Method to obtain PRP and L-PRP for the use in routine medical practice of orthopedic traumatologist

The paper presents the results of experimental study of two methods for blood processing to obtain platelet-rich plasma (PRP). Due to the widespread clinical use of platelet-rich plasma in orthopedics and traumatology, a differential approach to the use of PRP and leukocyte- and platelet-rich plasma (L-PRP) has become relevant. Most commercial kits for PRP preparation significantly increase the cost of treatment. In addition, the main attention is focused on the concentration of platelets in the final product and the marketing features of a particular kit (separating gel, tube shape, anticoagulant, activator, etc.). The role of leukocytes in the features of the regenerative effect of platelet-rich plasma stays out of focus. The blood of volunteers was processed in two different ways and the cellular composition of the obtained products was analyzed. Sample No. 1 was used as a control for the baseline blood cell composition. Sample No. 2 was centrifuged in a sterile plastic tube, platelet rich plasma was collected manually. Sample No. 3 was centrifuged in a sterile modified syringe, platelet rich plasma was collected using a closed technique by means of connectors together with a rich layer. The results of statistical analysis showed that there were obtained totally different biological products with different concentrations of platelets and leukocytes. The first sample (control) had platelet concentration of (228.69 ± 39.15) × 109/l and leukocyte concentration of (5.18 ± 1.32) × 109/l. In the second sample (tube + manual sampling), platelet concentration was (429.38 ± 79.92) × 109/l and leukocyte concentration — (0.85 ± 0.34) × 109/l. The third sample (syringe plunger + closed vacuum collection) had platelet concentration of (541.15 ± 85.49) × 109/l and leukocytes of (6.56 ± 1.92) × 109/l. The data are given without regard to –12.5% deviation on dilution with citrate dextrose. As the result of the work, orthopedic traumatologists can use the simplest non-commercial methods for obtaining PRP and L-PRP in their practice depending on nosology and clinical tasks.

Simple Tube Centrifugation Method for Platelet-Rich Plasma (PRP) Preparation in Catalonian Donkeys as a Treatment of Endometritis-Endometrosis

The aim of this study was to standardize a simple, manual platelet-rich plasma (PRP) protocol in Catalonian donkeys using single-spin tube centrifugation as a treatment for jenny endometritis. The objective was to obtain a blood product with a moderate concentration of platelets (2 or 3 times baseline physiologic values) and a low WBC (White Blood Cells) concentration. Blood was drawn from six Catalonian donkeys using acid citrate dextrose (ACD) as an anticoagulant, and then processed by single centrifugation at 133× g for two different centrifugation times (10 and 15 min). The PRP samples were evaluated by flow cytometry, and TGF-β1 (Transforming Growth Factor-Beta1) concentrations were determined by enzyme-linked immunosorbent assay (ELISA). The 10 min centrifugation protocol resulted in a slightly greater release of TGF-β1 (6044.79 ng/mL), a 2.06-fold increase in platelet concentration, and a 15-fold reduction in leukocyte concentration when compared to the initial values. The 15 min centrifugation time resulted in a 2.44-fold increase in baseline platelet concentration, a reduction in WBC count by a factor of 20, and slightly lower TGF levels (5206 ng/mL). We conclude that both protocols are adequate for the obtention of PRP, and both may have an acceptable therapeutic potential for use in this species, although this needs to be further validated.

Assessment of Storage Related Haematological and Biochemical Changes in Blood Units

Red blood cells are still the most widely transfused blood component worldwide and their story is intimately entwined with the history of transfusion medicine and the changes in the collection and storage of blood.1,2 At present, the most widely used protocol for the storage of red blood cells (for up to 42 days) is the collection of blood into anticoagulant solutions (typically citrate-dextrose-phosphate); red cell concentrates are prepared by the removal of plasma and, in some cases, also leukoreduction. The product is stored at 4 ± 2° C in a slightly hypertonic additive solution, generally SAGM (sodium, adenine, glucose, mannitol, 376 mOsm/L).1 The British obstetrician, Braxton Hicks in 1868, experimented with a solution of phosphate of soda, but this also proved toxic. Richard Lewinsohn, in 1915, of the Mount Sinai Hospital in New York is credited with introducing sodium citrate into clinical practice as an anticoagulant.3In fact, a 1% solution of sodium citrate was already widely used in laboratories as an anticoagulant. This high concentration was toxic to humans but, as Lewinsohn himself recalled, `Nobody had ever followed the simple thought of carrying out experiments to ascertain whether a much smaller dose might not be sufficient' for use as an anticoagulant.

Haematobiochemical changes of ovine (Ovis aries) blood during storage for transfusion

Haematobiochemical changes of ovine (sheep) blood were investigated during preservation and storage with Citrate Phosphate Dextrose Adenine-1 (CPDA-1) and Acid Citrate Dextrose (ACD) for transfusion. Twelve healthy sheep were selected and divided into two equal groups: group X (n=6) and group Y (n=6). Thirty-five ml of blood was collected from each animal and preserved with CPDA-1 in group X and ACD in group Y under 4°C in refrigerator for 28 days. Haematological changes viz., total erythrocyte count (TEC), total leukocyte count (TLC), haemoglobin (Hb), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC); and biochemical changes viz., total protein (TP) and pH were evaluated immediately after blood collection and thereafter on day-1, day-3, day-7, day-14, day-21 and day-28 for both groups. In ACD preserved blood; TEC, TLC, Hb and PCV decreased significantly (P<0.01) from day-14 onward, whereas in CPDA-1 preserved blood, these parameters decreased significantly (P<0.01) from day-21 onward. Blood preserved in ACD showed significant changes (P<0.01) in MCV, MCH and MCHC respectively from day- 7, day-14 and day-21 onward, whereas blood preserved in CPDA-1 showed no significant changes in the same parameters throughout the experiment. In both groups, no significant changes were noticed in TP but significant changes (P<0.01) were observed in pH with the progression of storage period. These findings elicited that both ACD and CPDA-1 exert certain haematobiochemical changes in stored sheep blood, however, CPDA-1 was more efficient than ACD in terms of maintaining proper levels of TEC, TLC, Hb., PCV, MCV, MCH and MCHC during preservation and storage of sheep blood for transfusion. Res. Agric., Livest. Fish.7(1): 113-120, April 2020

Changes of caprine (Capra hircus) blood during prolong storage for transfusion

Background: This experiment was performed to investigate the effects of acid citrate dextrose (ACD) and citrate phosphate dextrose adenine-1 (CPDA-1) on the keeping qualities of various haematological and biochemical parameters of caprine blood during long time preservation and storage for transfusion. Methods: Sixteen healthy goats were selected and divided into 2 equal groups (A, n=8 and B, n=8). Fifty ml of blood was collected from each goat and preserved with ACD for group A (n=8) and CPDA-1 for group B (n=8). All the samples were stored at 40C in refrigerator for 28 days. The recorded blood parameters include total erythrocyte count (TEC), total leucocyte count (TLC), haemoglobin (Hb), packed cell volume (PCV), total protein (TP) and pH. The blood parameters were analyzed immediately after collection and thereafter on day-1, day-3, day-7, day-14, day-21 and day-28 for both the groups. Results: In both groups, the TEC, TLC, Hb and PCV values were decreased gradually from day-1 onward. In ACD preserved blood, the control values of TEC (11.27±0.26 million/cumm), TLC (8.85±0.22 thousand/cumm), Hb (8.61±0.13 g/dl) and PCV (30.75±0.59%) were decreased to TEC (9.21±0.38 million/cumm), TLC (7.58±0.10 thousand/cumm), Hb (7.03±0.06 g/dl) and PCV (22.25±0.53%) respectively on day-7 which was statistically significant (p‹0.05). However, the gradual decrease in the parameters was also noticed from day-7 onward. On the other hand, in case of CPDA-1 preserved blood, the control values of TEC (11.88±0.28 million/cumm), TLC (8.91±0.26 thousand/cumm), Hb (8.91±0.42 g/dl) and PCV (32.13±0.79%) were found decreasing slightly with the progression of the preservation period, but the changes were statistically significant (p‹0.05) on day-21 [TEC (8.06±0.22 million/cumm), TLC (6.28±0.34 thousand/cumm), Hb (6.28±0.16 g/dl) and PCV (25.02±0.46%) respectively] and onward. Changes in the TP and pH values were also noticed in both the groups during the experiment but CPDA-1 group showed less alteration than ACD group as compared to the control values. Conclusion: The results of this study revealed that CPDA-1 can be used for storing caprine blood longer period for transfusion in comparison to ACD with greater RBC viability.

A Comparative Study of the Effects of Anticoagulants on Pure Platelet-Rich Plasma Quality and Potency

It is generally accepted that citrate or the A-form of acid-citrate-dextrose (ACD-A) are suitable for preparing platelet-rich plasma (PRP) for regenerative therapy. However, this is based on evidence from blood transfusions and not from regenerative medicine. Thus, we examined the effects of anticoagulants, such as ACD-A, ethylenediaminetetraacetic acid (EDTA), and heparin, on the regenerative quality of PRP to address this gap. The blood samples were collected in the presence of anticoagulants and were processed to prepare pure-PRP. Platelet size, activation status, and intra-platelet free Ca2+ concentration were determined while using a hematology analyzer and flow cytometer. Platelet-derived growth factor-BB (PDGF-BB) was quantified while using an ELISA. In pure-PRP samples, EDTA caused platelet swelling and activation, but yielded the highest number of platelets. Heparin aggregated platelets and disturbed the overall counting of blood cells. However, no significant differences in PDGF-BB levels were observed among the anticoagulants tested. Moreover, when considering the easy preparation of platelet suspensions, without the need for high-level pipetting skills, these findings suggest the comparable potency of EDTA-derived pure-PRP in tissue regeneration and support the use of EDTA in the preparation of pure-PRP. Further in vivo studies are required in animal models to exclude the possible negative effects of including EDTA in pure-PRP preparations.

Patrick Mollison CBE. 17 March 1914—26 November 2011

Patrick Mollison was a pioneer in blood transfusion, playing a major role in changing it from a risky procedure to one which is now extremely safe. The urgent need for blood during World War II provided a stimulus for the development of this important lifesaving measure. His first major contribution was to devise a mechanism whereby blood could be stored for more than just short periods. Mixing donated blood with acid–citrate–dextrose (ACD) became a standard procedure for almost 30 years and was used worldwide. He later took a special interest in haemolytic disease of the newborn (HDN), which was largely due to Rhesus incompatibility between mother and baby. He was also involved with work which eventually led to HDN becoming preventable with the use of anti-D treatment of mothers. He wrote the first standard textbook on blood transfusion; almost 70 years later it is in its eleventh edition and still bears his name in the title. He spent his working life in blood transfusion and the study of the scientific aspects of this subject, developing a university department at Hammersmith Hospital and publishing almost 200 scientific papers as well as the textbook. He was very much a clinical scientist rather than a front-line clinician, although he was physician to Her Majesty Queen Elizabeth and was present at the birth of all four of her children.