Individual Determinations

Test strips contain either individual tests or a combination thereof (e.g. for urinary tract infection or diabetes mel-litus compensation check), but they often contain all of the 10 most frequent parameters. The tests are outlined below – the principle of determination is mentioned only if it is important for interpreting results (relatively frequent false positive or false negative results).

5.4.1.1. Specific Gravity (Density)

Specific gravity expresses the density ratio of the sample to distilled water, and so is a dimensionless quantity. The specific gravity of plasma is 1.010, and typically ranges from 1.015 to 1.025 in the definitive urine of a healthy person (⁴). It is measured using physical (e.g. densitometer or refractometer) or chemical means (⁵). Individual principles of measurement do not provide comparable results; osmometer measurement is most accurate (⁶).

The specific gravity of urine reflects renal tubule functions (the secretion and absorption of ions and water, in particular) and may be one of the first signs of renal damage (in particular a loss of reaction to changes in fluid intake).

Indications: Distinguishing between pre-renal and renal cause of renal failure, test for renal concentrating ability (this indication requires more accurate measurement for urine osmolality, see also Kidney Function Tests).

For a pre-renal cause of renal failure (e.g. dehydration), a high specific gravity of urine can be expected (maximum concentration); with a renal cause, tubule functions are damaged and the kidneys produce less concentrated urine.

5.4.1.2. pH

Hydrogen ion concentration in urine reflects the balance of H⁺ production, metabolism and excretion; however, it may also be a sign of a kidney or urinary tract disease. First morning urine pH ranges from 5 to 6, but a random sample from a healthy person may also have a pH of 4.5 or 8 (more alkaline after a meal, more acidic after physical strain, di-etary influences…) (⁷). The reference range for urine pH is rather misleading, and the result should be evaluated along with other data (e.g. acid-base balance status). Acidic urine is found in acidosis and alkaline urine in alkalosis (compens-ation or correction) of the disorder by the kidneys; unless of course the kidneys are the cause of the acid-base balance disorder). Vegetable diets (low protein content, e.g. a vegetarian diet) usually lead to a lower basic production of H⁺ and more alkaline urine.

Indications: Primarily the diagnosis of urinary tract infection, but also the evaluation of acid-base balance

disor-4 It may also range from 1.003 to 1.035 (samples with specific gravity < 1.003 are probably not urine, > 1.035 might occur following i.v. administration of radiopaque substances). The very wide range is caused by the influence of hydration (fluid intake) on this parameter.

5 Or as osmolality, typically using a cryoscopic method (see Kidney Function Tests).

6 Osmolality only depends on the particle count in the solution. Density is also affected by particle size. There-fore, Na⁺ will increase specimen density less than a higher urea or glucose content. The chemical principle of specific gravity measurement (on a test strip) detects only ions and so it is not affected by the presence of urea, glucose or a contrast agent; however, the presence of protein (anion) leads to a false increase in the results. In clinical terms, these limitations are usually negligible due to the preliminary character of the examination.

7 Specimens of urine with pH < 4.5 and > 9 are an indication for a new sawmple collection (probably contamina-ted or artificial proliferation of bacteria with urease).

ders or the diagnosis and monitoring of urolithiasis treatment.

Urinary tract infections caused by bacteria with urease (e.g. Klebsiella) cause alkaline urine pH. The creation of renal calculi also depends on urine pH. Renal calculi (calcium oxalate) are typically formed in acidic urine and it is advi-sable to maintain a more alkaline urine pH (mainly by dietary measures) to prevent recurrence.

Falsely high values can be expected if the sample is supplied late (bacterial activity, see above); falsely low values may occur if the sample is contaminated by reagents from the adjacent reagent pad (typically in protein determination where the reaction takes place in a strongly acidic environment).

5.4.1.3. Leukocytes, Nitrites

The main purpose of both tests is to rule out/confirm suspected urinary tract infection (⁸) and, if needed, sub-sequent indication of microbiological urine culture, and identification of the pathogen and pathogen sensitivity to antibiotics. This test is not therefore used for the precise diagnosis and therapy of urinary tract infection (this is done based on microbiological examination). The leukocyte test detects one of the granulocytic leukocyte enzymes (estera-se). The test for nitrites utilizes the ability of the bacteria to reduce nitrates to nitrites, which is an ability possessed, for example, by enterobacteria such as E. coli or Proteus. The true positivity (sensitivity) of the test is conditional upon urine being in the urinary bladder for at least 4 hours (required to reduce a sufficient amount of nitrates) and the pati-ent having a sufficipati-ent amount of nitrates in the diet (⁹).

Indications: Diagnostics, urinary tract infection screening

5.4.1.4. Protein

The determination of protein (albumin) using a test strip is one of the most important basic urine examinations, since it can reveal a developing renal pathology at an early stage. A small amount of albumin (relative molecular weight is about 70 kDa) and all small proteins (microproteins) penetrate through a healthy glomerulus, although most of these physiologically filtered proteins are then reabsorbed by proximal tubule cells. In a renal disease, either the glomerular membrane (glomerular proteinuria) or tubular cells (tubular proteinuria) are damaged. The limit for proteinuria has been set to 150 mg/24 hrs.

Principle of determination: Acid-base indicator that changes colour in the presence of protein (particularly albu-min that has many binding sites for protons and may remove them from the indicator). To avoid colour changes due to changes in urine pH, the strip contains a buffer, ensuring a constant (acidic) pH of around 3 (see Figure 5.1). This method of determination is most sensitive to albumin (less so to other proteins). The strip usually captures albumin concentrations over 150 mg/l.

8 For the early detection of cystitis, especially in cases of inapparent clinical problems.

9 Vegetables are the main source of nitrates in the diet. This is why the urine nitrite test may be falsely negative in some inpatients (e.g. fasting due to surgery, on parenteral nutrition).

Figure 5.1. The principle of urine protein determinat ion using a diagnostic strip and potential false positive and false negative results

False positive results may be caused by strongly alkaline (pH > 8) and buffered urines. These can remove protons from the indicator (and change its colour) even in the absence of albumin. This can be solved by using another test (e.g. sulfosalicylic acid precipitation test) for strongly alkaline urines, or repeating the examination at a later time (e.g.

in non-complicated urinary tract infections with a high pH of urine, it is reasonable to repeat the determination after antibiotic treatment of the uroinfection).

False negative results can be expected in most pre-renal and tubular proteinurias (a relatively low protein concen-tration in urine and a low indicator sensitivity), including the Bence Jones protein (free light chains in the urine in mul-tiple myeloma). In addition, albumin in the microalbuminuria zone (30-150 mg/l) is usually not detected by standard test strips (refer to Diabetes for more information about microalbuminuria Diabetes/microalbuminuria).

There are also other ways of urine protein (albumin) determination using a test strip that do not suffer so much from pH interference (e.g. sulfosalicylic acid precipitation reaction or the newer chromogenic and immunochemical strip methods).

Depending on the location of the cause, proteinuria is usually divided (¹⁰) into the following types (see Figure 5.2):

• Pre-renal proteinuria (¹¹) – caused by an increased supply of microprotein (which also penetrates a healthy glomerulus) to tubules that do not manage to take it up. This usually involves a low amount of protein (under 1 g per day). Examples of such proteins are β2-microglobulin, α1-microglobulin, acute phase rea-ctants, Bence Jones protein, myoglobin and haemoglobin. The presence of these proteins does not prima-rily mean damaged renal function, and often it is not detected by the test strip at all. If Bence Jones protein is suspected, serum protein electrophoresis and urine immunofixation must be carried out;

• Renal proteinuria – the cause is kidney damage at the level of li or tubules. Glomerular proteinuria is caused by a higher permeation of the glomeru-lus (¹²) and occurs in many glomerulonephritides, diabetic nephropathy or renal amyloidosis;

10 A classical laboratory test to determine the type of proteinuria is SDS-PAGE electrophoresis, where proteins are sorted out by size (thereby easily determining their origin).

11 Sometimes also referred to as “overflow” proteinuria.

12 Can be further subdivided into selective (albumin and/or transferrin penetrate the glomerular membrane) and non-selective (even large proteins such as immunoglobulins penetrate the glomerular memberane) glomerular protein-uria. The prognosis and response to therapy are usually better in diseases with selective glomerular proteinprotein-uria.

Tubular proteinuria is subject to damage to tubules (proximal tubules in particular), for example, during poisoning by heavy metals (Hg, Cd), as an adverse effect of some drugs (gentamicin, cyclosporin, cisplatin, lithium …) or in some viral infections (¹³);

• Post-renal proteinuria – presence of proteins from efferent urinary tract (e.g. α2-macroglobulin or IgM), the cause being inflammation or bleeding, or a pre-analytical error (e.g. menstrual blood, prostatic secre-tion, sperm).

Figure 5.2. The distribution of proteins in urine by size during electrophoresis (takes place in polyacrylamide gel and proteins are usually sodium dodecyl sulfate-coated – SDS-PAGE)

Nephrotic syndrome

Nephrotic syndrome can be defined as a proteinuria capable of causing hypoalbuminaemia and oedemas. The amount of proteinuria may vary; usually it is > 3.5 g/24 hrs. Nephrotic syndrome may be caused by glomerulonephritis with minimal changes, proliferative glomerulonephritis or systemic lupus erythematosus.

U_protein/U_creatinine ratio

This ratio is evidently very useful and practical and can replace urine collection to quantify proteinuria. The ratio is usually expressed in mg (protein)/mmol (creatinine); numerically, the limit is 15 mg/mmol for proteinuria and 350 mg/mmol for nephrotic syndrome.

5.4.1.5. Blood

Principle of determination: Oxidation of chromogen (detection pigment) by haem (pseudoperoxidase activity of haem). The haem is detected, so the strip is sensitive both to erythrocytes (lysed in contact with the reagent pad) and haemoglobin or myoglobin (also contains haem). False positive results can be measured if the sample contains oxidi-zers (e.g. disinfectant residues from test tube decontamination); false negative results can be seen in classical tests in the presence of a high concentration of vitamin C (¹⁴).

13 The determination of relatively stable microproteins (cystatin C or α₁-microglobulin) in the urine is currently used to diagnose this type of proteinuria.

14 Note the resemblance of interferences with the determination of glucose. The basic principle in both is

chro-The following haematuria types are distinguished by intensity:

• Macroscopic haematuria (visible to the eye, urine is pink to red in colour, turbidity present – see Appea-rance of Urine for more details);

• Microscopic haematuria (detectable microscopically or chemically).

• The following haematuria types are distinguished by cause:

• Pre-renal haematuria – haemoglobin gets into the urine (due to the intravascular haemolysis of erythro-cytes, e.g. in haemolytic anaemia, during incompatible transfusion), or myoglobin enters the urine (e.g.

in extensive muscular trauma, burns, as a rare consequence of hypolipidaemic treatment by statins and fibrates), from the blood. The massive presence of each of the aforementioned proteins can cause acute renal failure (obstruction of tubules by precipitated protein) (¹⁵). Erythrocytes are not found in microscopic examination. Making the distinction between myoglobin or haemoglobin is based on patient history, the appearance of urine and serum (see Table 5.3) or other laboratory tests (high LD activity and immeasura-ble haptoglobin in haemolytic anaemia; high CK and AST in muscular damage; alternatively, both proteins can be distinguished in an immunoassay by determining their concentration);

• Renal sometimes also called glomerular haematuria, usually caused by glomerulonephritis. It can be distin-guished from non-glomerular haematuria using phase contrast microscopy. This method visually highlights the edges of erythrocytes in the urine – dysmorphic erythrocytes (¹⁶) (erythrocytes with “thorny” projec-tions, also called acanthocytes are typical – a consequence of passage through the glomerular sieve, see Figure 3) can be found in glomerular erythrocyturia. This type of haematuria is often accompanied by proteinuria and the presence of erythrocyte cylinders (see below);

Figure 5.3. Dysmorphic erythrocytes (acanthocytes) – diagram. Acanthocytes are typical of the glomerular origin of erythrocytes

• Subrenal sometimes also called non-glomerular haematuria, usually caused by bleeding in the urinary tract during urinary tract inflammation, urolithiasis, or urinary tract or renal tumour. Traumatic bleeding after catheterization (especially with concurrent anticoagulation therapy) is quite common. A relatively minimal proteinuria is typical of this type of haematuria. Normally shaped (biconcave, biscuit-shaped) erythrocytes can be found in the phase contrast image.

Sometimes even exercise-induced haematuria (temporary, following strenuous exercise, after becoming chilled) or artificial haematuria (the patient intentionally adds blood into the urine sample) may occur.

5.4.1.6. Glucose

Principle of determination: Chromogen (detection pigment) oxidation by hydrogen peroxide produced by enzyma-tic decomposition of glucose (glucose is oxidized by glucose oxidase to form gluconolactone and H₂O₂). The determi-nation is specific for glucose (other reducing carbohydrates such as galactose or fructose do not react) (¹⁷).

False positive results can be expected in the presence of oxidizers (e.g. some disinfectants used for test tube decon-tamination), false negative results are found in the presence of ascorbic acid (vitamin C) (¹⁸) or, most often, in samples

mogen oxidation (oxidizing agents will therefore cause false positive results and reducing agents false negative ones).

Newer strips prevent vitamin C interference by an added agent that oxidizes the vitamin C, thereby rendering it ineffec-tive.

15 The basic precaution (acute renal failure prevention) in massive myoglobinuria and haemoglobinuria is increa-sed fluid intake.

16 Acanthocytes disintegrate very easily, so the sample has to be delivered to the lab very early – ideally, the sam-ple should be taken there directly.w

17 The reaction with the Benedict’s reagent is still in use (CuSO4 in alkaline environment + heating -> Cu2O [pcipitate colour from blue-green to brick red]). It is of major importance in galactosemia screening and this test is re-commended in some countries in all children under 2 years of age.

18 Some manufacturers add a substance able to oxidize ascorbate into the reaction mix, which prevents

interfe-delivered late (bacterial decomposition).

Indications: In particular, primary diagnosis of diabetes mellitus.

Glucose filtered by the glomerulus of a healthy person is almost completely absorbed in the proximal tubule. If concentration in blood exceeds about 10 mmol/l (known as the renal threshold for glucose), tubular cells are no longer able to take it up and glucose will appear in the urine. Therefore, the causes of glycosuria can be as follows:

• Most commonly, exceeding the renal threshold for glucose (as is the case in diabetic patients);

• Lower renal threshold (Fanconi syndrome, also in healthy individuals) – referred to as renal glycosuria;

• During pregnancy when glomerular filtration is increased (therefore also an increased amount of glucose passing through tubules) and the renal threshold for glucose may be lowered.

Glycosuria may appear even in a healthy person after a meal rich in carbohydrates, so it is advisable to use the second morning urine for (preliminary) diagnostic purposes (¹⁹) and the patient should be fasting until the sample col-lection (the first morning urine may contain postprandial glucose from the evening meal). It should be recalled that the sensitivity of this test is not ideal (glucose appears in the urine when concentration in the blood exceeds 10 mmol/l);

the renal threshold for glucose can be highly individual and may vary in the course of diabetes mellitus. Glycosuria is therefore just a simple preliminary diagnostic test and is not suitable for the monitoring of diabetes compensation.

5.4.1.7. Ketone bodies

Principle of determination: Reaction of a keto group with nitroprusside (in an alkaline environment).

Ketone bodies are primarily produced by the incomplete oxidation of free fatty acids (from fats) in situations with a lack of glucose (e.g. fasting, diabetes mellitus – type 1 in particular, intensive physical strain, vomiting).Ketone bo-dies include β-hydroxybutyrate (BHB) and its oxidized form, acetoacetate (AcAc), the spontaneous decarboxylation of which generates acetone (²⁰) - see Figure 5.4.

Figure 5.4. Ketone bodies. Note the lilac-shaded acetoacetate and acetone keto group (reacting with nitroprusside). In β-hydroxybutyrate the keto group is replaced by the hydroxy group, so it does not react with nitroprusside

In quantitative terms, β-hydroxybutyrate is predominant in urine (about 80%), while acetoacetate forms the rest (the acetone amount is negligible). Only the acetoacetate and acetone keto group reacts with nitroprusside. For exam-ple, a significant increase in the BHB/AcAc ratio may occur in severe diabetic ketoacidosis accompanied by hypoxia and shock, or in alcoholic ketoacidosis. As BHB does not react with nitroprusside, a quantitative underestimation of results may occur in this (exceptional) situation.

Indications: Primarily the monitoring of diabetes mellitus compensation, insulinotherapy in type 1 diabetes pati-ents in particular. If ketone bodies occur in a type 1 diabetes patient’s urine (on insulinotherapy), it means they are lacking insulin and the insulin dose has to be adjusted.

rence with glucose determination.

19 The definitive diagnosis of diabetes mellitus is established from the plasma (see Diabetes Mellitus).

20 More information about ketone body pathophysiology can be found in Acid-Base_Balance_Preparation -> pro-duction of H⁺

5.4.1.8. Bilirubin, urobilinogen

Conjugated bilirubin in the blood (as opposed to non-conjugated bilirubin bound on albumin) freely penetrates into the urine, where it is detected in obstructive jaundice or liver damage, for example. Urobilinogen appears in the urine in liver damage or haemolytic anaemia, for example. Refer to the chapter Liver for more information.

In document Clinical Biochemistry Edited by (Stránka 51-57)