# Optimization of Storage Medium for Hematological Reference Samples in External Quality Assessment
## 3 Results
### Evaluation of Experimental Models  
To test the results of the model, experiments were conducted according to the set values to obtain the number of blood cells as predicted. The osmosis of the combined sample according to the optimized formula was 200 \(\pm\) 3 mOsmol/kg, and this osmotic condition was suitable for three kinds of RBCs (Table S3).  
The average numbers of blood cells obtained from experimental results were 4.21 \(\times\) 10\({}^{12}\) cells/L for human RBCs, 6.4 \(\times\) 10\({}^{9}\) cells/L for pseudo-leucocytes, 187 \(\times\) 10\({}^{9}\) cells/L for pseudo-platelets. The compatibility between experimental and theoretical values was 99.73%, confirming the accuracy of the model and the existence of optimal points.  
The results of the homogeneity evaluation of the stored samples from two different lots are shown in Table 4.  
This result indicated that there was no significant difference between storage conditions at \(\alpha\) = 0.05. The number of RBCs, WBCs, and PLTs was also identified every 5 days to determine their stability in the stored samples (Figure 4).  
It was shown that the \(\mid\)t\({}_{\text{Experiment}}\mid\) value for RBCs (2.04) was higher than the t\({}_{\text{Theory}}\) value (2.03) on the 100 th day. However, this \(\mid\)t\({}_{\text{Experiment}}\mid\) value was observed to be less than the t\({}_{\text{Theory}}\) value on the 95 th testing day. This result indicated that the number of RBCs in the stored samples was stable up to the 95 th storing day. Meanwhile, the \(\mid\)t\({}_{\text{Experiment}}\mid\) value for both WBCs and PLTs was found to be less than t\({}_{\text{Theory}}\) value up to 100 th testing day. Collectively, the stability of the samples that were stored at 2-8 \({}^{\circ}\)C could be maintained up to the 95 th storing day.  
In addition, the stability of RBC, WBC, and PLT number was simultaneously tested on different machine models, such as a CellDyn 1700 and Mindray BC 3000. The result was tested once a month for three months and these data are shown in Table 5.  
\begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline \multicolumn{2}{c}{**Factor**} & \multicolumn{3}{c}{**Level**} & \multicolumn{3}{c}{**Level of Influence**} \\ \hline  & & & \multicolumn{3}{c}{**Human RBCs**} & \multicolumn{3}{c}{**Pseudo-Leucocytes**} & \multicolumn{3}{c}{**Pseudo-Platelets**} \\ \hline
**Notation** & **Factor name** & **Low (-1)** & **High (+1)** & **Influence** & **Prob \(>\) F** & **Influence** & **Prob \(>\) F** & **Influence** & **Prob \(>\) F** \\ \hline X\({}_{1}\) & Serum (\%) & 45 & 65 & 1.833 \(\times\) 10\({}^{-4.8}\) & 0.0836 & 0.22 \({}^{\text{a}}\) & 0.0592 & 4.50 \({}^{\text{a}}\) & 0.0721 \\ X\({}_{2}\) & Neomycin sulfate (g/L) & 0.03 & 0.5 & 2.167 \(\times\) 10\({}^{-4.8}\) & 0.0478 & 0.22 \({}^{\text{a}}\) & 0.0592 & 6.63 \({}^{\text{a}}\) & 0.0137 \\ X\({}_{3}\) & Chloramphenicol (g/L) & 0.02 & 0.5 & 8.333 \(\times\) 10\({}^{-5}\) & 0.4051 & 0.05 & 0.6679 & 1.83 & 0.8008 \\ X\({}_{4}\) & Sodium azide (g/L) & 0.5 & 1.5 & \(-8.333\times\) 10\({}^{-5}\) & 0.4175 & \(-0.05\) & 0.6785 & 2.17 & 0.7725 \\ X\({}_{5}\) & Glycerol (\%) & 1 & 3 & 2.167 \(\times\) 10\({}^{-4.8}\) & 0.0478 & 0.25 \({}^{\text{a}}\) & 0.0350 & 7.83 \({}^{\text{a}}\) & 0.0069 \\ \hline \hline \end{tabular}  
\({}^{\text{a}}\) Statistically significant at \(\alpha\) = 0.1.  
\end{table}
Table 4: Variables in the Plackett–Burman matrix and their influences.  
Figure 4: The stability of samples containing RBCs, WBCs, and PLTs during storage time of 100 days.  
The result of ANOVA at \(\alpha\) = 0.05 showed a similar number of RBCs, WBCs, and PLTs in the stored samples when they were tested on three different machine models, including ABX, CellDyn1700 and Mindray BC3000. This result indicated that the hematological reference samples agreed well with different machine models and can maintain their stability up to the 3\({}^{\mathrm{rd}}\) month of storage time.