# Effects of sample age and storage temperature on the flow cytometric diagnosis of chronic lymphocytic leukaemia in South Africa

## TL;DR

This study shows that how blood samples are stored and for how long affects the accuracy of diagnosing chronic lymphocytic leukaemia using flow cytometry.

## Contribution

The study empirically demonstrates the impact of sample age and storage temperature on CLL diagnosis using flow cytometry in a South African setting.

## Key findings

- CD5, CD23, and CD200 antigen expression showed significant changes over time, with better stability at refrigerator temperatures.
- By 96 hours, most refrigerated samples showed a shift to atypical CLL diagnosis, highlighting the importance of timely analysis.
- Extended room temperature storage led to greater diagnostic uncertainty, altering Matutes scores and potentially misclassifying CLL cases.

## Abstract

Chronic lymphocytic leukaemia (CLL) is a haematological neoplasm with characteristic flow cytometric immunophenotyping. Pre-analytical variables impact the quality and reproducibility of flow cytometric data, which could alter the diagnosis from CLL to atypical CLL (aCLL).

This study investigated the effects of pre-analytical variables, specifically sample age and storage temperature, on the stability of key antigens used in the diagnosis of CLL.

Serial flow cytometric analyses were performed from January 2022 to March 2023 on blood samples of 10 CLL patients from the Universitas Academic Hospital Haematology Clinic in Bloemfontein, South Africa. Samples were stored at room and refrigerator temperatures and analysed at baseline, 24 h, 48 h, 72 h and 96 h. We recorded the percentage and intensity of antigen expression of CLL makers, including CD5, CD20, CD23, CD79b, CD200 and sIgM, and assessed whether these affected the adapted and modified Matutes scores.

Statistically significant changes were observed in CD5 (p = 0.028), CD23 (p = 0.003) and CD200 (p = 0.005) expression, with better stability at refrigerator temperature. Two samples showed changes in both Matutes scores by 24 h, irrespective of storage temperature. By 48 h, scores changed to aCLL in six room-temperature and four refrigerated samples. A majority shift in diagnosis to aCLL (modified Matutes: n = 8/10; adapted Matutes: n = 7/10) was observed at 96 h for refrigerated samples.

These findings indicate that pre-analytical variables influence antigen stability in CLL samples, with better preservation at refrigerator temperature, recommending analysis within 48 h of collection.

This study highlights the impact of pre-analytical variables on the flow cytometric diagnosis of CLL. Extended room temperature storage alters antigen expression, shifting Matutes scores and potentially affecting the final diagnosis. The findings emphasise optimised sample handling, for improved diagnostic accuracy in laboratory medicine.