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


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.


Laboratory Specimens

Most common Laboratory Specimen Types:

Blood Urine

Additional Laboratory Specimens:

Body fluids Sputum Stool Tissue samples Culture swabs


Coronary Disease

CK, LDH

Kidney Disease

BUN, Creatinine

Liver Disease

Bilirubin

Why do we analyze our blood?


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


Sampling

Types of samples :Serum,plasma, blood, urine, CSF...


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


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…..


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


What is Blood?

Blood CompositionPlasmaCells

0.1% LeucocytesWhite blood cellsProtection against

bacteriaviruses

0.9% ThrombocytesPlateletsCoagulation at injuries


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?


Phlebotomist draws sample

What samples do we analyze?


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.


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


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.


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


Pre-analyticsPre-Analytical


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


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


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

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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.


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.


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


Analytical


Analytical

Adequate test methodologyStandard Operating ProcedureUnderstandableTraceable

Routine test must beEasy to be executedReliableLow risk on failure


Statistical Quality Control

Samples with known concentrationLowMediumHigh

As part of the daily routineBegin of the runMiddle in the runEnd of the dayRandom


Quality control


Cumulative control


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


Analytical Results

Reference RangeNormal valuesBased on a large pool of healthy persons

Differences betweenChildren vs. adultsMale vs. femaleSerum vs. plasmaPopulationBiorhythm

Expected Values


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


PhotometryOptical Density A = - log 10 T

A= ε x L x C


Photometry


End Point there is final <<stable>> color


END POINT –linear calibration curve


CALCULATIONS


Rate Method


RATE or ZERO ORDER kinetics


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)


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)


Turbidimetric assays




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



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


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


Auto-validation

Limits defined by the lab

Delta checks

Quality control with Westgard rules

Messages from the systems

Automatic


RESULTS

Clinic

Technology

Clinical laboratory basic