AUBF LEC WEEK 2

Kidney function

Cortical – Cortex of the kidney

Juxtamedullar – Medula

1. Clear waste product

2. Reabsorption of nutrients
a. Per kidney contains approximately 1 to 1.5 million nephrons

KIDNEY FUNCTION

 Renal blood flow

 Glomerular Filtration
 Tubular reabsorption
 Tubular Secretion

Renal Blood Flow

Receives 25% of blood pumped by the heart.

 Process of blood entering the nephron

o Enters the afferent arteriole
o Then flows to the glomerulus
o Followed into efferent arteriole
o Before entering the renal vein, Blood from the renal arteriole will enter peritubular capillaries and vasa recta
will flow slowly to the cortex and medulla of the kidney.

Based on the average body size of 1.73m2 of surface total renal blood is approximately 1200mL/min and the renal plasma
flow ranges 600 to 700mL/min.

Glomerulus filtration

The glomerulus consists of a coil of approximately eight capillary lobes referred as glomerulus filtration barrier. It is
located at the bowman’s capsule which forms the beginning of the renal tubule.

 Its serves as a non-selective filter of plasma of substances with molecular weights less than 70,000.
 It includes the cellular structure of the capillary walls and bowman’s capsule, Hydrostatic pressure and oncotic
pressure the feedback mechanism of the renin-angiotensin-aldosterone system (RAAS)

Cellular Structure and the glomerulus

The plasma filtrate must pass through the three glomerular filtration barrier cellular layers : capillary wall membrane,
basement membrane (basal lamina), and the visceral epithelium of bowman’s capsule.

 Capillary wall differ from those in other capillaries by containing pores referred to as fenestrated.
 Further restriction of large molecules occurs as the filtrate passes through the basement membrane and the thin membrane
covering the filtration slits formed by the inter twinning foot processes of the podocytes of the thinner layer of Bowman’s
capsule.
 The barrier contains a shield of negativity that repels molecules with a positive charge.

Glomerular Pressure

The presence of hydrostatic pressure results from the smaller size of the efferent arteriole and it enhances filtration. This
pressure is necessary to overcome the pressure from the fluid within the bowman’s capsule and the Oncotic pressure of the unfiltered
plasma proteins in the glomerular capillaries.

 ↓of blood pressure = Dilation of afferent arterioles and constriction of efferent arterioles = Prevents a marked decreased
blood flowing through the kidney thus preventing the increase in the blood level of toxic waste product.
 ↑ of blood pressure = Constriction of the afferent arterioles to prevent over-filtration or damage to the glomerulus.
 NOTE: hydrostatic is inversely proportional to oncotic
RAAS Renin-Angiotensin-Aldosterone-System

Regulates the flow of the blood to and within the glomerulus. The system responds to changes in blood pressure and plasma
sodium content that is monitored by juxtaglomerular apparatus which consists of juxtaglomerular cells in the afferent arteriole and
the macula densa located at the distal convoluted tubule.

 Low plasma sodium content decreases water retention within the circulatory system
 When the macula densa senses such change , a cascade of
reactions within the RAAS occurs.
 Renin an enzyme produced by the juxtaglomerular cell
secretes and reacts with blood borne substrate
angiotensinogen → produce an hormone Angiotensin I,
passing through the alveoli of the lungs ACE angiotensin-
converting enzyme → changes it into a active Angiotensin II
correacts renal blood flow in the following ways: causing
vasodilation of the afferent arteriole and constriction of the
efferent arterioles, simulating reabsorption of sodium and
water in the proximal convoluted tubule → triggering the
release of aldosterone. Therefore angiotensin II produces
pressure within the nephron.
 Every minute approximately 2-3 million glomeruli filter
approximately 120mL of water-containing low molecular
weight substances.
 This filtration is non-selective the only difference between the composition of the filtrate and the plasma protein is the
absence of plasma protein.
 Analysis of the fluid as its leaves the glomerulus shows the filtrate to have a specific gravity of 1.011 and confirms that it is
chemically an ultra-filtrate plasma.

Tubular Reabsorption

The body cannot lose 120mL of water containing essential substances every minute. The ultrafiltrate enters the proximal
convoluted tubule which is the major site of reabsorption.

 Reabsorption Mechanism:
o Active Transport – to occur the substance to be reabsorbed must combine with a carrier protein contained in the
membrane of the renal tubular epithelial cells
 Reabsorbs glucose, amino acids and salts in the proximal convoluted tubule, chloride in the ascending
loop of henle, and sodium in distal convoluted tubule.
 From higher concentration to lower concentration.
o Passive Transport – Is the movement of molecules across a membrane as a result of differences in their
concentration or electrical potential on opposite sides of the membrane – physical difference gradients.
 Reabsorbs water in all parts except in the ascending loop of henle, Urea in proximal convoluted tubule
and the ascending loop of henle and sodium together with chloride in the ascending loop of henle.
 From lower concentration to higher concentration
 These mechanisms can be influenced by the concentration of the substance being transported.
 The filtrate concentration exceeds the maximal reabsorptive capacity ™

Tubular Secretions

Involves the passage of substances from the blood in the peritubular capillaries to the tubular filtrate.

Functions of tubular secretions

 Eliminating wastes products not filtered by the glomerulus and

 Regulating the acid-base balance in the body through the secretion of hydrogen ions.

Many foreign substances such as medication cannot be filtered by the glomerulus because they are bound to plasma proteins.

The major site of removal of these nonfiltered substances is the proximal convoluted tubule.
 To maintain normal blood pH 7.4, the blood must be buffered and eliminate the excess acid formed by dietary intake and
body metabolism. The buffering capacity of the blood depends on bicarbonate.

This process provides almost 100% reabsorption filtered bicarbonate and occurs primarily in the proximal convoluted tubule.

Resulting of the small molecular size, hydrogen ions are readily filtered and reabsorbed.

HISTORY

 Hippocrates—5th century BCE wrote book on “uroscopy”. During the Middle Ages, physicians concentrated their
efforts very intensively in usorcospy.
 Frederik Dekkers – Discovery in1964 albuminuria by boiling urine.
 Thomas Bryant -- These Charlantans “pissed prophets” became the subject book published in 1627, The revelation
of thus book inspired the passing of the first medical licensure laws in England.
 Thomas Addis – The intervention of microscope in the 17 th century led the examination of urinary sediment to the
development of methods for quantitating the microscopic sediment.
 Richard Bright – introduced the concept of urinalysis as part of the doctor’s routine examination in 1827.

Factors that affects the urine concentration:

 Dietary intake
 Physical activities
 Body metabolism
 Endocrine functions

Two unique characteristics of a urine specimen account for this continued popularity:

1. Urine is readily available and easily collected.

2. Urine contains information which can be obtained by inexpensive laboratory test about many of the body’s
major metabolic functions.

URINE COMPOSITON: Urine is normally 95% water and 5%solutes.

URINE VOLUME: Normal daily urine output is usually 1200 to 1500mL. A range of 600 to 2000 mL is considered normal.

Oliguria: Decreased in urine output, less than 1mL/kg/hr for infants, less than 0.5Mllkg/hr for children, less than
400mL/kg/hr for adults.

Anuria: Cessation of urine, serious damage to the kidneys, of decreased in blood flow to the kidneys.

Polyuria: An increased in daily urine volume, greater than 2.5L/day in adults, 2-3mL/kg/day in children. Often associated
with diabetes mellitus and diabetes insipidus.
RENAL FUNCTION TEST AND PHYSICAL EXAMINATION LECTURE

 GLOMERULAR FILTRATION TEST

o The standard test used to measure the filtering capacity of the glomeruli are termed as clearance test
o Clearance test – measures the rate at which kidneys are able to remove filterable substances from the blood.
o To ensure that glomerular filtration is being measured accurately, the substance must be one that neither reabsorbed
nor secreted by the tubules.
o Factors to consider in selecting a clearance test:
 Substance’ stability in the urine must stable for a 24 hour collection period.
 The substance availability to the body
 The availability of the test to analyze the substance.
 Plasma consistency.
 The substance is neither reabsorbed nor secreted by the tubules.
o Example:
1. Urea – the earliest glomerular filtration tests measures urea because its presence to the urine specimens and he
existence of routinely used methods of chemical analysis. 40% of the urine is reabsorbed, normal values were
adjusted reflect to the reabsorption and patients were hydrated to produce a urine flow of 2Ml/min ensure no
more than 40% of the urea was reabsorb.
2. Creatinine – is a waste product of muscle metabolism that is produced by creatinine phosphokinase from
creatine that links with ATP to produce ADP and energy.
a. The use of creatinine has several disadvantage and careful considerations should be given to
them:
i. Some creatinine is secreted by the tubules and secretions that increases as blood level rise.
ii. Chromogens present in human plasma react in the chemical analysis that helps in
counteracting the falsely elevated rates caused by tubular secretion.
iii. Medications, including gentamicin, cephalosporins, and cimetidine, inhibits tubular
secretion of creatinine resulting falsely low serum levels.
iv. Bacteria will breakdown urinary creatinine if specimens are kept in room temperature
for extended periods.
v. A heavy diet , like meat during 24hr urine specimen will influence the results if the plasma
specimen is drawn before the collection period = the increase intake of meat can raise the
urine and plasma levels of creatinine during 24 hour collection period.
vi. Measuring creatinine clearance is not reliable indicator in patients suffering from muscle
wasting diseases or person involve in heavy exercise or athletes supplementing with
creatinine.
vii. Accurate results depend on the accurate completeness of a 24 hour collection.
viii. It must be corrected for body surface area, unless normal is assumed and must always
corrected for children.
3. Inulin – a polymer of fructose, is an extremely stable substance that is not reabsorbed or secreted by the
tubules.
4. Beta2- microglobulin – molecular weight of 11,800 dissociates from human leukocyte antigen and a constant
rate and is rapidly removed from the plasma glomerular filtration. Sensitive method using enzyme
immunoassay are available for measuring beta2- microglobulin. A rise in the plasma level beta2-microglobulinn
= more sensitive indicator of decreased in GFR than creatinine clearance – not reliable for patients with
history of immunologic disorders or malignancy.
5. Cystatin C – is small protein(MW= 13,359) produced at a constant rate by all nucleated cells. It is readily
filtered by the glomerulus and reabsorbed and broken down by the renal tubular cells. Immunoassays are
available for measuring cystatin C. Monitoring of cystatin C is recommended for pediatric patients, elderly
and critically ill patients. The advantage of cystatin C is that it is independent of muscle mass.
6. Radionucleotides – exogenous procedures, more labor intensive, and costly, injecting radionucleotides such as
I-iothalamate provides for the method for determining glomerular filtration through the plasma disappearance
of the radioactive materials and enable visualization of the filtration of both or one kidney.

o PROCEDURE: greatest source of error in any clearance procedure using urine is the use of improperly timed urine
specimens.
: the GFR is reported in milliliters cleared per minute
: to calculate you will need:
  Urine volume in mL/min
 Urine creatinine concentration in mg/dL
 Plasma creatinine concentration in mg/dL
: normal creatinine balance 0.5 to 1.5 mg/dL: M: 107 to 139 mL/min F: 87 to 107 mL/min

o Clinical Significance of creatinine clearance

o Used to assess the functioning nephrons but also the functional capacity of these nephrons.
o Used to determine the extent of nephron damage in known cases of renal disease.
o Monitor the effectiveness of the treatment
o Feasibility of administering medications

 TUBULAR REABSORPTION TEST

o Test to determine the ability of the tubules to reabsorb the essential salts and water that have been nonselecctively
filtered by the glomerulus are called concentration test.
o Ultrafiltrate plasma gravity = 1.010
o Methods to produce water deprivation
 Fishberg: measured a specific gravity, patients were deprived of fluids for 24 hours
 Mosenthal : compared the volume and specific gravity of day and night urine samples.
 Osmometry : test for quantitative measurement fr renal concentration ability, reported in milliosmole
(MOSM).
o P-aminohippurc acid test (PAH)
 exogenous procedure
 non toxic substance is loosely bound to plasma proteins which permits complete removal as the blood
passes through the peritubular capillaries.
 All plasma PAH is secreted by the proximal convoluted tubule, therefore the volume of plasma through the
kidneys determines the amount of PAH excreted in the urine..
 The advantage, the chemical PAH meets the criteria needed to measure renal bloodflow.
 Most commonly associated with tubular secretion and renal blood flow.
o Phenolsulfonphhthalein test
 not yet currently performed
 standardization and interpretation of PSP result are difficult however because of interference by
medications, elevated waste products in patient’s serum.
 Colorless in acid solution and red in alkaline solution..
o Urinary ammonia and titratable acidity
 Determines the defective function in the ability of the kidney to produce acidic urine
 Factors involve to produce acidic urine
 TUBULAR SECRETION OF HYDROGEN ION
 PRODUCTION AND SECRETION OF AMMONIUM IONS
o A normal person excretes approximately 70mEq/day of acid in the form of titratable acid,
hydrogen phosphate ions or ammonium ions
o Diurnal variation affects urine acidity appears shortly after arising and postprandially
approximately 2pm and 8pm the lowest pH is found at night.
o The inability to produce an acid urine in the presence of metabolic acidosis is called
renal tubular acidosis.
 o SPECIMEN: fresh or toluene preserved urine specimens collected at 2hr intervals
from patients who have been primed with an acid load consisting of oral ammonium
chloride.

PHYSICAL EXAMINATON

 Determination of the urine color, clarity, and specific gravity

 It provides preliminary information concerning disorders such as glomerular bleeding, liver disease, inborn errors
metabolism and UTI.
 It aids in evaluation of renal tubular function
 It is use to confirm or to confirm things in the chemical and microscopic analysis.

COLOR

- Varies from colorless to black.

- These variations maybe due to normal metabolic functions, physical activity, ingested materials or pathologic conditions.
- Normal urine color – common description include pale yellow, yellow, dark yellow
o The specimen should be examine under a good light source, looking down through the container against a white
back ground
o Urochrome – responsible for the pigment yellow color of the urine
 Product of the endogenous metabolism and normal conditions of the body that produces at its
constant rate.
 The actual amount of the urochrome produced is dependent on the body’s metabolic state, with
increased amount produced thyroid conditions and fasting.
 Increases in urine that stands at a room temperature
 Dilute urine will be pale yellow and concentrated specimen will be dark yellow,
 Owing variations in the body’s state of hydration, these differences in the yellow color can be
normal
 Additional pigments uroerythrin and urobilin
 Uroerythrin – a pink pigment, most evident in specimens that have been refrigerated =
precipitation of amorphous urates
 Urobilin – oxidation product of the normal urinary constituent urobilinogen imparts the orang-
brown color urine that is not fresh.
- Abnormal urine color
o Dark yellow or amber urine not always signify normal concentrated urine but can be caused by presence of
abnormal pigment bilirubin
 Concentrated specimen
o Amber
 Dehydration from fever and burns
o Yellow orange
 Photooxidation of large amounts excreted urobilinogen and urobilin
 Presence of bilirubin, when the specimen is shaken yellow foam appears.
 Phenazopyridine (pyridium)
 Thick orange pigment that interferes in chemical test that are based in color reactions.
 It produces yellow foam
o Yellow green
 Photooxidation of bilirubin caused by the presence of biliverdin
o Brown/black
 RBC remaining in an acidic urine produces brown color due to the oxidation of hemoglobin to
methemoglobin.
 Glomerular bleeding
 Contains melanin or homogentisic melanogen, produced in excess when a malignant , melanoma is
present.
  Homogentisic acid a metabolite of phenylalanine, imparts black color to alkaline urine from persons with
the inborn-error metabolism called alkaptonuria,
 Other causes: levodopa, methyldopa, phenol derivatives and metronidazole.
o Blue
 Medications like methocarbamol (Robaxin), methylene blue, amitriptyline (Elavil)
o Green
 Ingestion of clorets
 Bacterial infections – pseudomonas species
 Phenol derivatives
 A purple staining may occur in catheter bags and is caused by indicant, Klebsiella and Providencia
species.
o Purple
 Indicanuria
 Bacterial infection
o Red
 Can be due to menstrual contamination, ingestion of highly pigmented foods, and medications, eating fresh
beets, rifampin, phenolphthalein, phenindione, phenothiazines.

o Port wine
 Due to oxidation of porphobilinogen to porphyrins

CLARITY

 General term that refers to the transparency or turbidity of urine specimen

 Precipitation of amorphous phosphates and carbonates may cause white cloudiness
 Common terminology use:
o Clear – no visible particulates, transparent
o Hazy – few particles print easily seen through urine
o Cloudy – many particulates, print blurred through urine
o Turbid – print cannot be seen through urine
o Milky – may precipitate or be clotted
 Amorphous phosphates and carbonates produce white precipitate in urine in alkaline pH
 Amorphous urates produce precipitate in acidic urine that resembles pink brick dust due presence of uroerythrin.
 RBC, WBC and bacteria is caused by infection or systematic organ disorder.
SPECIFIC GRAVITY

 Instruments use:
o Urinometer – consist of weighted float that displaces a volume of liquid equals to its weight.
 Disadvantages: less accurate, large volume needed and temperature correction needed, not recommended
by CSLI.
o Harmonic oscillation densitometry – based on the principle that the frequency of sound wave entering a solution changes in
proportion to density of the solution.
o Refractometer – refractive index is a comparison of the velocity of light in air with the velocity of light in a solution
 Advantages: small volume of urine needed, no temperature corrections
 Calibrators:
 water that should read 1.000
 Distilled 5% NaCl that should read 1.022. (+/- 0.001)
 9% Sucrose that should read 1.034 (+/- 0.001)
o Chemical reagent strip – based on the pka of a polyelectrolyte in alkaline medium.

CLINICAL SIGNIFICANCE

 The specific gravity entering the glomerulus is 1.010

 Isosthenuric –1.010
 Hyposthenuric – below 1.010
 Hypersthenuric – above 1.010
 Normal random urine – 1.003 to 1.035
 Below 1.003 is not a urine
 Above 1.035 seen IV pyelogram, dextran

ODOR

 Its not part of the routine urinalysis

CHEMICAL EXAMINATION PART 1

Reagent Strips – Provides simple, rapid means for performing medically significant chemical analysis of urine including pH,
proteins, glucose, ketones, blood, bilirubin, urobilinogen, nitrite, leukocytes, specific gravity.

Two major types of reagent strips are manufactured under

- Multistix: Siemens Healthcare Diagnostics, Deerfield , IN

- Chemstrip: Roche Diagnostics, Indianapolis IN

Reagent strips consists of chemical-impregnated absorbent pads attached to the plastic strip. A color producing chemical reaction
takes place when the absorbent pads comes in contact in urine

Reagent strip techniques:

1. dip the reagent strip briefly into a well-mixed uncentrifuged urine specimen
at room temperature.

2. Remove the excess urine by touching the edge of the strip to the container as
the strip is withdrawn.

3. Blot the edge of the strip on a disposable absorbent pad

4. wait the specified time for the reaction to occur

5. Compare the color reaction of the strip pads to the manufacturer’s color chart
in good lighting.

HANDLING AND STORING REAGENT STRIP

Reagent strips must be protected from deterioration caused by moisture volatile chemicals, heat and light. Reagent strips are packed
in opaque container with a desiccant to protect them from light and moisture.

Bottles should not be opened in the presence of volatile fumes. Manufacturers recommends that reagent strips must be stored at room
temperature below 37C but never refrigerated.

1. Store with desiccant in an opaque tightly closed container

2. Store below 30C; do not freeze
3. Do not expose to volatile fumes
4. Do not use past expiration date
5. Do not use if chemical pads become discolored
6. Remove strips prior to use

QUALITY CONTROL OF REAGENT STRIP

Reagent strip must be checked with both positive and negative control a minimum once in 24hrs. Several companies manufacture both
positive and negative controls. Distilled water is not recommended as a negative control.

Reagent strip are meant to perform at ionic concentration. All negative controls must result negative and positive control must follow
th published readings.

1. Test open bottles of reagent strips with known positive and negative control every 24hrs
2. Resolve control results that are out of range by further testing
 3. Test reagents used in back up tests with positive and negative controls
4. Perform positive and negative controls on new reagents and newly open bottles of reagent strips
5. Record all results and reagent lot numbers

CONFIRMATORY TESTING

Confirmatory testing are defined as a test using different reagents or methodologies to detect same substances as detected by the
reagent strips with same or greater sensitivity or specificity.

REAGENT STRIP REACTION AND PRINCIPLE

1. pH – the importance of pH is primarily as an aid in determining the existence of systematic acid-base disorders of metabolic
or respiratory origin and in the management of urinary conditions that require the urine to be maintained at a specific pH.
- Kidneys are the major regulators of acid-base content in the body by secreting hydrogen phosphates ions and ammonium and
week organic acids at the convoluted tubules
- Healthy person produces first morning specimen in slightly acidic a pH of 5.0 to 6.0. the normal random samples ranges 4.5
to 8.0
- Reagent strip reaction : Double indicator system of methyl red and bromthymol blue, Methyl red produces color change
from red to yellow, pH= 4.0 to 6.0. Bromthymol blue turns yellow to blue,

- Clinical Significance:
o In respiratory or metabolic acidosis is not related to renal function disorders, the urine is acidic; conversely, if
respiratory or metabolic alkalosis is present the urine is alkaline. = NOT ASSOCIATED WITYH RENAL FXN
DISEASES
o The precipitation of organic chemicals dissolves in the urine forms urinary crystals and renal calculi.
 Example: calcium-oxalate a frequent constituent of renal calculi precipitates primarily in acidic not alkaline
urine. Therefore, maintaining urine at alkaline pH discourages formation of calculi
 Knowledge of urinary pH is important in the identification of crystals observe during microscopic
examination of the urine sediment.
 Maintaining acidic urine can be valuable in treating urinary tract infection caused by urea-splitting
organisms they do not multiply as readily in an acidic medium
o Urinary pH is controlled by
 Dietary intake
 Medications
 Persons with high protein and high meat intake tend to produce acidic urine
 Vegetarians are most likely alkaline due to formation of bicarbonate
o Freshly excreted urine does not reach above 8.5pH in normal or abnormal conditions
o A pH above 8.5 is associated of improperly preserved specimen.

2. Protein – the most indicative of renal disease is the protein determination. Proteinuria is often associated with early renal
disease , making the urinary protein test important part of physical examination
- Normal urine contains very little amount of urine = 10mL/dL or 100mg per 24hrs is excreted.
- Reagent strip reaction: Protein error of indicator,
o This is because protein accepts hydrogen ions from the indicator. The test is more sensitive to albumin because
albumin contains more amino group to accept hydrogen ions other than proteins.
 o Depending on the manufacturer the protein area of the strip reagent contains :
 tetrabromophenol blue (Multistix)
 or 3’,3”,5’,5”-tetracholophenol, 3,4,5,6-tetrabromosulfonpthalein (Chemstrp)
o however the protein concentration increases , the color progresses through various shades of green and finally to
blue.
o Readings are reported of terms negative, trace 1+, 2+, 3+ and 4+ ; semiquantitative values of 30, 100, 300 or 2000
mg/dL
o Trace values are considered to be less than 30mg/dL.

- Clinical Significance:
o The normal urinary albumin is low because the majority of the albumin presented to the glomerulus is not filtered.
o Other proteins:
 Tamms-Horsfall (uromodulin) protein produce by renal tubular epithelial cells
 From prostatic, seminal, and vaginal secretions
o Clinical proteinuria is indicated at 30mg/dL or greater than 300 mg/Dl.
o The causes of proteinuria:
 Pre-renal proteinuria:
 is cause by conditions affecting the plasma prior to its reaching the kidney.
 Caused by increased level of low-molecular-weight plasma proteins such as hemoglobin and myoglobin and
the acute phase reactants.
 Suspected cases of myeloma must be diagnosed by performing serum electrophoresis and
immunoelectrophoresis – the screening test for Bence Jones
 Renal proteinuria:
 Associated with true renal disease may be the result of either glomerular or tubular damage.
 Glomerular proteinuria: this condition may be reversible, such as occurs during strenuous exercise and
dehydration or is associated with hypertension or exposure to cold. Proteinuria that occurs in pregnancy may
indicate pre-eclamptic state .
 Orthostatic (postural) Proteinuria: it occurs frequently in young adults, it occurs following period spent in
horizontal position is assumed.
 Tubular proteinuria: increased volume of albumin is also present in disorders affecting tubular reabsorption
because normally filtered albumin can no longer reabsorbed. Causes of tubular dysfunction includes exposure
to toxic substances, heavy metals , severe viral infections and Fanconi Syndrome.
 Postrenal proteinuria: protein can be added to urine specimen as it processed through the structures of the
lower urinary tract (ureters, bladder urethra, prostate, and vagina). Bacterial and fungal infections and
inflammations. The presence of blood is caused by menstrual contamination, large presence of prostate
fluid leads to increase amounts of protozoa.
- Confirmatory Test: SSA SALISYLIC ACID PRECIPITATION TEST
o Test is cold precipitation test that reacts equally in all form of protein.

3. Glucose – the glucose test is the most frequently performed chemical analysis in urine
- Reagent strip reaction: Double Sequential enzyme reaction
o The glucose oxidase procedure provides a specific test for glucose
o Reagent strips employ the glucose oxidase testing method by impregnating the testing area with a mixture of glucose
oxidase, peroxidase, chromogen, and buffer to produce double sequential reaction
o Step 1: Glucose oxidase catalyzes reaction between glucose and room air to produce gluconic acid and peroxide.
o Step 2: Peroxidase catalyzes reaction between peroxide and chromogen to form oxidized color compound that is
produce in direct proportion to the concentration of glucose
o Reagent manufacturers use several different chromogen, including
 potassium iodide, green to brown (MULTI STIX)
 tetramethylbenzidine, yellow to green (CHEMSTRIP.
o Glucose is reported in negative trace by : 1+, 2+. 3+, 4+

o Color charts : 100mg/dL to 2g/dL or 0.1% to 0.2%

o Sensitivity: Multistix = 75 to 125 mg/dL
Chemstrip = 40 mg/dL

- - Clinical Significance:
o Almost all the glucose filtered by the glomerulus is actively reabsorbed in the PCT, therefore urine contains only a
minute amounts of glucose
o Should the blood level of glucose become elevated (hypergylcemia) occurs in diabetes mellitus.
o Hyperglycemia that occurs during pregnancy and disappears after delivery is called gestational diabetes.
o Hyperglycemia of non-diabetic origin is seen in a variety of disorders and also produces glycosuria. Many of these
disorders are associated with hormonal function include pancreatitis, acromegaly, Cushing syndrome,
hyperthyroidism, pheochromocytoma, and thyrotoxicosis.
 The hormone glucagon, ephinephrine, cortisol, thyroxine, and growth hormone which is increased in
this disorder, work in opposition to insulin = hyperglycemia and glucosuria
  Whereas, primary function of insulin secretion is to convert glucose to glycogen for storage, opposing
hormones causes the breakdown of glycogen to glucose – In short yung presence ng mga elevated

hormones na yon d nagagawa ng isnulin yung main function niya 😊

 Epinephrine is also a strong inhibitor of insulin secretion and is increased when the body is subjected to
severe stress.
- Reaction interference:
o Because the glucose oxidase method is specific for glucose, false positive reactions are not obtained from other
urinary constituents including reducing sugars that may be present.
- Confirmatory test:
o Copper reduction test (CLINITEST): the test relies on the ability of the glucose and other substances to reduce
copper sulfate to cuprous oxide in the presence of alkali and heat.
 the tablets contain copper sulfate, sodium carbonate, sodium citrate and sodium hydroxide.
 An alternate method using two drops of five drops of urine can minimize the occurrence of “pass through”
 Clinitest subject to interference other reducing sugars including galactose, lactose, fructose, maltose,
pentoses, ascorbic acid, certain drug metabolites, and antibiotics = CLINITEST DOES NOT
PROVIDE CONFRIMATORY TEST FOR GLUCOSE
 Clinitest tablets are hydroscopic and should be stored in their tightly closed container. Strong blue color in
used tablets = deterioration due to moisture accumulation = vigorous fizzing
 Clinitest significance: in addition to glucose commonly found in reducing sugars include galactose,
pentose and lactose.

4. Ketones – The term ketones represents three immediate products of fat metabolism: acetone 2%, acetoacetic acid 20%, B-
hydroxybutrate 78%.
- Reagent strip reaction: Sodium Nitroprusside Reaction
o In this reaction acetoacetic acid in an alkaline medium reacts with sodium nitroprusside to produce a purple color.
o The test does not measure B-hydroxybutyrate and is only slightly sensitive to acetone when glycine is also present
o These compounds are derived from acetoacetic acid.
o Results are reported as: Small 1+ or Trace 5mg/dL
Moderate 2+ Small 15mg/dL
Large 3+ Moderate 40mg/dL
Large 80 to 160mg/dL
- Clinical Significance:
o Increased fat metabolism include the ability to metabolize CHO, as occurs in diabetes mellitus: increased in loss of
carbohydrates from vomiting and inadequate intake of carbohydrate associated with starvation and malabsorption.
o Testing for urinary ketones is valuable in management and monitoring of insulin dependent type-1 diabetes milletus.
o Ketonuria = deficiency in insulin
- Confirmatory test:
o Acetest tablets: provides sodium nitroprusside, glycine, disodium phosphate and lactose in tablet form
 The addition of lactose gives better color differentiation.

5. Blood – may be present in the urine either in the form of intact rbc (hematuria) or as the product of the rbc destruction
(hemoglobinuria)
- In the presence of free hemoglobin and myoglobin = uniform color ranging from negative yellow-green through green-
blue is strongly positive
- Intact RBC = are lysed when the com in contact on the pad
- Liberated hemoglobin = isolated reaction that results in a speckled pattern in the pad.
- Reagent Strip Reaction: Pseudoperoxidase activity of hemoglobin
o Chemical test blood use pseudoperoxidase to catalyzed a reaction between the heme component of both hemoglobin
and myoglobin and the chromogen tetramethylbenzidine to produce oxidize chromogen that has green-blue color.
o Sensitivity: Multistix= 5 to 20 RBCs/mL, 0.015 to 0.062mg/dL hemoglobin
Chemstrip = 5 RBCs/mL, 10 RBCs/mL hemoglobin
o Results are reported as: 1+, 2+, 3+

- Clinical significance: The finding of a positive reagent strip test result for blood indicates the presence of rbcs , hemoglobin,
myoglobin.
o Hematuria: Major causes of hematuria include renal calculi, glomerular diseases, tumors, trauma, pyelonephritis,
exposure to toxic chemicals and anti-coagulant therapy.
o Hemoglobinuria: Occurs in hemolytic anemias, transfusion reactions, severe burns, brown recluse bites, infection
and strenuous exercise.
o Myoglobinuria: trauma, crush syndromes, prolonged coma, convulsions, muscle wasting diseases, alcoholism,
heroin abuse and extensive exertion.
6. Bilirubin – appearance of bilirubin in the urine can provide an early indication of liver disease. It is often detected long
before patient exhibits (jaundice).
- Reagent strip reaction: Diazo Reaction
o Bilirubin combines with 2,4-dichloroaniline diazonium salt or 2,6—dichlorobenzene-diazonium-tetraflouroborate in
acid mediumto produce azodye with colors ranging increasing degrees of tan or pink, or pink to violet
o Results are reported as: negative small, moderate, large or 1+, 2+, 3+
o Sensitivity: Multistix = 0.4 to 0.8mg/dL bilirubin
Chemstrip = 0.5mg/dL bilirubin

- Clinical significance:
o Hepatitis – other liver diseases
o Cirrhosis – Biliary obstruction (gallstones, carcinoma)
- Confirmatory test:
o Icototest tablets – consist of testing mats and tablets containing p-nitrobenzenediazonium-p-toulenesulfonate, SSA,
sodium carbonate and boric acid.
7. Urobilinogen – Increased urobilinogen greater than 1mg/dL is seen in liver disease and hemolyzed disorders.
- Measurement of urine urobilinogen can be valuable in the detection of early liver disease
- This is frequently caused by constipation
- Reagent strip reaction: Ehrlich aldehyde reaction (Multi stix)
Azo-coupling reaction (Chemstrip)
o Multistix uses Ehrlich aldehyde, in which urobilinogen reacts with p-dimethylaminobenzaldehyde (E.reagent) to
produce color ranging from light to dark pink/red.
o RESULTS ARE WRITTEN IN: Ehrlich Unit (EU)
o Readings in : N: 0.2 and 1 to AN: 2,4,8
o Chemstrip uses an azo-coupling reaction using 4-methoxybenzene-diazonium-tetraflouroborate to react with
urobilinogen producing color ranging white to pink/red
o RESULTS ARE REPORTED IN: mg/dL
o Sensitivity: Multistix = 0.2mg/dL urobilinogen
Chemstrip = 0.4mg/dL urobilinogen

8. Nitrite – reagent strip test for nitrite provides a rapid test for the presence of urinary tract infection
- Reagent strip reactions: Greiss Reaction
o the chemical basis of the nitrite test is the ability of certain bacteria reduce nitrite, a normal constituent of urine to
nitrite which does not normally appear in urine.
o Nitrite at acidic pH reacts with aromatic amiine(para-arsanilic acid or sulfanilamide) to form diazonium compound
that then reacts with the tetrahydrobenzoquinolin compounds to = pink azodye
o Results are reported as : NEGATIVE OR POSITVE
o Sensitivity: Multistix = 0.06 to 0.01mg/dL nitrite ion
Chemstrip = 0.05mg/dL nitrite ion
9. Leukocyte esterase – the detection of increased urinary leukocytes required microscopic examination of the urine sediment.
- Reagent strip reaction: Hydrolysis of acid ester
o Uses the action of LE to catalyzed hydrolysis of an acid ester embedded on the reagent pad to aromatic compound
and acid.
o The aromatic compound then combines with a diazonium salt present on the pad to produce = purple azodye
o Results are reported as: small, moderate, large OR 1+, 2+, 3+
o Sensitivity: Multistix = 5 to 15 WBC/hpf
Chemstrip = 10 to 25 WBC/hpf

- Clinical significance:
o Normal values for leukocytes are based on he microscopic sediment examination and vary from 0 to2 – 0-5 per high
power field.

10. Specific Gravity

- The reagent strip reaction is based on the pKa (dissociation constant) of a polyelectrolyte in an alkaline medium.
- Reagent strip reaction: Incorporation of the indicator bromthymol blue on the reagent and pad measures the change in pH.
o As the specific gravity increases = the indicator changes in blue (1.000 alkaline)
o Results are reported in : Readings can be made in 0,005 intervals, Therefore adding 0.005 when the pH is 6.5 or
higher
o ↑ pH = ↓ Specific gravity = blue-green
o ↓ pH = ↑ Specific gravity = yellow green
o Sensitivity: 1.000 to 1.030
MIDTERM: MICROSCOPIC EXAMINATION PART I

Protocols have been developed to increased the standardization and cost effectiveness of microscopic urinalysis
Importance of microscopic analysis – to detect and identify insoluble materials present in the urine.
Formed elements seen in urine:
1. Rbc
2. Wbc
3. Epithelial cells
4. Cast
5. Bacteria, yeast, parasites
6. Mucus
7. Spermatozoa
8. Crystals
9. Artifacts
Disadvantages of microscopic analysis
- less standardized and most time consuming part of the routine urinalysis.

Macroscopic screening also referred as “chemical sieving”, to enhance the cost effectiveness of urinalysis
SEDIMENT CONSITITUENTS
1.RBC
2.WBC
3.Epithelial cells
4. Bacteria
5. Yeast
6. Parasites
7.Spermatozoa
8.Mucus
9.Casts
10. Crystals

RBC
 Appear in smooth, non-nucleated, biconcave
 Identified under HPO
 Dilute (hyposthenuria) urine the cells absorb water, swell, releasing hemoglobin
 Concentrated (hypersthenuric) cells shrink, loss of water and may appear crenated.
 Large empty cells are called ghost cells.
 Most difficult to recognized because:
o Lack characteristics structures
o Various in size
o Resemblance to other sediment consituents
 Adding of acetic acid to apportion of the sediment can help in identification
 RBC is isotonic in nature.
 Circular shapes will be gone because lysing cause by the acetic acid. Leaving the yeast, oil droplets, and WBCs intact.
 Supravital staining may also be helpful
 RBCs that vary in size, have circular portrusions or are fragmented called dysmorphic that have been associated with
glomerular bleeding.
 Further analysis containing dysmorphic RBC using Wright stain.
 CLINICAL SIGNIFICANCE:
o Associated with damage to the glomerular membrane or vascular injury within the genitourinary tract.
o The presence of not only RBC but also hyaline, granular, and RBC casts may be seen following
 strenuous exercise. That is applied more than the body’s limit, increased in hyaline cast, granular and RBC
casts.
o Possibility of menstrual contamination.
WBC
  Larger than RBC
 Identified under HPO
 The predominant WBC found in urine sediment is neutrophil.
 NEUTROPHIL
o Lyse in dilute alkaline urine
o Exposed to hypotonic urine absorbs water and swell
o Brownian movement (movement of granules) cause by the hypotonic diluted solution produces sparkling appearance
referred as “glitter cells”
o When stained with Sternheimer-Malbin stain these cells will stain light blue opposed to the violet color.
o Glitter cells are no pathologic significance.
 EOSINOPHIL
o Primarily associated with drug induce interstitial nephritis.
o Small numbers are seen in UTI and renal transplant rejection.
o Hansel and Wright stain is used for differentiation.
o Finding more than 1% is considered as significance.
 MONONULEAR CELLS
o Lymphocytes
 Smallest WBC
 They may resemble as RBC
 Seen in early stage of renal transplant rejection
o Monocytes, macrophages, histiocytes
 Larger cells
 Appear vacuolated or contain inclusions
 WBC most common sources of identification error is RTE CELLS
 An increased in urinary WBC is termed as pyuria
o Pyelonephritis – one or both kidney is infected
o Cystitis – inflammation of bladder
o Prostatitis – inflammation of prostate gland
o Urethritis – inflammation of urethra
o Non bacterial disorders:
 Glomerulonephritis
 Lupus erythematosus
 Interstitial nephritis
 Tumors

EPITHELIAL CELLS
 Derived from the linings

Rectangular shape: PCT

Round/oval: DCT
Cuboidal: Collecting ducts
2 or more RTE cells = tubular injury
  Directly related to the function of the kidneys

 CLINICAL SIGNIFICANCE:
o Most clinically significant epithelial cells
o Increased amount is indicative of necrosis (cell death) of the renal tubules with the possibility affecting all renal
function.
o Condition producing tubular necrosis include:
 Drug induced toxicity
 Hgb and Mgb
 Viral infections (Hepa-B)
 Pyekonephritis
 Allergic reactions
 Malignancy
 Salicylate poisoning.
 Allogenic transplant rejections
 OVAL FAT BODIES
o Lipid containing RTE cells
o Examine using Sudan III or Oil red O fat stains under polarized microscopy
 The droplets are composed of triglycerides, neutral fats, and cholesterol.
 Fat stain – triglyceride and neutral fats produces orange-red droplets
 Polarized light – cholesterol produces Maltese cross formation.
o Identified in HPO
o Non-lipid filled vacuoles are called Bubble cells. They appear to represent injured cells in which endoplasmic
reticulum is dilated.

BACTERIA
 Not normally present in the urine
 Unless specimens are collected under sterile conditions
 Present only in the form of cocci or bacilli
 Owing their small size they must e examined under HPO
 Presence of bacteria can be indicative of either upper or lower UTI
 The bacteria most frequently associated with UTI is Enterobacteriaceae – gram negative rods
 Staphylococcus and enterococcus are also capable of causing UTI

YEAST
 Appear as small, refractive oval structures that may or may not contain a bud
 Reported as rare, few, moderate, many per HPO
 Candida Albicans – are seen in diabetics immunocompromised patients and women with vaginal moniliasis
 A true yeast infection should be accompanied by the presence of WBC

PARASITES
 Trichomonas Vaginalis
o Most frequently encountered parasite in the urine
o Sexually transmitted pathogen associated with vaginal inflammation
o Trophozoite is pear shaped flagellate with an undulating membrane
o More difficult to identify because they may resemble as WBC, transitional or RTE cells
o Darting movement
o Reported per HPO
 Schistosoma haematobium
o Bladder parasite
o Seldom seen in US
o The most contaminant is ova from he pinworm Enterobius vermicularis

SPERMATOZOA
 Semen, urinary bladder = death
 Appear as oval, slightly tapered head and long flagella like tails
  Urine is toxic spermatozoa
 Clinically significant in case of male infertility or retrograde ejaculation
 Positive reagent test for protein when increased amount of semen is present
 Laboratory protocols vary with regard to reporting or not reporting the presence of spermatozoa in urine
MUCUS
 Protein material produced by the glands and epithelial cells of the lower genitourinary tract and RTE cells
 Major constituent is Tamm-Horsfall Protein
 Appears as thread-like structure
 Reported per LPO
 Uromodulin is major constituent of mucus
 Glycoprotein excreted by the RTE cells of the DCT and upper collecting ducts
 Frequently seen in female

CASTS
 Only urinary sediment that are unique to the kidney
 Their shape is the representative of the tubular lumen
 They are formed within the lumens of the DCT and collecting ducts
 It has parallel sides rounded ends and may contain other elements present in filtrate
 Examine under LPO
 Cast matrix dissolve easily in dilute alkaline urine.
 Major constituent of casts is Tamm-horsfall protein
 Other protein incorporated such as albumin and immunoglobulins
 Step by step formation of Tamm-horsfall protein matrix
o Aggregation of uromodulin protein into individual protein fibrils attached to the RTE cells.
o Interweaving of protein fibrils to form a loose fibrillar network (urinary constituent may become enmeshed in the
network at this time.
o Further protein interweaving to form a solid structure.
o Possible attachment of urinary constituents to the solid matrix
o Detachment of protein fibrils from the epithelial cells
o Excretion of casts
 The presence of urinary casts termed as cylinduria
 HYALINE CAST
o Most frequently seen in casts which consists most consists of uromodulin
o Increased number in:
 Strenuous exercise
 Dehydration
 Heat exposure
 Emotional stress
 Acute glomerulonephritis
 Pyelonephritis
 Chronic renal disease
o Colorless in unstained sediments
o Stained with Sternheimer Malbin
o Parallel sides and rounded ends
o Wrinkled or convoluted shapes indicates aging of the cast matrix
 RBC CASTS
o Presence of RBC cast us much more specific showing bleeding within the nephron
o Primarily associated with glomerulonephritis
o Seen also in individuals participating in strenuous exercise
o Detected under LPO
o In the presence of massive hemoglobinuria or myoglobinuria, homogenous orange-red brown cast may be observed
o They are more fragile irregular shape and may exists as fragments
 WBC CASTS+
o Presence signifies infection or inflammation within the nephron
o Associated with pyelonephritis
o Acute interstitial interstitial nephritis
 o Glomerulonephritis
o Visible using LPO and HPO
o Mostly compose of neutrophils
o Irregular borders
 BACTERIAL CASTS
o Commonly seen in pyelonephritis
o Resemble granular casts
o Considered when increase WBC, bacteria, WBC casts are seen
o Confirmatory test : Gram stain
 EPITHELIAL CELL CASTS
o Casts containing RTE cells represent advance tubular destruction
o Similar to RTE cells they are associated with
 Heavy metals and chemical toxicity
 Drug induced toxicity
 Viral infection
 Allograft rejection
 Pyelonephritis
 FATTY CASTS
o Seen in conjunction with oval fat bodies and free fat droplets
o Associated with
 Nephrotic syndrome
 Toxic tubular necrosis
 Diabetes mellitus
 Crush injuries
o Confirmatory test: Sudan III (triglycerides and neutral fats) or oil res using polarized microscope (cholesterol)
o Fats do not stain with Sternheimer Malbin
 MIXED CELLULAR CASTS
o Considering the variety of cells may be present in the urinary filtrate
o Encountered include RBC,and WBC casts, glomerulonephritis, RTE casts or WBC casts, bacterial casts –
pyelonephritis
o Staining or phase microscopy aids in identification
 GRANULAR CASTS
o Coarsely granular casts and finely granular casts, may be pathologic or non-pathologic
o Non-pathologic : LYSOSOMES excreted by the RTE cells during normal metabolism
o Pathologic:
 Disintegration of cellular casts and tubule cells
 Protein aggregates filtered by glomerulus
o Seen in conjunction with WBC casts
o Visualized under LPO and identified using HPO
o Source of identification error:
 Clumps of crystals
 Fecal debris
 Columnar RTE cells
o When remain to tubule for extended periods, the granules disintegrade and develop a waxy appearance
 WAXY CASTS
o Indicated chronic renal failure
o Brittle, highly refractive caused by the degeneration of hyaline cast matrix and any cellular elements granules
contained in the matrix
o Appear in fragmented with jagged ends and notched sides
o Stains dark pink in supravital stains
 BROAD CASTS
o Referred as renal failure casts
o Indicates destruction of the tubular walls
o Most commonly seen broad casts are granular and waxy.
CRYSTALS
 Crystal identification aim to detect abnormal types of crystals seen in pathologic disorders
 Reported as few, moderate, many per HPO
 Abnormal crystal may be average and reported per LPO
 Crystal formation:
o Are formed by the precipitation of urine solutes
o Precipitation factors:
 Changes in temperatures
 Solute concentration
o pH
 Solute precipitates more at low temperature
 As the concentration of solute increases their ability to remain in the solution decreases
 pH is valuable indentification of crystals

NORMAL CRYTALS SEEN IN URINE IN ALKALINE pH

 AMORPHOUS PHOSPHATES
o Granular in appearance
o Causes white precipitate in refrigerated sample
o Dilute in acetic acid
o It can be in alkaline or neutral pH
 TRIPLE PHOSPHATES
o Ammonium magnesium phosphates
o Coffin lead
o Birefringent under polarized light
o Associated with the presence of urea splitting bacteria
o Dilute in acetic acid
 CALCIUM PHOSPHATES
o Small, colorless, rectangular plates, flat or thin prism often in rosette form
o Confused with sulfonamides crystals
o Dilute in acetic acid
o Common constituent of renal calculi
 CALCIUM CARBONATE
o Small colorless, dumbbell or spherical
o Resemble amorphous materials
o Produce gas after addition of acetic acid
o Birefringent
o No clinical significance
 AMMONIUM BIURATE
o Yellow-brown color
o Thorn apples
o Dissolve at 60C and convert to uric acid upon addition of glacial acetic acid
o Encountered in old specimens
NORMAL CRYTALS SEEN IN URINE IN ACIDIC Ph
 URIC ACID
o Appear in rhombic, four sided plated, wedges, rosettes
o Yellow brown, colorless
o Birefringent
o Increased amount is associated with increase levels of purine and nucleic acid
o Alkali soluble
 AMORPHOUS URATES
o Brick dust or yellow brown granules
o Refrigerated specimen produces pink sediments by uroerythrin
o Appear in clumps
o Alkali and heat soluble
 CALCIUM OXALATE
o Seen acidic pH but may appear in neutral and alkali pH
o 2 types
 Dihydrate – easily recognized as a colorless, octahedral envelope or as two pyramids joined at their bases
 Monohydrate – oval, dumbbell shaped
o Birefringent
o Associated with:
 Renal calculi
 Foods high in oxalic acid
 Ethylene glycol poisoning
o Dilute in HCL

 CYSTEINE CRYSTALS
o Seen in patients with metabolic disorders cystinuria
o Colorless hexagonal plates may be thick or thin
o Difficult to differentiate from uric acid crystals
 CHOLESTEROL CRYSTALS
o Appear as colorless notched plate
o Associated causing lipiduria: Nephrotic syndrome
o Seen in conjunction with fatty cast and oval fat bodies.
o Birefringent
o Soluble in chromogen
 BILIRUBIN CRYSTALS
o Present in hepatic disorders
o Clump of yellow color needles or granules
o Seen in conjunction with positive chemical test for bilirubin
o Soluble to acetic acid, HCL, NaOH, ether and chloroform
 RADIOGRAPHIC DYE CRSYTALS
o Resemble as cholesterol crystals
o Birefringent
 o Comparison of urinalysis result and patient history for better differentiation
o Soluble in 10% NaOH
 LEUCINE CYRSTALS
o Yellow-brown spheres
o Concentric circles and radial striations
o Seen in liver disorders accompanied by tyrosine crystals
 TYROSINE CRYSTALS
o Appear as fine needles
o Form of clumps or rosettes
o Seen in conjunction with leucine and positive chemical test for bilirubin
o Associated with liver disorders and inherited disorders of amino acid metabolism
o Soluble in alkali or heat
 SULFONAMIDES CRYSTALS
o Seen in patients treated for UTI
o Appearance of sulfonamide crystals is fresh urine can suggest possibility of tubular damage
o Appear as needles, rhombic, whetstones, sheaves of wheat and rosettes with color
o Confirmatory test: Diazo reaction
o Soluble with acetone
 AMPICILLIN CRYSTALS
o Seen in massive doses of penicillin compound
o Appear as colorless needles form bundles following refrigeration
o Check for patient history

URINARY ARTIFACTS
 Starch
o Corn starch is use to powder gloves
o Highly refractile spheres with dimpled center
o Resembles as fat droplets
o Confused with RBC
 Oil droplets
o Sources can be from immersion oil or lotions and creams
o Resembles as RBC
o Air bubbles occur when the specimen is places under a cover slip
 Pollen Grains
o Appear as spheres with a cell wall and occasional concentric circles
 Fibers
o Mistaken as casts
o Longer and more refractile
o Use of polarized microscope for differentiation of fiber from casts
 Fecal contaminants
o Due to improperly collected specimens
o Appearance of plant and meat fibers
MIDTERM TOPIC 2
URINE SCREENING FOR METABOLIC DISORDERS
 Many of the abnormal results in routine urinalysis are related to metabolic rather than renal disease
 Observation of urine color and odor can be good indicator that there must be something wrong with the specimen.
 Often these substances ca be detected or monitored by additional screening test.
 From observation of abnormal specimen color by nursing staff and patients, clinical symptoms and family history are other
deciding factors.

OVERFLOW VS RENAL DISORDER

 Overflow – result from the disruption of normal metabolic pathway that causes increase plasma concentration.
 Renal type – caused by malfunction in the tubular reabsorption mechanism.
 Disruption of enzyme function can be caused by failure to inherit the gene produce a particular enzyme, referred as inborn
metabolism IEM
NEWBORN SCREENING TEST
 Primary goal is to detect and monitor newborns for IEM
 Many states currently require testing for many as 30 or more metabolic diseases
 Collection: infant heel puncture
 Testing is done with the mass spectrophotometry
 MS/MS is capable of screening infant blood samples for specific substances associated with particular IEMs

AMINO ACID DISORDERS

 Phenylketonuria
o most well known aminoaciduria
o occur in 1 of every 10,000 to 20,000 births
o failure to inherit h=the gene to produce enzyme phenylalanine hydroxylase
o causes mental retardation
o identified by Ivan Folling 1934, Mousy odor
o increase amount of keto acids
o produces fair complexion
o major constituent of milk from infant’s diet can prevent excessive buildup serum phenulalanine
o detected as early as 4hours after birth
o most well known blood test is microbial inhibition assay developed by Guthrie
o Modification of Guthrie test can be also used for MSUD homocystinuria, tyrosinemia, histidemia. Valinemia,
galactosemia.
o Urine test are based upon ferric chloride reaction

 Tyrosyluria
o May be result of inherited or metabolic
o Excess tyrosine in the plasma
o seen is transitory tyrosinemia in premature infants, which is caused by underdevelopment of liver
o acquired severe liver disease also produces tyrosyluria
o Type I – caused by deficiency of the enzyme fumarylacetoacetate hydrolase
 Generalized renal tubular disorder and progressive renal failure in infants as soon after birth
o Type II – caused by lack of enzyme amino transferase
 Person develop corneal erosion and lesion on the palms, fingers, soles of the feet caused by
crystallization tyrosine in the cells
o Type III – caused by lack of p-hydroxyphenylpyruvic acid dioxygenase
 Results in mental retardation, if dietary rstriction of phenylalanine and tyrosine are not
implemented.
 o Screening test using MS/MS are available for tyrosinemia types I, II, III
o Nitroso-Napthol Test for tyrosine
 5 gtts of urine in tube
 1mL of 2.63N nitric acid
 1 gtts of 21.5% sodium nitrite
 0.1 mL of nitroso-2-napthol
 Mix
 Wait 5 mins
 Observe for orange red color

 Melanuria
o Melanin - the pigment responsible for the ddark color of hair, skin and eyes.
o Deficient production of melanin results in albinism
o Increased urinary melanin produces darkening of urine upon exposure to air
o Elevate urinary melanin is serious finding that indicates proliferation of the normal melanin-producing cells
(melanocytes) producing a malignant melanoma (secretes 5,6-dihydroxyindole to melanogen to melanin
o Test done is sodium nitroprusside positive result is red color
 Alkaptunuria
o One of the six original inborn errors of metabolism described by Garrod in 1902
o Urine darkened after becoming alkaline
o 3rd major defect in phenylalanine-tyrosine patheway
o Failure to inherit the gene that produce homogentisic acid oxidase
o Observation of browned stained or black stained and disposable diapers have reported
o A high percentage of persons with alkaptonuria develop liver and cardiac disorders.
o Reacts with ferric chloride = deep blur
o Clinitest = yellow precipitation
o alkalization of fresh urine = darkening of color due to increased large amount of ascorbic acid
o paper and thin chromatography procedures for quantitating homogentisic acid
 4mL of 3% silver nitrate
 0.5 mL of urine
 Mix
 10% NH4OH by drops
  Observe for black color
 BRANCHED CHAIN AMINO ACID DISORDERS
o MSUD
 Caused by an EIM inherited as an autosomal recessive trait
 The amino acid involve leucine, isoleucine, and valine
 Transamination – transfer an amino group to a ketoacid to form new amino acid
 Liver – a keto isovaleric, a ketoisocaproic, a keto B methylvaleric
 Failure to inherit the gene for enzyme necessary to produce oxidative decarboxylation.
 The urine screening test is most frequently performed for ketoacids is the 2,4-dinitrophenylhydrazine
 Yellow turbidity or precipitate
o Organic acidemia
 Generalized symptoms are metabolic acidosis, hypoglycemia, ketonuria and increased serum ammonia
 Detected using MS/MS
 The three most frequently encountered disorders
 Isovaleric
 Propionic
 Methylmalonic acidemia
 Propionic is an intermediate precursor of methylmalonic

 TRYPTOPHAN DISODERS
o Indicanuria
 5-hydroxyindoleacetic acid
 Increase of metabolites indicant and 5 hydroxyindoleacetic acid
 Associated with intestinal disosrder
 Hartnup disease or blue diapr syndrome
 Urinary indicant reacts with acidic ferric chloride
 Indican excreted in urine I colorless until oxidized to indigo blue by exposure to air

o 5-hrydroxyindoleacetic acid
 Degradation product of serotonin carried by
platelets
 Increased when carcinoids tumors involving the
argentaffin enterochromaffin cells develop
 Reacts with nitrous acid and 1-nitroso-2napthol =
black/purple urine
 Normal daily excretion of 5-HIAA is to 2 to 8 mg
 If a 24 hr sample is used it must be preserved with
HCl or boric acid
 Serotonin is major constituent of foods such as
bananas, pineapple and tomatoes
 Medication, including phenothiazines and
acetanilids also cause interference.

 CYSTINE ORDRES
o Cystinuria
 The inability of the renal tubules to reabsorbed cysteine filtered by glomerulus
 Demonstration that not only cysteine but also lysine, arginine and ornithine are not reabsorbed has ruled out
the possibility of an error metabolism
 A chemical screening: cyanide nitroprusside
 3mL urine
 2 mL sodium cyanide
 Wait 10mins
 5 gtts 5% sodium nitroprusside
 Observe for red-purple color
 o Cystinosis
 2 general categories
 Nephropathic
 Infantile and late onset cystinosis
 Renal tubules , particularly the proximal convoluted tubules , are effected by the deposits of
cysteine that interfere with reabsorption
 If untreated, results in renal failure in early life
 Non-nephpropathic
 Relatively benign but may cause some ocular disorders
o Homocystinuria
 Defects in metabolism of the amino acids methionine produce an increase in homocysteine throughout the
body
 Silver nitroprusside for homocysteine
 1mL urine
 2gtts concentrated NH4OH
 0.5 mL 5% silver nitrate
 Wait 10 mins
 5 gtts sodium nitroprusside
 Observe for red purple color

PORPHYRIN DISORDER
 Are the intermediate compounds in the production of heme
 Disorders of porphyrin metabolism are collectively termed as porphyrias
 Observation of a red or port wine color to the urine after exposure to air
 Two screening test: erlich reaction and fluorescence under ultraviolet light in the 550 to 600 nm range

MUCOPOLYSACCHARIDE DISORDERS
 Consist of a protein core with numerous polysaccharidoses:
o Hurler syndrome
 Mucopolysaccharides accumulate in the cornea of the eye
o Hunter Syndrome
 Inherited as sex-linked recessive and rarely seen in females
o Sanfilippo syndrome
 Mental retardation
 Result is obtained in the presence of lead poisoning unless ALA is first converted to porphobilinogen
 The most frequently used screening test:
o Acid albumin and cetyltrimethylammonium bromide (CTAB) = white thick turbidity forms
o The turbidity is usually graded on a scale 0 to 4 after 30 minutes with acid albumin and after 5 minutes CTAB
o Blue spots

PURINE DISORDERS
 Lesch-nyhan disease that is inherited as a sex linked recessive results in massive excretion of urinary uric acid crystals
 Failure to inherit the gene to produce the enzyme hypoxanthine guanine phosphoribosyltransferase
 Patients suffer from sever motor defects , mental retardation, a tendency toward self destruction, gout, and renal calculi
 Orange sand in diapers

CARBOHYDRATES DISORDERS
 Galactosuria
o Indicating the inability to properly metabolize galactose to glucose
o Caused by deficiency in any of the three enzyme
 Galactose-1-phosphate uridyl transferase GALT
 Galactokinase
 UDP-galactose-4-epimerase
 Lactosuria
o Seen in during pregnancy and lactation
 Fructosuria
 o With parenteral feeding
 Pentosuria
o Ingestion of large amount of fruit
o One of Garrod original six IEM