MLS 220

Three phases of total testing process

1. Pre-Analytical

2. Analytical

3. Post-Analytical

Pre-analytical

-prior to testing

-most errors

-examples: test ordering, phlebotomy, specimen collection, sample labeling, checking patient identifiers (need at least 2)

Analytical phase

-testing sample

-least errors, less opportunity for human error due to automation

Post-Analytical Phase

-after testing is completed

-examples of errors: manual result entry, test result interpretation

Quality Assurance (QA)

Policies, procedures, and practices necessary to make sure that a laboratory's results are reliable

QA examples include

-record keeping

-calibration and maintenance of equipment

-quality control

-proficiency testing

-training

QA Program

-Plan to carry out policies and practices necessary to comply with QA standards set by accreditation agencies

-CLIA requirement

-evaluate entirety of the TTP

Define Delta checks

-checks difference between most

Current result and previous results

When will you get a delta check?

If there's a change between past 2-3 results that exceed a specified cutoff

Proficiency Testing (PT)

-CLIA requirement

-program enrollment: CAP, CDC, some state health departments

Quality Control

-Analytical phase of Total Testing Process (TTP)

Goal of Quality Control

-detecting and correcting errors, alert analyst when there may be potential problem with a testing procedure

-generate high-quality results that are accurate, precise, and reliable

Two types of quality control

Internal QC

And External QC

Internal QC (statistical)

Evaluated the daily precision of an assay to make sure it's working

External QC

Evaluated the accuracy of an assay measurements

-submission of internal QC results and specified control specimens

-proficiency testing

QC Errors include

1. Systematic

2. Random

Systematic error (inaccurate)

-Caused by the same factor producing reproducible errors in one direction from the true value

-can be detected and corrected

-causes inaccurate assay results

-results are inaccurate by the same amount

Ex: instrument calibrated incorrect

Random Error (imprecise)

1. Caused by an accident that can be difficult to identify

2. Causes imprecision of results

3. Not reproducible

Ex: inaccurate pipetting

Define accuracy

-the closeness of the result obtained to the true or actual value

-how close we are to the center of that bulls eye, how good we are getting that target

Define Precision

-repeatability or reproducibility in obtaining the same value in subsequent tests in the same sample

-how good we are at shooting and same spot repeatedly

Accuracy in lab

1. Standardize Procedures

2. Comparing new methods vs old methods

3. Use of controls (sample with known values)

Precision in the lab

1. Proper inclusion of standards/reference samples

2.valid replicate determinations of a single sample

3. Duplicate determination of sufficient numbers of unknown samples

4. Day-to-day and between-run precision assessed by inclusion of control specimens

Accuracy vs Precision

Calibration Standard

-highly purified substances of a known composition

-purchase from a manufacturer

-used to established reference points to construct graphs or a test result

Calibration curve

-Compares an instrument measurement/reading to a known concentration (standard)

-established before any testing is completed

Sensitivity

The proportion of cases having a specific disease or condition that gives a positive test result

Represents how much of a given substance is measured

-the more sensitive the test the smaller amount of assayed substance is measured

Specificity

The proportion of cases with a sense of a specific disease or condition that gives a negative result

Represent what is being measured

-the more specific the tests, the smaller number of analytes being measured

-the test only measure the specific

Analyte in question

Positive predictive value

Indicates the number of patients with an abnormal test result who have the disease compared with all patients with an abnormal result

Negative predictive value

Indicates the number of patients with a normal test result who do not have the disease, compared with all patients with a normal (negative) test result

PPV

True positives/ (true positives+false positives)

NPV

True negative/ (True Negatives+False Negatives)

Standard deviation

Measure of the spread, or variability in a data set

Precision -> how good are we hitting that mean every single time we run test

When is standard deviation used to determine?

Reference range +/- 2 SD

Coefficient of variation

Calculate the SD as a "percent of mean"

When is CV useful?

When comparing two or more groups of data to determine which has the greater precision

- the lower the CV, the higher the precision

How is acceptable range calculated?

-Run level 1 control sample daily for 15-25 days

-treat QC material exactly like a patient

-calculate mean and SD

-acceptable QC ranges will be +/- 2SD. 95% of results should fall within the acceptable range.

Mean - (+/-)SD

Level-Jennings chart

Daily results plotted onto graphical representation

-manual vs. automated

-automated warning limits: +/- 2 or +/-3 SD

Levey-Jennings chart terms include

-shift

-trend

-dispersion

Define shift

Sudden and sustained change in one direction in control sample values

Shift causes

Sudden malfunction with instrumentation, QC contamination, etc.

Define Trend (aka Drift)

Gradual change in the control sample

Results

Control value direction moves progressively in one direction from the mean for at least 3 days

Trend causes:

Deterioration of reagents, control, or instrument (ex. a lamp starting to go out)

Define Dispersion

When random error or

Lack of precision increases

Dispersion causes:

Instability problems with instrumentation

Westward Rules

-Rules formulated to analyze data in control charts based on statistical methods

-define performance limits for a particular assay

-detect random and systematic error

1_2s

Violated when one or the two control results fall outside of ±2 SD

◦ Warning rule – lets you know of a possible problem with control data
◦ Specimens not rejected

1_3s

Violated when any QC result falls outside of ±3 SD

◦ Random error
◦ Beginning of larger systematic error?

2_2S

Violated when 2 consecutive control values for the same side fall outside of ±2 SD in the same direction, or when both control levels in the same test batch exceed
±2 SD
◦

Detects systematic error (only)
**Patient results cannot be reported**
◦ Requires immediate corrective action

3_1s

Violated when 3 consecutive control results are greater than 1SD on the same side of the mean

◦ Within one level of control, indicates systematic bias
◦ Across control levels, indicates systematic error over broader range of
concentrations

4_1S

Violated when four consecutive control results are greater than 1 SD and
on the same side of the mean

◦ Within one level of control, indicates systematic bias
◦ Across control levels, indicates systematic error over broader range of concentrations

R_4S

Violated when One Control Measurement exceeds the +2 SD and the other exceeds the -2 SD control limit

◦ Must be interpreted within a run, not between runs
◦ 4 SD difference
◦ Random error
**Patient results cannot be reported **

10_X

Violated when 10 consecutive QC results for one level of assay control are
on one side of the mean OR both levels of control have five consecutive results
that are on the same side of the mean

**Patient results cannot be reported **

N_X

violated when 7, 8, 9, 10, or 12 control results are on the same side of the mean

◦ Within one level of control → indicates systematic bias
◦ Across control levels → indicates systematic error

Multi-Rule QC

• Uses multiple rules in tandem
• Decreases chance of false rejection
• 1_3𝑠 and 𝑅_4𝑠 → detect random error
• 2_2𝑠 and 4_1𝑠 and 10x → detect systematic error

No analytical Factors in Quality Assessment

1. Qualified Personnel

2. Established Laboratory Policies

3. Laboratory Procedure Manual

4. Testing Requisitioning

5. Patient identification,

Specimen procurement, and labeling

6. Proper Procedures for specimen collection and storage

7. Specimen transportation and processing

8. Preventative maintenance of equipment

9. Appropriate Methodology

Test Requisitioning

• Laboratory test requested by physician
• Electronic or paper

• Must include…
Patient identification data
Time and date of specimen collection
Specimen Source
Tests to be completed

• Information on specimen must match information on test

Serum

Liquid portion of blood from a sample that has clotted

Plasma

Liquid portion of blood from a sample that has not clotted

PlasmAnticoagulant

What does anticoagulants do?

Chelating (binding) or precipitating calcium (calcium needed for coagulation process)

Inhibiting the formation of thrombin

Under filled tube

More anticoagulant than blood-> clotting of blood is prolonged

Over filled tube

More blood than anticoagulant -> clot faster

Anticoagulants include

1. EDTA

2. Citrate

3. Heparin

4 Oxalate

5. Special-Use

Red Top Tubes

• “Plain” or silica coated
• Serum as no anticoagulants present

EDTA (ethylenediaminetetraacetic acid)mechanism of action

Removed ionized calcium through chelation.

Forms calcium salt

Pink or Purple Tubes

Common EDTA test

•Common Tests → Hematology

-complete blood count (CBC)

-erythrocyte Sedimentation Rate (ESR)

-blood type/antibody IDs and Screens

Citrate - Sodium Citrate

-Removes calcium by precipitating it into an unusable form

-9:1 ratio of blood to anti coagulant

Light Blue

Citrate common test

Common Tests → Hemostasis

-prothrombin time (PT)

-activated partial thromboplastin time (aPTT)

-fibrinogen

Heparin Cap Color

Lithium Heparin -> light/mint green (most common)

Sodium heparin -> dark green

Ammonia heparin (least common)

Heparin mechanism of action

-prevents thrombin formation

-interrupts clotting cascade

Heparin common test

Common Tests → Chemistry

Electrolytes

pH

Enzymes

Oxalate cap color

Gray

Precipitates calcium

Oxalate common test

Chemistry -> Glucose levels

Acid citrate dextrose cap color

- ACD

- yellow

•Mechanism of Action
Acid citrate binds calcium
•Common Tests
DNA Testing
HLA Typing

ACD mechanism of action

Binds calcium

Citrate Phosphate Dextrose cap color

-CDP

-yellow top or bag

CDP mechanism of action

-binding calcium

-dextrose provides cells with energy to keep them alive

-phosphate stabilizes pH

CDP common uses

Blood collection units

Sodium polymers sulfonate (SPS)

-SPS

-yellow top

- binds calcium

- reduces complement -> allows bacteria to grow

- slows down phagocytosis

- reduces activity of certain antibiotics

sodium polyanethol sulfonate does

Blood cultures

Antiglycolytic Agents

•Prevents glycolysis (breakdown of glucose by blood cells)
•Sodium Fluoride
•Preserves glucose for up to 3 days
•Commonly used with potassium oxalate

Clot Activators

Substance that enhances coagulation in tubes used to collect serum specimens

Clot activator cap color

Yellow, red, black and red

Clot activator mechanism of action

provide more surface for platelet activation

-glass (silica) Celite (inert clay)

thrombin

-clotting factor

Common tests for clot activators

Chemistry

-BUN

-creative

-electrolytes

Gel separator

Thixotropic Gel

-density is between cells and serum/plasma

-provides physical barrier

Gel separator cap color

- gold evacuated tube (SST:serum separator tube)

- Mint evacuated tube (PST: Plasma separator tube)

Trace Element-Free Tubes

-royal blue top tubes (metal free)

Made of materials as free of trace element contamination as possible
•Trace elements (lead), toxicology studies, nutrient determination

After centrifugation 3 layers are

Formed

-plasma (liquid portion of blood that has been mixed with anticoagulant. 55% of total blood volume)

-Buffy coat: gray/white layer composed of white blood cells and platelets

-erythrocytes: RBC, 45% of total blood volume

Unacceptable Specimens

Potential Reasons for Unsatisfactory Specimens
• Incorrect tube/specimen container for sample or test
• Specimen is labeled incorrectly
• Tube is under/over filled
• Specimen not tested in timely manner
• Sample is hemolyzed

Two types of blood draws

Venipuncture

-vacutainer

-syringe method

-butterfly

Capillary stick

-finger

-heel

Venipuncture-vacutainer supplies

-gloves

-tourniquet

-alcohol wipe

-gauze

-tubes

-vacutainer needle

-vacutainer hub

-test/patient labels (order)

-bandage or coban wrap

Vacutainer needles

-the larger the number, the smaller the gauge

Ex: 21 G needle is smaller than s 18G needle

Vacutainer hub

-engineering control

-protects against accidental needle stick

-single use

Venipuncture-syringe method supplies

-gloves

-tourniquet

-alcohol wipe

-gauze

-tubes

-syringe needle or butterfly

-syringe

-transfer device

-test/patient labels (order)

-bandage or coban wrap

Venipuncture-butterfly method

-smaller gushed needle

-follow syringe method

-typically used for patient with small veins, elderly, children, or puncturing somewhere other than the arm (hand, foot, etc.)

Two preferred veins to use while performing venipuncture

Antecubital fossa (AC)

- in front of the elbow + "shallow depression"

Antecubital Veins

-H pattern->Median cubital vein (70%)

-M Pattern ->median basilic vein and median cephalic vein (30%)

Venipuncture general procedure

1. Introduction of phlebotomist and procedure

2. Patient identification

-two patient identifiers given verbally

3. Gather appropriate supplies

4. Perform hand hygiene, put on gloves

5. Apply tourniquet and find draw sites

6. Alcohol draw sites, allow to dry

7. Anchor the vein

8. Insert the needle, fill tubes

9. Release the tourniquet (do this before removing the needle to avoid a hematoma)

10. Remove the needle

11. Apply gauze and pressure

12.apply bandage or coban (patient preference)

13. Clean up supplies

14. Label tubes

-in the presence of the patient

-double check patient labels with patient identification (either verbally or armband)

-hand write date, time, phlebotomist identifier on label

15. If drawing in a patient's room, leave jr how it was found

16. Send/deliver blood to lab

Capillary puncture

-finger or heel stick

-neonatal testing

-point of care testing

-hard stick

Capillary puncture tube

- microtainer

- same color-coded system as full-sized tubes