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Stumped by CDP


Bijoux71

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Good afternoon,  I hoped to weaken or destroy unexpected (w+) reactivity observed with [2 of 6] cells at LISS-IAT in allo-adsorbed plasma.  The CDP (Chloroquin Diphosphate) treated (120 min) cells appeared to have enhanced the plasma reactivity to  (2+) at LISS-IAT.  Obviously, there is known info about antigens sensitive to CDP treatment but I have not found any mention of blood group antigens enhanced with this chemical.  I may try to repeat the CDP treatment with a shorter incubation time, or a 1:3 (cell: CDP) ratio in case it is an overtreatment issue.......Any thoughts or suggestions?

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On 12/16/2022 at 11:14 AM, Bijoux71 said:

Good afternoon,  I hoped to weaken or destroy unexpected (w+) reactivity observed with [2 of 6] cells at LISS-IAT in allo-adsorbed plasma.  The CDP (Chloroquin Diphosphate) treated (120 min) cells appeared to have enhanced the plasma reactivity to  (2+) at LISS-IAT.  Obviously, there is known info about antigens sensitive to CDP treatment but I have not found any mention of blood group antigens enhanced with this chemical.  I may try to repeat the CDP treatment with a shorter incubation time, or a 1:3 (cell: CDP) ratio in case it is an overtreatment issue.......Any thoughts or suggestions?

Hello, can I ask if the patient's DAT was positive for IgG? 

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I believe the primary action of CDP is as a mild acid, thereby eluting bound IgG (reducing DAT) and denaturation of HLA/Bg antigens (removal of "unexpected reactivity").

Perhaps the prolonged treatment time removed some of the sialic acid on the red cells and has inadvertently created cells that are easier to agglutinate, i.e., the equivalent of enzyme-treatment ?

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I got the chloroquine method from the most recent AABB Technical manual. Maybe it can help you. Good luck. 

 

 

METHOD 2-20. DISSOCIATING IgG BY CHLOROQUINE FOR ANTIGEN TESTING OF RED CELLS WITH A POSITIVE DAT RESULT

 

Principle

Red cells giving a positive direct antiglobulin test (DAT) result cannot be tested accurately with blood typing reagents that require an indirect antiglobulin technique. Under controlled conditions, chloroquine diphosphate dissociates IgG from the red cell membrane with little or no damage to its integrity. Use of this procedure permits complete phenotyping of red cells coated with warm-reactive autoantibody, including tests with reagents solely reactive by indirect antiglobulin techniques.

 

Specimen

Red cells with a positive DAT resulting from IgG coating.

 

Reagents

1.       Chloroquine diphosphate solution prepared by dissolving 20 g of chloroquine diphosphate in 100 mL of saline. Adjust to pH 5.1 with 1 N NaOH, and store at 2 to 8 C.

2.       Control red cells carrying a single-dose expression of antigens for which the test samples are to be phenotyped.

3.       Anti-IgG antiglobulin reagent.

 

Procedure

Step

Action

1

To 0.2 mL of washed IgG-coated cells, add 0.8 mL of chloroquine diphosphate solution. Similarly treat the control sample.

 

 

2

Mix and incubate at room temperature for 30 minutes.

 

3

Remove a small aliquot (eg, 1 drop) of the treated test cells and wash them four times with saline.

 

 

4

Test the washed cells with anti-IgG.

 

5

If this treatment has rendered the cells nonreactive with anti-IgG, wash the total volume of treated test cells and control cells three times in saline and make a 2% to 5% suspension in saline to use in subsequent blood typing tests.

 

 

6

If the treated red cells are reactive with anti-IgG after 30 minutes of incubation with chloroquine diphosphate, Steps 3 and 4 should be repeated at 30-minute intervals (for a maximum incubation period of 2 hours), until the sample tested is nonreactive with anti-IgG. Then proceed as described in Step 5.

 

 

Notes

1.       Chloroquine diphosphate does not dissociate complement proteins from the cell membrane. If red cells are coated with both IgG and C3, only anti-IgG should be used in tests performed after chloroquine treatment.

 

2.       Incubation with chloroquine diphosphate should not extend beyond 2 hours. Prolonged incubation at room temperature or incubation at 37 C may cause hemolysis and loss of red cell antigens.

 

3.       Some denaturation of Rh antigens may occur.

 

 

 

 

4.       Many serologists test chloroquine-treated control cells for each antigen of interest. Select control cells that are positive for the antigen corresponding to the antisera that will be used to type the patient’s cells.

 

 

 

5.       Chloroquine diphosphate may not completely remove antibody from sensitized red cells. DAT results on red cells from some persons, particularly those with a strongly positive initial test result, may only be diminished in strength.

 

 

6.       In addition to its use for removal of autoantibodies, this method can be used for removal of Bg (HLA)-related antigens from red cells. Appropriate Bg controls should be used.

 

7.       If a commercial kit is used, manufacturer’s instructions should be followed for testing and controls.

 

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