Internal quality control (IQC) is essential for ensuring the accuracy of results in clinical trials. However, there is a significant shortage of commercial quality control materials for IQC in flow cytometry. This study aimed to develop cost-effective in-house-made whole blood materials from clinically discarded samples to serve as IQC in clinical flow cytometry analysis. Discarded clinical whole blood samples were collected to prepare red blood cell (RBC) suspensions through density centrifugation. White blood cell (WBC) suspensions were prepared using made in-house (MiH) lysing buffer, followed by fixation with the MiH fixative solution. The in-house-made whole blood materials were then prepared by mixing the RBC and WBC suspensions. These mixtures were stored under controlled temperature conditions to ensure long-term stability. These materials are intended for use in internal quality control (IQC) for clinical flow cytometry analysis. The recovery rate of RBC suspensions achieved through density centrifugation was 95%. Different blood type RBC suspensions were effectively preserved in Alsever's solution via plasma washing and re-mixing. The average viability of MiH WBC suspensions was 97%, with a recovery rate of 84%, both significantly higher than those observed with ACK (p <
0.001). Among the in-house-made whole blood material samples, sample a1-which included plasma, Alsever's solution, RBC suspension, WBC suspension, and Proclin 300-exhibited the best stability in flow cytometry, demonstrating stable expression of cell antigens for over 5 months. The in-house-made whole blood materials proved to be cost-effective and suitable for use in IQC for clinical flow cytometry analysis.