The samples were lyophilized and tested for homogeneity and stability. Homogeneity and stability testing results for lyophilized and frozen control samples for creatinine, total cholesterol and AST showed no significant difference in 20 days across six assessment time points (p > 0.05). The results also indicated that despite the acceptable stability performance within 20 days. The procedure for production of lyophilized quality control material for several clinical chemistry tests showed initial success.
Trang 1PRODUCTION OF LIOPHILIZED QUALITY CONTROL SAMPLES
FOR SEVERAL CLINICAL CHEMISTRY TEST
Nguyen Quynh Giao 1 , Dang Quang Huy 1 , Pham Thi Huong Trang 2 , Dang Thi Ngoc Dung 3 , Trinh Thi Phuong Dung 1
1 Faculty of Medical Technology 2
Quality Control Center for Medical Laboratory
3 Department of Biochemistry
An essential part of medical laboratory quality assurance is statistical quality control (SQC) which requires the laboratory to analyse quality control materials Our research focused on lyophilized quality con-trol material that can be produced using materials from laboratory Plasma samples, anticoagulated by hepa-rin, that had common clinical chemistry parameters including creatinine, total cholesterol and AST, were collected from the Department of Medical Laboratory at the Hanoi Medical University Hospital All parame-ters were within the normal reportable range The samples were lyophilized and tested for homogeneity and stability Homogeneity and stability testing results for lyophilized and frozen control samples for creatinine, total cholesterol and AST showed no significant difference in 20 days across six assessment time points (p > 0.05) The results also indicated that despite the acceptable stability performance within 20 days The procedure for production of lyophilized quality control material for several clinical chemistry tests showed initial success
Keywords: Statistical quality control, quality control material, homogeneity, stability
Corresponding author: Nguyen Quynh Giao, Faculty of
Medical Technology
Email: quynhgiao83107@gmail.com
Received: 15/11/2017
I INTRODUCTION
Statistical quality control (SQC) - an
essen-tial part of medical laboratory quality
assur-ance - is a procedure that requires quality
con-trol materials Quality concon-trol materials are
often commercially bought from various
manu-facturers at relatively high cost by laboratories
Self-production of quality control materials
using samples collected by laboratories is
an-other method of obtaining quality control
mate-rial that is commonly used by many
laborato-ries around the world This method helps
labo-ratories reduce cost and improve the
availabil-ity of qualavailabil-ity control material At the moment,
there is no Vietnamese laboratory able to self-produce lyophilized plasma samples for Statis-tical quality control The aim of this research project was to produce lyophilized quality con-trol materials, following the standard guide-lines from the World Health Organization
II METHODS
1 Study setting
Department of medical laboratory of Hanoi Medical University Hospital (for sample collection), Department of Histology and Embryology (for lyophilization), and National Geriatric Hospital (for samples measurement) from March to May of 2016
2 Materials and methods
Trang 2contain-Laboratories in Hanoi Medical University
Hos-pital and National Geriatric HosHos-pital
Selection criteria for subjects were as
fol-lows: common chemistry parameters including
creatinine, total cholesterol and AST that are
within normal reportable range
Exclusion criteria for this study were as
follows: samples that test positive for HIV,
HBV, and HCV, haemolysed samples,
sam-ples with elevated bilirubin levels, cloudy
ap-pearance, or insufficient amount of plasma
2.1 Homogeneity testing
By the time the sample lot had just been
produced, at least 10 samples (or 10% of the
total number of samples, whichever was
greater) were selected randomly to be
analysed twice The homogeneity assessment
was done either by determining the
between-sample variability using ANOVA-test and t-test
with 95% confidence interval and comparing it
to the assessment criteria (ISO 13528), or by
using statistical comparison tests (Guide 35),
or by using the combination of statistical
criteria and objectives (IUPAC) [2 - 4] (Refer to
the Appendix 1)
2.2 Stability testing
After confirming the homogeneity of the
produced samples, the process of stability
assessment was done following ISO 13528
guideline One random sample is selected
from the sample lot and analysed repeatedly
95% confidence interval, the collected results
are statistically compared with the results
gathered from the homogeneity assessment
step, which are considered to be the initial
concentration of the sample If the results
show no significant difference, the sample is still stable In this study, for each selected point of time within the assessment period with the interval of 4 days, one frozen and one ly-ophilized samples were selected for testing These 2 samples were then repeatedly tested
2.3 Measures and instruments
We used the LY3-TTE/DM8 lyophilizer, Panasonic refridgerator and Abbott Architect Ci4100 for automatic chemistry analysis to conduct our study Analytes were chosen based on several different measurement meth-ods A one-point enzymatic kinetic test was used to measure plasma creatinine, an end point colorimetric test was used to measure total cholesterol and multi-point enzymatic kinetic tests were used to measure AST levels
3 Statistical analysis
Collected data was analyzed using Micro-soft Office Excel 2013, SPSS 20 The protocol for evaluation of stability and homogeneity of samples was determined according to ISO
13528 (2005), IUPAC Harmonized protocol
cri-teria for homogeneity was p > 0.05 for both ANOVA and t-test The acceptable criteria for
4 Research ethics
The study was carried out in laboratory environment, Heparin samples were collected from the Medical laboratory department of the Hanoi Medical University hospital The study did not use for interfere with any private infor-mation that might violate human rights and regulations from the ethical committee
Trang 3III RESULTS
1 Quality of lyophilized plasma-based QC sample
Figure 1 Serology sample before and after freeze-dried
The lyophilized samples was soft and dry before reconstitution Post-reconstitution samples were light yellow colour and clear, with no precipitate or scum formed
2 Homogeneity testing
Table 1 Creatinine concentration results from the homogeneity test of lyophilized and
frozen samples
Sample
p > 0.05
No
Trang 4Graph 1 Creatinine level of lyophilized and frozen QC samples at different points of time
The stability test results gathered from day 4, day 8, day 12, day 16 and day 20 showed no significant difference compared to the initial concentration (p > 0.05) The same results were ob-tained for AST and total Cholesterol
Table 1 shows the creatinine concentration from the homogeneity testing of frozen and lyophi-lized sample There was no significant difference in the within-sample (2 replicates for 1 sample)
lyophilized and frozen samples in term of Creatinine, Total Cholesterol and AST concentrations were also insignificant (p > 0.05)
3 Stability test
Table 2 Creatinine concentration of lyophilized and frozen QC samples at 6 different
points of time
Time
period
Trang 5Table 3 Cholesterol concentration from homogeneity testing
of frozen and lyophilized samples
Measure
Table 4 AST concentration from homogeneity testing of frozen and lyophilized samples
Trang 64 Lyophilization performance
Table 5 describes the lyophilized performance The average evaporation was 95% of the total weight, which satisfied the requirement of maximum 5% water retained in the sample after lyophi-lization Therefore, the lyophilization performance was consistent throughout the lot
Table 5 Lyophilization performance
Sample Vial
(x)
Vial + liquid plasma (y)
Liquid plasma (y - x)
Vial + Ly-ophilized plasma (z)
Lyophilized plasma (z - x)
∆ H 2 O (y - z)
%H 2 O (y-z)/(y-x)
Trang 7IV DISCUSSION
Procedure for production of
plasma-based lyophilized QC material for clinical
chemistry test
The study used pooled plasma sample that
was clear, with no precipitate and had light
yellow colour, which satisfied the requirement
for lyophilisation It was also important to
monitor the performance of the lyophilisation
process The ratio of evaporated water
re-flected the lyophilisation performance
consis-tency of the samples within one lot; it also
helped reassure the homogeneity of the initial
pooled plasma The criterion for the remaining
water within product is no more than 5%.The
lyophilisation vials were weighted on an
elec-trical balance (0.0005g accuracy) before and
after the freeze-dried process The results
showed that the average evaporated water
was 95% in weight Therefore, the samples
within this lot were consistent in lyophilization
performance The final product was soft, dry,
and satisfied requirements for analysis
post-reconstitution [1] This result demonstrated
that the procedure taken was appropriate and
the plasma sample lot was completely
lyophi-lized and was ready for the homogeneity and
stability test
Homogeneity
In this study, there was no significant
differ-ence in the Creatinine concentration
within-sample (2 repeats for 1 within-sample) and
between-sample (1 run for 10 between-samples) (p > 0.05) The
similar results were obtained for Cholesterol
and AST Therefore, it was concluded that the
lyophilized samples were completely
homoge-nous and could be tested for stability
Creatinine concentration changes through out the assessment period compared to the initial value showed that despite the fact that both types of preservation indicated accept-able stability at least 20 days
Based on the result, lyophilized samples were proven to have high stability for Creatinine This report is similar to the results obtained by Rixin Jamtsho and his research team [5]
Total cholesterol results showed similar results regarding the stability of both lyophi-lized and frozen samples within the period of
20 days, with very low variable across different points of time (variable of 0 - 0.2%) This pa-rameter, however, indicated that the lyophi-lized samples were more stable
According to J Maurukas, the concentra-tion of Cholesterol in lyophilized serum were
The finding from this study is also in line with Maurukas’s results [6]
The results of changing AST activity in comparison to the initial value demonstrated that although both frozen and lyophilized sam-ples were stable within the period of 20 days The lyophilized showed less variation
P.D.Divya and K.K Jayavardhannan, activity AST in goat serum stable up to 11 11 days
[7]
The research only covered a short period
of time and had not considered different stor-ing conditions for both freeze and lyophilized samples Further research with larger number
of samples and scales are needed to provide
Trang 8V CONCLUSION
The procedure for production of lyophilized
quality control material for several clinical
chemistry tests showed initial success Further
research on longer period for stability test is
needed to improve upon and confirm the
cur-rent data
REFERENCES
1 Ngo Thin Duy, Trinh Binh, Pham
Duong Tuan, Do Phan Trung (2007)
Research on development of fresh plasma
lyophilization for clinical treatment using
LY3-TTE/DM8 lyophilizer Journal of Medical
Research, 49(3)
2 ISO 15328 (2015) Statistical methods
for use in proficiency testing by interlaboratory
comparison
3 Thompson M., Stephen LR Ellison.,
Wood R (2006) The international harmonized
protocol for the proficiency testing of analytical chemistry laboratories (IUPAC Technical
Report) Pure and Applied Chemistry, 78(1),
145 - 196
4 ISO Guide 35 (2006) Reference
materi-als -General and statistical principles for certi-fication
5 Jamtsho R (2013) Stability of
Lyophi-lized Human Serum for Use as Quality Control
Material in Bhutan Indian J Clin Biochem, 28
(4), 418 - 421
6 Maurukas J (1978) Process for
prepar-ing biological compositions for use as refer-ence controls in diagnostic analyses
7 Divya PD., Jayavardhanan KK (2010)
Effect of Temperature and storage time on Hepatobiliary enzyme activities in Goat serum
Veterinary World, 3(6), 277 - 279
Trang 9Plasma pool 1
Defreeze, mix, filter
20 eppendorf tubes
V = 0.5 mL
Prepare frozen sample,
Randomly select 10 samples,
run 2 repeats per sample
Perform homogeneity test
Take 1 sample every 4
days, run 10 repeats
Perform stability test
Compare
Compare
Selected plasma
Plasma pool 2
Re-analyze
biochemis-try parameters
Defreeze, mix, filter
Plasma pool 3
Divide the plasma pool
20 glass vials V = 5 mL
Prepare lyophilized
Randomly select 10 sam-ples, reconstitute, run 2 repeats per sample
Perform homogeneity test
Take 1 sample every 4 days, run 10 repeats
Perform stability test
Appendix 1 Research process