Farhah Izzati Urinalysis Lab 020212

8/3/2019 Farhah Izzati Urinalysis Lab 020212

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MED 2074 URINARY SYSTEM LABORATORYWorksheet Exercise Batch 2010

MED 2074 URINARY SYSTEMLABORATORY

2ND FEBRUARY 2012

NAME : FARHAH IZZATI BINTI MANSORMATRIC NO : 1008-1673GROUP : M18LECTURERSNAME : DR MOHD HAFIZ NGOO, DR FAHMI

MAHMOUD, AP DR SWE SWE WIN, MRSAID AL-GHORA

Feb 2/CLASS 2012/SEM4/YR2/MHN 1


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Objectives:

After carrying out the activities in the laboratory, the student should be able to: -

1. determine and justify the urine production rate after consumption of different types of

drinks.2. prepare the urine samples for microscopic analysis.

3. identify the composition of urine by performing physical and chemical tests.

4. identify the different types of cellular components, casts, crystals and artifacts found in

urinary sediments.

5. interpret and report the results of the examination carried out.

Introduction

Urine is produced by the kidney to maintain constant plasma osmotic concentration; to regulate

pH, electrolyte and fluid balances and to excrete some 50 grams of waste solids (mostly urea and

sodium chloride). It also contains other substances that are absorbed readily from the

gastrointestinal tract. Contents of urine therefore indicate the condition of internal environment of a

human body, and are very much dependent on the dietary intake of an individual. This includes the

types of drinks consumed by an individual.

Urinalysis is a routine laboratory procedure that allows examination of the urine in a noninvasive

way. This diagnostic tool uses a very cost-effective device consisting of paper or plastic

impregnated with a microchemistry system, allowing for a quick (one minute or less) qualitative

and semi-quantitative analysis. A simple observation of the color changes on each segment of the

dipstick, which when compared to a chart for interpretation, gives accurate results if done carefully.

It will also give clues to a various systemic diseases and urinary tract infections.

Microscopic analysis on the urine specimen serves to detect for crystals, casts, cells or microbes.

The types of substances observed are indicative of the internal body environment, and therefore

can help the physician to diagnose the present of diseases. It allows the physician to find diseases

that may otherwise go unnoticed, such as glomerulonephritis, renal failure, hypertensive

nephropathy, diabetes mellitus or chronic urinary tract infections.

General protocol

1. All students must do analysis on their own urine samples.

2. Selected male students will become subjects of an experiment to determine the effect of

drinking solution on volume of urine produced.

3. All students must analyze the lab data.

4. The final lab report is to be submitted by the SCTL group leaders (M01 M22).



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Materials and Equipment

1. Isotonic water

2. Coffee (Nescafe) without sugar

3. Sugar (50%) solution4. Drinking water provided in sealed bottles5. Beakers (50 ml, 100ml)6. Urine Collecting Containers7. Urinary test strip8. Refractometer9. Light microscope10. Microscopic slides clean and dry11. Cover slips12. Sterile capillary tubes

Experimental (Multipurpose Lab)

Part I (Urinalysis): To determine urine production rate after consumption of different types

of drinking fluids.

Methodology

1. The students are divided into 4 groups with each group containing 5 healthy males.

2. Before the start of the experiment, the selected individual is required to empty his bladder

completely, and the urine collected in container provided. Record the urine volume and

time collected as in Table 1.

3. All males students from each group perform the following:

4. The time each male student starts drinking the solution is recorded immediately. The

students will not be informed of the type of drink they consume.

5. Urine samples from each male student are collect 30, 60 and 120 minutes from the time of

drinking the solution and the volume obtained (individually) is measure. This is recordedas during the experiment data in Table 1. Calculate the urine production rate (ml/hr) foreach student during the experiment.

6. Identify the types of drinks consumed by each group in bottles A, B, C and D (drinking

water, 50% sucrose solution, isotonic drink and coffee without sugar, not necessarily in

that order) consumed.

Feb 2/CLASS 2012/SEM4/YR2/MHN

Group 1 Each drink 500mL from Bottle A

Group 2 Each drink 500mL from Bottle B

Group 3 Each drink 500mL from Bottle C

Group 4 Each drink 500mL from Bottle D

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Sediment Type Estimation

Crystals

ammonium biurate, uric acid, bilirubin,sodium urate, calcium carbonate,cholesterol, calcium oxalate, cystine,calcium phosphate, sulfonamide, leucine,tyrosine, triple phosphate, amorphouscrystals (phosphate or urate)

Present or absent

CastsHyaline, cellular, granular, waxy casts, fineand coarse granular casts and artifacts

Present or absent

Microorganisms

Bacteria- Coccus, bacilli, chains, clusters or

singles

Yeasts & Molds Budding cells, hyphalfilaments

Present or absent

Sperm Normal/abnormal morphology Present or absent

Blood cells White blood cellsRed blood cells

< 5 cells = +5 10 cells = ++> 10 cells = +++

Epithelial cells Transitional or squamous < 5 cells = +5 10 cells = ++> 10 cells = +++

Others Oval fat bodiesMucous threads

Present or absent

Results should agree within + 10% or + 2 cells, whichever is larger. Observations should becarried out by at least two persons.

Methodology: Part III. (Identification)

There will be slides show (OSPE style) during the laboratory time. The slides are photographstaken from microscopic examinations of urine sediments.



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TABLE 1. DATA FOR URINE PRODUCTION BEFORE AND AFTER CONSUMPTION OF DIFFERENT TYPES OF FLUIDS

Before experiment During experiment

Gp

Student

Micturition

time

Totalurine

collected(ml)

Startdrinkin

g[0min]

(time)

1stmicturition[30min]

(time)

1sturinevolume (ml)

Refractometerreadi

ng

2ndmicturition [60min]

(time)

2ndurine

volume(ml)

Refractometer

reading

3rdmicturit

ion[120min]

(time)

3rdurine

volume(ml)

Refractometer

reading

1

1 8.30am 34 8.45am 9.15am 24 1.028 9.45am 17 1.026 10.45am 26 1.028

2 8.30am 28 8.45am 9.15am 20 1.024 9.45am 20 1.022 10.45am 19 1.031

3 8.30am 36 8.45am 9.15am 24 1.02 9.45am 28 1.018 10.45am 24 1.0225

4 8.30am 31 8.45am 9.15am 82 1.004 9.45am 170 1.003 10.45am 62 1.011

5 8.30am 130 8.45am 9.15am NIL NIL 9.45am NIL NIL 10.45am NIL NIL

Total 259 150 235 131

Mean 51.8 30 47 26.2

2

1 8.30am 52 8.53am 9.23am 60 1.016 9.53am 44 1.014 10.53am 74 1.014

2 8.30am 29 8.53am 9.23am 19 1.035 9.53am 16 1.027 10.53am 14 1.027

3 8.30am 22 8.53am 9.23am 30 1.03 9.53am 22 1.031 10.53am NIL NIL

4 8.30am 32 8.53am 9.23am 22 1.022 9.53am 16 1.024 10.53am 14 1.021

5 8.30am 65 8.53am 9.23am 36 1.025 9.53am 27 1.026 10.53am 38 1.026

Total 200 167 125 140

Mean 40 33.4 25 28

3

1 8.44am 171 8.57am

9.30

am 59 1.013

10.00

am 91 1.006

11.00

am 79 1.014

2 8.45 am 52 8.57 am

9.30

am 18 1.028

10.00

am 11 1.025

11.00

am 34 1.015

3 8.43 am 32 8.57 am

9.30

am 19 1.032

10.00

am 11 1.029

11.00

am 32 1.025

4 8.43 am 30 8.57 am

9.30

am 18 1.032

10.00

am 12.5 1.028

11.00

am 25 1.028

5 8.43am 31 8.57am

9.30

am 18 1.03

10.00

am 38 1.01

11.00

am 28 1.018

Total 316 132 163.5 198

Mean 63.2 26.4 32.7 39.6

4 1 8.46am 26 8.55am 9.25am 22 1.022 9.55am 62 1.006 10.55am 46 1.016

2 8.46am 164 8.55am 9.25am 150 1.004 9.55am 90 1.004 10.55am 50 1.002

3 8.46am 86 8.55am 9.25am 15 1.028 9.55am 11 1.024 10.55am 14 1030



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4 8.46am 66 8.55am 9.25am 36 1.022 9.55am 32 1.022 10.55am 46 1.018

5 8.46am 30 8.55am 9.25am 21 1.022 9.55am 30 1.008 10.55am 31 1.015

Total 372 244 225 187

Mean 74.4 48.8 45 37.4

MEAN URINE PRODUCTION

Volume (ml)

Time (minutes)



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Group 1 2 3 4

Total volume of urine(ml) 516ml(second highest) 432ml(least) 493.5ml(third highest) 656ml(highest)

Final Urine Production rate(ml/hr)

(150+235+131)ml / 2hr=258ml/hr

(167+125+140)ml / 2hr=216ml/hr

(132+163.5+198)ml / 2hr=246.8ml/hr

(244+225+187)ml / 2hr=328ml/hr

Type of fluid consume Isotonic Coffee without sugar Sugar (50%) solution Water Justification Isotonic solution contain waterand electrolyte, it is much likelylike water, does it effects just likewater but much lesser thanwater.

Coffee is a diuretic agent. Thisgroup supposed to get the mosturine production, but they get theleast urine production maybebecause the students weredehydrated, thus drinking coffeeis such drinking hypertonicsolution, where increase theosmolari ty of ECF. Bodycompensate by increase ECFvolume, thus decrease urineoutput.

This is a hypertonic solution. Itincreased the osmolarity of ECF.Homeostasis occurs by retainingwater, thus decrease urineoutput.

Water increased the volume ofECF and decreased osmolarity.To compensate, it increase urineoutput.



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Answer the following questions:

1. Which of the groups produced the most urine at 30, 60 and 120 minutes and which the least?

What could cause the difference, if any?

Urine produced/ time

(minutes)

30 60 120

Most 4 1 3

Least 3 2 1

From the table it shows at first 30 minutes, group 4 produced the most urine, while group3 produced the least urine.

On the 60 minutes, group 1 produced the most urine, while group 2 produced the leasturine.

On 120 minutes, group 3 produced the most urine, while group 1 produced the leasturine.

There are few factors that cause difference in urine output.

Firstly in this experiment, the amount and concentration of fluid consume. Secondly, the amount of ADH and aldosterone circulating in body.

Thirdly, the health of students.

2. Is there any difference in the urine volume produced by each experimental group at 30, 60 and

120 minutes? (Effect of time on same treatment group). Justify your result.

Yes.

At 30 minutes, the highest urine output is produced by Group 4, followed by Group 2,Group 1 and lastly Group 3.

At 60 minutes, the highest urine output is produced by Group 1, followed by Group 4,Group 3 and lastly Group 2

At 120 minutes, the highest urine output is produced by Group 3 followed by Group 4,Group 2 and lastly Group 1

After consuming each fluid kidney will try to regulate the volume and osmolarity in theECF.

For example, group 4 which consuming water will have high volume and low osmolarity.For the first 30 minutes, it has increased in GFR thus increase in urine output, whilegroup 3 which consuming glucose (50%) solution will have high osmolarity by lowvolume. The kidney will regulate the osmolarity and volume in ECF by retaining water inECF, thus decrease urine output.

The mean of urine production is reduced time by time as regulation in the kidney proceed

to maintain the volume and osmolarity of ECF.

3. Is there any difference in the urine volume produced by each experimental group at 30, 60 and

120 minutes? (Effect of drinking solution at each time interval). Justify your result.

Yes.

At 30 minutes, the highest urine output is produced by Group 4, followed by Group 2,Group 1 and lastly Group 3.

At 60 minutes, the highest urine output is produced by Group 1, followed by Group 4,Group 3 and lastly Group 2

At 120 minutes, the highest urine output is produced by Group 3 followed by Group 4,Group 2 and lastly Group 1

There are differences in the urine volume produced effecting by type of solution in eachtime interval.



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A human body regulates its' internal environment within narrow limits. This is calledhomeostasis. When the body consumed the fluid, the brain will detect the concentrationof solution and stimulate the aldosterone to produce more or less anti-diuretic hormone(ADH), depending on the concentration and osmolarity of the blood. ADH is a hormonethat maintains the osmolarity of the blood by controlling the permeability of the walls ofthe collecting duct, thus enabling the regulation of the amount of water reabsorbed from

the glomerular filtrate back into the blood capillaries.

4. Compare the rate of urine production in different experimental group. Explain your result.

Group 1 2 3 4

Final UrineProductionrate (ml/hr)

(150+235+131)ml /2hr=258ml/hr

(167+125+140)ml /2hr=216ml/hr

(132+163.5+198)ml /2hr=246.8ml/hr

(244+225+187)ml /2hr=328ml/hr

From the table above, group that consume coffee (group2) has the lowest rate of urineproduction followed by group 3 (glucose), group 1 (isotonic) and lastly group 4 (water).

The student who consumes the coffee will produce more ADH to lower his/her bloodconcentration thus, a lower rate of urine formation will be obtained.

On the other hand, when a student consumes a low amount of solute such as the wateror an isotonic drink, his/her brain will detect the drop in blood-sugar concentration andinhibits the production of ADH by the aldosterone, making the wall of the collecting duct tobe more impermeable to water thus, more water will be loss through the urine, increasingthe student's rate of urine formation.

5. How is rate of urine produced affected by the type of drinks consumed in this experiment?

Rate of urine produced is affected by concentration of fluid consume as homeostasis willcontrol the rate of urine formation to maintain an optimum volume and osmolarity of ECF.

Isotonic 100plus

Slight increase/normal in ECF volume and osmolarity which does not

disturb normal reabsorption and secretion normal urine output

normal urine production rate

Coffee withoutsugar

Diuretic effect increase impermeability tubular membrane to water

decrease in water reabsorption increase urine output increase

urine production rate.

*In this experiment, the students might be dehydrated, therefore the

diuretic effects of the coffee only appear in the first 30 minutes of the

experiment.

Glucose (50%)solution

Increase in ECF osmolarity, decrease ECFvolume decrease GFR

stimulates release of renin and release of ADH increase water

reabsorption decrease urine output decrease urine production

rate

Water Increase ECF volume, decrease ECF osmolarity increase GFR

stimulates the release of ANP decrease secretion of ADH

Decrease water reabsorption increase rate of urine output.



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6. How is the urine production regulated? Relate the mechanism to the volume of urine produced by

subjects in each experimental group.

Urine production is regulated through osmolarity and volume of extracellular fluid via ADHand aldosterone.

Group 1 and group 4, which consume isotonic 100 plus and water, contain less glucose, lessconcentrate. The brain will detect the drop in blood-sugar concentration and inhibits theproduction of ADH by the aldosterone, making the wall of the collecting duct to be moreimpermeable to water thus, more water will be loss through the urine, increasing thestudent's rate of urine formation.

While, for group 2 and group 3 which consume coffee and glucose solution, cause high ECF

volume but not ECF osmolarity. This will increase the production of ADH to increase thepermeability of distal tubule and collecting duct in the presents of aquaporin-2, thus increasewater reabsorption thus decrease urine production

7. Explain the mechanism involved in the production of urine at 30, 60 and 120 minutes.

The formation of urine depends on 3 processes, which are filtration, reabsorption and

secretion.

Urinary excretion rate = Filtration rate Reabsorptionrate +Secretion rate

Filtration

The blood is filtered by nephrons, the functional units of the kidney. Each nephron begins in

a renal corpuscle, which is composed of a glomerulus enclosed in a Bowman's capsule.

Cells, proteins, and other large molecules are filtered out of the glomerulus by a process of

ultrafiltration, leaving an ultrafiltrate that resembles plasma (except that the ultrafiltrate has

negligible plasma proteins) to enter Bowman's space. Filtration is driven by Starling forces.

The ultrafiltrate is passed through, in turn, the proximal convoluted tubule, the loop of Henle,

the distal convoluted tubule, and a series of collecting ducts to form urine.

Reabsorption

Tubular reabsorption is the process by which solutes and water are removed from the tubular

fluid and transported into the blood. It is called reabsorption (and not absorption) because

these substances have already been absorbed once (particularly in the intestines).

Reabsorption is a two-step process beginning with the active or passive extraction ofsubstances from the tubule fluid into the renal interstitium (the connective tissue that

surrounds the nephrons), and then the transport of these substances from the interstitium into

the bloodstream. These transport processes are driven by Starling forces, diffusion,

and active transport.

Indirect reabsorption

In some cases, reabsorption is indirect. For example, bicarbonate (HCO3-) does not

have a transporter, so its reabsorption involves a series of reactions in the tubule

lumen and tubular epithelium. It begins with the active secretion of a hydrogen ion

(H+) into the tubule fluid via a Na/H exchanger.

In the lumen

The H+ combines with HCO3- to form carbonic acid (H2CO3)

Luminal carbonic anhydrase enzymatically converts H2CO3 into H2O and CO2Feb 2/CLASS 2012/SEM4/YR2/MHN 11


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CO2 freely diffuses into the cell

In the epithelial cell

Cytoplasmic carbonic anhydrase converts the CO2 and H2O (which is

abundant in the cell) into H2CO3

H2CO3 readily dissociates into H+ and HCO3

-

HCO3- is facilitated out of the cell's basolateral membrane

Hormones

Some key regulatory hormones for reabsorption include:

aldosterone, which stimulates active sodium reabsorption (and water as a

result)

antidiuretic hormone, which stimulates passive water reabsorption

Both hormones exert their effects principally on the collecting ducts.

Secretion

Tubular secretion is the transfer of materials from peritubular capillaries to renal tubular

lumen. Tubular secretion is caused mainly by active transport.

Usually only a few substances are secreted. These substances are present in great excess,

or are natural poisons.

8. Can urine from a dehydrated person show a reading of 1.010 for specific gravity? Explain your

answer.

No. reading 1.010 for specific gravity indicates the person is hydrated. In dehydrated person, it willproduce concentrated urine, thus increase specific gravity reading.

9. What cause a false increase in the specific gravity reading without actual increase in osmolality?

There are appreciable quantities of larger molecules in the urine, such as glucose, radiocontrastmedia, or the antibiotic carbenicillin. In these settings, the specific gravity can reach 1.030 to 1.050(falsely suggesting a very concentrated urine), despite a urine osmolality that may be only 300mosmol/kg.

10. Which method of specific gravity determination is most accurate?

refractometer

11. If a marathon runner is asking for your advice, what would you advice the marathon runner to

drink during the race? Justify your answer.

Isotonic. During exercise, electrolyte loose through transpiration, by drinking isotonic drinkselectrolytes, to restore bodys supply. Also the isotonic drink will maintain the osmolality of blood sothat less urine is produced (making rehydration more effective).



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Conclusion:

12. What can you conclude from this experiment?

In this experiment, we can conclude that types of drink can affect urine volume production andrate of production. Hifh concentrated type of drink will produce less urine and concentrated urine(high specific gravity reading) while less concentrated fluid will increase urine production with lowspecific gravity reading.



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TABLE 2. RESULTS OF URINALYSIS (OWN URINE)

Substance Reading time usingdipstick (after first

dip)Results

Glucose 30 -ve

Ketones 40 -ve

Specific gravityreading 45

Dipstick Refractometer

1.025 1.020

*Amount of solidpresent (grams

per Litre g/L) nil 66.5 53.2

pH 60 6.50

Protein 60 0.30

Blood 60 -ve

Urobilinogen 60 -ve

Nitrite 60 -ve

Leukocytes 120 -ve

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* multiply the last two digits of reading obtained with Longs Coefficient 2.66 to get amount ofsolids present in the urine sample.


Color Concentrated yellow

Turbidity Clear/transparent

Sediments Negative

Odor Mild aromatic

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Answer the following questions.

Q10. Explain the biochemistry (of the urinary strip) for detection of:

a. Blood

This test is based on the peroxidase-like activity of hemoglobin, which catalyzes thereaction of diisopropylbenzene dihydroperoxide and 3,3',5,5'-tetramethylbenzidine.The resulting color ranges from orange through green; very high levels of blood maycause the color development to continue to blue.

The presence of large numbers of RBCs in the urine sediment establishes thediagnosis of hematuria. If the dipstick is more strongly positive than would beexpected from the number of RBCs, then the possibility of hemoglobinuria ormyoglobinuria should be considered.

b. Glucose

This test is based on a double sequential enzyme reaction. One enzyme, glucoseoxidase, catalyzes the formation of gluconic acid and hydrogen peroxide from the

oxidation of glucose. A second enzyme, peroxidase, catalyzes the reaction ofhydrogen peroxide with a potassium iodide chromogen to oxidize the chromogen tocolors ranging from green to brown.

In general the presence of glucose indicates that the filtered load of glucose exceedsthe maximal tubular reabsorptive capacity for glucose. In diabetes mellitus, urinetesting for glucose is often substituted for blood glucose monitoring.

c. Protein

This test is based on the protein-error-of-indicators (tetrabromphenol blue) principle.At a constant pH, the development of any green color is due to the presence ofprotein. Colors range from yellow for negative through yellow-green and green togreen-blue for positive reactions.

Heavy proteinuria usually represents an abnormality in the glomerular filtrationbarrier. The test is more sensitive for albumin than for globulins or hemoglobin.

d. Ketones

This test is based on the development of colors ranging from buff-pink, for a negativereading, to purple when acetoacetic acid reacts with nitroprusside.

Urine testing only detects acetoacetic acid, not the other ketones, acetone or beta-hydroxybuteric acid. In ketoacidosis (insulin deficiency or starvation), it can bepresent in large amounts in the urine before any elevation in plasma levels.

Q11. What does a positive nitrite test indicate?

Positive nitrite indicates bacteriuria containing gram negative bacteria that producenitrite reductase enzyme which give positive result.

Q12. State one possible cause for an individual to produce urine with high pH (> 8.0),excluding the possibility of alkali supplements ingestion.

Vegetarian

Q13. What further test is needed to confirm the diagnosis stated in Q12?

Use pH indicator paper strip



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References

1. Urinalysishttp://library.med.utah.edu/WebPath/TUTORIAL/URINE/URIN072.html.

2. Medical Technology, Clinical Laboratory Science Urinalysishttp://www.irvingcrowley.com/cls/urin.htm

3. Urinalysis: Pitfalls and Pearls Eileen D. Brewerhttp://www.baylorcme.org/renal/presentations/brewer/brewer.pdf

4. Continuing Medical Education: Urinalysis Part II. The Urine Microscopic Examination

http://www.medicine.uiowa.edu/cme/clia/modules.asp?testID=20#4_5AbnormalCrystals

Submission of laboratory worksheet.

Please submit INDIVIDUAL WORK on Table 1 & Table 2, stating objectives of each experiment,and answers to the all questions. This worksheet exercise soft copy must be handed in onWednesday, 8 February 2012 at 8am to the lecturer (Dr. Mohd Hafiz Ngoo) via Team email.

[email protected] , and/or

[email protected]

http://library.med.utah.edu/WebPath/TUTORIAL/URINE/URIN072.htmlhttp://www.irvingcrowley.com/cls/urin.htmhttp://www.baylorcme.org/renal/presentations/brewer/brewer.pdfhttp://www.medicine.uiowa.edu/cme/clia/modules.asp?testID=20#4_5AbnormalCrystalsmailto:[email protected]:[email protected]://library.med.utah.edu/WebPath/TUTORIAL/URINE/URIN072.htmlhttp://www.irvingcrowley.com/cls/urin.htmhttp://www.baylorcme.org/renal/presentations/brewer/brewer.pdfhttp://www.medicine.uiowa.edu/cme/clia/modules.asp?testID=20#4_5AbnormalCrystalsmailto:[email protected]:[email protected]

Farhah Izzati Urinalysis Lab 020212

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