Title page CM

Dr. Benjamin Q. Bengco III Medical & Surgical Clinic St. Jude Village Brgy Alfonso Concepcion,
Tarlac Clinical Laboratory

STANDARD OPERATING PROCEDURES

FOR

CLINICAL MICROSCOPY
VERSION 2.0

Written by

RHENZ ALFRED D. GALDONEZ, RMT Medical technologist

Date:

March 2, 2020

Reviewed by

EMIL BRYAN M. GARCIA, MD

Pathologist

Date:

March 2, 2020
1. ROUTINE URINALYSIS
   
    1.1 Principle
       The microscopic examination is a vital part of the routine urinalysis. It is a valuable
diagnostic tool for the detection and evaluation of renal and urinary tract disorders as well as
other systemic diseases. Urine Microscopy will only be performed per physicians/primary care-
giver request for clinical management or per study protocol requirements.   
    
1.2 Specimen Collection and Handling
         1.2.1 Use fresh well-mixed urine collected by clean-catch method into a sterile container.
         1.2.2 The specimen should be unpreserved and uncentrifuged.
         1.2.3 All urine specimens should reach the laboratory within one (1) hour after collection.
        1.2.4 Urine specimens should be tested within 1 hour after collection. If urine cannot be
tested within one (1) hour, it may be stored for up to four (4) hours at 2 to 8°C. (The specimen
must be brought to room temperature before testing.)
        1.2.5 The following urine samples are not satisfactory for testing:
           1.2.5.1 Specimens received over two hours after collection.
           1.2.5.2 Mislabeled samples.
           1.2.5.3 Improperly collected samples. For example, urine samples with
preservatives, specimens collected in non-sterile containers, or specimens collected in
containers with soap or detergent residues will not be accepted.
          1.2.5.4 QNS (Quantity Not Sufficient) - The recommended minimum volume is 12
mL's. The required minimum volume for microscopic examination is 0.50 ml.
          1.2.4.5 In the event that an unacceptable sample is received, another sample
must be requested.

      1.3 Materials & Equipment

          1.3.1 Clinical Centrifuge
          1.3.2 Microscope
          1.3.3 Microscope Slides
          1.3.4 Microscope Coverslips.
          1.3.5 Reagent strips
1.4 PROCEDURE
    1.4.1 Mix urine specimen by swirling.
    1.4.2 Pour 5 ml of urine into a test tube.
    1.4.3 Centrifuge for 5 mins at 1,500 rpm.
    1.4.4 Pour off the supernatant fluid, sufficient urine remains in the test tube to suspend
the sediment.
    1.4.5 Mix the test tube and transfer a drop on the slide.
    1.4.6 Examine under the microscope using LPO then HPO.

1.5 EXPECTED/CRITICAL RESULTS & REPORTABLE RANGE:

    1.5.1 The following bolded analytes require a result entry during the microscopic results
even if they are not seen during the microscopic exam. Report "none" or 0-1 or 0-2 as indicated.
All other analytes are optional entry if seen during the microscopic exam.

ANALYTE EXPECTED RESULT FOR REPORTED RANGE

ALL AGES
Hyaline cast 0-1
Granular cast None 0-1,1-3,3-5,5-10,10-25,25-50,
or greater than (>) 50/LPF.
Cellular cast (RBC, WBC, None Quantify each cast type
ETC) separately
Waxy cast None
Epithelial cells 0-2
Leukocytes (White Blood 0-2 0-2, 2-5, 5-10,10-25, 25-50,
Cells) or greater than (>) 50/HPF
Red Blood Cells 0-2
Bacteria None-Few None, Few, Trace, Moderate
or Many
Mucus None-Light Light, Moderate or Heavy
Crystals None Few, Moderate, or Many for
each crystal type
Spermatozoa Males only: Few Few, Moderate or Many
Yeast None Light, Moderate, or Many.
Report any budding yeast or
hyphae seen using
comments as noted below
Trichomonas None Few, Moderate, or Many.
May only be reported if
motile.

1.6 REPORTING OF RESULTS

     1.6.1 Record the color of urine as Straw, Light Yellow, Yellow, Dark yellow, Amber, Red,
Orange, etc.
     1.6.2 Record the turbidity of urine as; clear, hazy, slightly turbid, or turbid.
     1.6.3 Note for the presence of Blood and Protein based on the urine strip, it is graded as
followed: 1+, 2+, 3+.
     1.6.4 The pH of the urine is graded based on the alkalinity/acidity of urine: from 5.0--9.0.
     1.6.5 Specific gravity should also be noted. Noted as 1.005-1.030
     1.6.6 Presence of RBC/WBC should be noted based on the average number per 10 hpfs
(i.e. "WBC=2-5/hpf, RBC=0-1/hpf”).
     1.6.7 Bacteria is recorded as rare, few, moderate or many per hpf
     1.6.8 Epithelial cells is correlated based on the clarity of the urine, it should be reported
as Rare, few, moderate, or many per lpf.
     1.6.9 Amorphous vary depending on the ph of the urine. If the urine is acidic, it should be
reported as Amorphous urates: Rare/lpf, if alkaline Amorphous phosphates: Rare/lpf.
     1.6.10 Mucus Threads are reported as rare, few, moderate, or many per hpf.
     1.6.11 Presence of Yeast, Trichomonas should be reported as rare, few, moderate or
many per hpf.
     1.6.12 Presence of casts should also be noted as average number per lpf (i.e. Hyaline
cast: 1-3/lpf, etc).

1.7 PROCEDURAL NOTES:

     1.7.1 Normal Characteristics of Urine:

The yellow color of the urine is due largely to the pigment urochrome and small amounts
of urobilin and uroerythrin. Normal urine is essentially clear, and the presence of particulate
matter in uncentrifuged urine needs to be explained microscopically. Normal urine has a faint,
aromatic odor of undetermined source.

1.7.2 Microscopic Sediment:

RBCs: Normally 0-2 RBCs/HPF may be seen in urine from males and non-menstruating
females. Increased numbers may indicate renal hematuria.

WBCs: Normally 0-5 WBCs/HPF may be seen in urine of normal males with slightly
higher ranges in females. Increased numbers may indicate renal disease or acute infection.

Epithelial cells: A few epithelial cells are normal and indicate normal sloughing off of
aging cells. Increased numbers may indicate renal disease, urinary tract infection, or poor
technique in specimen collection.

Casts: 0-1 hyaline cast/LPF is found in normal urine. Increased numbers or more
advanced types indicate proteinuria.

Bacteria: A few bacteria are normally seen due to poor technique in collection of the
specimen. Increased numbers may indicate kidney, bladder, or urinary tract infection.

Crystals: The following crystals may be seen in normal or abnormal urine as noted
below. Use urine pH and solubility information to aid in identification, as needed. Also use
appropriate image and literature resources to assist with identification.

NORMAL ABNORMAL
Acidic Urine Alkaline Urine Acidic Urine Alkaline Urine
Amorphous urates Amorphous Cystine None
phosphates
Uric Acid Triple phosphates Tyrosine
Calcium oxalate Ammonium biurates Leucine
Calcium phosphates Sulfonamide
(Sulfadiazine)
Calcium carbonates

Mucus: Light mucus is normally found in urine and can sometimes be confused with
hyaline casts.
 Spermatozoa: A few are normally found in urine from normal males. Increased numbers
are found in prostatic disease.

Trichomonas: Normally not found in urine. Presence of trichomonas is abnormal and

may indicate infection.

Yeast: 0-1/HPF or few per high power field are normal in females. Increased numbers
indicate infection.
Casts: are classified according to their matrix, inclusions, pigments and cells present.

1.7.3 Cast Matrices:

Hyaline casts: Are translucent cylindrical structures composed of mucoprotein.

Increased numbers are seen with renal diseases and transiently with exercise, fever, congestive
heart failure, and diuretic therapy.

Waxy casts: These differ from hyaline casts in that they are easily visualized because of
their high refractive index. Waxy casts are homogeneously smooth in appearance. Their
margins are sharp, their ends are blunt, and cracks or convolutions are frequently seen along
the lateral margins. Waxy casts are commonly associated tubular inflammation and chronic
renal failure. They are also found during acute or chronic renal allograft rejection.

1.7.4 Cast Inclusions:

Granular casts: are semitransparent cylinders containing small (fine) or large (coarse)
granules. These granules represent plasma protein aggregates. Granular casts appear with
glomerular or tubular diseases.

Fatty casts: Are semitransparent or granular cylinders containing large highly refractive
vacuoles droplets. Visible fat droplets are triglycerides or cholesterol esters. These are
commonly seen when there is heavy proteinuria and are a feature of the nephrotic syndrome.

Crystal Casts: Crystalline inclusion in a semitransparent or granular cylinder These

casts indicate disposition of crystals in the tubule or collecting duct.

1.7.5 Cellular Casts:

Erythrocyte (Red Blood Cell) casts: Semitransparent or granular cylinders containing

distinct erythrocyte. Disorders reflected in the presence of erythrocyte casts in the sediment
may include acute glomerulonephritis, IgA nephropathy, lupus nephritis, subacute bacterial
endocarditis, and renal infarction.

Leukocyte (White Blood Cell) Casts: Semitransparent or granular cylinders containing

leukocytes. They may be seen pyelonephritis, glomerular diseases, interstitial nephritis, lupus
nephritis, and nephrotic syndrome.
2. QUALITATIVE TEST FOR URINE GLUCOSE
    
2.1 Principle
            Reducing sugars under alkaline condition tautomerize and form enediols. Enediols are
powerful reducing agents. They can reduce cupric ions (Cu2+) to cuprous form (Cu+), which is
responsible for the change in color of the reaction mixture. This is the basis of Benedict's test.
When the conditions are carefully controlled, the coloration developed and the amount of
precipitate formed (Cuprous oxide) depends upon the amount of reducing sugars present.

       2.2 Composition and Preparation of Benedict's Reagent

           One liter of Benedict's solution can be prepared from 100 g of anhydrous sodium
carbonate, 173 g of sodium citrate and 17.3 g of copper (II) sulfate pentahydrate.

CONSTITUTENT AMOUNT FUNCTIONS

Copper sulphate 17.3 gm Furnishes cupric ions (Cu++)
Sodium carbonate 100 gm Makes medium alkaline
Sodium citrate 173 gm Complexes with copper (II) ions so that they do not
deteriorate to copper (I) ions during storage
Distilled water Up to 1000 Solvent

Quality Checking: Benedict's solution is blue in color. In order to check purity of Benedict's
solution take 5 ml of Benedict's solution in test tube and heat it. If it does not change color, it
means it is pure.
     2.3 Procedure Benedict's test
        2.3.1 Pipette 5 ml of Benedict's reagent in a test tube (20x150mm).
        2.3.2 Add 8 drops of urine to the Benedict's reagent.
        2.3.3 Heat carefully on a flame of a gas burner or place in a boiling water for 5-10
minutes.
2.3.4 Cool under tap water or by placing in a beaker containing tap water.
2.3.5 Observe the color change and precipitate formation and analyze the test result.

2.4 Result Interpretation

The color of the mixture serves as a guide to the amount of sugar in the urine. Remove the
tubes and examine the solution in each tube for precipitate and change of color. Report the
sugar concentration as follows:

COLOR APPROXIMATE GLUCOSE INDICATION

mg/dl
Blue solution Nil
Green solution <500 mg/dl Trace
Green ppt 500-1000 mg/dl +
Yellow ppt 1000-1500 mg/dl ++
Orange ppt 1500-2000 mg/dl +++
Red to Brick red ppt >2000 mg/dl ++++

3. URINE PROTEIN SULFOSALICYLIC ACID PRECIPITATION TEST (SSA)

       3.1 Principle

             There are two basic approaches available for measuring protein in urine, the
turbidimetric method and colorimetric reagent strip. Sulfosalicylic acid method comes under
turbidimetric method. Protein is denatured by acid so that it becomes less soluble and is
precipitated.

       3.2 Procedure

          3.2.1 Centrifuge urine if cloudy or hazy.
          3.2.2 Fill 10x75 mm tube 1/3 full with urine supernatant.
          3.2.3 Forcibly squirt in 3% SSA until the tube is 2/3 full.
          3.2.4 Cover with parafilm and invert to mix.    
          3.2.5 Read and report the results as follows:

Negative no precipitate (no clinically

significant protein present)
Trace faint white precipitate (1-10
mg/dL)

1+ (image) turbid, but can see lines and

read print through tube (15-
30 mg/dL)
2+ (image) cannot read through but can
see lines (40-100 mg/dL)

3+ (image) cannot see through, fine

granules may be present
(150-350 mg/dL)

4+ (image) flocculent precipitate or

gelled tube (greater than 500
mg/dL)
  

   

II. ROUTINE FECALYSIS

      Fecalysis, or a stool exam, is a series of tests conducted on a stool sample. This procedure
detects bacteria and parasites that are causative agents of diseases.
     Aside from detecting living organisms, a fecalysis test can also detect substances such as
blood, bile, or sugar, which are not normally found in the stool.
      With a fecalysis, results can also indicate and detect Colon Cancer. Thus, the results of the
fecalysis is a great help to doctors in the diagnosis of the disease of a patient.

   3.1 Specimen Collection

         Before you begin the specimen collection, you will need a few things: a stool sample
container from the laboratory, a pair of gloves, and a clean bedpan or plastic container.

           1. To prevent toilet water from contaminating the stool sample, it is important to defecate
on a clean, dry bedpan or plastic container.
           2. Collect parts of the stool that contain blood or mucus and put it in the small container
provided. The sample should at least be peanut size or the size of your thumbnail at most.
            3. Label the container with the patient's full name, as well as the date and time of
collection.

After collection, it is important to submit the stool sample to the laboratory as soon as possible.
This urgency is because some parasites; especially those found in watery stool samples may be
difficult to find after 30 minutes has passed.