Exam 2 review

check that the testing system is in control

• water quality (type 1 control)
• preventative maintenance
• daily recording of temp and humidity

ensure internal quality control-accurate control solutions (minimum of two levels is typically assayed)

Usually analyzed every 30 days; ~5% inaccuracy allowed (2SD)

Frequency distribution:

• Gaussian
• 68.2% +/- 1SD; 95.5% +/- 2SD; 99.7% +/- 3SD

Monitors several aspects of control quality:

• accuracy, precision, S&R error

Must be recorded, usually on a Levey-Jennings chart

Used to display control data

shows fluctuations of control data over time

One control value exceeds +/- 2SD

Considered first before any other rule

Serves as a warning to trigger careful inspection of the control data by the other rules

(If +/- 2SD is used instead of multirule, there's a high level of false rejections)

One control value exceeds +/- 3SD

It detects random error

Rejection rule

Two consecutive control values excee +/- 2SD

Detects systematic error

Rejection rule

When the range or difference between 2 control results EXCEEDS 4 SD

Detects random error

Rejection Rule

4 consecutive contrl results fromt he same control exceed the same 1SD limit

OR

Consecutive control observations from 2 different control levels exceed +/- 1SD

Detects systematic error

Rejection rule

10 consecutive control results on the same side of the mean

Detects systematic error

Rejection rule

1_2s

1_3s

2_2s

R_4s

4_1s

10x

Delta checkd-review for changes from previous results

alert checks/technical flags-critical values must be reported

regional QC program

proficiency testing

Calculations

Patient test report

run controls

compare them to expected results

to enhance diagnostic accuracy

reveal genomic defects

used as prognostic indicators

Immunocytochemistry

electron microscopy

fluorescent techniques

molecular techniques

cellular image analysis

immunomagnetic bead techniques

proteomics

beam of electrons illuminates teh specimen

provides information regarding tissue organization, individual cells

viscualization of molecules and atom

used most often for the diagnosis of poorly differentiated neoplasms (a tumor that has an unknown origin or cause)

used for teh ultrastructural labeling of cytokines, oncogenes, and growth factors

projects electrons through a very thin slice of tissue

provides a 2-D image on a phosphorescent screen

a protein (ab) is used to locate specific cellular ag in tissue-immunoperoxidase method most common

applications- surgical pathology, diagnosing abnormal cells in cancer, targeted therapies, cell proliferation/apoptosis, infectious processes

uses ag-ab rxn to locate a target ag

tissue sections or cell smears are stained w/abs that are specifically targeted against ags

ag-ab rxns are visualized using a microscope (populare in pathology)

primary ab is conjugated prior to application to the tissue

(used in fluorescence)

fixation/pretreatment-steps vary according to:

• type of specimen
• ab
• is rxn in cytoplasm or nucleus

blocking

• reduces non-specific binding
• decreases background staining

1. primary ab
2. secondary ab conjugated to biotin
3. enzyme conjugated streptavidin (HRP or alkaline phosphotase)
4. chromogen- colored rxn represents the presence of ag
5. counterstain-minimizes background stain and preserve the specimen

formalin-fixed paraffin-embedded tissue is the preferred sample

protocols mus be validated

controls must be used every test, every ab

careful selection of panel of abs

disadvantage: lack of standardization within and across labs, difficult to compare results

ab concentration

lack of specificity of ab

ab cross-reactivity

non-specific ab binding

inappropriate fixation

misinterpretation of cells

immunofluorescence

flow cytometry

fluorescence in situ hybridization

fluorochromes are conjugated directly to abs

allows visualization of objects that are too small to see with the light microscope

commonly used fluorochromes:

• fluorescein
• fluorescein isothiocyanate (FITC)
• rhodamines/texas red
• phycoerythrin

proper fixation is essential

fluorochromes can directly label the primary ab or indirectly label the secondary ab

multiple labeling is possible

cell membranes must be "permeable" to allow optimal ab/ab binding

clinical microbiology

• primary id of microorganisms
• final id of bacteria
• id organisms that can't be cultured

earlier diagnosis

increased sensitivity

"instrument designed to detect and enumerate cellular elements in suspension"

cells must be alive and in suspention (no clumps)

characterization of single cells as they pass at high speeds thru a laser beam

light scatters and excites fluorescent molecules used to label the cells

cells are characterized individually by physical and chemical properties

1-2 lasers

forward scatter-proportional to cell cize

side scatter -90 degree angle proportional to cytoplasmic granularity

series of filters and mirrors

several photomulitplier tubes (PMT)

live cells in suspension

staining w/fluorochrome

laser activates the dye

fluorescence is emitted

information is transmitted to a computer

used to diagnose and classify hematologic neoplasms (leukemias/lymphomas)

extreme sensitivity in detecting ag expression and can id small, clonal populations

can analyze fluids, CSF, bone marrow, blood, FNA's, lymph node biopsy specimens

advantages:

• multiple stains per cell
• rapid turnaround
• quantitave

limitations:

• large # of cells required
• doesn't provide morphologic detail

uses fluorescently labeled nucleic acid "probes" that hybridize to specific chromosomal loci

allows the detection of chromosomal alterations in cells

detection of cancer cells in cytologic specimens

provides info that allows the prediction of tumor behavior and appropriate treatment

detection of microorganisms

FDA approved FISH test

detection of urothelial carcinoma in the bladder in voided urine

detection of recurrent tumor in patients with a history of bladder cancer

evaluation of patients with hematuria for bladder cancer

isolate cells from urine and place on slide

prepare cells for FISH hybridization (pre-hybridization)

hybridize FISH probes to cells overnight

wash to remove unbound probe

DAPI counterstain and coverslip

access cells for nuclear & chromosomal abnormalities using fluorescence microscope

cells must be visible and adequate in number

crowded cells w/ overlapping nuclei do not yield adequate results

different tissue types have different characteristics

used to analyze minimal sample volumes

applicaitons:

• diagnosis and classification of tumors
• microbiology
• mutational analysis
• patient management

PCR

ISH

FISH

high snesitivity

accurate

potential for simple, rapid, standardized procedures

added diagnostic utility

generates many copies of DNA/RNA

• contamination
• false-positive of other specimens

expensive

ethical issues (privacy, discrimination)

inability to recognize unusual situations questions about the biological significance of a positive result

1. denature sample
2. hybridize-allow annealing
3. extension-making copies

any type of sample can be analyzed

extremely sensitive

id of organisms

charact. of antibiotic resistance

cancer diagnosis and prognosis

det. of circulating tumor cells and min. residual disease

Uses RNA probes, anti-DNA/RNA hybrid antibodies,a nd chemiluminescent detection

Detects 13 high risk types of HPV

Capable of quantitating viral load

Doesn't test for all high-risk types

Doesn't identify the specific HPV genotype

Lacks an internal control (very problematic)

Cross-reactivity of probes may occur

Uses a nucleic acid probe to locate a gene or an mRNA molecule in a cell or tissue

Preserves cell morphology

Allows correlation of cytology with biopsy

Automation results in multiple testing capabilities

• baking-adhere tissue to slide
• deparaffinization-remove wax for reagent penetration
• pretreatment-allows probe to reach target
• denaturation-frees target dna strand
• hybridization-joins probe to target
• wash-remove probe
• detection-links visual molecule to probe
• counterstain-add "backdrop" color
• coverslip-Protect staining

• Identifies specific HPV genotypes (16 and 18)
• has an internal control
• detects specific nucleic acid sequences
• structure specific recognition and cleavage with clevase enzyme
• doesnt require PCR
• fluorescent detection is proportional to DNA