Best poster award winner, Charlotte Bunnage, GSK, UK, with her diploma congratulated by Ofer Doron, Lahav CRO, Israel, on behalf of the MRF Steering Committee. See the poster on page 23. Seafood at the gala dinner night on Thursday at the Olympic Harbour of Barcelona: Delicious food, however maybe “a little” too much: Three more servings went down! (Less food in 2019: we promise…!) Mark your calendar: MRF 2019 The 13th MRF from 22 to 24 May 2019 in Vienna, Austria Follow MRF on The MRF group on LinkedIn is an informative and useful platform where minipig users connect, interact and share results & experiences. Apply for MRF LinkedIn group membership by sending us an email: contact@minipigresearchforum.org or click here: https://www.linkedin.com/groups/4219925 22
MRF 2018 Best Poster Investigating the the mechanical fragility of of minipigs erythrocytes, in in comparison with other species Charlotte Bunnage and Fiona McClure, Comparative and Translational Sciences (CTS), Translational Medicine and Comparative Pathophysiology (TMCP), UK Charlotte Bunnage and Fiona McClure, Comparative and Translational Sciences (CTS), Translational Medicine and Comparative Pathophysiology (TMCP), UK All animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Animals The All human animal biological studies were samples ethically were reviewed sourced and ethically carried and out their in accordance research use with was Animals in accord (Scientific with the Procedures) terms of the Act informed 1986 and consents the GSK under Policy an on IRB/EC the Care, approved Welfare protocol. and Treatment of Animals The human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents under an IRB/EC approved protocol. May 2018 May 2018 Introduction Introduction Mechanical fragility of red blood cells (RBCs) refers to the degree or Mechanical proportion of fragility haemolysis of red that blood occurs cells (RBCs) when RBC’s refers are to the degree subjected or proportion to mechanical of haemolysis stress. that It is occurs thought when that RBC’s minipigs, are when being subjected handled to for mechanical in vivo research, stress. It undergo is thought a degree that minipigs, of stress when which being leads handled to a decreased for in vivo research, resistance undergo and therefore a degree higher of stress fragility which of leads their to RBCs a decreased [1,2]. The resistance increased and use therefore of minipigs higher in research fragility corresponds of their RBCs to [1,2]. an observed The increased increase use in of the minipigs number in of haemolysed research corresponds blood samples to an (figure observed 1) received increase for in clinical the number of pathology haemolysed analyses blood compared samples (figure with dogs 1) received and rats. for Haemolysis clinical is known pathology to interfere analyses with compared measurement with dogs of chemistry and rats. biomarkers Haemolysis is including known sorbitol interfere dehydrogenase with measurement (SDH) of and chemistry Aldolase biomarkers (ALD) (table including 2). sorbitol dehydrogenase (SDH) and Aldolase (ALD) Published (table 2). data shows a difference between pig, sheep, human, cow Published and rabbit data erythrocyte shows a difference fragility [1,2] between and has pig, been sheep, focused human, on osmotic cow and fragility. rabbit Some erythrocyte investigations fragility [1,2] date and back has to 1969 been focused [3] and on may osmotic not be fragility. consistent Some with investigations current scientific date rigour. back to No 1969 recent [3] and published may not data be consistent could be found with current showing scientific a comparison rigour. No of the recent mechanical published fragility data could of multiple be found species, showing which a comparison could explain of the the incidence mechanical of haemolysed fragility of multiple samples species, observed. which could explain the incidence of haemolysed samples observed. The aim of this experiment is to compare the mechanical fragility of The multiple aim species, of this experiment to propose is an to answer compare to the the mechanical question, are fragility minipig of multiple red blood species, cells to propose more fragile an answer than to other question, species? are This will minipig be achieved red blood by inducing cells more haemolysis fragile than over other a 30 minute species? period This via will agitation be achieved with a by steel inducing ball and haemolysis measuring over the haemolysis a 30 minute both period visually via agitation and by with measuring a steel the ball concentration and measuring of the plasma haemolysis both haemoglobin visually and (PHb) by measuring on the Advia the concentration 1800 chemistry of plasma analyser. haemoglobin (PHb) on the Advia 1800 chemistry analyser. Figure 1. Haemolysis index from Advia 1800 showing incidence Figure 1. Haemolysis index from Advia 1800 showing incidence and degree of haemolysis from minipig study samples. and degree of haemolysis from minipig study samples. Materials • • 5ml 5ml lithium lithium heparin heparin samples samples obtained obtained from from each each of of 3 3 minipigs, minipigs, 3 rats, 3 rats, 3 human 3 human and and 3 dogs 3 dogs from from GSK GSK Stevenage/Ware Stevenage/Ware UK. UK. • • Denley Denley TechSpiramix TechSpiramix 5: 5: Used Used to to rock rock 500ul 500ul aliquots aliquots of of blood blood with with or or without without a steel a steel ball. ball. • • Steel Steel ball ball bearing: bearing: 6.5mm 6.5mm diameter diameter steel steel ball ball used used to to mechanically mechanically haemolyse haemolyse samples. samples. • • Pasteur Pasteur pipette: pipette: used used in in preference preference to to pipettes, pipettes, due due to to their their wider wider based based tips, tips, minimising minimising the the potential potential for for haemolysis haemolysis caused caused when when transferring transferring samples samples through through restricted restricted space. space. • • Heraeus Heraeus multifuge multifuge 3 L-R: 3 L-R: After After being being rocked rocked for for the the relevant relevant time time period period at at a speed a speed of of 3000g 3000g for for 10 10 minutes, minutes, samples samples were were spun spun • • Siemens Siemens Advia Advia 1800 1800 Chemistry Chemistry system: system: Used Used to to measure measure plasma plasma haemoglobin haemoglobin levels, levels, indicating indicating level level of of haemolysis haemolysis • • Siemens Siemens Advia Advia 2120 2120 Haematology analyser: analyser: Used Used to to measure measure haematocrit haematocrit (Hct) (Hct) and and Mean Mean Cell Cell Volume Volume (MCV) (MCV) of of each each baseline baseline sample. sample. Methods Samples Samples were were split split in in to to 7 x 7500ul x 500ul aliquots. Aliquots Aliquots were were rocked rocked with with one one 6.5mm 6.5mm steel steel ball ball for for pre-specified time time points: points: 2, 2, 5, 5, 10, 10, 20, 20, 30 30 minutes. At At the the appropriate timepoint, an an aliquot aliquot from from each each animal animal was was removed from from the the rocker rocker and and centrifuged for for 10 10 minutes at at 3000g, following which which the the plasma was was separated into into Starstedt tubes, tubes, assessed visually and and measured for for plasma plasma haemoglobin. A base A base line line sample (no (no rocking) and and a a control sample (rocked for for 30 30 minutes with with no no steel steel ball) ball) were were also also included. Visual Inspect ion: ion: Figure 2. 2. Haemolysis grading sheet, used used to to visually inspect samples (0-7) (0-7) Plasma haemoglobin: ADVIA 1800 1800 Chemistry Instrument, a a quantitative measure. All All samples, standards and and Quality Controls (QCs) (QCs) were were lysed lysed with with saponin. Hb Hb oxidised to to MetHbin in the the presence of of Drabkin's alkaline potassium ferricyanide: MetHb MetHb + KCN + KCN CyanmetHb The The absorbance of of the the sample at at 540 540 nm nm is is proportional to to Hb Hb concentration. Haematocrit and MCV values: measured on the Advia 2120. Hct ranging Haematocrit from 37.8% and MCV to 48.5%. values: Samples measured were on not the normalised Advia 2120. for Hct Haematocrit. ranging from Mean 37.8% MCV to 48.5%. values Samples are also were shown not (table normalised 1). for Haematocrit. Mean MCV values are also shown (table 1). Clinical Chemistry: Clinical chemistry analysis was performed on the plasma Clinical samples Chemistry: using Clinical the Siemens chemistry Advianalysis 1800 Chemistry was performed system on the (A1800), plasma with samples methodology using the available Siemens [5]. Advia 1800 Chemistry system Parameters (A1800), with included: methodology aldolase, available alanine [5]. aminotransferase, aspartate aminotransferase, Parameters included: glutamate aldolase, dehydrogenase, alanine aminotransferase, lactate dehydrogenase, aspartate urea, aminotransferase, SDH, Na, K, Cl glutamate and plasma dehydrogenase, haemoglobin. lactate dehydrogenase, QCs urea, included SDH, Na, BL1, K, BL3 Cl and (manufactured plasma haemoglobin. by Randox Laboratories). SDH1 QCs and included SDH2 BL1, (manufactured BL3 (manufactured by Sekisui by Diagnostics) Randox Laboratories). were QCs for SDH SDH1 measures and SDH2 and (manufactured pre-specified PHb by Sekisui concentrations Diagnostics) (0.2, were 1, QCs 5 g/ul) for (manufactured SDH measures in-house) and pre-specified were QCs PHb for haemoglobin concentrations measurements. (0.2, 1, 5 g/ul) (manufactured in-house) were QCs for haemoglobin measurements. Results Results Plasma Haemoglobin Mean Fold Change Plasma Haemoglobin Mean Fold Change 16,00 16,00 14,00 14,00 12,00 12,00 10,00 10,00 8,00 8,00 6,00 6,00 4,00 4,00 2,00 2,00 0,00 0,00 Baseline 2 Min 5 Min 10 Min 20 Min 30 Min Baseline 2 Min 5 Min 10 Min 20 Min 30 Min Minipig Rat Dog Human Minipig Rat Dog Human Figure 3. Mean plasma haemoglobin fold change comparison between minipig, Figure rat, 3. Mean dog and plasma human haemoglobin rocked at varying fold change time comparison points between minipig, rat, dog and human rocked at varying time points 5 Plasma Haemoglobin (g/L) Plasma Haemoglobin (g/L) 4 3 2 1 0 5 Plasma Haemoglobin (g/L) Plasma Haemoglobin (g/L) 4 3 2 1 0 Plasma Haemoglobin Raw Change Occasion 1 Plasma Haemoglobin Raw Change Occasion 1 5 4 3 2 1 0 Baseline 2 min 5 min 10 min 20 min 30 min Baseline 2 min 5 min 10 min 20 min 30 min Dog 1 Dog 2 Dog 3 Dog 1 Dog 2 Dog 3 Plasma Plasma Haemoglobin Haemoglobin Raw Raw Change Change Occasion Occasion 2 2 5 4 3 2 1 0 Baseline 2 Min 5 Min 10 Min 20 Min 30 Min Baseline 2 Min 5 Min 10 Min 20 Min 30 Min Dog 1 Dog 2 Dog 3 Dog 1 Dog 2 Dog 3 Figure Figure 4/5. 4/5. Mean Mean plasma plasma haemoglobin haemoglobin raw raw change change of of dog dog 1, 1, dog dog 22 and and dog dog 3 3 rocked rocked at at varying varying time time points points on on occasion occasion 11 and and 22 Animal Animal MCV MCV PHb PHbfold fold change change at at 30 30 mins mins Human Human 89.1 89.1 14.2 14.2 Dog Dog 69.3 69.3 11.0 11.0 Minipig Minipig 51.4 51.4 8.8 8.8 Rat Rat 51.1 51.1 5.6 5.6 Table Table 1. 1. Correlation between mean species MCV and and PHbfold change at at 30 30 minutes time time point point Example of of positive and negative interference as as a a result of of haemolysis in in minipig blood sample. Animal Parameter 0 0 min min no. no. (U/L) 22 min 55 min 10 10 min 20 20 min 30 30 min 1 1 ALD 14 14 15 15 17 17 21 21 33 33 33 33 2 2 ALD 13 13 14 14 18 18 26 26 70 70 55 55 3 3 ALD ALD 14 14 15 15 17 17 22 22 28 28 29 29 1 1 SDH 5.3 5.3 -2.5 -2.5 -10.3 -20.1 -32.8 -53.5 2 2 SDH SDH 2.4 2.4 -3.6 -3.6 -8.1 -8.1 -11.5 -17.4 -30.3 3 3 SDH SDH 4.2 4.2 -1.3 -1.3 -6.3 -6.3 -12.6 -27.7 -34.6 Table 2. 2. Aldolase (ALD) and and Sorbitol Dehydrogenase (SDH) activities from from minipig blood samples at at varying time time points of of rocking Discussion Discussion This investigation compared the haemolysis of erythrocytes from minipig, This investigation rat, dog and compared humans the which haemolysis underwent of erythrocytes mechanical stress from for minipig, up to 30 rat, minutes dog and using humans Plasma which Hbunderwent and visual mechanical inspection as stress endpoints. for up to 30 Other minutes chemistry using Plasma parameters Hb and were visual measured inspection but not as all presented endpoints. here. Other This chemistry investigation parameters showed were that measured minipig but not all erythrocytes presented here. were This not investigation more susceptible showed to mechanical that minipig stress than dogs erythrocytes although were they not appear more to susceptible be more susceptible to mechanical than stress rat than erythrocytes. dogs although Human they appear samples to be appeared more susceptible to be most than susceptible rat to mechanical erythrocytes. stress. Human Susceptibility samples appeared to mechanical to be most fragility susceptible appeared to to mechanical correlate with stress. MCV Susceptibility values (table1), to mechanical i.e. small erythrocytes fragility appeared appear to correlate to be with less MCV fragile values than (table1), larger ones, i.e. small which erythrocytes has been noted in appear the literature to be less previously fragile than [4]. larger Plasma ones, Hb results which has from been the Advia noted 1800 in the and literature visual previously haemolysis [4]. scoring Plasma correlated, Hb results making from the results Advia more 1800 reliable and visual and haemolysis confirm that scoring visual correlated, assessment making is adequate results for determination more reliable and of degree confirm of that haemolysis. visual assessment Due to technical is adequate issues, for human determination results presented of degree of were haemolysis. not performed Due to on technical the same issues, day as the human other results species. presented However, were the not same performed dog samples on the were same day as measured the other species. on both occasions: However, the Figures same 4 dog and samples 5 demonstrates were the consistency measured on between both occasions: occasions Figures for the 4 dog. and 5 demonstrates the consistency between occasions for the dog. Table 2 shows the minipig ALD and SDH results when you induce haemolysis Table 2 shows in samples. the minipig This ALD table and emphasises SDH results the when interference you induce that haemolysis haemolysis in samples. can have This on table chemistry emphasises endpoints the reiterating interference the importance that haemolysis of minimising can have disruption on chemistry in these endpoints samples. reiterating Other the chemistry importance parameters of minimising also disruption showed interference in these samples. effects Other but have not chemistry been presented parameters here. also showed interference effects but have not been presented here. From results displayed and previous processing investigations, looking From results at standard displayed centrifugation and previous and processing storage conditions,[6] investigations, the prevalence looking at standard of haemolysis centrifugation in minipig and samples storage received conditions,[6] for analysis the prevalence of haemolysis in minipig samples received for analysis cannot be attributed to the fragility of the minipig erythrocyte or cannot be attributed to the fragility of the minipig erythrocyte or post sampling processes. post sampling processes. Conclusions • Red blood cells of minipigs were found not to be more fragile • Red blood cells of minipigs were found not to be more fragile than dogs or humans but more fragile than rats, consistent with than dogs or humans but more fragile than rats, consistent with previous literature [1,2] previous literature [1,2] • Fragility of erythrocytes correlates with the MCV consistent • Fragility of erythrocytes correlates with the MCV consistent with previous literature [4]. with previous literature [4]. • There is still further investigation required to reduce the • There is still further investigation required to reduce the incidence incidence of of haemolysis haemolysis observed observed in in minipig minipig blood blood samples, samples, possible possible investigations investigations could could include: include: • • Influence Influence of of bleed bleed sites sites • • Correlation Correlation of of incidence incidence of of haemolysis haemolysis with with cortisol cortisol or or adrenaline. adrenaline. • • Influence Influence of of acclimatisation acclimatisation and and training training Acknowledgements Aubrey Swain, Rebecca Grice, Mitul Gandhi - Clinical Pathology and Diagnostics, TMCP, IBPS, GSK UK Neil Young, Matt Gillespie, Matt L'Affineur – In Vivo Science Delivery Team, IBPS, GSK UK Blood Donation Unit, GSK Stevenage References [1] Oyewale, J. J. (1992). Changes in Osmotic Resistance of Erythrocytes of of Cattle, Pigs, Rats and Rabbits During Variation in Temperature and pH. Journal of Veterinary Medicine Series A, 39(1-10), pp.98-104. [2] Brzezinska-slebodzinska,E., 2003. Species differences in the susceptibility of erythrocytes exposed to free radicals in vitro. Veterinary Research Communications, 27 (3), 211-217 [3] Coldman MF, Gent M, Good W (1969) The osmotic fragility of mammalian erythrocytes in hypotonic solutions of sodium chloride. Comp Biochem Physiol. 1969 Nov 15;31(4):605-9 GSK Internal Communication- Charlotte Bunnage and Fiona McClure [4] Betticher DC1, Geiser J Resistance of mammalian red blood cells of different size to hypertonic milieuComp Biochem Physiol A Comp Physiol. 1989;93(2):429-32 [5] GSK SOP: Operation and Maintenance of the Siemens Clinical Chemistry System (Advia 1800), UK Clinical Pathology and Diagnostics SOP_49272 [6] GSK poster: Charlotte Bunnage/ Fiona McClure, "Effect of processing conditions on erythrocyte haemolysis in minipigs" Created by: Digital Media Services 23
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