Confirmatory tests

Introduction

The aim of these tests is to confirm the identity of crystals. These tests are rarely used, and only if indicated by a clinical situation.
Many confirmation tests can easily be done using routine reagents (uric acid, cholesterol, phosphates, calcium...). However, some tests need special reagents rarely used for other reasons. In these cases, reagents will probably have to be freshly made.
To be sure that the reaction is due to crystals, it is wise to do two tests. The first test is done with the centrifuged supernatant and the second one is done with the dispersed sediment. The color difference between the two tests is then due to solid matters. A good strategy is often necessary with mixed crystalluria.
Since the sediment's volumes are normally small, it is necessary to have a good idea on the identity of the crystals. Before making tests to identify rare crystals, the possibility of a rare form of a usual crystal should be considered and excluded.

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Cystine

From: Ringsrud KM, Linné JJ: Urinalysis and Body Fluids

Reagents

Ammonia 10%

17 ml of concentrated ammonia in 100 ml H2O

Sodium cyanide 5%

5 g of sodium cyanide in 100 ml H2O + 200 ul NH4OH 28%

Sodium nitroprusside 5%

0,5 g of sodium nitroferricyanide in 10 ml H2O. stable 1 week refrigerated

Procedure

1. Three drops of dispersed specimen : Tube A
2. Three drops of supernatant : Tube B
3. To both tubes add one drop of ammonia 5%
4. To both tubes add one drop of sodium cyanide 5%. Wait 5 minutes
5. To both tubes add two drops of sodium nitroprusside 5%
6. Compare colors ( positive = red purple color)

NB: Similar reagents are included in kits for urinary calculus identification.

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Hemosiderin

Hemosiderin can be demonstrated by the Roux reaction ( Prussian blue). This stain reacts with the iron depot of a cytospun specimen. These depots can be free (amorphous mass), within macrophages, within tubular cells, and embedded in casts.

Reagents

Potassium ferricyanide 2%

2 g of potassium ferricyanide diluted in 100 ml H2O

HCl 1%

5 ml concentrated HCl diluted in 500 ml H2O

Working reagent

mix ( 5:5) the potassium ferricyanide 2% and the HCl 1%
prepare freshly before use

Procedure

1. Three drops of dispersed specimen
2. Add 1 ml of working reagent. Mix
3. Wait 10 minutes
4. A blue color indicates iron.
5. Centrifuge or cytospin the mix.
6. Read under the microscope for blue granules.

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Sulfamide

From: Ringsrud KM, Linné JJ: Urinalysis and Body Fluids

Reagents

HCl 10%

50 ml concentrate HCl in 500 ml H2O

Sodium nitrite 0,1%

0,1 g of sodium nitrite in 100 ml H2O. Stable 2 weeks, refrigerated.

Ammonium sulfamate 0,5%

0,5 g of ammonium sulfamate in 100 ml H2O

Diazo reagent 0,1%

0,1 g of N(1-naphtyl ethylene-diamine di-HCl) in 100 ml H2O

Procedure

1. Three drops of dispersed specimen : Tube A
2. Three drops of supernatant : Tube B
3. Two drops of HCl 10%. Wait 5 seconds
4. Two drops of sodium nitrite 0,1%. Wait 60 seconds
5. Two drops of ammonium sulfamate 0,5%
6. Two drops of the diazo reagent 0,1%
7. compare immediately ( positive = magenta )

NB: Similar reagents can be found in some LAP kits.

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