of Shaoxing maternal and child health hospital from January 1, 2019 to June 30, 2021 were divided into male groups (19 cases) and female groups (120 cases) according to the neonatal gender. 9360 cases from September 2020 to September 2021 were enrolled as clinical validation cases.First step, 200 μl plasma from a 47 years-old non-pregnant healthy women was used as a matrix. Different amounts (0.1, 0.2, 0.5, 2.5, and 5 μl) of positive DNA from fetal chromosome aneuploidy (T21, T18, T13) detection kit were added. The appropriate volume of positive DNA was 0.5 μl according to the test results. Second step,Plasma in male and female group was treated as matrix. 0.5 μl positive DNA was added per 205 μl. Plasma matrix from female group showed good repeatability and the sensitivity was 100%.Third step, evaluate the self-made plasma quality control material, including storage stability, matrix uniformity and repeatability, and the effect of different batch numbers of positive DNA, by calculating Z score and the CV of fetal DNA concentration (FF).Results:Plasma matrix from female group showed good repeatability and the sensitivity was 100%, while the sensitivity of male group was only 84%. The CV of FF in female matrix was 3.9% in the repetitive experiments. After adding 0.5 μl positive DNA, the mean FF of self-made positive plasma quality control was 5.63%±0.42%, Z values>6, and the CV was 7% after storage of three months. Considering the concentration variation of positive DNA in different lots, 1 μl of positive DNA should be added when the FF of positive DNA is lower than 10%.Used in 9360 clinical cases from September 2020 to September 2021, all positive plasma quality control materials showed positive results, and the positive predictive value of trisomy 21 was 100%.Conclusions:The NIPT self-made positive plasma quality control material has been successfully developed in this study. The preliminary experimental results show that it has good repeatability and stability, which is suitable for clinical application.

Retention of hemostatic and immunological properties of frozen plasma and COVID-19 convalescent apheresis fresh-frozen plasma produced and freeze-dried in Canada.

BACKGROUND: Randomized clinical trial data show that early plasma transfusion may save lives among trauma patients. Supplying plasma in remote environments is logistically challenging. Freeze-dried plasma (FDP) offers a possible solution. STUDY DESIGN AND METHODS: A Terumo BCT plasma freeze-drying system was evaluated. We compared pooled frozen plasma (FP) units with derived Terumo BCT FDP (TFDP) units and pooled COVID-19 convalescent apheresis fresh-frozen plasma (CC-AFFP) with derived CC-TFDP units. Parameters measured were: coagulation factors (F) II; V; VII; VIII; IX; XI; XIII; fibrinogen; Proteins C (PC) and S (PS); antithrombin (AT); α2 -antiplasmin (α2 AP); ADAMTS13; von Willebrand Factor (vWF); thrombin-antithrombin (TAT); D-dimer; activated complement factors 3 (C3a) and 5 (C5a); pH; osmolality; prothrombin time (PT); and activated partial thromboplastin time (aPTT). Antibodies to SARS-CoV-2 in CC-AFFP and CC-TFDP units were compared by plaque reduction assays and viral protein immunoassays. RESULTS: Most parameters were unchanged in TFDP versus FP or differed ≤15%. Mean aPTT, PT, C3a, and pH were elevated 5.9%, 6.9%, 64%, and 0.28 units, respectively, versus FP. CC-TFDP showed no loss of SARS-CoV-2 neutralization titer versus CC-AFFP and no mean signal loss in most pools by viral protein immunoassays. CONCLUSION: Changes in protein activities or clotting times arising from freeze-drying were <15%. Although C3a levels in TFDP were elevated, they were less than literature values for transfusable plasma. SARS-CoV-2-neutralizing antibody titers and viral protein binding levels were largely unaffected by freeze-drying. In vitro characteristics of TFDP or CC-TFDP were comparable to their originating plasma, making future clinical studies appropriate.

Biochemical and cellular markers differentiate recovered, in-line filtered plasma, and plasma obtained by apheresis methods.

BACKGROUND AND OBJECTIVES: Assessment of plasma quality often focuses on the common safety tests for minimizing the risk of transmitting blood-borne pathogens. Little attention is paid to the possible quality attributes that ensure a consistent biochemical composition of plasma for fractionation. We therefore investigated the suitability of selected biochemical and haematological attributes that could be used as markers of plasma quality obtained by different separation and pre-treatment procedures. MATERIAL AND METHODS: We characterized six plasma types, including source plasma, plasma recovered by classic means and in-line filtered plasma, by determining the analytical attributes protein content, coagulation factors and markers of coagulation, contact and complement activation. Residual cell content and cell-specific variables were also measured. RESULTS: We found relevant differences between the plasma types in complement activation, as indicated by C3a measurements, while thrombin antithrombin complex values and, to a minor extent, activated factor XII concentrations indicated only moderate differences in activation levels of coagulation and contact systems. The most striking differences, however, were detected in residual cell content and concentrations of the platelet-associated proteins, platelet factor 4 and ß-thromboglobulin. We showed that leucocyte reduction filters disrupt cells. This includes platelets, thereby releasing the platelet-associated proteins platelet factor 4 and ß-thromboglobulin, and leucocytes as demonstrated by the release of elastase from polymorphonuclear leucocytes. Furthermore, the filtration processing of whole blood can lead to activation of the complement system. CONCLUSION: Our results show that biochemical and cellular surrogate markers are valuable discriminators of plasma types.

Challenging the 30-min rule for thawed plasma.

BACKGROUND AND OBJECTIVES: Frozen plasma (FP) is thawed prior to transfusion and stored for ≤5 days at 1-6°C. The effect of temperature excursions on the quality and safety of thawed plasma during 5-day storage was determined. MATERIALS AND METHODS: Four plasma units were pooled, split and stored at ≤-18°C for ≤90 days. Test units T30 and T60 were exposed to 20-24°C (room temperature [RT]) for 30 or 60 min, respectively, on days 0 and 2 of storage. Negative and positive control units remained refrigerated or at RT for 5 days, respectively. On Day 5, test units were exposed once to RT for 5 h. Quality assays included stability of coagulation factors FV, FVII, FVIII, fibrinogen and prothrombin time. Bacterial growth was performed in units inoculated with ~1 CFU/ml or ~100 CFU/ml of Serratia liquefaciens, Pseudomonas putida, Pseudomonas aeruginosa or Staphylococcus epidermidis on Day 0. RESULTS: Testing results of all quality parameters were comparable between T30 and T60 units (p < 0.05). Serratia liquefaciens proliferated in cold-stored plasma, while P. putida showed variable viability. Serratia epidermidis and P. aeruginosa survived but did not grow in cold-stored plasma. Positive and negative controls showed expected results. Overall, no statistical differences in bacterial concentration between T30 and T60 units were observed (p < 0.05). CONCLUSION: Multiple RT exposures for 30 or 60 min do not affect the stability of coagulation factors or promote bacterial growth in thawed plasma stored for 5 days. It is therefore safe to expose thawed plasma to uncontrolled temperatures for limited periods of 60 min.

Extending the pre-processing holding time of whole blood beyond 48 h reduces coagulation FVIII activity and immunoglobulin G content of recovered plasma.

BACKGROUND: Plasma obtained via whole blood (WB) donation may be used either for transfusion or as recovered plasma (RP) for pooling and fractionation. In Canada, transfusable plasma must be processed within 24 h of phlebotomy, while the limit for RP processing is 72 h. We assessed the quality of RP produced by two WB processing methods and as a function of processing time. STUDY DESIGN AND METHODS: RP units produced via the buffy coat method (BCM, n = 26) or whole blood filtration (WBF, n = 52) were tested for: the activities of prothrombin, fibrinogen, von Willebrand Factor (VWF), FV, FVII, and FVIII; the prothrombin time (PT); and total protein and IgG concentration. WBF RP units were evenly divided between those processed <48 h of phlebotomy (shorter-processed) or 48-72 h after phlebotomy (longer-processed). RESULTS: WBF-RP did not differ significantly from BCM-RP in any tested parameter except for FV and FVIII, which exhibited mean reductions of 10.2% and 20%, respectively. Longer-processed WBF-RP did not differ significantly from shorter-processed WBF-RP in any tested parameter except for FVIII activity and IgG concentration, which exhibited mean reductions of 30.1% and 14.3%, respectively. CONCLUSIONS: Canadian RP is currently fractionated into IgG, albumin, fibrinogen, and FVII/VWF concentrates irrespective of its method or time of processing. Our results supported the current approach of fractionating both BCM- and WBF-derived RP, but suggest that greater yields of immunoglobulin and FVIII/VWF products could be obtained if the maximum processing time was reduced from 72 h to 48 h.

Quantification of protein markers monitoring the pre-analytical effect of blood storage time before plasma isolation using 15 N metabolically labeled recombinant proteins.

In the hospital, blood samples are collected to monitor patients' health states, and thus various protein-based clinical methods have been developed. However, some proteins are found to change in abundances during the process of blood collection and storage. In order to account such pre-analytical effects, we performed liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) on 15 selected proteins in plasma samples prepared by varying storage time and temperature of whole blood prior to plasma isolation. Two cytosolic proteins, profilin-1 (PFN1) and thymosin beta-4 (TMSB4X), were absolutely quantified using 15 N-labeled recombinant proteins spiked externally. The other 13 proteins were quantified in a relative way compared with the two reference proteins. Triplicated LC-MRM-MS measurements showed that the median CV of MRM peak areas was 5.7%. The amounts of PFN1 and TMSB4X increased rapidly depending on the storage time between blood collection and plasma preparation. It indicates the leakage of cellular components into the plasma fraction. Relative quantification further revealed that five proteins including PFN1, S10A8, S10A9, S10A11, and TMSB4X showed significant difference (P < 0.05). We further monitored PFN1 and TMSB4X on 40 samples collected for protein diagnostics under a typical clinical study condition. Compared with the plasma samples prepared within a day, the level of both PFN1 and TMSB4X increased in the plasma samples prepared from the blood collected the day before and kept overnight at 4°C (0.51 to 3.11 µg/mL for PFN1 and 0.98 to 5.36 µg/mL for TMSB4X in average). Our result suggests an effort of assuring plasma quality for accurate protein-based diagnosis or biomarker discovery and validation.

Quality of plasma samples and BD Vacutainer Barricor tubes: Effects of centrifugation.

BACKGROUNDS: The BD Vacutainer® Barricor™ Plasma collection tube (BD Barricor) uses an innovative non-gel separation method. This study compared the plasma residual cell count (PRCC) obtained from BD Barricor and from BD PST II plasma tubes. METHODS: Four BD Barricors and one BD PST II were collected from 40 donors. BD PST II was centrifuged at 1300g/10 min, while the BD Barricors were centrifuged at 1800g/10 min, 4000g/3 min, 4000g/7 min and 4000g/15 min. PRCC was evaluated measuring white blood cells (WBC), red blood cells (RBC) and Platelets (PLT) counts by Siemens ADVIA 2120. Cell-free hemoglobin was quantified by haemolysis index (HI) by Roche Cobas c501. RESULTS: BD PST II Median WBC, RBC and PLT counts were 0.38 (109/L), 0.0291 (1012/L) and 113.5 (109/L), respectively. Considering the BD PST II as reference, PRCC differences were expressed as median bias percentage. WBC showed a significant reduction at all the conditions (p < 0.01), being the reductions: 63.9% (1800g/10 min), 69.9% (4000g/3 min), 75.0% (4000g/7 min) and 82.7% (4000g/15 min). RBC reductions 29.7% (1800g/10 min), 33.8% (4000g/3 min), 39.6% (4000g/7 min) and 66.4 (4000g/15 min) were all significant (p < 0.01). PLT reductions were 1.6% at 1800g/10 min (p = ns), 1.2% at 4000g/3 min (p = ns), 27.1% at 4000g/7 min (p = 0.046) and 46.6% at 4000g/15 min (p = 0.005). BD Barricor centrifuged for 7 and 15 min at 4000g showed an increased haemolysis. CONCLUSIONS: BD Barricors plasma quality improved with increasing the centrifugation times but already at 4000g/3 min, the suggested centrifugation condition, a significant improvement was achieved.

Manufacturing of Plasma-Derived Medicinal Products: Qualification Process of Plasma Suppliers.

Manufacturers of human plasma-derived products ensure, through their qualification departments, the quality and safety of human plasma-the biological starting material of the industrial fractionation process. The qualification department has established written procedures to approve the plasma supplier (i.e., initial qualification) according to current regulations and to the manufacturer's plasma specifications. Once the plasma supplier is approved, a periodical assessment is necessary (i.e., continuous qualification) to guarantee the level of compliance. In addition, a signed quality agreement between the plasma supplier and the manufacturer defines the duties and the responsibilities of both parties. The qualification department implements the following requirements to ensure the quality of plasma from suppliers: (i) a regular audit program to confirm the satisfactory initiation of the quality arrangements and (ii) monitoring of the quality and safety of plasma including critical quality parameters. For several years, the Grifols Qualification Department has worked with several plasma suppliers of the European Union (EU) and has performed a detailed, continuous assessment of the audits, deviations, operational incidences, epidemiological data, and quality controls. In this article, we will report data from this Grifols assessment from 2010 through 2013 on plasma suppliers from four EU countries. In the future, additional data will be collected and studied to confirm and verify the conclusions and trends observed in this study.

Quality of frozen transfusable plasma prepared from whole blood donations in Canada: an update.

BACKGROUND: Transfusable plasma is obtained by processing whole blood donations, by apheresis, or as solvent/detergent plasma (SD plasma), a pooled pathogen-reduced plasma product. The quality of plasma is typically assessed by testing the activities of multiple coagulation-related plasma proteins, due to a lack of clinical trial data linking plasma composition to clinical endpoints. We sought to update previous quality surveys of Canadian frozen plasma (FP; manufactured from single donor whole blood donation and frozen within 24h of phlebotomy), to provide transfusionists with a more complete picture of its characteristics. STUDY DESIGN AND METHODS: FP units (n=131) were tested for: the activity of factors V, VII, VIII, X, and XI, protein S (PS), α2-antiplasmin (AP), and fibrinogen; and the activated partial thromboplastin (APTT) and prothrombin (PT) times. Comparisons were made to: previous Canadian FP surveys; and to studies of single-donor plasma and SD plasma from other nations. RESULTS: Mean FVIII, fibrinogen, or APTT values did not differ from the previous annual survey of Canadian FP; FV activity was increased and PT values decreased. FP produced with or without leukoreduction differed only in mean APTT. Canadian FP exhibited generally similar quality to that reported by other organizations in Europe and Asia for similarly manufactured single-donor plasma, but contained notably higher PS and AP (≈ four-fold) activities than did SD plasma. CONCLUSION: Our results indicate that Canadian FP is of similar quality to single-donor products produced in other jurisdictions. While it is of arguably superior in vitro quality to an SD plasma product recently licensed in Canada, these differences are highly unlikely to have clinical significance for most indications for plasma transfusion.