| Summary: | Hematological reference samples for external quality assessment (EQA) in hematology are essential for many laboratories in Vietnam, however, the prices are high along with short storing time. This study attempted to establish an optimal formula for the manufacture of hematological reference samples for hematological EQA programs. Human red blood cells (RBCs) were mixed with goose RBCs (pseudo-leucocytes) and goat RBCs (pseudo-platelets) as alternatives for white blood cells (WBCs) and platelets (PLTs), respectively, in different formulas of storage media. The optimal formula was obtained using response surface methodology–central composite design (RSM-CCD). Three main factors affecting the concentrations of RBCs were determined by Plackett–Burmann matrix. The optimal concentrations determined by RSM-CCD were 46% serum, 0.44 g/L neomycin sulfate, and 2.5% glycerol. The model predicted that the maximum number of human RBCs was 4.1 × 10<sup>12</sup> cells/L, pseudo-leucocytes was 6.4 × 10<sup>9</sup> cells/L, and pseudo-platelets was 188 × 10<sup>9</sup> cells/L. The practical concentrations of RBCs, pseudo-leucocytes, and pseudo-platelets were 4.21 × 10<sup>12</sup> cells/L, 6.4 × 10<sup>9</sup> cells/L, and 187 × 10<sup>9</sup> cells/L, respectively, which showed 98.93% similarity with theoretical data. This result could be a premise for further study to improve manufacturing of hematological reference samples for hematology EQA programs.
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