Clinical laboratory basic
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Transcript of Clinical laboratory basic
Clinic Lab : Basic Overview
Training Design: Dorra Hung
Page 2 Mar-07 Dorra Hung
Laboratory Roles & Responsibilities
The clinical lab provides diagnostic test data to aid in the detection, diagnosis and treatment of disease. Data is used by physicians, nurses, pharmacists and other healthcare professionals.
The responsibilities of the clinical lab include:
Correct identification, collection and processing of patient specimens Accurate performance of testing Timely reporting of results Communication with physicians and other healthcare professionals
Analyst testing is used to help diagnose, monitor or treat disease
Page 3 Mar-07 Dorra Hung
Laboratory Workflow
There are six main steps in how a sample flows through the lab from order creation to final test result.
1. Test is ordered. 2. Sample is collected 3. Sample is delivered to the lab. 4. Sample is processed. 5. Sample is analyzed. 6. Results are reported.
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Laboratory Specimens
Most common Laboratory Specimen Types:
Blood Urine
Additional Laboratory Specimens:
Body fluids Sputum Stool Tissue samples Culture swabs
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Coronary Disease
CK, LDH
Kidney Disease
BUN, Creatinine
Liver Disease
Bilirubin
Why do we analyze our blood?
Page 6 Mar-07 Dorra Hung
Medical Examination Overview
Medical Examination Pre-diagnoseAnamnesis
Enquiry
Examination RequestAnalytical-Analytical Sensitivity-Method Specificity-Statistical Quality Control
Pre-Analytical-External Influences-Patient Sampling-Sample Transport-Sample Preparation
Analytical Result (Value and Unit)
Patient Result
Diagnose
Post-Analytical Phase-Plausibility Check-Alarm Values-Trend Check-Constellation Check
Reference Interval(Normal Values)
Diagnostic Sensitivityand Specificity
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Sampling
Types of samples :Serum,plasma, blood, urine, CSF...
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Medical Examination
Objective is to get an answer about the health status of a patient
The physician determines on the basis of the anamneses, his clinical examination and on the basis of additional known information an Enquiry Examination Request
This is followed by the necessary preparation of the patient and blood sampling
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Pre-Analytical
Common material for examination
Venous Blood (Serum or Plasma)Capillary bloodUrine (Single shot or 24 hour collection)Cerebrospinal fluid (CSF)Puncture FluidsOthers, such as Faeces, Saliva, Gastric acid, Hair…..
Page 10 Mar-07 Dorra Hung
What is Blood?
Blood CompositionPlasmaCells
55% PlasmaYellow, sticky liquidTransport of
Nutrients (proteins, fats, carbon hydrates)Hormones
44% ErythrocytesRed blood cellsContain Haemoglobin
O2 and CO2 transport
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What is Blood?
Blood CompositionPlasmaCells
0.1% LeucocytesWhite blood cellsProtection against
bacteriaviruses
0.9% ThrombocytesPlateletsCoagulation at injuries
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Red top Vacutainer® collection tubes are used for
serum determinationsin chemistry.
They contain NO coagulant.
The grey and red speckled SST™tube at left (“tiger top”) contains a polymer gel for serum separation
and has a Hemoguard™ tube closure.
The Vacutainer® at right has a conventional tube stopper,
Tubes are now made of plastic to help protect personnel from injury
and bloodborne pathogens.
Vacutainer® and Hemogard™ are trademarks of Becton, Dickinson & Company.
Tube size (mm)ID x Height
Draw Volume(mL)
13 x 75 3.513 x 100 4.0, 5.0,16 x 100 5.0, 6.0, 7.0, 8.016 x 100 8.5
Stoppercolor
Coagulant Use
Lavender EDTA HematologyLight Blue Sodium citrate Coagulation
Green Lithiumheparate
Plasmachemistry
Light Green Lithiumheparate + gel
Serumchemistry
Grey Sodium citrate Glucosetesting
Sample containers - What do we use?
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Phlebotomist draws sample
What samples do we analyze?
Page 14 Mar-07 Dorra Hung
Plasma versus Serum
Blood to which an anticoagulant has been added will not clot. Blood cells will settle to the bottom of the tube leaving plasma at the top of the tube.
Blood to which no anticoagulant has been added will clot. Blood cells get caught in the clot leaving serum behind.
Page 15 Mar-07 Dorra Hung
Pre-Analytical
Possible Influences
Age, genderGenetic influencesNutritional influencesPregnancyBiorhythm (diurnal rhythm causing analytical fluctuations)Muscular mass, body weightPhysical activity or inactivityPsychological stress (fear for blood collection, surgery)Use of medicines
Page 16 Mar-07 Dorra Hung
Pre-Analytical
Disturbing Influences
Sample collection (body position, venous congestion, ….)Sample condition (haemolytic, lipemic, icteric) Normal serum obtained from an individual in good health is usually clear, pale yellow in color. However, the color of the patient’s serum may appear different for various reasons such as disease or improper handling of the blood specimen.Lipemia (Lipe) results from increased levels of lipoproteins associated with triglycerides, and it can cause the serum to appear white.Hemolysis (Heme) is caused usually by the release of hemoglobin from ruptured red blood cells during sample collection and/or sample handling. This interference can cause the serum to appear red.
Page 17 Mar-07 Dorra Hung
Pre-Analytical
Icterus (Icte) is the result of increased levels of bilirubin, and it can cause the serum to appear yellow.
Centrifugation, DeproteinizationChromatographyElectrophoresis,....
Separation of samples
Pre-Analytical
After the centrifugation if the sample was without anticoagulant the supernatant fluid is SERUM otherwise is plasma .As anticoagulants they use EDTA K3 , EDTA K2 , Heparin, Citric acid 9:1, Citric Acid 4:1 NaF and others.
If we use plasma we must know the type of the anti-coagulant due to different interferences f.e. Ca , Na , Fe , ALP ...
Pre-Analytical
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Pre-analyticsPre-Analytical
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Some photometric assays may be influenced by the presence of these abnormal serum colors and the reliability of the test results may be decreased.
haemolysis can cause analytical interferences such as high K+ caused by release from erythrocytes, or can interfere with the measuring technique (photometry)
Inadequate sample transportWrong centrifugationInadequate sample storage (Bilirubin)
Pre-Analytical
Page 22 Mar-07 Dorra Hung
Pre-Analytical
Different type of sample collection in commercially available blood collection systems (Beckton Dickinson Vacutainer, Sarstedt Monovetten, …..)
GreyGlucose, LactatePlasmaNa-Fluoride / K-Oxalate
BleuCoagulation testsPlasmaCitrate
LilacHaematology, Special Chemistry, Immunochemistry
PlasmaEDTA
GreenClinical ChemistryPlasmaHeparin
RedClinical Chemistry, Serology, Immunochemistry
SerumWhole blood (without agent)
Colour-coding
ApplicationExtraction ofTube with additional Anti-Coagulation agent
Page 23 Mar-07 Dorra Hung
Pre-Analytical
Serum30-45 minutes clothing (preferably in the dark)10-15 minutes centrifugation@ 1000-1500 g
PlasmaImmediate 10-15 minutes centrifugation @ 1000-1500 g
Page 7 Jan-07For internal use only
EU Sales TrainingVelemirov / Twisk
Plasma versus Serum
Blood to which an anticoagulant has been added will not clot. Blood cells will settle to the bottom of the tube leaving plasma at the top of the tube.
Blood to which no anticoagulant has been added will clot. Blood cells get caught in the clot leaving serum behind.
Page 24 Mar-07 Dorra Hung
Pre-Analytical
Sample transport and storage
Properly packedTransport must be save
Bio hazardous material 4 hours stable @ 15-25 oC
Closed to avoid evaporation24 hours stable @ 4-8 oC
Dry ice, cool packs, refrigerator, etc.
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Pre-Analytical
Example: PotasiumPlasma is recommended for rapid centrifugation
Use only serum or plasma from single patientsSample preparation (heparin plasma)
Centrifuge within 30-45 minutes after collectionErythrocytes produce Homocysteine, which continues after sampling
Store on ice if centrifugation within 30-45 minutes is not possibleStore plasma at -20 oC if sample can not be measured within 48 hours
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Analytical
Page 27 Mar-07 Dorra Hung
Analytical
Adequate test methodologyStandard Operating ProcedureUnderstandableTraceable
Routine test must beEasy to be executedReliableLow risk on failure
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Statistical Quality Control
Samples with known concentrationLowMediumHigh
As part of the daily routineBegin of the runMiddle in the runEnd of the dayRandom
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Quality control
Page 30 Mar-07 Dorra Hung
Cumulative control
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Patient Result
Demographic informationPatient name, Patient ID, Lab numberSample matrix, Visual distortionsDate, Time
Sample collection, Arrival in the lab, Time of analyses
Analytical resultsTest name, Unit, Reference values, Comments (high/low, diluted, duplicates, ……)
Test Report
Page 32 Mar-07 Dorra Hung
Analytical Results
Reference RangeNormal valuesBased on a large pool of healthy persons
Differences betweenChildren vs. adultsMale vs. femaleSerum vs. plasmaPopulationBiorhythm
Expected Values
Page 33 Mar-07 Dorra Hung
Diagnose
After checking the reliability of the analysis
Analytical rangeStatistical Quality controlPre-analytical and analytical disturbancesPlausibility of the result
Compared with previous resultsFit with the situation of the patient
DIAGNOSE
Photometry ChemiluminencePotentiometry (ISE)ElectrophoresisNephelometryγ- COUNTER Mass absorption OsmometryHPLCTLCCoagulation...
Methods of Clinical Chemistry
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PhotometryOptical Density A = - log 10 T
A= ε x L x C
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Photometry
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End Point there is final <<stable>> color
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END POINT –linear calibration curve
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CALCULATIONS
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Rate Method
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RATE or ZERO ORDER kinetics
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RATE , ZERO ORDER
Decrease :
340 nm AST/GOT-ALT/GPT,LDH P--L,ALDOLASE
FACTOR or FV =
(Vtotal x 1000) / (Vsample x Light Path xMEC)
Page 43 Mar-07 Dorra Hung
RATE or ZERO ORDER kinetics
Increase :
340 nm : LDH L->P, CK , CKMB , HBDH , ELASTASE , LAP405 nm : ALP(AMP) , ALP(DEA) , ACP , NP ACP , AAMY , PAMY
FACTOR or FV =
(Vtotal x 1000) / (Vsample x Light Path xMEC)
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Turbidimetric assays
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Direct potentiometry : This is the simplest method of making ion-selective electrode measurements. The electrodes are immersed ina test solution and the electrode potential is measured directly with a millivolt meter. The concentration is then related directly to this measurement by reading the answer from a calibration graph of concentration versus millivolts.
Indirect potentiometry : Dilution of the sample (less volume, less problems, less interventions
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Pre-analytical factors that affect serum proteins concentrations
1)Time of the day2)Position3)Exercise4)Fasting vs non fasting5)Medications6)Time of year (season)7)Age and gender8)Geographic location9)Venipuncture technique10)Sample handling and storage
Page 50 Mar-07 Dorra Hung
Patient Result
Demographic informationPatient name, Patient ID, Lab numberSample matrix, Visual distortionsDate, Time
Sample collection, Arrival in the lab, Time of analyses
Analytical resultsTest name, Unit, Reference values, Comments (high/low, diluted, duplicates, ……)
Test Report
Page 51 Mar-07 Dorra Hung
Auto-validation
Limits defined by the lab
Delta checks
Quality control with Westgard rules
Messages from the systems
Automatic
Page 52 Mar-07 Dorra Hung
RESULTS
Clinic