AUBF- Outline - Lecture notes 11 PDF

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Urinalysis and Body Fluid outline...

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AUBF OUTLINE RENAL PHYSIOLOGY  

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1M-1.5 Nephrons/Kidney Nephrons comprised of small coiled tubes which starts from glomerulus down to collecting tubules. Each of tubes have their own function; filtration, reabsorption, and secretion. Entry of the plasma in the kidney through renal artery. Nephrons serve as filtration unit. Two types of nephrons; Cortical and Juxtamedullary Cortical is responsible for the removal of waste products and reabsorption of nutrients. Juxtamedullary is for the concentration of urine

Hormonal Effects 

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ADH is increased = Urine concentration increase, increased water reabsorption, blood pressure decreases ADH is decreased = urine concentration decrease, decreased water reabsorption, blood pressure increases Aldosterone when present = cause the absorption of Na and Cl ions Aldosterone when absent = release of Na and Cl ions Aldosterone secreted by adrenal gland ADH secreted by posterior pituitary gland. Glucose and Protein are reabsorbed in urine formation. These substances are reabsorbed in the bloodstream because they have use in the body.

Urinalysis Blood Glucose High and Urine Glucose Positive = Diabetes Mellitus Blood Glucose High Urine Glucose Negative = Glucose Normal Level Blood Glucose Normal Urine Glucose Positive = physician requests another examination. Possibly the patient is not on fasting. Patient had breakfast (Coffee with sugar). Supper may contain high sugar or carbohydrates. No basis for Diabetes Physician does the interpretation. Not the MedTech.

Routine Urinalysis   

Yellow Form = Urinalysis Blue Form = Fecalysis Pink Form = Blood Examination

1. Physical or Macroscopic Examination  Color, Character (Clarity, Transparency), Specific Gravity, pH  Normal Urine Color = Straw to Amber Yellow, because of urochrome, substance responsible for urine color  Variation in urine color due diet  Abnormal color of urine = does not mean diseases/abnormality  Example green color medicine can cause green color urine. Not all abnormal color urine has medical significance and not all normal color of urine means absence of disease.  Measure accurately. No estimation.  Shake the container of the urine sample before pouring into the test tube.  Examine the color by holding the test tube at eye level.  For clarity, hold the test tube, vortex it a little, we can see visible sediments by tapping it. (Turbid/ Cloudy/Hazy). If very cloudy, maybe there is defect in filtration process. Normal urine is clear.  For color in first morning urine, usually darker compared to urine excreted after it. Slightly cloudy too.  When in fasting, after supper, no eating already and do not take in something in the morning. This sample is called urine fasting sample.  Specific gravity is high in first morning urine, concentrated urine. Usually preferred specimen.  Specific gravity high = concentrated urine  Physical examinations are correlated with chemical and microscopic examinations. 2. Chemical Examination  Ketone, Bilurubin = Special examinations, another payment  Glucose (Sugar), Protein/Albumin = Routine Urinalysis  Cloudy urine usually positive for protein tests/albumin.

If negative for albumin for cloudy urine, microscopic examination must be done because cloudiness was not caused by protein/albumin. Cloudy urine and negative albumin can be caused by: a. Squamous epithelial cells - can be avoided by proper collection procedure such as midstream collection and washing of genitals.

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3. Microscopic Examination  After examinations, MedTech can correlate the three examinations in urinalysis to determine if it is correct.  MedTech must learn how to prepare reagents. Its proportions and composition.  Some reagents are not commercially prepared, some MedTechs still prepare it.

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Rounded bulb has granular appearance (mercury), while the stem has calibration from 1.000 Requires sufficient amount of urine to fill ¾ of urinometer 20-30 ml of urine was required amount but today, can be much lower due to dipsticks If the urine is insufficient, you can add it with equal amount of distilled water. In measuring, the reading of the last two digits must be multiplied by 2 For every 3 degrees difference from standard room temperature, you are going to make correction of 0.001. If lower, subtract, if higher, add. Standard room temperature is 25 degree Celsius. When the reading is corrected, you must label it with corrected specific gravity.

Procedure: PHYSICAL EXAMINATION OF URINE pH of urine - is a reflection of the ability of the kidney to maintain normal hydrogen ion concentration in plasma and extracellular fluid. pH determines whether urine is acidic, basic, or neutral.  

Conventional method = Litmus paper Modern Way = Dipstix

Specific gravity - is the ratio of the weight of a substance to the weight of an equal volume of reference substance. (General Term) Urine Specific Gravity - it is a measure of the concentration of solutes and the concentrating and diluting power of the kidney, a very complicated function involving osmosis, counter current, ultrafiltration, and secretion mechanisms. So, in order to determine the specific gravity of the urine we have different apparatuses: 1. Urinometer - this apparatus is a floating gadget calibrated from 1.000 and up. this particular gadget is composed of a cylinder but comparing it to the graduated cylinder, the urinometer is a little bit stouter and has no calibrations. But together with it, it has a floating gadget.  Distilled water has specific gravity of 1.000.  It has a floating gadget, elongated stick at the end with a rounded bulb

Slightly mix the contents of the sample, then pour the urine into the cylinder of the urinometer (3/4) then insert the floating gadget, twist it before leaving it in the urine sample When it stops- it does not settle at the bottom it just float, at the center of the sample not touching the side of the cylinder- check the level of the urine sample versus on the calibration on the stem of the floating gadget  Must have sufficient sample- 10 to 15mL  Colorless solution = Lower meniscus  Colored solution = Upper meniscus  Urine is colored solution 2. Refractometer – has ocular or eyepiece but not vertically standing, slightly horizontal, attached to a stand with a wire for the connect to electricity  Has an ocular, just like microscope  Requires only 1 drop of urine  Calibration 1.000 – 1.005 -1.010 – 1.020 = means their specific gravity Procedure: On the ocular tube part Place only one drop of the sample- then peep through the ocular (you will see a rounded field) you will see a portion of lighted and shadow (dark) covering portion In the middle portion has calibration- starting from 1.000 – going up add (.001)- look at the edge of the dark portion

Collection of Specimens Avoid contaminations 1. Midstream clean catch  Preferred time of collection: Morning 2. Timed collection  12 hours  24 hours 3. Random – collected anytime 4. Suprapubic Aspiration 5. Three-glass collection Preserving Specimen 1. Formalin – must not be used if there is sugar test because formalin contains sugar. 2. Dry Ice (Refrigeration) – slows the chemical reactions in urine. Dipstix 1. Albustix - 1 parameter – test only albumin/protein 2. Clinistix – for glucose 3. Uristix – glucose and protein 4. Combistix – glucose, protein, pH 5. Hemacombistix - glucose, protein, pH, occult blood 6. Labstix - glucose, protein, pH, occult blood, ketone 7. Bililabstix - glucose, protein, pH, occult blood, ketone, bilirubin 8. Multistix - glucose, protein, pH, occult blood, ketone, bilirubin, eurobilinogen Components          

Specific gravity – bromthymol blue Bilirubin – 2,4-dichloroaniline diazonium salt WBC/Leukocytes – pyrrole amino acid ester, diazonium salt Nitrite – p-arsenilic acid, 1,2,3,4Tetrahyrdrobenzo(h)-quinolin-3-ol Blood – Diisopropylbenzene dehydroperoxide, tetramethylbenzidine Urobilinogen – p-dimethylaminobenzaldehyde Occult Blood - Ortho-toluidine peroxide, buffer Protein – Tetrabromphenol blue, Citrate buffer Glucose – Glucose oxidase, Peroxidase, tetramethyl benzidine or potassium iodide pH – Methyl red and Bromthymol blue

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Contamination – if the sample was contaminated, the urine may contain too many bacteria. For diabetic patient, urine has high glucose but when left for too long the bacteria breaks down the sugar making the test inaccurate.

MICROSCOPIC EXAMINATION OF URINE 1. Shake the container the of the specimen. Not vigorous, just enough to shake the sediments. 2. Pour ¾ to the test tube 3. Do the macroscopic and chemical examination using dipsticks. 4. After that, you can centrifuge it. You can use water in centrifuge to balance the machine. Remove spilled urine inside. 5. Usually 5-10 mins is utilized, for urine 3-5 mins of centrifuge. Very clear urine still has sediments. 6. Decant the supernatant liquid leaving behind 1-2 drops of sediments. Pour the supernatant liquid in container with disinfectant/antiseptic. 7. Invert immediately. do not stop. just let it flow freely. Straighten again the tube 8. Shake the tube to mix the sediment and transfer it to the center of glass slide 9. While pouring, use the brim of the tube to spread the center of the slide in a circle smear. 10. For beginners, you need to use cover slip to cover the smear so as to avoid the drying of the smear. Once dried, the sediments cannot be identified properly. 11. Place distance between stage and objective. 12. Place the glass slide in the stage. 13. Lower the LPO using course adjustment knob to close the distance between the glass slide/stage and body tube/objective. 14. Peep through, to check if there is light. Adjust the amount of light. 15. Adjust the iris diagpragm if the light is on but still dark on eyepiece. 16. Slowly raise the body tube until you see something in eyepiece. Use the coarse adjustment knob to put distance between glass slide and LPO. 17. If the object is blurred, use the fine adjustment knob. Turn it slowly until see something clearly. 18. If already clear, you scan start to scan. You can do it left to right or right to left. If you had reached the end, move it downward a little

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then go opposite direction of how you first scanned the glass slide. Start counting per field. You can report the objects you see by words (occasional, few, several, many) or numbers (0 to 3 LPF or HPF) Switch the objective from LPO to HPO if finished. Do not move anymore the coarse adjustment knob. Use the revolving nosepiece to switch to HPO and see if it does not hit the glass slide. Adjust the source of light because HPO is a little longer than LPO. If blurred, use fine adjustment knob slowly until you see things clearly. Just like LPO, scan also the field but unlike LPO, using HPO search for something you had not found during LPO.  Usually RBC, looks shrinked which is called crenated RBC.  In a manner of reporting do not use description, just count the objects/cells.  Pus cells is slightly bigger than RBC. Have granules and nucleus.  Renal epithelial cells have round shape. Much bigger that WBC/Pus cells. Contains nucleus that has the size of a WBC. Observe for other cells too such bacteria which is not normally seen in urine sample.  If there is a large number of bacteria, ask the patient for the manner of collection. If the patient says it is done correctly, maybe the bacteria is the one causing the patient disease.  Report the bacteria using general name (bacteria). Do not use the exact name of bacteria such as E. Coli.  The doctor may want to know the identity of the bacteria. He/she may request urine culture (Bacteriology/Microbiology section). Gram staining is the last step for urine culture which determine the identity of the bacteria.  Takes about 1 weeks for culture and sensitivity test.  We can also find parasites such as Trichomonas vaginalis, Schistosoma hematobium, Enterobius vermicularis and include it in results reporting.

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Indicate the specific name such as Trichomonas vaginalis in reporting. Can be reported through words or numbers. Include casts in your reporting. Whether hyaline cast, RBC cast and many more. Usually, casts are already visible in LPO. Organized sediments are cells (RBC, WBC, bacteria, yeast cells or fungi which indicates the presence of sugar.) Yeast cells have lollipop-shaped or just round without the stick. Can cause confusion from RBC. Use HPO to differentiate RBC from yeast cells. Also correlate glucose examinations with it. Yeast cells usually buds (Budding) Female patients usually have squamous epithelial cells in their urine sample due to their genitals. Mucous threads are colorless lines, thread-like, in the background. If this appears, indicates irritation. Clumps (RBC, WBC). Few of clumps – manner of reporting. Use estimation for bacteria in manner of reporting because bacteria is too small even using HPO. Numbers (+1,+2,+3,+4) example +3 is ¾ field. Unorganized sediments are crystals. Very common amorphous urates crystals. Normal but if too many may signify stone formation. No definite shape. Granular. Amorphous urates are colored, appear yellowish brown, orange. Appear in acidic urine Amorphous phosphates are granular but no color. Appear in alkaline urine. Correlate the result with pH. Urates appear colorless in alkaline urine.

CHEMICAL EXAMINATION OF URINE (CONVENTIONAL METHODS) Chemicals for urine chem analysis (conventional method):   

Benedict's Test Fehling’s Test Acetic Acid Test

Other Computations:  

computation for total solids (not done with routine urinalysis; special exam) collect timed specimen for this

Smelling of specimen is not recommended (done by others for correlation purposes) For Chem Exam: -Only detect for Glucose and Protein (routine urinalysis) CHEMICAL EXAM

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Color can change right away During the test, use equal vol of A and B and mix with urine sample in the test tube before heating

Reagents must be readily available for CONVENTIONAL METHODS - used kapag nadisgrasya ang dipstick  Medtechs prepare the reagents even the stains (kaya we should have the knowledge of the basics)  always label after preparing, tsaka ang expiration date ilagay (discard if expired na)

Dipstick  

wait for 30 seconds after dipping sa urine; then compare with the chart may indicated time limitation for the reading ng dipstick (read and follow the container)

In F2F -after using the materials, wash and clean then return sa basket -readily prepared na ang mga gamit sa table -buy 2 test tubes (same physical characteristics)

For Benedict's Tests Detects glucose in urine the reagent is already prepared Avoid contamination of reagent (use pipette for each reagent)  use a clean dropper for urine  If the Benedict’s Reagent is contaminated, it will change its color already  Use 5 cc of BensReagent then use a dropper to drop 8 drops of urine sample, mix.  After adding be sure to mix by tapping the bottom against your palm (palms open);  Then do the heating (pwede sa alcohol lamp directly or by water bath)  After heating, observe the changes in the color; if there is no glucose-blue color remains blue Blue green -- trace glucose Greenish Yellow -- positive 1 Yellow Green -- positive 2 Yellow to orange -- positive 3 Brick Red/Rusty -- positive 4 (highest)   

**Benedict's R and Fehlings R both contains copper sulfate **Fehling’s A and B are prepared in two containers Why is Fehling A and Fehling B separated?  

Fehling’s Reagent is too sensitive; if it's mixed early, premature reduction can occur

Detection of Glucose and Protein in Urine  pinaka important kaya included siya sa Routine Urinalysis Glucose in urine  doesn't always indicate Diabetes Mellitus (can be affected by the last meal);  must be correlated with the blood sugar;  many factors should be considered before final concluding if its disease related Protein in urine  (Normal amount in urine --- 10-100 mg especially in a 24 hr urine sample)  In the case of Albumin… Albumin in urine = albuminuria Protein in urine = proteinuria Types of Proteinuria: 1. Pre-renal Proteinuria- congestive heart failure 2. Renal Proteinuria- correlated with kidney; Glomerulonephritis (Bright’s disease) 3. Post Renal Proteinuria- inflammation in the urinary tract 4 kinds of Albuminuria: 1. Physiological Albuminuria – can be due to excessive exercise; very cold bath; too much ingestion of protein

2. Accidental Albuminuria - because of contamination (rbc, wbc, pus cell etc.); could be due to chronic vaginitis in females; doesn't really indicate abnormality 3. Postural/Orthostatic Albuminuria- kapag nakatayo ng mahabang oras; must rest first before taking the exam para di magfalse positive 4. Pathologic Albuminuria - renal albuminuria na; something is wrong with the urinary system especially sa kidney ***May mga special exams sa urinalysis na medyo the same sa clinical chem* - May principles of each test sa urinalysis (know by heart ang principles) Principles of Urinalysis  Principle of Benedicts test and  Principle of Heat and Acetic Acid Test Routine Qualitative Test Principles BENEDICT'S TEST PRINCIPLE  Cupric ion is reduced to cupric oxide by glucose and other reducing substances; The color of the final solution is roughly proportional to the amount of the reducing agent present o Reducing subs is not only glucose (ex. Vitamin C, lactose, homogentisic acid) o If urine specimen is preserve chemically, especially formalin (it contains sugar, so pwedeng mag false positive)

Procedure ng HAAT:  Clear urine on test tube (only half or a little above the half of the test tube; wag punuin ang test tube)  Technique: position the test tube holder sa top portion ng test tube para pag iincline, makita ang urine sa gitna vs sa bottom part ng tube  keep only the upper part of the urine  when inclined, heat only the upper portion of the urine; do not position the flame sa bottom part; do not boil  when heating, wag igalaw ang tube, steady lang; Limit the movement  If there’s smoke it’s already heated, remove the tube  then straighten the tube, drop at least 3-5 drops of acetic acid on the heated urine sample and observe if there's turbidity; DO NOT MIX; o if may turbidity or wala, itake note na.  then bring it back to the flame (the same heating process nung una) heat ulit then observe  compare the heated upper portion to the unheated bottom part; basis for comparison ang bottom part -trace protein (sa upper lang ang turbidity) -heavy population protein present (upper and lower part ang turbid tapos may precipitate pa)  

Other causes of urine turbidity (beside protein) can disappear if acetic acid is added; If the turbidity is persistent, yung protein or albumin na ang cause MICROSCOPIC EXAMINATION

HEAT AND ACETIC ACID TEST PRINCIPLE  Protein is coagulated by heat, dilute acetic acid is added to dissolve the precipitated phosphate and enhance coagulation of protein HEAT AND ACID TEST  HAAT determine the presence of protein specifically albumin  urine sample needed should be clear; if cloudy, must be filtered and use the urine filtrate; o pwede din icentrifuge para maging clear

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we need to use the sediment see to it that the container is mixed before transferring sa test tube do the macroscopic then dipstick then centrifuge the urine after centrifugation, transfer the clear to another test tube (for HAAT sa conventional method) while the sediment obtained is used for microscopic exam pour the sediment sa glass slide kapag ang amount is 1-2 drops; ...