False positive and Negatives Flashcards
Estimation of proteins in urine, false positive and negatives
Above 150mg/24hrs is proteinuria
Talk about pre renal proteinuria, renal and post renal.
Lab estimation is based on test with sulfosalicylic acid(denaturation) and test strips
Protein error of acid base indicator (tetrabromophenol blue). if a protein is present in the sample, it binds with its amino groups to the indicator. colour intensity depends on the protein concentration and is evaluated visually or instrumentally.
False positive: alkaline pH or if the urine is highly concentrated (due to exhaustion of the buffer)
contamination of sample vessel with disinfectants based on quaternary ammonium salts binds on the indicator in the same way that proteins do
FALSE NEG: Globulins and light chains are difficult to detect (test is sups specific for albumin)
SUBJECTIVELY: compare with colour on scale on tube
OBJECTIVELY: By reflection photometer
Estimation of bile pigments in urine, false positive and negatives
Only conjugated bilirubin is found. unconju bound to albumin can’t cross glom filtrate blah blah
Tests for bilirubin become pos when 30-34 micro mol/l
Presence of higher amount-> dark-brown colour.
Tests based on bill oxidation to green biliverderin or blue bilicyanin (Rosin test)
For urobilinogen: 4-methylaminobenzaldehyde in acidic medium producing a colour condemnation product.
Test strips based on azocoupling bullshit (stabilised diazonium salt), pink or red dye develops if urobilinogen also present its orange.
FALSE POSITIVE: Exogenous substances in urine that have red colour or turn red in strong acid
FALSE NEGATIVE: high contents of nitrites, light exposition (would cause oxidation of bilirubin)
Presence of ascorbic acid
UROBILINOGEN:
Bacterial degradation of bili, 17micromol is upper limit
Its a sensitive marker of liver overload. Over production of bill or damage to the hepatocyte(hepatocellular icterus)
On the other hand, total absence-> blockage of biliary ducts
Diagnostic strips: azocoupling bs. turns red or pink. Weak pink is physiological excretion of urobilinogen.
FALSE POS: Exogenous substances in urine that have red colour or turn red in SA.
FALSE NEG: Formaldehyde, light exposition, old urine.(oxidation of urobilinogen to urobilin)
Estimation of sugars in urine
9-10 mmol/l is renal threshold. Talk about glom filtrate, tubular reabsorption. Glycosuria. Talk about glycosuria in presence of hyperglycemia and normoglyc(disorder of glucose reabsorption in the renal tubular cells)
Method: Either the non specific reactions, based on reducing properties of monosaccharides. So they would reduce cupric ions to cuprous ions forming green yellow, yellow or even brick red ppt.
Fehlings: reduction of Cu2+
Benedict: Red of Cu2+
Nylonder: Reduction of Bi3+
In lab, fehlings solutions was used which is a mix of Fehlings(copper sulcate 1) and feelings sol 2 (NaK.tartate with NaOH)
So mixed with urine and then boiled if presence of reducing sugars, colour change depends on concentration.
Green tint: 8-15mmol/l, green ppt 25 greenish brown 50 brownish red 100 red ppt if more than 150
STRIPS: GOD and Peroxidase bullshit.
Only specific for D glucose not fructose galactose or lactose.
FALSE POS: Contamination of sample vessels with oxidising disinfectants
FALSE NEG: Ascorbic acid exert an inhibitory effect
Other reducing substances(DOPA)
Urinary infection
Estimation of ketone bodies in urine
Acetoacetic acid, beta hydroxybutyric acid and acetone. Made from acetyl Coa in liver, formed in the oxidation of lipids. When biochemical energy is NOT produced from conversions of glucose, metabolism shits to ox of lipids.
60-70% of beta 30-35% of aa, and 2% of acetone. Important for type 1 diabetes. Presence suggests metabolic ketoacidosis and hyperglyc
However small amount can result from weight reduction, vomitting, excessive physical exercise and during starvation.
Legal and lestradets:
Legal: 1)Few crystals of sodium nitropruss +distilled water
2) Three drops added +alkalized with NaOH
3) Few drops of acetic acid
IF colour changes to yellow: creatinine, if red violet KETONE BODIES
Lestradets: Circular paper, wet with distilled water
2) Small amount of powder placed in middle of watch glass
3) add few drops of urine, colour changes to purple if KETONE BODIES are present.
Diagnostic test: Aa and acetone with sodium nitroprusside produced a purple coloured complex.
FALSE POS:
Substances with free sulfhydryl groups, some drugs based on phenolphthalein or sulfophthalein can stain due to alkaline reaction. phenylpyruvic acid
FALSE NEG: beta hydroxy doesn’t react.
Estimation of blood, hemoglobin and leukocytes in urine.
Blood and Hemoglobin: Hb catalyses the decomposition of H2O2, the nascent o2 is able to oxidise suitable substrates. It has a pseudoperoxidase activity. The diagnostic strips for detection of blood in urine is based on the principle: The reagent zone contains the chromogen together with a stabilised hydrogen peroxide.
So reduced chromogenic substrate+ H2O2 catalysed by Hb gives oxidised chromogen and water.
In the lab, we did Heitz boyer (oxidation of reduced phenolphthalein) Mix urine and heitz, then add H2O2, red violet ring will develop if Hb is present and Benzidine (Oxidation of o tolidine or tetramethyl benzidine) Dissolve o tolidine in 2ml of ethanol and acidify with conc acetic acid. Add H2O2 and add urine, blue or blue green develop if Hb is present.
Evaluation: Hemoglobin can occur in urine either free (hemoglobinuria) or bound inside the red blood
cells (hematuria, erythrocyturia). From quantitative point of view we can distinguish
macroscopic and microscopic hematuria. The macroscopic hematuria is visible by bare
eyes, since it gives the urine sample pink or red color1. For detection of the microscopic
hematuria chemical and microscopic examination of urine is needed.
Hematuria is a finding that should always be given an adequate attention, because it can
indicate a severe disease of the kidney or urinary tract. In general the blood in urine can be
either of renal or extrarenal origin. It is a sign of inflammatory kidney diseases such as
glomerulonephritis or pyelonephritis. Another source of erythrocytes in the urine can be
rupture of small blood vessels or bleeding into urinary tract due to tumors or traumatic
injury to kidney or other parts of the uropoetic system; and also urolithiasis.
Hemoglobinuria occurs in intravascular hemolysis in hemolytic anemias, transfusion of
incompatible blood, or paroxysmal hemoglobinuria. Chemical tests cannot differentiate
between the presence of erythrocytes and free hemoglobin in urine; rather, examination of
urinary sediment provides the answer, since in case of hemoglobinuria no erythrocytes are
seen in the sediment.
FALSE POS : microbial peroxidases, contamination of vessels with oxidising disinfectants
FALSE NEG: High conc of nitrites or ascorbic acid
Leukocytes:
Chemical detection of leukocytes with the diagnostic strip is based on demonstration of enzymes
esterases present in granulocytes. The esterase hydrolyses ester of indoxylcarbonic acid to indoxyl,
which reacts with a stable diazonium salt yielding the corresponding azo dye (Fig. 1). In normal
(negative) reaction the strip zone is colored creamy yellow; in case of positive reaction it turns pink or
violet. The chemical demonstration of leukocytes cannot substitute the microscopic examination of
urinary sediment. On the other hand, however, the biochemical examination can also find lysed
leukocytes (e.g. in hypotonic urine), which is not possible with microscopy.
Leukocyturia comes generally as a sign of kidney or urinary tract inflammation. Majority of positive
findings are caused by a bacterial infection of the urinary tract. In case of a positive leukocyte test it is
recommended to perform also examination of proteinuria, hematuria, nitrituria, examination of the
urinary sediment and microbiological examination.
FALSE POS: Formaldehyde, alkaline pH, high specific gravity of urine
FALSE NEG: Ascorbic Acid, some drugs
Results of biochemical examination of urine in urinary infections, urolithiasis and tumors of the urinary tract.
Urinary infections: Bacteria, nitriles, leukocytes, glucose, ammonia conversion, microscopy and cultivation, pH, proteinuria
Urolithiasis: Uric acid and pH, , cysteine
Tumors: Bence-jones proteins, microscopy
Examination of urinary sediment. Determination of type of hematuria.
Not routine, only used when signs point to kidney or urinary tract disease, test for leuko positive, pathological components such as blood, protein, nitrite found in urine.
Equivalent for microscopic examination: Hb, blood to erythrocyte casts, leuko to leuko casts, protein to casts granular, wax, hyalite, nitrites to bacteria.
So First introductory examination with diagnostic strips: erythrocyturia, leuko and nitrites
Then microscopy to find, erythrocytes, leuko, squamous epithelium, transitional epithelium, renal tubular cells, casts (cell free, cellular), bacteria, crystals, yeast, artifacts.
Erythrocytes: stick on the matrix surface degeneration of erythrocytes can transform them into haemoglobin casts (PROOF OF HEMATURIA OF RENAL ORIGIN, because casts are formed only in renal tubule)
Leukocytes: Surface of hyaline matrix holds mostly granulocytes. (indicative of inflammatory kidney diseases)
Squamous epithelium: surface of hyaline matrix bears epithelial cells peeled from renal tubuli (occurs in patients with tubular damage)
Casts: cellular are erythro, leuko, epithelial and bacterial.
Cell free: Hyaline: formed only by tamm hors fall protein, poor light refraction, not always absorb stain (could be proteinuria or normal people following unusual physical exercise)
Granualar: Origin from deg of cells (occurs in proteinuria/damage to tubular cell patients)
Wax: From granular cast by complete decomposition of cell debris. (hallmark of renal failure or renal insufficiency)
Bacterial: Markedly granular, (proof of renal origin of bacteria)
HEMATURIA:
The appearance of urinary red blood cells can be indicative of their origin; most importantly it is
possible to assess whether their source is renal (passage through glomerular membrane), or
subrenal (bleeding into the urinary tract). If the glomerular membrane is severely damaged, not
only proteins, but also erythrocytes can pass into the filtrate. The passage of an erythrocyte through
the glomerular membrane, however, alters its shape and structure. Erythrocytes that display
deviations from the ordinary discoid shape are referred to as dysmorphic. Some may take the shape
of the ‘tyre’ (called annular erythrocytes); or their membranes form ‘blebs’, in this case they are
called acanthocytes. Significant proportion of dysmorphic erythrocytes is distinctive for affection
of kidney glomeruli. If more than 80 % of urinary erythrocytes are dysmorphic, it is conclusively a
glomerular hematuria; simultaneously a proteinuria is found. In contrast, if more than 80 % of red
blood cells in the sediment are isomorphic (normal shape), it is a non-glomerular hematuria
whose source is bleeding into urinary tract due to e.g. kidney/urinary tract tumor, or urolithiasis.
The hematuria originating in the urinary tract is not associated with proteinuria. For correct
identification of dysmorphic erythrocytes the microscopic examination under phase contrast is
required.
Causes of hematuria must always be elucidated; in particular a tumor or a severe glomerulopathy
(glomerulonephritis) must be excluded.
An increased number of red blood cells in the urinary sediment can also be caused by an extreme
physical labor, therapy with anticoagulant drugs or admixture of menstruation blood.
Physiological values: roughly < 5 erythrocytes/visual field
< 5 erythrocyte/ 1 μl urine
Estimation of pH, specific gravity and osmolality of urine.
Relative specifc gravity:
Relative specific gravity (formerly relative density) is determined by mass concentrations of all
dissolved substances excreted into urine.1 Unlike osmolality it depends on the number of
dissolved particles as well as on their molecular masses. High-molecular-weight compounds
affect density more than electrolytes. In case of marked glucosuria or proteinuria the relative
specific gravity of urine increases. Protein concentration of 10 g/l adds 0.003 to the urinary
relative specific gravity; glucose concentration 10 g/l adds 0.004. The relative specific gravity
of urine also significantly depends on temperature.
Under physiological condition the specific gravity of urine during 24 hours ranges from 1.015 to
1.025. In dilution and concentration tests the limit values obtainable are 1.003, and 1.040,
respectively.
Usually in low diuresis spec gravity increases, except in the case of diabetes a high volume and spec gravity is seen.
The specific gravity enables assessment of concentration ability of the kidneys. Values above 1.020
indicate good renal function and ability of kidney to excrete excess of solutes. Highly concentrated
urine suggests a substantial decrease in the circulating blood volume.
Inability of kidney to concentrate urine is called hyposthenuria. The patient needs more water to
excrete the same amount of solutes. Extremely diluted urine can be a sign of impaired kidney
concentration ability, such as in cases of diabetes insipidus (lack of ADH), or side effects of some
drugs. Combination of hyposthenuria with polyuria indicates damage to the renal tubular system
with relatively intact glomerular filtration. A serious sign of kidney damage is isosthenuria. The
kidneys lose any ability to concentrate or dilute; and excrete urine of the same specific gravity as
the glomerular filtrate. The relative specific gravity remains permanently rather low, around 1.010.
Simultaneous finding of isosthenuria and oliguria indicates a severe renal insufficiency. Elevation
of the urinary relative specific gravity – hypersthenuria – results from proteinuria or glycosuria.
ESTIMATION OF RELATIVE GRAVITY OF URINE:
1) Urinometer: Simple device for estimation of spec gravity, has to be calibrated for temperature corresponding to the temperature of urine standing one hour at room temperature (around 15C) need 10-15mL
2) Refractometer: Measures urine density on the basis of the index of light refraction. Only needs 1-2 drops
3) Diagnostic strips: indication zone contains a polyelectrolyte acting as an ion exchanger and bromthymol blue as an acid base indicator. Based on exchange of urinary cations such as Na K and NH4, basically the H+ released acidifies the the indicator previously present in alkaline unprotonated form. This changes the colour of the bromthymol blue.
FALSE NEG: insensitive to presence of non electrolytic substances such as glucose, urea, creatinine, some others
OSMOLALITY:
Depends on the amount of osmotically active particles. It depends on the kidneys ability to dilute and concentrate urine normally ranges from 300-900. Can be from 50 to 1200. Iso, hypo and hyperosmolal urine (in relation to blood)
Theoretically, the final urine is formed from an isoosmolal GF to which the renal tubule add or take clear.
Examination:
1) osmometer: based on the principle that dissolved substances lead to modification of solvent prop (decreased freezing point, increased boiling point, decreased pressure of solvent vapours above the solution)
2) approx calc based on molar conc of Na, K and NH4
3) approx calc from relative spec gravity
Read also on kidneys ability on concentration
PH OF URINE:
So kidney regulates acid base balance based on H+ excretion. the pH of GF is same as plasma then in PCT, hydrogen carbonates are absorbed and H is secreted. In the distal tubules H K exchange.
NORMAL : 5-6.5, 4.5-8 is extreme.
Ph depends on food composition, acid base balance (disorders in acid base which the kidney compensates e.g., aciduria results from metabolic or reap acidosis), infections of kidney or urinary tract(bacteria produce urease, hydrolysis of urea yield ammonia which alkalises urine.), renal tubular acidosis, distal type. Used in diagnostics of and treatment of urinary infection and urolithiasis.
In acidic urine the concrements usually contain calcium oxalate; they can also readily originate
from uric acid. Alkalisation of urine to pH above 7.0 can achieve gradual dissolution of the
concrements from uric acids and prevent their further formation. Acidic pH also favors
precipitation of cystine.
In alkaline urine the stones are made of phosphates. If pH is above 7.0, ammonium-magnesium
phosphate (struvite – MgNH4PO4·6H2O), calcium phosphate, and calcium carbonate precipitate
from the solutions.
EXAMINATION: Using diagnostic strips, indic zone has suitable acid base indicators i.e. methyl red and bromthymol blue. For precise measurement pH meters are used.
Read also on acidication ability of kidney.
