This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
1
CLINICAL MICROSCOPY
1 Urine 53%
1.1 Anatomy and physiology of the kidney, formation of urine 5%
1.2 Macroscopic examination 10%
1.3 Chemical analyses 18%
1.4 Microscopic examination 15%
1.5 Pregnancy testing 2%
1.6 Renal calculi 3%
2 Feces 3%
3 Other body fluids 21%
3.1 CSF 5%
3.2 Seminal fluid 5%
3.3 Amniotic fluid 3%
3.4 Gastric fluid and duodenal content 2%
3.5 Sputum and bronchial washings 2%
3.6 Synovial fluid 2%
3.7 Peritoneal, pleural and pericardial fluids 2%
4 Collection, preservation and handling of specimens 10%
5 Microscope, automation and other instruments 5%
6 Quality assurance and laboratory safety 8%
TOTAL 100%
CLINICAL MICROSCOPY NOTES URINALYSIS
PHYSICAL EXAMINATION OF URINE
I. VOLUMENormal range (24o): 600 to 2000 mLAverage volume: 1200 to 1500 mLNight:day ratio________
LABORATORY CORRELATIONS IN URINE TURBIDITYAcidic urine Amorphous urates, radiographic contrast media
Alkaline urine Amorphous phosphates, carbonates
Soluble with heat Amorphous urates, uric acid crystals
Soluble in dilute acetic acid RBCs, amorphous phosphates, carbonates
Insoluble in dilute acetic acid WBCs, bacteria, yeast, spermatozoa
Soluble in ether Lipids, lymphatic fluid, chyle
APPEARANCE AND COLOR OF URINEAppearance Cause Remarks
Colorless Very dilute urine Polyuria, D. insipidus
Cloudy Phosphates, carbonates Sol in dilute acetic acidUrates, uric acid Dissoves at 60C and in alkaliLeukocytes Insol in dilute acetic acidRed cells (“smoky”) Lyse in dilute acetic acidBacteria, yeasts Insol in dilute acetic acidSpermatozoa Insol in dilute acetic acidProstatic fluid
1
Appearance Cause Remarks
Mucin, mucous threads May be flocculentCalculi, “gravel” Phosphates, oxalatesClumps, pus, tissueFecal contamination Rectovesical fistula
Milky Radiographic dye In acid urineMany neutrophil (pyuria) Insol in dilute acetic acidFatLipiduria, opalescentChyluria, milky
Nephrosis, crush injury, sol in etherLymphatic obstruction, sol in ether
Emulsified paraffin Vaginal creams
Yellow Acriflavine Green fluorescence
Yellow-orange Conc urine Dehydration, feverUrobilin in excess No yellow foamBilirubin Yellow foam, if sufficient bilirubin
Red Hemoglobin Pos. rgt strip for bldErythrocytes Pos. rgt strip for bldMyoglobin Pos. rgt strip for bldPorphyrin May be colorlessFuscin, aniline dye Foods, candyBeets Yellow alkaline, geneticMenstrual contam Clots, mucus
Red-purple Porphyrins May be colorless
Red-brown ErythrocytesHgb on standingMethemoglobin Acid pHMyoglobin Muscle injuryBilifuscin (dipyrrole) Result of unstable hemoglobin
Brown-black Methemoglobin Blood, acid pHHomogentisic acid On standing, alkaline; alkaptonuriaMelanin On standing, rare
Blue-green Indicans Small intestine infectionsPseudomonas infectionsChlorophyll Mouth deodorants
V. SPECIFIC GRAVITYDensity of solution compared with density of similar volume of dist water at a similar temperature
Influenced by number and size of particles in solution
DETERMINATION1.Refractormetry (TS meter)
Indirect mtd based on RI
Compensated to temp (15-38oC)
Requires corrections for glucose and proteino 1 g/dL Glucose ________o 1 g/dL Protein ________
Calibrationo Distilled water ________o 5% NaCl ________
1
o 9% Sucrose ________
2.Urinometry Requires temp correction
0.001 must be subtracted from the reading every 3oC that the sp temp is below the urinometer calibration temp0.001 must be added from the reading every 3oC that the sp temp is above the urinometer calibration temp
Require corrections for glucose and protein
3.Reagent strip Prin. pKa change of a polyelectrolyte
Rgt sensitive to number of ions in the urine specimen; indicator changes color in relation to ionic concentration
Manufacturers recommend adding 0.005 to sp gr reading when pH is 6.5 or higher due to interference with the bromthymol blue indicator
Urine Specific Gravity Reagent Strip SummaryReagents Mutistix: Poly (methyl vinyl ether/maleic anhydride) bromthymol blue
Chemstrip: 6 mg/dL albuminSources of error/interference False-positive:
Highly buffered alkaline urinePigmented specimens, phenozopyridineQuaternary ammonium compounds (detergents)Antiseptics, chlorhexidineLoss of buffer from prolonged exposure of the reagent strip to the specimenHigh specific gravity
False-negative: proteins other than albuminCorrelations with other tests Blood
NitriteLeukocytesMicroscopic
SULFOSALICYLIC ACID PRECIPITATION TESTCold precipitation test that reacts equally ith all forms of protein
Grade Turbidity Protein range (mg/dl)
Negative No increase in turbidity <6
Trace Noticeable turbidity 6-30
1+ Distinct turbidity with no granulation 30-100
2+ Turbidity with granulation, no flocculation 100-200
3+ Turbidity with granulation and flocculation 200-400
4+ Clumps of protein >400
II. GLUCOSERenal threshold: 160 to 180 mg/dLOther sugars in urine
FructoseGalactoseLactosePentose
CLINICAL SIGNIFICANCE OF URINE GLUCOSE
HYPERGLYCEMIA ASSOCIATEDDiabetes mellitus PancreatitisPancreatic cancer AcromegalyCushing’s syndrome HyperthyroidismPheochromocytoma StressCentral nervous system damage Gestational diabetes
Interference False-positive: Contamination by oxidizing agents and detergentsFalse-negative:
High levels of ascorbic acidHigh levels of ketonesHigh specific gravityLow temperaturesImproperly preserved specimens
Correlations with other tests Ketones
COPPER REDUCTION TEST (Clinitest)Test relies on the ability of glucose and other substances to reduce copper sulfate to cuprous oxide in the presence of al-
kali and heatA color change progressing from a negative blue (CuSO4) through green, yellow and orange/red (Cu2O) occurs when the
1+ positive Negative Small amount of glucose present
4+ positive Negative Possible oxidizing agent interference on reagent strip
III. KETONESResults from INCREASED FAT METABOLISM due to inability to metabolize carbohydrate, as occurs in DM, increased loss of carbohydrate from vomiting, and inadequate intake of carbohydrate associated with starvation and malabsorption
False-negative:Nonreductase-containing bacteriaInsufficient contact time between bacteria and urinary nitrateLack of urinary nitrateLarge quantities of bacteria converting nitrite to nitrogenHigh concentrations of ascorbic acidHigh specific gravity
Correlations with other tests Protein LeukocytesMicroscopic
VIII. LEUKOCYTESignificance:
UTI/InflammationScreening of urine culture specimens
Primary function of AF is to provide a protective cushion for the fetus and allow movement
The amount of amniotic fluid increases throughout pregnancy, reaching a peak of approximately 1 L during the third trimester, and then gradually decreases prior to delivery.
DURING THE FIRST TRIMESTER, the approximately 35 mL of amniotic fluid is derived primarily from the maternal circula-tion. During the latter third to half of pregnancy, the fetus secretes a volume of lung liquid necessary to expand the lungs with growth. During each episode of fetal breathing movement, secreted lung liquid enters the amniotic fluid, as evidenced by lung surfactants that serve as an index of fetal lung maturity.AFTER THE FIRST TRIMESTER, fetal urine is the major contributor to the amniotic fluid volume. At the time that fetal urine production occurs, fetal swallowing of the amniotic fluid begins and regu-lates the increase in fluid from the fetal urine.
Increased AF _________________________________
Decreased AF ________________________________
1
Collection of amniotic fluid ____________________Maximum of 30 mL of AF is collected in sterile syringes
Second trimester amniocentesis _________________
Third trimester amniocentesis ___________________
Differentiation between AF and maternal urineAnalyte Amniotic Fluid Maternal Urine
Latex agglutination and ELISA for detection of bacterial antigensVDRL – neurosyphilis (recommended by CDC)
SEMINAL FLUID
Reasons for AnalysisFertility testingPostvasectomy semen analysisForensic analyses
Physiology
Semen is composed of four fractions that are contributed
1
by:
1. ________________________ ___________
2. ________________________ ___________
3. ________________________ ___________
4. ________________________ ___________
Collection
Abstinence for _________________________________
Analysis should be done after liquefaction
Specimen awaiting analysis should be kept at 37oC
Semen Analysis
AppearanceGray-white, translucent
Inc white turbidity
Red coloration
Yellow coloration
Volume
NV:
Increased volume
Decreased volume
Viscosity
Normal: Pour in droplets
Increased viscosity
pH
NV:
Increased pH
Decreased pH
Sperm Concentration
NV:
1
1. Improved Neubauer Counting Chamber
2. Makler Counting Chamber
Sperm Count
NV:
Calculation:
Sperm Motility
Evaluated in approximately __________
NV:
Sperm Motility GradingGrade WHO Criteria
4.0 a
3.0 b
2.0 b
1.0 c
0 d
Source: Urinalysis and Body Fluids, 5th edition by Strasinger and Di Lorenzo, p203
Sperm Morphology
Routine criteria:
Kruger’s strict criteria:
Normal Values for Semen AnalysisVolume 2 – 5 mL
Viscosity Pour in droplets
pH 7.2 – 8.0
Sperm concentration >20 million/mL
Sperm count >40 million/ejaculate
Motility >50% within 1 hr
Quality >2.0 or a,b,c
Morphology >30% normal forms (routine criteria)>14% normal forms (strict criteria)
Round cells <1.0 million/mL
Source: Urinalysis and Body Fluids, 5th edition by Strasinger and Di Lorenzo, p201
Sperm Viability
______________________________________
______________________________________
______________________________________
1
Seminal Fluid Fructose
___________________________________________
_________________________________
Antisperm Antibodies
Detected in semen, cervical mucosa or serum
1. Mixed Agglutination Reaction (MAR)
Detects presence of IgG antibodies
Semen sample + AHG + latex particles or
treated RBCs coated with IgG
Normal: <10% motile sperm attached to the particles
2. Immunobead TestDetect the presence of IgG, IgM and IgA antibodies and will demonstrate what area of the sperm (head, neck, tail) the autoantibodies are affectingNormal: presence of beads on less than 20% of the sperm
Routine aerobic and anaerobic cultures and tests for C. trachomatis, M. hominis and U. urealyticum
Postvasectomy Semen Analysis
____________________________________________
____________________________________________
_____________________________
SYNOVIAL FLUID
Viscous fluid in cavities of movable joints o Lubricates jointso Reduce friction between boneso Provides nutrient to the articular cartilageo Lessen shock of joint compression occurring during activities such as walking or jogging
Macrophage Large mononuclear leukocyte, may be vacuolated
NormalViral infections
Synovial lining cell Similar to macrophage, may be multinucle-ated, res. mesothelial cell
Normal
LE cell Neutrophil containing ingested round body LE
Reiter cell Vacuolated macrophage with ingested neutrophils
Reiter’s syndromeNonspecific inflammation
1
Cell/Inclusion Description Significance
RA cell Neutrophil with dark cytoplasmic granules containing immune complexes
RAImmunologic inflammation
Cartilage cells Large multinucleated cells Osteoarthritis
Rice bodies Macroscopic: res. polished riceMicroscopic: show collagen and fibrin
TB, septic and RA
Fat droplets Refractile intracellular and extracellular globulesStain with Sudan dyes
Traumatic injury
Hemosiderin Inclusions within clusters of synovial cells Pigmented villonodular synovitis
Crystal IdentificationCauses of crystal formationo Metabolic disorderso Decreased renal excretion that produce elevated blood levels of crystallizing chemicalso Degeneration of cartilage and boneso Injection of medications (corticosteroid)
A. Monosodium urateB. Calcium pyrophosphateC. Hydroxyapatite (calcium phosphate)D. CholesterolE. Corticosteroids
Fluid is examined unstained under polarized and COMPENSATED POLARIZED LIGHT for detection of MSU and CPPD crys-tals
Chemistry Tests
Glucose
Lactate
Protein
UA
Microbiology testsCommon organisms that infect synovial fluid Staphylococcus Streptococcus Haemophilus Neissreria gonorrhoeaeRoutine bacterial cultures should always include enrichment medium such as CAP
Serologic TestsAutoantibody detection: SLE and RAAntibody detection: Lyme disease
Early detection of gastrointestinal bleeding, liver and biliary duct disorders, maldigestion syndromes and inflammation
Normal: 100 to 200 g of stool passed per day
Steatorrhea Mushy, foul smelling gray stool that floats on water
Constipation Small, firm, spherical masses of stool (scybala)
Spastic bowelRectal narrowing or stricture
Narrow, ribbon-like stool
1
Blood from lower gutBeets
Red
Bleeding from upper GITBismuth, iron, charcoal
Black
Spinach and other green vegetables or calomel, or presence of biliverdin
Green
Presence of mucus in stool is abnormal and should be reportedSpastic constipation or mucous colitis Translucent gelatinous mucus clinging to the surface of formed
stoolNeoplasm or inflammatory process of the rectal canal Bloody mucus clinging to fecal mass
Ulcerative colitis, bacillary dysentery, ulcerating diverticulitis and intestinal tuberculosis
Mucus associated with pus and blood
Villous adenoma of the colon Copious quantity of mucus (3 or 4 L in 24 hours)
Patients with chronic ulcerative colitis and chronic bacillary dysentery frequently pass large quantities of pus with the stool
Determination of peroxidase and pseudoperoxidase activity of red blood cells including hemoglobin
Indicators include guaiac, orthotoluidine, orthodinisidne and benzidine
TESTS FOR STEATORRHEA
Screening tests Microscopic examination of feces for fat globules
Determination of serum carotenoid
Definitive test Fecal fat determination
Titrimetric method (Van de Kamer) Definitive diagnosis of steatorrhea
TESTS FOR REDUCING SUBSTANCES IN FECES
Stool suspension + Clinitest tablet
1
Normal: ≤0.25 g/dL
Suspicious: 0.25g to 0.5 g/dL
Abnormal: >0.5 g/dL
FECAL SREENING TESTS
TEST METHODOLOGY/PRINCIPLE/INTERPRETATIONExam.for neutrophils Microscopic count of neutrophils in smear stained with methylene blue, Gram stain or
Wright’s stain Three per hpf indicates condition affecting intestinal wall
Quali. fecal fats Microscopic examination of direct smear with Sudan III – 60 large orange-red droplets indi-cates malabsorption
Microscopic examination of smear heated with acetic acid and Sudan III – 100 orange-red droplets measuring 6-75 µm indicates malabsorption
Occult blood Pseudoperoxidase activity of hemoglobin liberates oxygen from hydrogen peroxide to oxidize guaiac reagent
Blue color indicates gastrointestinal bleedingAPT test Addition of sodium hydroxide to hemoglobin-containing emulsion determines presence of mater-
nal or fetal blood Pink color indicates presence of fetal blood
Trypsin Emulsified specimen placed on x-ray paper determines ability to digest gelatin
Inability to digest gelatin indicates lack of trypsinClinitest Addition of Clinitest tablet to emulsified stool detects presence of reducing substances
Reaction of 0.5 g/dL reducing substances suggests carbohydrate intolerance