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.

Diagnosis and Monitoring of Cystinosis Using Immunomagnetically Purified Granulocytes

BACKGROUND Cystine determination is a critical biochemical test for the diagnosis and therapeutic monitoring of the lysosomal storage disease cystinosis. The classical mixed-leukocyte cystine assay requires prompt specialized recovery/isolation following blood drawing, providing cystine concentrations normalized to total protein from assorted types of white blood cells, each with varying cystine content. METHODS We present a new workflow for cystine determination using immunomagnetic granulocyte purification, and new reference ranges established from 47 patient and 27 obligate heterozygote samples assayed. Samples were collected in acid-citrate dextrose tubes and their stability was proven to allow for overnight shipping before analysis. Cystine was quantified by LC-MS/MS. RESULTS The new method was reproducible (<15% root mean square error) and specific, assaying purified granulocytes from blood samples that no longer required immediate preparation and therefore allowing for up to 30 h before processing. There was a nearly a 2-fold increase in the therapeutic target (1.9 nmol half-cystine/mg protein) range, established using distributions of patient, obligate heterozygote, and control samples. The 2.5–97.5 percentile ranges (−2 SD to +2 SD around mean) for these cohorts were 0.67–6.05 nmol/mg protein for patients, 0.33–1.35 nmol/mg protein for obligate heterozygotes, and 0.09–0.35 nmol/mg protein for controls. CONCLUSIONS The intracellular cystine determination method using immunopurified granulocytes followed by LC-MS/MS analysis improves the inherent variability of mixed leukocyte analysis and eliminates the need for immediate sample preparation following blood draw.

Implications of anticoagulants and gender on cell counts and growth factor concentration in platelet-rich plasma and platelet-rich gel supernatants from rabbits

SummaryObjectives: Our objectives were as follows: 1) to validate a protocol for producing rabbit platelet-rich plasma (PRP); 2) to determine the influence of two anticoagulants, sodium citrate and acid-citrate-dextrose solution A, and gender on cell count in PRP and growth factor concentration in pure platelet-rich gel supernatants; 3) to correlate the variables evaluated.Methods: Whole blood from 18 New Zealand rabbits (9 males and 9 females) was obtained with sodium citrate and acid- citrate-dextrose solution A for processing PRP fractions (A and B), which were evaluated for haematology. The PRP fractions were either activated with calcium gluconate or lysated with a detergent. The concentrations of transforming growth factor beta 1 and platelet-derived growth factor BB were assayed by ELISA.Results: The sodium citrate PRP-B had significantly higher counts of platelets in comparison to PRP-A and whole blood obtained with the same anticoagulant and the homologous acid-citrate-dextrose solution A PRP fraction. The sodium citrate PRP-A had a significantly higher count of leukocytes compared to the homologous acid-citrate-dextrose solution A fraction. All the PRP fractions had a significant leuko-reduction when compared to whole blood. The sodium citrate PRP-A fraction from female rabbits had significantly lower platelet counts and significantly higher leukocyte counts than the same acid-citrate-dextrose solution A fraction. Growth factor concentration was not affected by the type of anticoagulant or gender.Clinical significance: The type of anticoagulant and gender affected the cell counts in PRP, but they did not influence the growth factor concentration. More complete rabbit PRP studies should be performed before evaluating this type of substance in models of disease.