An Experimental Study on RBC Count and Serum Potassium Concentration Changes During Compression Transfusion of WBC-removal Whole Blood☆

Jin-Lin Cheng，Shi-Fn Hn，Y-Qin Li，Yin-Ping Chu，Yu-Mei Sun，Jin-Feng Guo

aFirst Hospital of Shanxi Medical University，Taiyuan，Shanxi 030001，China

bShanxi Provincial People's Hospital，Taiyuan，Shanxi 030001，China

cThe Hospital of Yangquan Coal Industry（Group）CO，LTD，Yangquan，Shanxi 045000，China

An Experimental Study on RBC Count and Serum Potassium Concentration Changes During Compression Transfusion of WBC-removal Whole Blood☆

Jin-Lian Chenga*，Shi-Fan Hana，Ya-Qin Lia，Yin-Ping Chub，Yu-Mei Suna，Jin-Feng Guoc

aFirst Hospital of Shanxi Medical University，Taiyuan，Shanxi 030001，China

bShanxi Provincial People's Hospital，Taiyuan，Shanxi 030001，China

cThe Hospital of Yangquan Coal Industry（Group）CO，LTD，Yangquan，Shanxi 045000，China

A R T I C L E I N F O

Article history：

Accepted 18 June 2015

Published 20 June 2015

Compression transfusion

Blood

Intravenous detaining needle

Pressure

RBC count

Serum potassium

RBC Morpheus

Objective:To observe changes in RBC count，changes，RBC morphology，and serum potassium during compressed transfusion of WBC-removal whole blood.

Methods:Prepared human WBC-removal whole blood and connected transfusion apparatus with different sizes of intravenous detaining needles（18G，20G，22G and 24G）.Observed RBC count and serum potassium concentration under different pressures（100 mmHg，200 mmHg，and 300 mmHg）as blood flowed out of the pinhead end of the intravenous detaining needle.Samples obtained with the 20G needle were smeared on glass slides，and RBC morphologic changes were observed under an oil immersion lens.

Results:RBC count and serum potassium changed slightly under different pressures with different sizes of intravenous detaining needles as blood flowed through the transfusion apparatus.In addition，the observation of blood samples under a common light microscope revealed that coarseprick，oblong，and spindle cell counts in the visual fields increased gradually as the pressure increased.Additionally，a portion of cells had undergone splintering.

Conclusions:While applying 18G，20G，22G and 24G intravenous detaining needles for blood transfusion under less than 300 mmHg of pressure，no significant RBC count change was found in blood samples in the short term.However，there were significant RBC morphologic changes.The results could offer more basis to ensure the clinical safety of patients undergoing blood transfusion.

ⓒ2015 Shanxi Medical Periodical Press.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license（http://creativecommons.org/licenses/by-nc-nd/4.0）.

1.Introduction

Blood transfusion is an important procedure for rescue and disease treatment of critically ill patients and is widely used in clinical practice.In certain circumstances，a blood infusion is urgently needed to correct hypovolaemia to ensure blood perfusion and improve the body's blood circulation as quickly as possible.However，due to the viscosity of blood products，the flow resistance of the blood increases with the viscosity increment of the blood，so that regular transfusion methods cannot satisfy a patient's needs.Therefore，compression，haemodilution and warming have been adopted to accelerate blood transfusion in clinical practice to quickly achieve the required blood volume in the patient.1-3Most reports do not mention the extent of the effect of change in the blood composition and biochemical indices on the human body after using a pressurizer.This study analyzed changes in RBC count，morphology，and serum potassium and the degree of blood composition during compressed transfusion of human WBC-removal whole blood in vitro using a transfusion apparatus with different sizes of intravenous detaining needles.The objective of this report is to provide an experimental basis for selecting the optimal compression transfusion method to ensure the clinical safety of patients undergoing blood transfusion.

2.Materials and Methods

2.1.Blood specimen selection

The material is human WBC-removal whole blood mixed with a mixture of healthy blood and anticoagulants CPDA （Citrate-phosphate-dextrose solution with adenine），which stored between 2℃ and 4℃ within the period of validity.The blood volume in each bag was 400 mL.

2.2.Methods

2.2.1.Experimental instruments and equipment

The experimental instruments and equipment included the following：100 mL measuring glasses，a blood transfusion apparatus（provided by Shandong Weigao Group Medical Polymer Co.，Limited），closed intravenous detaining needles（18G，20G，22G，24G）（provided by Suzhou Bidi Medical Instrument Company），stopwatch，test tubes（produced by United States of America BD Company），pressurizers（CFUSORTM 500 medexine，USA），electronic swing metre，100 mL bags of sterile saline solution，10 mL syringes，and infusion support.

2.2.2.Experimental methods

Put one bag of 400 mL blood at room temperature for 30 min and then placed it on the electronic shaker to shake slightly for 10 min until the colour is evenly distributed.Connected a number 12 puncture needle to the bag，and poured the blood into four small bags，approximately 100 mL per bag，which were marked as 1，2，3，and 4 in order.The blood sample was collected using 10 mL syringes and then injected into two test tubes as the basic control.Then，the test tubes were connected to the blood transfusion apparatus，intravenous detaining needle and exhaust using conventional methods.The height of the transfusion support was 80 cm with the clamp adjusted to the highest setting.The blood was collected into the measuring glass.Blood infusion was performed using the blood samples with numbers 1，2，3 and 4 in order.No pressure was exerted on sample no.1 during the infusion process（P0）.For sample numbers 2，3 and 4，the infusion process is performed with pressure maintained at 100 mmHg（P1，1 mmHg=0.133 kPa），200 mmHg（P2），and 300 mmHg（P3）respectively.The pressurizer was wound around the blood bag，and the inflatable bag was placed in the centre of the blood bag.After every infusion，8 mL of blood was collected from the glass and mounted into two test tubes for determination of terminal values for this experiment. The collected samples were sent for inspection in a timely manner to determine the changes in RBC count，mor-phology and serum potassium concentration.Additionally，the required times of infusion for the four small bags of blood were recorded as the blood flows through the transfusion apparatus and indwelling needles，and the blood flow velocities with the different sizes of indwelling needles and under different pressures were calculated.

2.2.3.Observation parameters and measurement methods

2.2.3.1.RBC count

The KX-21N Sesmex instrument（model G6020）produced in Japan and provided by Jinan Sysmex Corporation was applied for the measurement of RBC count.

2.2.3.2.Ordinary light microscope slide production

Use a 20G needle to take blood sample，and to coat the surface of a glass slide in the routine manner.The Wright-Giemsa staining method was performed for 10 min with a buffer solution that is a mixture of KH2PO4and NaHPO4.Afterwards，the slide was washed with cool running water and air dried it.Due to the thickness of the blood membrane on the entire glass slide，three visual fields were selected at the end of the slide to observe the RBC morphological changes under an oil immersion lens（magnification 10×100）.

2.2.3.3.Serum potassium concentration

Serum potassium concentrations were measured in the hospital biochemical examination room using the KX21N Sesmex instrument（provided by Jinan Sysmex Corporation，and produced in Japan）.

2.2.4.Statistical analysis

The blood flow velocities were expressed as mean values.RBC counts and serum potassium concentrations were expressed as means±standard deviation（x± s）.Then，for the dependent variables that differed under different transfusion conditions，variance analysis was performed and then to check the distribution of the residual error.SAS software was used for further analyses.

3.Results

3.1.Blood flow velocity during compressed transfusion of WBC-removal whole blood using different sizes of intravenous detaining needles（Table 1）

Table 1 Blood flow velocity during compressed transfusion of WBC-removal whole blood using different sizes of intravenous detaining needles（x）.mL/min.

3.2.Changes in RBC count under different pressures and with different sizes of intravenous detaining needles（Table 2）

Table 2 Changes in RBC count under different pressures and with different sizes of intravenous detaining needles（x±s）.×1012.

Differences in dependent variables between before and after transfusion were used for the variance analysis. The data were not normally distributed，with approximately 15%of data values being outliers after checking the residuals.Therefore，the classic variance analysis method could not be applied，and the robust method of M-estimate was applied as an alternative that is less affected by outliers using SAS software.

The results showed that RBC count was not statistically significantly different（P=0.196 3）under different pressures with the same size needle.Thus，RBC count does not change significantly with different pressures below 300 mmHg when using the same size intravenous detaining needle.

3.3.Observations under the ordinary light microscope

When blood is not pressurized，less poikilocytes appear in the field of vision during blood transfusion through the transfusion apparatus and needles.With increased pressure，the number of coarse，spiny，ovalshaped，spindle-shaped cells in the field of view gradually increased；some cells were even teardrop-shaped，and some cell debris was observed.The mean poikilocyte counts of samples obtained through a 20G intravenous detaining needle were determined using a light microscope and are presented in detail in Table 3.

Table 3 Mean poikilocyte counts of samples obtained through a 20G intravenous detaining needle determined using a light microscope（x）./HP.

3.4.Changes in serum potassium concentration under different pressures with different sizes of intravenous detaining needles（Table 4）

Table 4 Changes in serum potassium concentration under different pressures with different sizes of intravenous detaining needlesmmol/L.

Table 4 Changes in serum potassium concentration under different pressures with different sizes of intravenous detaining needlesmmol/L.

Needle types 　Items 　P0　P1　P2　P318G　Base value　30.084±6.138　30.550±5.448　30.111±5.59530.113±5.807 Terminal value　30.187±6.080　31.311±5.851　30.375±5.781　30.685±5.331 Difference　0.734±0.728　0.761±0.440　0.264±0.409　0.573±0.802 20G　Base value　17.800±3.068　16.768±2.980　17.556±3.106　17.449±3.334 Terminal value　18.796±2.249　16.696±2.927　17.925±3.164　17.547±3.433 Difference　0.996±1.874　0.201±0.282　0.369±0.219　0.049±0.136 22G　Base value　17.255±9.181　17.746±8.107　14.904±8.244　14.956±8.072 Terminal value　17.393±8.122　16.870±8.603　15.196±8.529　15.954±8.318 Difference　0.138±0.122　-0.876±3.328　0.293±0.323　0.998±2.015 24G　Base value　38.083±4.484　38.330±4.302　36.115±4.799　36.475±5.178 Terminal value　38.753±4.833　38.934±4.641　37.844±4.192　38.403±3.788 Difference　0.670±0.495　0.604±0.369　1.729±0.6431.928±1.556

The differences in the potassium concentrations of samples obtained through different sizes of detaining needles under the same pressure were examined by analysis of variance（F=2.408，P=0.076）.The differences in the potassium concentrations of samples obtained through the same size detaining needle under different pressures were examined by analysis of variance（F=0.996，P=0.401）.

4.Discussion

4.1.Impact of compressed transfusion on RBCs

Blood is composed of visible and invisible components，namely，cellular and non-cellular components respectively.The cellular components include red blood cells，white blood cells and blood platelets，in which the red blood cell is the largest component of blood.Blood transfusion is important for rescue and treatment of some diseases for critically ill patients in clinical practice. Compressed transfusion is a conventional rescue treatment for patients with haemorrhagic shock and aims to satisfy the amount of body blood infusion needed within the short-term.Due to the particularity of blood，the red blood cells in blood are influenced by many factors，such as pressure and temperature.Red blood cells have particular plasticity and brittleness，and blood flow can have an effect on red blood cell activity.Thus，if red blood cells are damaged before entering the body，the potassium ions in those cells can be released，resulting in an elevated serum potassium concentration.When a large amount of blood containing high concentrations of potassium enters the body，it can cause a rise in the body's blood potassium concentration within a short period of time and can even cause hyperkalemia.Reports in the literature have shown that storage of banked blood at temperatures between 4℃ and 2℃degrees Celcius and prolonged storage time cause changes in blood electrolyte content，especially in regards to the potassium ion.Therefore，a large input of banked blood in short period of time may increase the risk of hyperkalemia and electrolyte disturbance.4Zhang et al.5divided patients into 2 red blood cell suspension transfusion groups to examine blood specimens collected from the blood junction transfusion tube and the scalp needle and found that the RBC count was significantly reduced after the drip compared to before the drip（P＜0.05）and that the serum potassium concentration was increased obviously after the drip compared to before the drip（P＜0.05）.These findings suggest that when blood runs through the transfusion apparatus，the red blood cells are damaged due to shock and friction，resulting in potassium releasing from the cells；this presents as a decreased red blood cell count and an increased serum potassium.By determining RBC count and serum potassium content in 2 mL of whole blood that had passed through the transfusion apparatus and scalp needle，Cao et al.6achieved the same results as Zhang et al.：the RBC count obviously decreased（P＜0.05），and the serum potassium content was significantly increased（P＜0.05）.Li et al.7found that with compressed transfusion by squeezing the blood transfusion apparatus，serum potassium was significantly higher after compressed infusion than before compressed infusion（P＜0.01），and RBC count was significantly lower after infusion than before infusion（P＜0.01）. However，that study did not have a strict limitation on compression time.With regards to the safety margin of pressure degree，relevant studies on retrieval have not been conducted.In the current study，after blood flowed through the transfusion apparatus with the same size intravenous detaining needle，the differences in RBC count and serum potassium content in the blood bag under different pressures were not statistically significant（P＞0.05）. As pressurization was conducted on approximately 100 mL of blood in this experiment，the time of blood transfusion was no more than 2 min.Thus，pressure on blood bags may have little effect on red blood cell damage over a relatively short period of time.However，the mean serum potassium of the blood in the bag tended to be lower than that in the blood that passed through the needle，indicating that the red blood cells still obtained some damage.

4.2.Effect of different pressures on RBCs and serum potassium

Red blood cells form the major visible component of the blood and have a life span of 120 days.During the gradual process of cell senescence，the activity of red blood cell enzymes decrease resulting in a decrease in the energy supply available for the physiological function of the erythrocyte membrane.In addition，membrane lipid composition changes to decrease membrane deformability，making the membrane easier to damage and，thus，affecting the physiological performance of the cell. The method of Watch-style sphygmomanometer cuffmeasurement was adopted by Liu et al.8to observe the RBC count changes in 20 bags of whole blood from before to after 30 min-pressurization with 36 kPa（270 mmHg）.The statistical analysis showed that the difference in the red blood cell count from before to after pressurization was not statistically significant（P＞0.05），indicating that the red blood cells in the blood bag had not been obviously damaged by pressurization.However，on average，the red blood cell count tended to drop from the day before to after pressurization.Li et al. used a pressurized blood transfusion apparatus and randomly selected 20 samples of blood in bags for examination before and after pressurization.They found an RBC count of the blood in the bag before compression of 3.52×1012/L±0.25×1012/L and an RBC count of the blood in the bag after pressurization with 13.3 kPa of 3.49×1012/L±0.37×1012/L.The difference in RBC count from before to after compression was not statistically significant（P＞0.05）.This indicates that during the process of blood transfusion，the change in the RBC count in the blood as it runs through the transfusion apparatus under normal transfusion conditions or a particular low pressure.Namely，the compression blood transfusion apparatus itself causes little damage to red blood cells in blood.However，Chen10reported a statistically significant difference（P＜0.05）in RBC count between before and after processing with a pressurizer（Vital Signs，Britain）.They proposed that the pressurized blood transfusion apparatus causes a certain extent of red blood cell damage.Thus，during pressurization，red blood cell damage should be better supplement the blood volume and correct haemorrhage.This precaution would also prevent the haemolytic reaction caused by red blood cell damage.

The experimental results showed that the RBC count underwent little change with different pressures on the blood bag as the blood flowed through the transfusion apparatus with intravenous detaining needles.However，from before to after transfusion，the mean RBC count tended to decrease，and the mean serum potassium tended to increase.Therefore，some of the red blood cells in the blood had burst，resulting in the rise in potassium content.

Many factors cause red blood cell damage during transfusion：（1）The eddy current formed during the compressed transfusion increases the shear stress，and when red blood cells are exposed to powerful shear stress，their fragility rises and may result in cell destruction and haemolysis.（2）Shear stress and efflux of the blood stream can injure red blood cells as blood flows through small-bore needles.（3）Due to the incomplete removal of glucose from the tube wall by washing with saline，red blood cell aggregation occurs in the tube，resulting in reduction of the cell survival rate.RBC counts were observed before and after compressed transfusion by Li and Han.9，11Controlled observation of red blood cell morphology and other visible components of the blood was carried out under a microscope and revealed no obvious morphological changes or cell destruction. The results of that experiment may be associated with the lower pressure（100 mmHg）used than that used in our experiment.In our experiment，under pressurization，the number of poikilocytes in the blood samples flowed through the transfusion apparatus with an intravenous detaining needle were greater than those of the inner bag（not pressurized），and this phenomenon became more obvious with increasing pressure.

4.3.The limitations of this experimental study

Due to the limitation of blood source，the experimental sample size was relatively small，and the experiment results may have been affected by blood quality（viscosity，density，storage time，and temperature）. Thus，in future studies，larger sample sizes should be used.More attention should be paid to the influence of pressure time on the experimental results.In addition，it should be noted that this was an in-vitro experiment，but in clinical practice，physical factors of patients have to be considered to fully evaluate the advantages and disadvantages of blood transfusion for each patient individually and to ensure that blood transfusion is safe and effective.

5.Conclusions

There was no significant RBC count change in blood samples in the short term while applying 18G，20G，22G and 24G intravenous detaining needles for blood transfusion under less than 300 mmHg of pressure. However，there were significant RBC morphologic changes.

Conflicts of interest

All contributing authors declare no conflicts of interest.

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8 March 2015

in revised form 8 June 2015

☆This work was supported by the Shanxi Science and Technology Development Fund（No.200233）.

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E-mail address：cjl030001@163.com（J.-L.Cheng）.

Peer review under responsibility of Shanxi Medical Periodical Press.

http：//dx.doi.org/10.1016/j.cnre.2015.06.002

2095-7718/ⓒ2015 Shanxi Medical Periodical Press.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license（http：//creativecommons.org/licenses/by-nc-nd/4.0）.