Many methods are utilized to destroy mononuclear cells (primarily lymphocytes) either for neoplasm or immunosuppression. Neither radiation nor chemotherapy are truly selective or completely successful. The concept of “Double-Dye” treatment of blood for transfusion has been developed in order to inactivate parasites, bacteria and viruses (

J Thromb Haemost 2003; 1 Supplement 1 July: P1114
). In recent studies, it has been observed that this same “Double-Dye” concept presents the possibility of very selectively eliminating lymphocytes (mononuclear cells) without affecting neutrophils in whole blood.

To demonstrate the selectivity of dyes for lymphocytic/mononuclear cell types, two sets of experiments were performed. In the first, 0.3% (w/v) of the “Double Dye’ solution was added to several normal citrated whole blood samples to assess the effect on normal cells, compared to an untreated control. At 24hours post treatment, the lymphocyte count in the treated sample had dropped more than 80%, while little effect on neutrophils was noted. The control counts showed little change for either lymphocytes or neutrophils.

Table 1.

Lymphocyte reduction by 0.3%(w/v) Double-dye solution (units: cells/mm3).

0hrControl0.3% Dye
WBC 6850 ± 560 6920 ± 630 
Neut 5030 ± 375 5100 ± 420 
Lymph 1485 ± 220 1490 ± 265 
24hr Control 0.3% Dye 
WBC 6700 ± 480 5300 ± 505 
Neut 5025 ± 360 5025 ± 420 
Lymph 1474 ± 240 275 ± 75 
0hrControl0.3% Dye
WBC 6850 ± 560 6920 ± 630 
Neut 5030 ± 375 5100 ± 420 
Lymph 1485 ± 220 1490 ± 265 
24hr Control 0.3% Dye 
WBC 6700 ± 480 5300 ± 505 
Neut 5025 ± 360 5025 ± 420 
Lymph 1474 ± 240 275 ± 75 
View Large

In the second series of experiments, 0.3%(w/v) “Double-Dye” solution or 0.15%(w/v) Crystal Violet or 0.15%(w/v) Methylene Blue were added to two T-cell leukemia lines (Jurkat, L1210), with a non-malignant, non-lymphocytic cell line (WISH) for the control. The combination of dyes showed the most potent activity against the lymphocytic lines, while the control was virtually unaffected.

Table 2:

Viability of cell lines after 24 hour exposure to dye solutions.

JurkatL1210WISH
Control 100% ± 2% 100% ± 4% 99% ± 2% 
0.3% Double Dye 37% ± 5% 12% ± 4% 88% ± 9% 
0.15% Meth. Blue 58% ± 12% 52% ± 9% 100% ± 3% 
0.15% Cr. Violet 90% ± 12% 92% ± 10% 99% ± 4% 
JurkatL1210WISH
Control 100% ± 2% 100% ± 4% 99% ± 2% 
0.3% Double Dye 37% ± 5% 12% ± 4% 88% ± 9% 
0.15% Meth. Blue 58% ± 12% 52% ± 9% 100% ± 3% 
0.15% Cr. Violet 90% ± 12% 92% ± 10% 99% ± 4% 
View Large

The novel use of these dyes reported here coincided with the recent interest in utilizing methylene blue to increase transfusion safety, but recognizing that the concurrent need to photoactivate was too toxic to certain proteins and didn’t inactivate all pathogens (

Transfusion 2003; 43(9): 1238–47
). Studies investigating the in-vivo efficacy of these novel immunosuppressive and chemotherapeutic methods are currently underway.

Topics:
dyes, transfusion, methylene blue, sodium bicarbonate, chemotherapy regimen, gentian violet, immunosuppressive agents, leukemia, t-cell, neoplasms, therapeutic immunosuppression

Disclosure: No relevant conflicts of interest to declare.

Author notes

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Corresponding author

2006, The American Society of Hematology
2006
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