urinalysis & body fluids bachelor intro

7/29/2019 Urinalysis & Body Fluids Bachelor Intro

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Urinalysis & Body FluidsHLB 31203

Quality Assurance & Safety



WHAT IS QUALITY?

Conformance to the requirements of

user or customers

Satisfaction of the needs andexpectations of users or customers



FIVE ‘Q’ Framework

Quality Planning

Quality Lab Processes

Quality ControlQuality Assessment

Quality Improvement Goals

Objectives

Quality Requirements



• QP - PROVIDES THE PLANNING STEP.

• QLP - ESTABLISHES STANDARD PROCESSFOR DOING THINGS.

• QC & QA - PROVIDES MEASURES FOR

CHECKING , HOW WELL THINGS

ARE DONE.

• QI - PROVIDES MECHANISAM FOR ACTING

ON THESE MEASURES



QUALITY ASSURANCE

REQUIRES

1. CAUSES OF PROBLEMS BE

IDENTIFIED AND ELIMINATED

2. DETECTION OF THE PROBLEMS

EARLY ENOUGH TO PREVENT THEIRCONSEQUENCES



ELEMENTS OF QUALITY

ASSURANCE

1. COMMITMENT

• Dedication to quality service must becentral. A true commitment isrequired by Lab Directors,

• Managers and Supervisors if the

efforts of the lab personnel are to besuccessful.



2.FACILITIES AND RESOURCES

• Lab must have the administrative support

necessary to provide the quality of servicesthat is desired.

• This means having ,

• adequate space,

• equipment,

• materials,

• supplies,

• staffing,

• supervision and

• budgetary Resources.

• These resources provide the basis upon which

quality services can be developed andmaintained.



3. TECHNICAL COMPETENCE

• High quality personnel are essential for high quality services. The educational

background and experience areimportant. In service training candevelop and maintain skills.



4. TECHNICAL PROCEDURES

• Good technical procedures are necessary

• Control of preanalytical conditions or variablessuch as

• Test requests

• Patient preparation• Patient identification

• Specimen acquisition

• Specimen transport

• Specimen processing

• Specimen distribution

• Preparation of work lists and logs

• Maintenance of records



• Control of analytical variables, which includes

• Analytical methodology

• Standardization and calibration procedures

• Documentation of analytical protocols

and procedures

• Monitoring of critical equipment and

materials

• Monitoring of analytical quantity by theuse of statistical methods and control

charts.



CONTROL OF PREANALYTICAL VARIABLES

• The responsibility for accurate and timely testreports generally lies with the laboratory butmany problems can arise prior to and after

the analysis of the submitted specimens.

• So it is essential to perform a systemanalysis of the laboratory and to identify thetype of preanalytical variables.



LABORATORY TESTING PROCESSES AND THEIRPOTENTIAL ERRORS

PRE ANALYTICAL ERRORS

PROCESS POTENTIAL ERRORS

Test ordering inappropriate test

Handwriting not legible

Wrong patients IDSpecial requirements not specified

Specimen acquisition

Incorrect tube or container

Incorrect patient IDInadequate volume

Invalid specimen

(hemolysed or diluted)Collected at wrong time

Improper transport conditions



ANALYTICAL ERRORS

Analytical Instrument not calibratedMeasurement correctly

Specimens mix – up

Incorrect volume of specimenInterfering substances present

Instrument precision problem

Test reporting Wrong patient IDReport not legible

Report delayed

Transcription error



POST ANALYTICAL ERRORS

Test interpretation Interfering substance notrecognized

Specificity of the test not understoodPrecision limitation not recognized

Analytical sensitivity not appropriate

Previous values not available for comparison



HOW TO CONTROL THESE ERRORS?

PRE ANALYTICAL VARIABLES

It is very difficult to establish effective methods for

monitoring and controlling preanalytical variables

because may of the variables are outside the laboratory

areas.

Requires the coordinated effort of many individuals and

hospital departments



Patient Identification

The highest frequency of errors occurs

with the use of handwritten labels and

request forms. The use of bar code

technology has significantly reduced ID

problems.



Turnaround time

Delayed and lost test requisitions, specimens

and reports can be major problems for labs.

Recording of the actual times of specimen

collection, receipt in the lab and reporting of

results with use of computers will solve these

problems



Transcription error

A substantial risk of transcription error exists frommanual entry of data even with the double checking of

results, computerization will reduce this type of transcription error.



Patient preparation

Lab tests are affected by many factors,such as, recent intake of food, alcohol, or

drugs smoking, exercise,stress

sleep or posture during specimen

Collection.

The lab must define the instructions and

procedures compliance with these instructions

can be monitored directly efforts should be

made to correct non compliance



CONTROL OF ANALYTICAL VARIABLES

There are many analytical variables thatmust be carefully controlled –

Water quality

Calibration of analytical balances

Calibration of volumetric glasswareand pipets

Stability of electrical power

Stability of temperature of heatingbaths, refrigerators, freezers and

centrifuges



The procedure Manual should contain thefollowing

Procedure name

Clinical significance

Principle of methodSpecimen of choice

Reagents and equipments

Procedure

Reference values

Comments

References



CONTROL OF THE ANALYTICAL QUALITYUSING STABLE CONTROL MATERIALS

• The performance of analytical methods can bemonitored by analyzing specimens whoseconcentrations are known and then bycomparing the observed values with known

values.• The known values are usually represented by

an interval of acceptable values, or upper andlower limits for control (control limits)



• When the observed values fall within

the control limits – analysis is workingproperly

• When the observed value fall outside

the control limits the analyst should be

alerted to the possibility of problems in

the analysis.



DEFINITIONS

• Accuracy

• Precision



Precision and Accuracy• Accuracy: it is the

agreement of a

particular value with thetrue value.

• Precision: it is the

agreement among

several measurementsof the same quantity.

Uncertainty in measurement

Not Accurate

Not precise

Not Accurate

But precise

Accurate

and precise



Errors

• Random error : it means that

a measurement has an

equal probability of beinghigh or low.

• Systematic error : error accurse in the same

direction each item.

Uncertainty in measurement

Large Random

Error

SmallRandom

and Large

Systematic

Error

Small Random

And NO

Systematic

Error



Good Accuracy

Good Precision

Good

PrecisionOnly

Neither Good

precision Nor Accuracy



MICROSCOPY



Introduction

• The microscope is designed to makeobjects visible that are too difficult or toosmall to see with the unaided eye.

• There are many different kinds of lightmicroscopes, including phase-contrast,dark field, polarizing, and UV.

• These differ primarily in the source andmanner in which light is passed throughthe specimen to be viewed



• The microscopes in biology lab are usually compoundbinocular or monocular light microscopes, some of which

may have phase-contrast attachments.

Compound

• means that the scopes have a minimum of twomagnifying lenses (the ocular and the objective lenses).

• Binocular microscopes have two eyepieces,

• Monocular have only one eyepiece,

• Light refers to the type of illumination used, that is,visible light from

lamp.



Types of Light Microscopes

• Bright-field – most widely used; specimen isdarker than surrounding field; live and preservedstained specimens

• Dark-field – brightly illuminated specimens

surrounded by dark field; live and unstainedspecimens

• Phase-contrast – transforms subtle changes inlight waves passing through the specimen into

differences in light intensity, best for observingintracellular structures



Parts of the Microscope



Parts of a Microscope

• Eyepiece

• Arm

• Nosepiece

• Stage

• Base



Parts of a Microscope: Eyepieces

Binocular eyepieces

Telescoping eyepiece



Parts of a Microscope: Nosepiece

turret

objectives



Parts of a Microscope: Stage Elements

Stage clip

Aperture(below)

Stage adjustment

knobs

Backwards andforwards

Side to side



Parts of a Microscope: Power, Light, and

Focus

Power switch

Light adjustment wheel

Power cord

Coarse focus

Sub stage lamp

Fine focus



Stage Clips & Objectives

• Stage clips hold the

slide in place

• Low power objective

is used to focus themicroscope (short &

fat)

• High power

objective is used to

view details of a

specimen



Coarse Adjustment, Fine Adjustment, &

Base

• Coarse adjustment

focuses adjustment

• Fine adjustment finetunes & gives detailed

focus(usually smaller

than coarse

adjustment knob)• Base is where the

microscope rests



Stage

• Stage is part where

the slide rests

• Mirror (or light

source) directs lightupwards onto the

slide.



Diaphragm

• Diaphragm allows

light in



Nosepiece

• Nosepiece is the

rotating device that

holds the objectives

(lenses)



Arm

• Arm is the part

where you carry the

microscope



Can you name the parts of a Compound

Microscope?



Answers

1) base2) mirror (light source)

3) diaphragm

4) stage

5) stage clips6) low power objective lens

7) high power objective lens

8) nosepiece

9) arm

10) fine focus knob11) body tube

12) coarse focus knob

13) eyepiece



Characteristics

Key characteristics of a reliable microscope

are:• Magnification – ability to enlarge objects

• Resolving power – ability to show detail

Magnification in most microscopes results from interactionbetween visible light waves and curvature of the lens.

– angle of light passing through convex surface of glasschanges

– Depending on the size and curvature of the lens, theimage appears enlarged.

– extent of enlargement - magnification



Principles of Light Microscopy

• Magnification occurs in two phases –

– The objective lens forms the magnified real image.

– The real image is projected to the ocular where it is

magnified again to form the virtual image.• Total magnification of the final image is a product of the separate

magnifying powers of the two lenses.

power of objective X power of ocular = total magnification



Resolution defines the capacity to distinguish

separate two adjacent objects.Resolving Power:

RP measures the ability to distinguish small

objects close together



RP is also the minimum distance (d) between objects that

reveals them as separate entities

RP = d = 0.5 / n sin

d is the minimum resolving distance

• λ is the wavelength of the light being used

• n is the index of refraction of the embedding medium

• θ is the acceptance angle of the objective lens

• nsinθ is the Numerical Aperture of the objective being

used



nmeasures the slowing of the velocity of light by a substance.

ndetermines the direction & magnitude of bending of light at the

interface between the 2 substances.

n sin is called numerical aperture (NA), NA of a microscope objective is a measure of

its ability to gather light and resolve fine specimen detail at a fixed object distance.



Function of wavelength of light that forms the

image along with characteristics of objectives

• Visible light wavelength is 400 nm – 750nm.

• Numerical aperture of lens ranges from

0.1 to 1.25.

• Oil immersion lens requires the use of oil to

prevent refractive loss of light.

• Shorter wavelength and larger numerical

aperture will provide better resolution.



The ability of a microscope objective to capture deviatedlight rays from a specimen is dependent upon both the

numerical aperture and the medium (e.g. Immersion

oil) through which the light travels.

Immersion oil allows you to control and actually "bend"

more of the light passing through the subject into the

lens so that you can see.



Compound Microscope

Objectives: Specifications and Identification



Objectives: Specifications and Identification

Key Microscopy Terms



Key Microscopy Terms

Working distance

Distance from the objective lens to the top of thecover slip when the sample is in focus.

Aberration-flaws in images due to flaws in the

objective lensTwo main types:

Geometrical or spherical-flaws in the shape of the

lens

Chromatic-color flaws due to differences in indices

of refraction

I i di dd di l ti th t li b t



Immersion media-add diagram solution that lies between

the objective lens and the cover slip e.g. air, oil, water, and

glycerin.

Mounting media solution in which the specimen is

suspended in.

Numerical aperture indicates the light acceptance angle of the objective. Indicates the amount of light entering, the

resolving power, and the depth of field of the objective.

Types of Microscopes



Types of Microscopes

• Upright

- Ideal for microscope slides and thin specimens

• Stereo

- Doesn’t require light to be passed through thesample for imaging

- Good for imaging opaque and larger objects

• Dissecting- Low magnification scope with large working

distance for manipulating small specimens

Köhler illumination



Köhler illumination

• Early 20th Century Professor Köhler developed

the method of illumination still called “Köhler illumination”

• Köhler recognized that using shorter

wavelength light (UV) could improve resolution.• Köhler illumination creates an evenly illuminated

field of view while illuminating the specimen with

a very wide cone of light



• Most manufacturers of modern compound microscopes use a



p p

variation called pseudo-Köhler illumination in their designs



Light Pathway

Compound - upright



Light Pathway



Fluorescence Microscope

• Modified compound microscope with an

ultraviolet radiation source and a filter that

protects the viewer’s eye

• Uses dyes that emit visible light whenbombarded with shorter UV rays -

fluorescence

• Useful in diagnosing infections



• Dark-Field Microscope



Dark Field Microscope

As a consequence, the dark-field microscope shows a light silhouette

of an organism against a dark background.

An opaque disk in the condenser blocks all light passing in a straight

line from the condenser, through the specimen, into the objective

lens.

• Phase-Contrast Microscope Employed to view free, unfixed microorganisms with good details.

It converts slight differences in refractive index & cell density in easily

detected variations in light intensity.

• Transmission Electron Microscope (TEM)



• Transmission Electron Microscope (TEM)

TEM transmits electrons through the specimen thus revealing

internal structures, e.g., such as the structures within cells.

Specimens must be cut very thinly and treated with substances(metals) that scatter electrons.

Viruses can be viewed without being thinly cut (because they are

already so small/thin).

TEM of a cyano bacterium

• Scanning Electron Microscope (SEM)

SEM of E. coli

A heavy metal must be deposited on the surface of the specimen andit is actually this metal whose image is formed.

Supplies less magnification but is good for observing the surface of

relatively large objects (i.e., thin sectioning is not necessary).

.



Electron Microscopy

• Forms an image with a beam of electrons thatcan be made to travel in wavelike patterns when

accelerated to high speeds

• Electron waves are 100,000 times shorter thanthe waves of visible light.

• Electrons have tremendous power to resolve

minute structures because resolving power is a

function of wavelength.

• Magnification between 5,000X and 1,000,000X

Focusing a Microscope



Focusing a Microscope

1. Rotate the turret so that the 4X objective clicks into

place.2. Place the slide on the stage. Pull out the slide clip, slip

the slide in place and gently release the slide clip. Theslide should be held under tension in place.

3. Center the specimen over the aperture.4. Using the coarse focus raise the stage until you reacha stop. Watch from the side to make sure that the objective does

not smash into the slide!

5. Look through the right eyepiece only and lower thestage using the coarse focus until the specimen isfocused. NEVER RAISE THE STAGE TO FOCUS!



Focusing a microscope

6. Adjust with the fine focus knob until specimen is sharpand clear.

7. Now adjust the binocularity by rotating the eyepiecesso that they match the distance between your eyes

and you see one circle with both eyes.8. Cover your left eye and bring the specimen in focuswith the fine focus knob.

9. Cover your right eye and adjust the sharpness byrotating the telescoping knob on the eyepiece.

10. Your specimen should be in focus for your eyes.



Care of microscope• Carrying a microscope: one hand grabs the arm

and the other hand supports the bottom of thebase.

• Lens care

– DO NOT TOUCH THE LENS! The oil from your hands can etch the glass.

– CLEAN THE LENS WITH LENS PAPER ONLY! Other paper has fiber that can scratch the lens.

• Putting away the microscope: rotate to the 4X

objective and roll the nosepiece away from thestage so that the space between the stage andnosepiece is at a maximum.

C C



Carry a Microscope Correctly



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