Lecture Notes and Case Studies Compilation PDF

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Clinical Microscopy| BS MEDTECH 4Fritzl Joy Tumampos MagdadaroURINALYSISUrine Formation In the normal adult, approximately 1,200 ml/min of blood perfuse the kidneys each minute, which accounts for about 25% of the cardiac output. Total renal plasma flow: 600 to 700 ml/min.1. Glomerular FiltrationGlo...

Description

Clinical Microscopy- is the study of non-blood body fluids like urine, stool, seminal fluid, serous fluid, pleural fluid, pericardial fluid, peritoneal fluid, synovial fluid, amniotic fluid, and other body fluids.

Microscopy| Clinical Microscopy |BS MEDTECH 4 Fritzl Joy Tumampos Magdadaro

URINALYSIS Urine Formation In the normal adult, approximately 1,200 ml/min of blood perfuse the kidneys each minute, which accounts for about 25% of the cardiac output. Total renal plasma flow: 600 to 700 ml/min. 1. Glomerular Filtration

The glomerulus functions as a sieve or filter. Although it serves as a nonselective filter of plasma substances with Molecular Weight: sodium > potassium. Small or trace amounts of many additional inorganic chemicals are also present in urine.

Methods of Collection 1. 2. 3. 4.

Midstream - routine screening, bacterial culture Catheterized - bacterial culture Suprapubic aspiration - bladder urine for bacterial culture, cytology Glass technique  Three glass technique – prostatic infection

Quantitative cultures are performed on all specimens, and the first and third specimens are examined microscopically. In prostatic infection, the third specimen will have a white blood cell count and bacterial count 10 TIMES that of the first specimen. Macrophages containing lipids may also be present. The second specimen is used as a control for bladder and kidney infection. If it is positive, the results from the third specimen are INVALID, because infected urine has contaminated the specimen. 5. Pediatric specimen Use of soft, clear plastic bag with adhesive Sterile specimen obtained by catheterization or suprapubic aspiration 6. Drug specimen collection

CHAIN OF CUSTODY (COC): Step-by- step documentation of the handling and testing of legal specimens. It is the process that provides this documentation of proper sample identification from the time of collection to the receipt of laboratory results.

Take Note: DONOR – individual from whom a specimen is collected

Volume Temperature

30-45 ml urine 32.5°C to 37.7°C

If the specimen temperature is NOT within range, the temperature should be recorded and the supervisor or employer must be contacted immediately.  Urine temperatures outside of the recommended range may indicate specimen contamination.  Recollection of a second specimen as soon as possible will be necessary. RECALL: A midstream clean-catch urine is submitted to the laboratory for routine urinalysis and culture. The routine urinalysis is done first, and after 30 minutes, the specimen is sent to the microbiology department for culture. The specimen should be:  Reject the specimen. Because in the hospital, it should be sent first in the microbiology department before doing the routine urinalysis. It is prone to contaminants.  Optional: But if done both routine and culture, you can separate the urine sample in 2 test tubes.

RECALL: Which of the following is inappropriate when collecting urine for routine bacteriologic culture? A. The urine must have an additives when testing is delayed B. The sample must be held at 2-8°C for up to 72 hours prior to plating C. The collected must be plated within 2 hours unless refrigerated D. Two of these

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Clinical Microscopy |BS MEDTECH 4 Microscopy| Fritzl Joy Tumampos Magdadaro Types of Urine Specimen 1. Occasional/single/random – routine screening 2. Timed – quantitative chemical tests    

24 hour Ex: Creatinine clearance test 12 hour Afternoon specimen (2pm – 4pm) Ideal for the determination of Urobilinogen content 4 hour

3. First morning – routine screening, pregnancy tests, orthostatic protein 4. Fasting/second morning

Changes in Unpreserved urine INCREASED: “PBAON-C”    



Unpreserved urine that is stand for more than the required time in room temp.

Protein Bacteria Odor foul odor Nitrite due to bacterial multiplication Color dark color because of prolong standing

DECREASED: “RWUB Ke GC”   

Clarity Glucose Ketones

 

Bilirubin due to exposure or oxidation Urobilinogen RBC/WBC dissolved (also with the casts) when unpreserved becomes alkaline instead of acidic



instead of clear, it becomes turbid because of prolonged standing the bacteria will utilize the glucose as food/energy in the urine (glycolysis)

volatilization

Preservation 

REFRIGERATION – Most Common Preservation

Following collection, specimens should be delivered to the laboratory promptly and tested within 2 hours. A specimen that cannot be delivered and tested within 2 hours should be refrigerated or have an appropriate chemical preservative added. 1. Physical a) Refrigeration b) Freezing 2. Chemical a) Formalin b) Thymol c) Boric acid d) Toluene/Toluol also used in gen path laboratory which serves as clearing agent e) Sodium fluoride/benzoic acid sodium fluoride is also an additive for vacutainer tube (glucose analysis)

f) g) h) i)

Phenol HCl Sulfuric acid Saccomano’s fixative

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Microscopy| Clinical Microscopy |BS MEDTECH 4 Fritzl Joy Tumampos Magdadaro Urine Preservatives Preservatives

Advantages

Refrigeration

Does not interfere with chemical tests

Thymol

Preserves glucose and sediments well

Boric acid

Preserves protein and formed elements well  Does not interfere with routine analyses other than pH Excellent sediment preservative

Formalin (formaldehyde)



Toluene

Sodium fluoride

Phenol

Commercial preservative tablets

Urine collection kits Gray C&S tube

Yellow plain UA tube Cherry red/yellow top tube

Saccamano’s fixative

Does not interfere with routine tests

Prevents glycolysis  Good preservative for drug analyses Does not interfere with routine tests



Convenient when refrigeration  Have controlled concentration to minimize interference Contains collection cup, C&S preservative tube or UA tube Sample stable at room temperature for 48 hours, preserves bacteria Use on automated instruments Stable for 72 hours at RT; instrument compatible 

Preserves cellular elements

Disadvantages Raises SG by hydrometer  Precipitates amorphous phosphates and urates Interferes with acid precipitation tests for protein May precipitate crystals when use in large amounts 

Acts as a reducing agent interfering with chemical tests for GBLC: Glucose, Blood, Leukocyte, and Copper reduction Floats on surface of specimens and clings to pipettes and testing materials Inhibits reagent strip tests for GBL: Glucose, Blood, and Leukocyte Causes an odor change May contain one or more of the preservatives including sodium fluoride

Must refrigerate within 2 hours Bilirubin and Urobilinogen may be decreased if specimen is exposed to light and left RT

Additional Information Prevents bacterial growth in 24 hours

Keeps pH at about 6.0

Rinse specimen container with formalin to preserve cells and casts

May use sodium benzoate instead of fluoride for reagent strip testing Use 1 drop per ounce of specimen Check tablet composition to determine possible effects on desired tests

Preservative is boric acid and may not be used for UA Round or conical bottom Preservative is sodium propionate; conical tube Used for cytology studies

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Microscopy| Clinical Microscopy |BS MEDTECH 4 Fritzl Joy Tumampos Magdadaro Physical Examination of Urine I. Volume  Normal range (24hr): 600 to 2000 mL  Average volume: 1200 to 1500 mL  Night: day ratio: 1:2 or 1:3 1. Polyuria - an increase in daily urine volume greater than 2.5 L/day in adults and 2.5 to 3 mL/kg/day in children.  Diuresis (increase urine volume)  Increased fluid intake  Diuretic medication  Diuretic drinks (coffee, tea, or alcohol)  Nervousness  Diabetes mellitus  Diabetes insipidus 2. Oliguria - a decrease in urine output which is less than 1 mL/kg/hr in infants, less than 0.5 mL/kg/hr. in children, and less than 400 ml/day in adults  Calculus or kidney stones  Dehydration 3. Anuria - complete cessation of urine  Complete obstruction (stones, carcinomas)  Toxic agents 4. Nocturia - >500 mL urine output at night time with SG: less than 1.018  Pregnant

II. Color  Roughly indicates the degree of hydration, and should correlate with urine specific gravity 3 Pigments: (3 UR’s) 1. Urochrome – MAJOR pigment (YELLOW)  Actual amount produced is dependent on the body’s metabolic state with increased amounts produced in thyroid conditions and fasting states.  Also increases in urine that stands at room temperature 2. Uroerythrin  Attaches to the urates producing a PINK color to the sediment 3. Urobilin  An oxidation product of the normal urinary constituent urobilinogen imparting ORANGE-BROWN color to the urine that is NOT fresh NORMAL: Colorless to deep yellow 7|M TAP

Microscopy| Clinical Microscopy |BS MEDTECH 4 Fritzl Joy Tumampos Magdadaro COLOR Colorless Pale yellow

Dark yellow Amber Orange

Yellow green Yellow brown Green Blue green

Pink Red

Brown Black

DISEASE - Recent fluid consumption - Polyuria - Diabetes mellitus - Diabetes insipidus - Concentrated specimen - Bilirubin - Acriflavine - Pyridium - Nitrofurantoin - Phenindione - Bilirubin oxidized to biliverdin - Pseudomonas infection - Clorets - Indican - Methylene blue - Phenol - RBCs - Hemoglobin - Myoglobin (25 mg/dL) - Porphyrin - Beets - Rifampin - Menstrual contamination - RBCs oxidized to methemoglobin - Homogentisic acid - Melanin or melanogen - Methyldopa or levodopa - Metronidazole (Flagyl)

A purple staining may occur in catheter bags and is caused by indican in the urine or a bacterial infection, frequently caused by Klebsiella or Providencia species.

Urine Color and Clarity Procedure 1. Evaluate and adequate volume of specimen NOTE: when you receive a urine specimen for urine analysis, make sure that it has sufficient amount of volume to do the examination

2. Use a well-mixed specimen NOTE: Before you transfer the urine specimen to the test tube for analysis, you have to mix it first so that if ever there are sediments that are settled at the bottom of the test tube, they will be included in the examination.

3. View the urine through a clear container NOTE: Regarding the color, when you get the color of the urine, you have to make sure to view it in a clear container so that it will be clear enough to read it.

4. View the urine against a white background using adequate room lighting 5. Maintain adequate room lighting 6. Evaluate a consistent volume of urine 8|M TAP

Microscopy| Clinical Microscopy |BS MEDTECH 4 Fritzl Joy Tumampos Magdadaro Urine Color changes with commonly used Drugs COLOR Pale, diuresis Reddish, alkaline; yellow-brown, acid Red Red Orange, red Brown Blue Brown on standing Red then brown; alkaline Yellow Green-brown Darken; if oxidizing agents present, red to brown Blue, blue-green Darkening, reddish brown Brown-yellow Orange-red, acid pH Orange, alkaline; color disappears when acidified Brown; oxidized to quinines (green) Red-purple, alkaline pH Pink-red, alkaline pH Bright orange-red Bright yellow Orange-yellow, alkaline pH

DRUG Alcohol, ethyl Anthraquinone laxatives (senna, cascara) Chlorzoxazone (Paraflex) - muscle relaxant Deferoxamine mesylate (Desferal) – chelates iron Ethoxazene (Serenium) – urinary analgesic Furazolidone (Furoxone, Tricofuro) – an antibacterial, antiprotozoal nitrofuran Indigo carmine dye – renal function, cytoscopy Iron sorbitol (Jectofer) – possibly other iron compounds forming iron sulfide in urine Levodopa (L-dopa) – for parkinsonism Mepacrine (Atabrine) – antimalarial, intestinal worms, Giardia Methacarbamol (Robaxin) – muscle relaxant Methyldopa (Aldomet) - antihypertensive Methylene blue – used to delineate fistulas Metronidazole (Flagyl) – for Trichomonas infection, amebiasis, Giardia Nitrofurantoin (Furadantin) - antibacterial Phenazopyridine (Pyridium) – urinary analgesic Phenindione (Hedulin) – anticoagulant *IMPORTANT TO DISTINGUISH FROM HEMATURIA Phenol poisoning Phenolphthalein – purgative Phenolsulfonphthalein Rifampin (Rifadin, Rimactane) – tuberculosis theraphy Riboflavin (multivitamins) Sulfasalazine (Azulfidine) – for ulcerative colitis

III. Clarity/Transparency/Turbidity The transparency of the urine is the degree of clarity of the urine

In routine urinalysis, clarity is determined in the same manner that ancient physicians used by: visually examining the mixed specimen while holding it in front of a light source. NABECULAE– faint cloud in urine after standing due to WBCs, epithelial cells, and mucus 9|M TAP

Microscopy| Clinical Microscopy |BS MEDTECH 4 Fritzl Joy Tumampos Magdadaro TERMINOLOGY CLEAR HAZY CLOUDY TURBID MILKY

Transparent, No visible particulates Few particulates, print easily seen through urine Many particulates, print blurred through urine Print cannot be seen through urine May precipitate or clot

PATHOLOGIC CAUSES OF TURBIDITY 1. 2. 3. 4. 5. 6. 7. 8.

RBCs WBCs Bacteria Yeast Non-squamous epithelial cells Abnormal crystals Lymph fluid Lipids

NON-PATHOLOGIC CAUSES OF TURBIDITY 1. 2. 3. 4. 5. 6. 7. 8.

Squamous epithelial cells Mucus Amorphous crystals Semen, spermatozoa Fecal contamination Radiographic contrast media Talcum powder Vaginal cream

Appearance and Color of Urine Appearance Colorless Cloudy

Milky

Yellow Yelloworange Yellow-green Yellow-brown Red

Red-purple

Cause Very dilute urine Phosphates, carbonates Urates, uric acid Leukocytes Red cells (“smoky”) Bacteria, yeasts Spermatozoa Prostatic fluid Mucin, mucous threads Calculi, “gravel” Clumps, pus, tissue Fecal contamination Radiographic dye Many neutrophil (pyuria) Fat Lipiduria, opalescent Chyluria, milky Emulsified paraffin Acriflavine Concentrated urine Urobilin in excess Bilirubin Bilirubin-biliverdin Bilirubin-biliverdin Hemoglobin Erythrocytes Myoglobin Porphyrin Fuscin, aniline dye Beets Menstrual contamination Porphyrins

Remarks Polyuria, Diabetes insipidus Soluble in dilute acetic acid Dissolves at 60°C and in alkali Insoluble in dilute acetic acid Lyse in dilute acetic acid Insoluble in dilute acetic acid Insoluble in dilute acetic acid May be flocculent Phosphates, oxalates Rectovesical fistula In acid urine Insoluble in dilute acetic acid Nephrosis, crush injury,soluble in ether Lymphatic obstruction, soluble in ether Vaginal creams Green fluorescence Dehydration, fever No yellow foam Yellow foam, if sufficient bilirubin Yellow foam “Beer” brown, yellow foam Positive reagent strip for blood Positive reagent strip for blood Positive reagent strip for blood May be colorless Foods, candy Yellow alkaline, genetic Clots, mucus May be colorless 10 | M T A P

Microscopy| Clinical Microscopy |BS MEDTECH 4 Fritzl Joy Tumampos Magdadaro Red-brown

Brown-black

Erythrocytes Hemoglobin on standing Methemoglobin Myoglobin Bilifuscin (dipyrrole) Methemoglobin Homogentisic acid

Melanin Blue-green

Indicans Pseudomonas infections Chlorophyll

Acid pH Muscle injury Result of unstable hemoglobin Blood, acid pH On standing, alkaline; alkaptonuria (disease) Darkens after prolonged standing On standing, rarer; melanuria (disease) Darkens after exposure to air Small intestine infections Mouth deodorants

REMEMBER LeSBaY: Insoluble with dilute acetic acid

IV. Specific gravity  Density of solution compared with density of similar volume of distilled water at a similar temperature  Influenced by number and size of particles in solution  Normal random specimens may range from approximately 1.002 to 1.035, depending on the patient’s amount of hydration  Specimens measuring lower than 1.002 probably are not urine 3 terms: Hyposthenuria SG= 1.010

Determination 1. Refractometry (Total Solids meter) - Indirect method based on Refractive index (RI) - NO TEMPERATURE CORRECTION - Compensated to temperature (15-38°C) - Requires correction for GP: Glucose and Protein  1 g/dL Glucose = -0.004  1 g/dL Protein = -0.003 - Calibration  Distilled water = 1.000  5% NaCl = 1.022 ±0.001  9% Sucrose = 1.034 ±0.001

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Microscopy| Clinical Microscopy |BS MEDTECH 4 Fritzl Joy Tumampos Magdadaro 2. Urinometry/Hydrometer - Requires TEMPERATURE CORRECTION  0.001 must be subtracted from the reading every 3°C that the

standard temperature is BELOW the urinometer calibration temperature  0.001 must be added from the reading every 3°C that the standard temperature is ABOVE the urinometer calibration temperature - Requires correction for GP: Glucose and Protein - DISADVANTAGE: higher urine volume needed (10-15 mL) - Calibrated with: potassium sulphate (SG = 1.015) 3. Reagent strip - Principle: pKa change of polyelectrolyte - Reagent sensitive to number of ions in the urine specimen; indicator changes

color in relation to ionic concentration - Manufacturers recommend adding 0.005 to specific gravity reading when

pH is 6.5 or higher due to interference with the bromthymol blue indicator - NOT AFFECTED BY GPR:

  

G – Glucose P – Protein R – Radiographic contrast media

Urine Specific gravity Reagent strip summary Multistix: Poly (methyl vinyl ether/maleic anhydride) Reagents Bromthymol blue Chemstrip: Ethyleneglycol-Bis (aminoethylether) Bromthymol blue Sensitivity 1.000 – 1.030 Interference False positive: High concentration of protein False negative: Highly alkaline urine (>6.5) 4. Harmonic oscillation densitometry - Frequency of sound wave entering a solution will change in proportion to the density of the solution This is not common. Usually, reagent strip is being used Summary of Urine specific gravity measurements METHOD PRINCIPLE Urinometry Density Refractive Index Refractometry Density Harmonic Oscillation Densitometry Reagent Strip pKa change of polyelectrolyte RECALL: A urine specimen was tested for specific gravity using the reagent strip. 2 g/dl of glucose and 1 g/dl of protein was noted in her urine. As SG reading of 1.030 was reported, the standard temperature was 20°C but upon checking the temperature of the urine was 26°C. What is your final urine SG that you will report? Ans: 1.030 12 | M T A P

Microscopy| Clinical Microscopy |BS MEDTECH 4 Fritzl Joy Tumampos Magdadaro RECALL: A urine specimen was tested for specific gravity using the refractometer. 1 g/dl of glucose and 2 g/dl of protein was noted in her urine. An SG reading of 1.025 was reported, the standard temperature was 24°C but upon checking the temperature of the urine it was 21°C. What is your final urine SG that you will report? Ans: 1.015 RECALL: A urine specimen was tested for specific gravity using the hydrometer. 2 g/dl of glucose and 2 g/dl of protein was noted in her urine. As SG reading of 1.015 was reported, the standard temperature was 24°C but upon checking the temperature of the urine, it was 30°C. What is your final urine SG that you will report? Ans: 1.003

V. pH  Normal: pH 4.5 to 8.0 (random)  First morning specimen with a slightly acidic pH of 5.0 to 6.0  More alkaline pH is found following meals due to ALKALINE TIDE Refers to a condition normally encountered after eating a meal. Where during the production of HCl by the parietal cells in the stomach. The parietal cells will secrete carbonate ions across their basolateral membranes and into the blood that causes a temporary increase in the pH of the urine.

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