Leukocyte Acid Phosphatase (TRAP) Kit

Paraffin sections are not ideal controls for use with either the 386A or 387A TRAP kits. When sections are cut from a bone, the bone must first be fixed, then decalcified in EDTA, and finally, paraffin embedded. While the Tartrate Resistant Acid Phosphatase (TRAP) enzyme is relatively hardy, it may not always survive decalcification and paraffin processing. The most effective method of decalcification involves removing calcium with EDTA solutions. Harsh acids such as HCL, Sulfuric Acid, and Formic Acid typically deactivate the TRAP enzyme, isoenzyme 5.

Suitable controls recommended for use with the kit are either peripheral blood smears or buccal smears. It is not known if buccal smears from animals are TRAP positive.
With a peripheral blood smear, Beaker A slides should be positive, and Beaker B slides should be negative.
With a buccal smear, both Beaker A and Beaker B slides should be positive.
If peripheral blood smears or buccal smears are negative for both Beaker A or Beaker B, there may be a problem with the kit or possibly with the staining technique.

In such cases, it is advisable to contact Technical Service for troubleshooting or to file a complaint.

Beaker A stains all isoforms of Acid Phosphatase, while Beaker B stains only the Tartrate Resistant Acid Phosphatase (TRAP) isoenzyme of Acid Phosphatase. Beaker A serves as a control and it is recommended to run both Beaker A and Beaker B. Running only Beaker B makes it impossible to determine if the kit is working properly, whether the enzyme is present, or if the procedure was performed properly.

If staining is present with slides for Beaker B, staining should also be present for the slides stained in Beaker A. However, if only Beaker B is utilized and no staining is present, the problem could be with the kit, the specimen, or how the procedure was performed. Therefore, Beaker A should always be run.

The 387A kit does not have a published protocol for staining cultured cells. The kit is approved for human In Vitro Diagnostic Use and was primarily intended for detecting TRAP positive cells in peripheral blood or bone marrow, particularly for the detection of TRAP positive cells associated with Hairy Cell Leukemia. However, the procedure included with the kit can be used.

First, remove the media, rinse briefly in isotonic PBS or suitable cell culture media, and then air dry the cells. Once dried, the cells can be fixed onto chamber slides, microtiter plates, or culture flasks. After fixing, the cells are washed.

The procedure mentions staining slides for Beaker A and Beaker B. When staining cultured cells, think of staining duplicate wells.
One set of cells will be incubated in the presence of tartrate (Beaker B), and the second set will be incubated without tartrate (Beaker A), which serves as the positive control.

Staining cells without tartrate is essential for ensuring the kit is performing to expectations. The kit was designed to stain an equal number of slides/wells with and without tartrate, and there is a separate product number (387-3) for the tartrate reagent, which may be ordered separately.

The kit instructions call for preparing a specific volume of staining reagent for Beaker A and Beaker B. If needed, the volume of reagent can be scaled down, but once prepared, the staining solution is for one-time use only and cannot be stored for later use.

When staining osteoclasts, both Beaker A and Beaker B are recommended. Beaker A will stain all 7 different isoenzymes of Acid Phosphatase. No tartrate is added to the staining solution for Beaker A. Beaker B includes tartrate. The only cells that will stain in the presence of tartrate are the hairy cells of hairy cell leukemia and differentiated osteoclasts (isoenzyme 5). The remaining cells are inhibited by the presence of tartrate and will not display positive staining.
Please see the link below to review the protocol for this kit:
https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/539/315/387.pdf

The 386A and 387A kits are approved for In Vitro Diagnostic Use on peripheral blood smears and bone marrow smears. However, there are no claims for these kits working on paraffin sections, and they are not intended or approved for use with human bone.

It is uncertain whether 4 weeks in 10% EDTA is too much time for decalcification. The most accurate methods for checking whether decalcification is complete typically involve using an X-ray or a chemical endpoint determination for the removal of calcium.

A buccal smear from the inside of the human cheek makes an excellent positive control.  The epithelioid cells are TRAP positive.

Both kits stain for Leukocyte Acid Phosphatase. The 386A kit was developed first. Some of the reagents are packaged in gelatin capsules. The capsules must be broken open to dissolve the reagents. The gelatin capsules will not dissolve in the staining solution in any reasonable period of time. Once dissolved the staining reagent should be viewed as unstable and the slides should be stained as soon as the temperature reaches 37°C. The 386A kit uses a Citrate Acetone fixative. The 387A kit, however, is formulated with all liquid reagents; there are no gelatin capsules. As a result the 387A kit is considered more user friendly. Once the reagents are prepared as outlined in the package insert, the working solution is again considered unstable and the slides should be stained as soon as the temperature reaches 37°C. The pricing of the kit is slightly less, but the expiration dating may also be slightly less. With either kit, it is recommended that the customer check expiration dating before ordering. The 387A kit uses a Citrate Acetone Formaldehyde fixative. Labs wishing to avoid use of formaldehyde should consider the 386A kit.

Yes.  The product insert contains information regarding only the approved in vitro diagnostic use of the product. Researchers can and do use the kit to stain cultured cells.  The kit is most commonly used to determine when various cell types differentiate into osteoclasts.

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