QUALITY ASSURANCE

Clinical Chemistry

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• Many of the quality assurance and quality control procedures and techniques already discussed were first developed for the chemistry laboratory and later applied to other areas of the clinical laboratory• Shewhart and Levey-Jennings control charts, • statistical multi-rule evaluation of control results, • evaluation and comparison of analytical methods, • procedure manuals, • and preventive maintenance procedures have long been familiar

to technologists in the chemistry laboratory

• To avoid unnecessary duplication, only those topics that are specific for the clinical chemistry laboratory will be discussed in this chapter

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Specimen Collection and Sample Quality• Serum or plasma collected by venipuncture or capillary

puncture is the sample of choice for the majority of chemical methods• specimen must be collected without an anticoagulant, producing serum• other methods require that a preservative or anticoagulant be used to

• Other tests on other fluids are also ordered• chemical analysis of urine• a limited number of tests are being applied to cerebrospinal fluid and

other body fluids• for example, glucose and protein tests

• Regardless of the specimen, it should be properly collected, preserved, and transported to the laboratory

• The specimen should be accurately identified with the patient's name, history number, the date and time of collection, and the initials of the person collecting the sample

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Collection & Transportation• Sodium fluoride

• For samples which is necessary to completely stop glycolysis

• Urine samples (24 h)• Preservation with boric acid or hydrochloric acid to prevent loss of

various metabolites

• Transportation of samples to Lab. should be as soon as possible• Some samples should be transported on ice

• blood gases (O2 levels decrease while CO2 levels increase)

• Some plasma constituents are photosensitive• Bilirubin measurement

• Excessive vibration or rough handling• Hemolysis

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Separation of Serum or Plasma

• Once sample is received, it should immediately

centrifuged to separate serum or plasma

otherwise many changes can occur:

• RBCs loose their integrity and intracellular components

start to leak

• Glucose and O2 concentration decrease

• CO2, lactic acid increase while pH becomes more acidic

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The Effects of Hemolysis• Causes release of RBCs components which will increase

the plasma concentrations of:• Potassium• Magnesium• Inorganic phosphorus• Copper• Lactate dehydrogenase• Free Plasma Hb

• Which will interfere with bilirubin determinations with any measurements made at 540 nm

• Aspartate transaminase• Alanine aminotransferase• Creatinine Kinase• Acid phosphatase

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Changes due to storage of whole blood at RT

Constituent Change in concentration

Ammonia

Marked increase

Total CO2

Carbonic acid

Ketone bodies

Lactic acid

Magnesium Mild increase

O2 Marked decrease

Bilirubin Mild to marked decrease

Plasma enzymes Mild to marked decrease in activity

Glucose Decreases

Protein, sodium, chloride, Creatinine, Calcium

Unchanged

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Quality Control• To start a quality control program for a chemistry method

follow these guidelines:1. Purchase the appropriate levels of control in a batch large enough to

last a year or more

2. Analyze it for a minimum of 20 consecutive working days on the method for which it is to be used.

• Carefully monitor the method for proper operation, using assayedstandards or a previous lot of control material.

3. At the end of the 20-day period calculate a mean, standard deviation

4. Set up the evaluative procedure (multi-rules) using the determined mean and standard deviation and setting up control ranges and tolerance limits

5. Analyze the controls at a frequency that is sensitive to the method's degree of imprecision.

• It is recommended that controls be analyzed with every batch or testrun, or in large batches analyzed every 20 samples.

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Do's And Don'ts Of Survey Samples

1. Don't give survey samples "special" treatment. • Do treat survey samples as routine specimen, exposing them

to the same environmental conditions as the average patient sample, such as temperature and light.

2. Don't analyze a survey sample several times and "report the average.

• Do analyze the survey sample the same number of times that the average patient sample is analyzed.

3. Don't always analyze survey samples directly after recalibration.

• Do place the survey samples randomly in the daily workload so that any systemic drift will be accurately reflected

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Do's And Don'ts Of Survey Samples

4. Do keep copies of all instrument printouts and survey reporting forms to check for the occurrence of transcription errors in case a survey evaluation report comes back as unacceptable.

5. Do keep the survey samples for reanalysis after summary reports are received.

• Most chemistry survey samples can be frozen and can be used either to investigate an unacceptable result on the evaluation or as a secondary standard to investigate instrument problems

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Sources of Error in Clinical Chemistry

1. Specimen mix-upA. A Specimen drawn from wrong patient

B. Specimen labeled with the wrong name or accession numbers

C. Serum transferred into mislabeled tubes

D. Improper cup placed into a tray position and the results reported on the wrong patient

2. Allowing evaporation of a sample while it sits on the analyzer waiting to be analyzed

• This will concentrate the sample, resulting in higher values

3. Dilution and calculation errors a) Incorrect dilution of sample

b) Failure to correct for dilution

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Sources of Error in Clinical Chemistry

4. Sampling errors• Partially clotted specimen • Short sampling • Air bubble in the bottom of the cup • Fibrin clot in the sample probes

5. Transcription errors

6. Instrument losing calibration due to:• dirty reaction cuvettes, • or worn pump tubing;

7. Instrument is not recalibrated when new reagents are placed on the instrument

• This can result in a shift in the calibration

QUALITY ASSURANCEpH and Blood Gas Determinations

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Blood Gas Measurements• Blood gas measurements are used to evaluate a person's

lung function and acid/base balance• They are typically ordered if someone is having worsening

symptoms of a respiratory problem, such as difficulty breathing or shortness of breath, and others

• Blood gases may also be used to monitor treatment for lung diseases and to evaluate the effectiveness of supplemental oxygen therapy

• Blood gases are used to detect an acid-base imbalance, such as can occur with kidney failure, heart failure, uncontrolled diabetes, severe infections, and drug overdose

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Sample collection• Arterial or venous blood samples are

used • Glass or oxygen - impermeable syringe

containing a small amount of heparin • Labeling "patient's name, hospital ID,

date and time of collection, and site of collection

• Sample transported on in ice (1-5oC) to reduce cellular respiration

• Transported immediately to the laboratory

• Analyzed without delay

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Calibration Of Blood Gas Instruments

• Buffers and calibrators used for calibration of pH and blood gas analyzers should be of the highest purity and quality

• A two point calibration of all parameters should be performed periodically

• One calibrator should have low pO2 and pCO2

concentrations • The other should have much higher concentrations

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Internal Quality control • Commercially available controls for pH and blood gas

analyzers are aqueous and blood-based controls • Controls are available in 3 levels:

• Resembling acidotic • Normal • Alkalotic patient

• The use of all three levels of control is important in monitoring the performance of the analyzer in the medically important ranges

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Aqueous Controls • Buffered water solutions with each level being titered to a

specific pH and saturated to specific pO2 and pCO2 levels

• Less expensive than blood - based controls • Have longer shelf- life • Stored at room temperature • Do not require warming

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Blood - based controls • Similar to patient blood compared to aqueous controls • Available at acidic, normal, and alkalotic concentrations • Controls are more sensitive to change in instrument

performance than aqueous controls due to their hemoglobin and protein content

• Stored in refrigerator • Warmed to 37oC and mixed before analyzing

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Sources Of Error In Ph/Blood Gas Measurements

A- Errors in the Collection of Samples • Collection container:

• inappropriate plastic syringe used instead of glass or gas impermeable plastic syringe

• Long delay • >1 hour in transporting the specimen to the laboratory or delay in

analyzing it

• Air bubble in sample • Improper anticoagulant used or inappropriate amount of

anticoagulant resulting in a clotted specimen • Dilution of the blood specimen with indwelling arterial catheter flush

solution • Specimen not placed on ice

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Sources Of Error In Ph/Blood Gas Measurements

B- Errors with the Instrument• Bubble in measuring chamber • Bacterial growth in measuring chamber on electrode membranes• Protein coating membrane • Instrument temperature not maintained at a consistent or correct

value • Contaminated buffers and calibrating gases • Defective, aging or improperly maintained electrodes

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Quality Assurance of Instrumentation

• pH and blood gas analyzers are sensitive instruments that require regular performance and function verification and routine preventive maintenance

• Analyzers contain five components:1. A constant temperature circulating water bath

2. pH electrode and reference electrode

3. pCo2 electrode

4. pO2 electrode

5. Millivolt meter used to measure the voltages generated by all three electrodes

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Quality Assurance of Instrumentation

• The manufacturer's instrument manual is used to develop an instrument verification and performance procedure for each instrument

• Performance verification of these analyzers involves the analysis of quality control specimens

• Function verification involves the calibration of an analyzer at intervals through out the day

• Blood gas analyzers require extensive maintenance checks for their components

QUALITY ASSURANCE

Coagulation

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• Prothrombin time (PT) and Activated partial thromboplastin time (APTT) evaluate the ability of the plasma to form a fibrin clot• They are used to measure the effects of anticoagulant therapy

• Other tests measure the concentration of specific clotting factors such as fibrinogen, Factor V or factor VIII

• Other tests measure platelet numbers and function

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Specimen Quality Assurance

• The specimen of choice for most coagulation testing is sodium citrated anticoagulated plasma• A 1:9 ratio of Na- citrate to blood is required

• The cells and the plasma should be separated as soon as possible

• If delay occurs, the byproducts of cellular metabolism alter the plasma, making it too acidic, the clotting time will be longer as the enzymes necessary for such process are sensitive to acidic pH

• The citrated blood specimen should be protected from overheating as some coagulation factors (V, VIII) are heat labile

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Specimen Quality Assurance • Hemolyzed specimens are not recommended as RBCs

contain activating factors for the intrinsic portion of the coagulation cascade... • these may shorten the APTT

• Specimen should be centrifuged to get platelet - poor plasma (RCF of 1000 x g for 10 minutes)

• Plastic tubes are recommended as contact with glass may result in initiation of coagulation cascade and the shortening of APTT

• The plasma should be processed within 30 min or stored at 4oCin a tightly stoppered tube

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Quality Control • lyophilized citrated plasma samples are available for quality control of

coagulation testing

• These controls are available at 3 levels: • Normal

• Mildly prolonged

• Abnormally long time for PT and APTT

• The mildly prolonged control should provide a clotting time in the therapeutic

ranges in response to heparin and coumadin therapy for the APPT and PT

• Controls are reconstituted with deionized water, the sample will be stable for

4-8 h at 2-8oC

• Multi-rule evaluation with two levels of controls is used to evaluate the

results of the control runs

• The participation in a regional quality control program will give a precision

performance comparison with other laboratories using the same method /

reagent

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External Quality Control• CAP offers lyophilized samples for proficiency testing • Provides the laboratory with an indication of its analytical

accuracy by comparison with other laboratories with same and similar methods

• Lyophilized samples that are reconstituted and assayed• Results are returned to CAP, group mean and standard

deviation is calculated for each sample• Acceptable performance is indicated with a standard

deviation index (SDI)

SDI= Individual lab. Result – Group mean

Group standard deviation

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Sources of Error in Coagulation Testing

1. Improper concentration of calcium solutions• This may be the result of making the solution improperly • or allowing it to concentrate through evaporation• The end result is prolonged times for the APTT and other

procedures requiring, re-calcification

2. Inaccurate or imprecise pipettes used to deliver reagents or sample

3. Incorrectly reconstituting reagents and controls

4. Overfilling or underfilling of the vacuum collection tube with blood

5. Not obtaining platelet-poor plasma for the APTT

6. Failure to perform preventive maintenance and verification checks of coagulation instrumentation

QUALITY ASSURANCEUrinalysis

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Introduction

• Urinalysis seems to be an ideal test• The sample is easy to collect; • the test procedures are simple and uncomplicated

• Unfortunately the development of a quality assurance program for urinalysis has lagged behind other areas of the laboratory

• Because of its simplicity and low regard in the laboratory it is not unusual to find that the least experienced and trained technologist and technicians are assigned to this workstation

• These individuals more often than not do not understand the importance of adhering to test procedures and fail to recognize or correctly count the formed elements in the urine sediment

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Introduction• The macroscopic and microscopic examination of urine

can reveal important insights to a patient's physical condition and state of health

• Until recently little effort has been spent on the standardization of this widely practiced procedure

• The quality of the laboratory result can be improved and maintained with a small amount of effort placed in assuring: • sample integrity • standardization of procedures• and correct interpretation and reporting of the urine sample

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Major Errors In Urine Testing

1. Failure to test a fresh specimen

2. Inadequate communication between the laboratory and the patient or nursing staff for collecting a urine specimen properly

3. Use of unclean collection containers, tubes, and slides

4. Inadequate care of specimen

5. Improper recording of information and test results

6. Poor or inconsistent technique in using the reagent dipstick

7. Inadequate understanding of interfering substances

8. Failure to mix urine specimen (blood, bacteria, casts) before pouring---a-portion for the microscopic analysis

9. Incomplete training of personnel to perform properly the technical aspects of the urinalysis or to identify elements in the sediment

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Specimen Collection

• Most routine urinalysis is performed on a freshly collected random urine

• Regardless of when the sample is to be obtained, clear and concise written instructions are necessary for both patients and nursing personnel

• The validity of a test result is no better than the quality of the specimen

• Once the urine specimen has been collected it should be processed immediately to prevent deterioration

• If samples can not be processed within 1-2 h of collection, they should be refrigerated or preserved

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Specimen Collection

Types of Urine Specimens

• Random specimen: • A urine specimen collected at any time by either clean catch,

catheterization, or suprapubic aspiration• Random specimens are collected most often for routine urinalysis

• First morning specimen: • A urine specimen collected upon rising from sleep in the morning• First morning specimens are also used for routine urinalysis

• Postprandial specimen: • A urine specimen collected at a specific interval after eating• This type of sample is used to evaluate a diabetic condition, either as part

of a glucose tolerance test, or for the control of a diabetic state

• Specially timed specimen (2-hour and 24-hour specimen):• Involve the collection of all urine in the specified time period• used in various renal function tests such as creatinine clearance

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• It is important to recognize which preservative is necessary for which test

• Type of preservative should me marked on the container to ensure safety

• Some of the more common methods of preservation include:• Freezing

• Destroys cells and some components

• Refrigeration• Preferable method, sample should come to RT before analysis

• Commercial preparation tablets• For routine urinalysis in absence of refrigeration

• Formalin, Toluene, Chloroform, Phenol & other chemicals• used mostly for 24-hour specimens

Specimen Collection

Preservation of Urine Specimens

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The Decomposition Of Urine • Urine undergoes these changes after collection if proper

steps are not taken to preserve it:

• Ascorbic acid: Decreases and may disappear

• Ammonia: Increases • Glucose: Decreases and disappears • Bacteria: increase • Nitrite: Appears • Casts: Disappear • Red blood cells: Hemolyze • Occult Blood: Reactivity appears or

increases

• pH: May increase • Color: Darkens • Odor: Becomes smelly • Turbidity: Develops • Protein: May appear or

disappear • Bilirubin: Decreases and

disappears • Urobilinogen: Decreases and

disappears • Ketone bodies: Decrease and

disappear

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Procedure Manual• Should pay special attention to the collection procedure• Should specify which preservatives are appropriate for which

tests and should detail the proper labeling of these sample containers

• Definition of the various formed elements in the sediment should be clear and methods of reporting should be well defined

• Arbitrary terms such as small, moderate, and large should be avoided and semiquantitative terms such as trace, 1+ , and 2+ should be defined to help reduce reporting inconsistencies between individuals

• Instructions for the preparation of control samples should be included in the urine procedure

• The control tolerance limits should be clearly displayed and specific actions to be taken when limits are exceeded

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Standardization Of The Urinalysis Procedure

• The routine urinalysis is divided into two parts:• the chemical urinalysis • and the microscopic urinalysis

• Until recently the chemical urinalysis was performed manually with: • Reagent strips

• The determination of the final color of the strip pad is a subjective one and two persons may interpret the same test in different ways

• The timing between immersion and reading the strip is another source of variation between one individual and another

• The longer the reaction is allowed to proceed before reading, the darker the color development• Consistent timing is important for precision and accuracy

• Or reagent tablets• The reagent tablets are also colorimetric reactions that require

timing and interpretation by technologists

Positive

Negative

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• The manual performance of the chemical portion of the urinalysis is subject to imprecision and inaccuracy due to shortcuts and inconsistent performance

• The introduction of automated readers has improved the precision and accuracy of the chemical portion

• Light is directed to the pad and the intensity of the reflected light is measured

Standardization Of The Urinalysis Procedure

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• The introduction of standardized systems for performing the microscopic examination reduces another variable in the urine procedure

• These systems standardize: • the volume of urine used to collect the sediment• the force of centrifugation• the volume of urine in which the sediment is resuspended• and the volume of sediment examined for cells and casts

Standardization Of The Urinalysis Procedure

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Quality Control • Commercially prepared controls for the chemical portion

of the routine urinalysis are available for the reagent strips and tablets

• Lyophilized urine samples or reagent sticks with impregnated chemicals that dissolve in measured amount of water

• Positive and negative controls are available to check the sensitivity of the reagent system

• Both positive and negative controls should be run daily and when opening a new bottle of reagent sticks or tablets

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Reagent And Instrumentation Quality Assurance

• Reagent sticks and tablets should be stored under conditions specified by the manufacturer to prevent the absorption of moisture and prevent exposure to light that may degrade some of the chemicals in the reagent pads

• Written maintenance procedures and schedules for all instrumentation in the urinalysis laboratory should be kept current and reviewed for compliance by the laboratory director or his designee