Renal Stone Analysis (Urinary Stones Analysis, Renal calculi, Nephrolithiasis), and Procedure for stone analysis

Sample

1. Renal stones passed in the urine.
2. Renal stones were taken at the time of surgery.

Indications

1. This analyses the constituents of renal stones.
2. It helps to treat the underlying cause of stone formation.
3. For the prevention of future stone formation.

Pathophysiology

1. In the USA about 5% of women and 20% of men may develop renal stones during their life.
2. About 20% of the people with stones have elevated serum calcium concentration due to hyperparathyroidism.
3. Prolonged deposition causes irreversible damage to kidneys.
4. If we get stone passed in the ureter or by surgery may be checked and can guide the patient for diet.
5. The composition of stones:
   1. calcium oxalate stones are 80%
   2. Calcium phosphate is 10%
   3. Uric acid is 9%.
   4. Rest 1% is cysteine or ammonium acid urates or magnesium ammonium phosphate.etc.
   5. These substances crystalize within the organic matrix.

Chemicals of stone	% occurrence of the chemical in stone
Calcium	97
Phosphate	88
Oxalate	65
Magnesium	25
Ammonium	20
Urate	15
carbonate	12
Cystine	2
Xanthine	0.5
Sulfate	rare
Cholesterol	rare
Iron	rare

6. Mechanism of stone formation Depends upon various factors like:
   1. Urine output depends upon fluid intake.
   2. Excretion of relatively insoluble substances.
   3. The absence of a substance which inhibits the stone formation.
      1. Stones form by concentric deposition of the poorly insoluble substance around some nuclei.
         1. This nucleus may be blood clots, bacteria, fibrin, or sloughed epithelial cells.
      2. The precipitation of the insoluble substances initiated by infection, dehydration, urinary obstruction, or excessive intake or production of the compound.
      3. Once the stone forms, then it starts to grow by accretion unless they dislodge or removed by surgery.

Renal stones type	The pH of the urine	Etiology
Calcium oxalate	Variable but mostly in acidic	Milk-alkali syndrome, Concentrated urine, Hypercalciuria (vitamin D intoxication, sarcoidosis, hyperparathyroidism) Osteoporosis, Renal tubular acidosis, hyperoxaluria, and idiopathic hyperoxaluria.
Uric acid	Acidic	Gout, a high purine diet, hyperuricemia, hyperuricaciduria, and urinary hyperacidity.
Calcium phosphate	Alkaline	Renal tubular acidosis, alkali ingestion,  and infection with urea-splitting bacteria.
Cystine stone	acidic	Cystinuria
Magnesium ammonium phosphate	alkaline	infection

Procedure for the stone analysis  (Renal stone analysis):

Physical examination:

1. Describe the shape of the stone.
2. Weigh and measure the size.
3. Note the color, surface appearance, and consistency.

Physical features of the stones:

1. Calcium stones are in the form of:
   1. Calcium stones are  65%.
      1. Calcium oxalate stones = 30%
      2. calcium phosphate = 10%
      3. Calcium mixed (oxalate+phosphate) = 25%
         1. These are hard, small to medium size and often multiple.
         2. An x-ray will show the radiopaque shadow.
2. Uric acid 
   1. These are 5%.
      1. These are small, yellow, friable.
      2. These may be large staghorn shape.
      3. On the  X-ray are radiolucent.
3. Magnesium-ammonium phosphate
   1. These are 25%.
      1. These may be large.
      2. These may be staghorn.
      3. These are on nX-ray are radiopaque.
4. Cystine
   1. These are 2%.
      1. On X-ray radiolucent.
      2. These may be brown in color.
      3. These may be large and may be staghorn.

Reagents needed:

1. Sodium carbonate solution.
   1. 2 mol/L. Dissolve 20 grams Na_²CO₃ in 100 mL of distal water.
2. Phosphotungstic acid.
   1. 50 grams of molybdenum- free sodium tungstate in 350 mL of water, add 20 mL of 85% H₂PO₄, and reflux for 2 hours.
   2. Add 1 drop of liquid bromine, cool to room temperature, make up to 1 L volume with water.
3. Ammonium Molybdate in HNO_3^. 
   1.  Dissolve 5 grams ammonium molybdate in 123 mL water and add 12 mL concentrated HNO₃ with stirring.
4. Sodium cyanide, 50 grams/L.
   1. Dissolve 5 grams NaCN in 100 mL water and add 0.2 mL in concentrated NH₄OH.
5. Ammonium hydroxide in concentrated form.
6. Nitric acid (HNO₃) concentrated needed.
7. Sulphuric acid.
   1. Concentrated H₂SO₄ is needed.
8. Hydrochloride (HCL).
   1. 1 mol/L. Add 8 mL concentrated HCl to water and makeup to 100 mL volume.
9. Sodium nitroferricyanide.
   1. Dissolve 5 grams in 100 mL. Discard when the color fades.
10. Ammonium Thiocyanate.
    1. Dissolve 3 grams in 100 mL water.
11. Sodium oxalate.
    1. Saturate 100 mL water with sodium oxalate by adding 5 grams of salts. shaking well, and allow it to settle.
12. Sodium hydroxide (NaOH) 5 mol/L.
    1. Dissolve 20 grams of NaOH in water and makeup to 100 mL volume when it is cool.
13. Manganese Dioxide (MnO₂)  powdered reagents.
14. Titan yellow.
    1. Dissolve 0.1 gram in 100 mL water. Make alkaline by the addition of 3 drops (5 mol/L).
    2. Store it in an amber bottle and prepare every 30 days.
15. Alkaline Hypochlorite.
    1. 0.5 mol/L NaOH, and 30 mmol/L of NaOCl.
    2. Dissolve 25 grams NaOH in 600 mL water, when it cools down, then add 43 mL of commercial-grade Hypochlorite solution (52 grams/L NaOCl).
    3. The reagent is stable for at least 3 months when protected from light and stored at 4 to 8 °C.
16. Phenol-Nitroferricyanide.
    1. 0.5 mol/L phenol and 0.8 mmol/L sodium nitroferricyanide.
       1. Add 50 grams reagent grade phenol to 500 mL water in a flask, and add 0.25 grams of sodium nitroferricyanide.
       2. Dilute to the mark with water.
       3. The reagent is stable for at least 2 months when stored at 4 to 8 °C.
17. Sodium nitrite (NaNO₂).
    1. 0.1 gram/dL in water.
    2. Prepare just before the use.
18. N-(1-naphthyl) Ethylenediamine Dihydrochloride.
    1. Dissolve 0.1 gram /100 mL of the above reagent in water.
    2. Prepare fresh solution on the day of the procedure.
19. Acetic anhydride.
20. Chloroform.
21. p-Methylaminophenol sulfate.

Chemical analysis

1. Calcium oxalate is the most common stone and it from in 80% of the population and when urine is acidic (low pH).
2. Calcium phosphate stone forms when urine is alkaline (high pH).
3. Uric acid is 5 to 10% and they form when urine is persistently acidic.
4. Cystine stones are rare and are a genetic disorder.

Procedure:

1. The stone is pulverized in the mortar if it is larger than 25 mg and crushed in a test tube.
   1. If this is less than 25 mg then crush it in a test tube with a glass rod.
   2. Dissolve the stone in 1 mol/L HCl.
2. Add 2 mL of HCl 1 mol/L, stir to dissolve.
3. Take the supernatant for the analysis.
5. Calcium
   1. 3 drops of the supernatant on a slide or plate.
   2. 4 drops of sodium oxalate.
   3. 2 drops of NH₄OH.
      1. Result: White precipitate positive for calcium.
6. Oxalate
   1. Take a pinch of MnO2 and add to the supernatant fluid.
   2. If there is the release of tiny gas bubbles.
      1. Result: Positive for oxalate.
7. Urate
   1.  Stone powder or residue adds one drop Na₂CO_3.
   2. Add 2 drops phosphotungstate solution.
      1. Result:  Positive prompt blue color develops.
8. Carbonate
   1. Observe the supernatant fluid for bubbles when the acid is added.
      1. Result: Positive when you see effervescence.
9. Phosphate
   1. Place a few mg of the crushed stone on the plate (or slide).
      1. For very small stone can use 3 drops of the supernatant.
   2. Add 2 drops of the ammonium molybdate.
   3. Add 2 drops of p-Methylaminophenol sulfate.
      1. Result: Positive deep blue color.
         1. You can run the blank (negative control) with the test.
10. Magnesium
    1. Place 3 drops of the supernatant on a white spot plate or slide.
    2. Add 3 drops of NaOH.
    3. Add one drop of Titan yellow solution.
       1. Result: Positive when seeing a red precipitate.
11. Ammonia
    1. Take 3 drops of the supernatant of the white spot plate or slide.
    2. Add 2 drops of NaOH.
    3. Add one drop of phenol-nitroferricyanide solution.
    4. Add one drop of alkaline hypochlorite solution.
    5. Mix and place in 37 °C incubator for 3 to 5 minutes.
       1. Result: Positive blue color develops.
12. Cystine
    1. Few mg of stone powder on a spot plate or slide.
       1. Or can take the residue from the acid solution.
    2. Add one drop of NH₄OH.
    3. Add one drop  NaCN.
    4. Wait for 15 minutes.
    5. Add 2 drops of sodium nitroferricyanide.
       1. Result: Positive is a brick red color.
13. Cholesterol
    1. Take small powder in the small test tube.
    2. 5 drops of chloroform and stir it.
    3. add 10 drops of acetic anhydride.
    4. One drop of concentrated sulphuric acid.
       1. Result: Positive when bluish-green color develops.
14. Xanthine
    1. Place the stone powder in the evaporating dish.
    2. Add 0.5 mL of concentrated HNO₃.
    3. Heat to dryness in the fumes hood.
    4. Add concentrated NH₄OH to a residue in the dish.
       1. Result: Positive when the yellow residue turns orange on the addition of NH₄OH.
15. Iron
    1. Make powder of the stone.
    2. Add 3 drops of HNO₃.
    3. Add 3 drops of NH₄SCN.
       1. Result: Positive when red color develops.

Chemicals	% occurrence of the chemical
Calcium	97
Phosphate	88
Oxalate	65
Magnesium	25
Ammonium	20
Urate	15
carbonate	12
Cystine	2
Xanthine	0.5
Sulfate	rare
Cholesterol	rare
Iron	rare

Calcium oxalate stones

1. Calcium oxalate stones are the most common stones. These may be associated with concentrated urine or consistently increased excretion of urinary calcium or oxalate.
2. kidney stones may be small as 1mm in diameter and larger more than 2.5 cm in diameter.
3. Some time small stones may pass into the ureter from the kidney and from there to the urinary bladder and ultimately through the urethra goes out.
4. Stones produce obstruction and pain.
5. The outcome of stones;
   1. < 5 mm in D stones have high chances to pass out.
   2. Stones of 5 to 7 mm in diameter has a 50% chance to pass out.
   3. Stones of > 7 mm in diameter needs treatment.
6. 90% of the stones are seen on X-Ray.
7. Renal stones formation promoters are:
   1. Albumin.
   2. Globulin.
   3. Matrix substance A.
8. Renal stone inhibitors are:
   1. Magnesium.
   2. Citrate.
   3. Glycoprotein (Tamm-Horsfall).
   4. Pyrophosphate.
   5. RNA.
9. Predisposing factors are:
   1. Pre Urinary are:
      1. More common in the female sex.
      2. Hot climate. This is due to decreased fluid intake.
      3. Stress.
      4. Immobilization.
      5. Protein-rich diet.
   2. Urinary factors are:
      1. increased Calcium.
      2. Increased oxalate.
      3. Increased urate.
      4. Decreased citrate.
      5. Decreased Magnesium.
      6. Increased pH.
      7. Decreased volume.
   3. Metabolic abnormalities are:
      1. Milk-alkali syndrome.
      2. Renal tubular acidosis.
      3. Primary hyperparathyroidism.
      4. Cushing’s disease.
      5. Hereditary hyperoxaluria.
      6. Medullary sponge kidney.

Calcium Oxalate stone formation:

1. There is a possibility that a person is excreting an excess of Calcium or oxalate.
2. Or there is a very small amount of citrate which binds the calcium and does not form the stone.
   1. An inherited tendency to absorb more than the normal amount of calcium from the diet, leading to hypercalciuria.
   2. Taking food with a high concentration of calcium or oxalate leading to an increased amount of these substances into the urine.
   3. Inflammatory bowel disease or intestinal surgery may lead to a nutrient imbalance of absorption and may result in excess urinary calcium.
   4. Raised level of parathyroid hormone leads to an increased level of calcium in the urine and blood.
   5. Metabolic disorders leading to metabolic acidosis causes an increased amount of calcium excreted in the urine.

Calcium Phosphate stone formation:

This has the same factors as calcium oxalate stones.

Uric acid stone formation:

1. This is due to increased production of uric acid or excess secretion, seen in the following conditions:
   1. Gout.
   2. Disorders of uric acid metabolism.

Cystine stone formation:

This is due to increased production and excretion of cystine, This is a hereditary disease.

The patient follow-up with stone formation:

1. Other tests needed to evaluate the renal stone formation includes:
   1. Urinalysis to see the pH, presence of RBCs or WBCs, and type of crystals.
   2. Blood and Urine collected for 24 hours to evaluate the amount of calcium, uric acid, and creatinine. It can evaluate oxalate, phosphates, citrate, or cystine.
   3. Complete blood count to rule out acute infection.

Diagnosis of the stones in a patient:

1. Urine analysis.
2. Ultrasound
3. CT scan
   1. A blood test to guide the patient in the formation of stones.

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