Laboratory practice

Question 1

How many grams of sodium chloride are added to 1000ml of water to make a 0.9%
solution?

Answer 1

9 grams (+)
0.09 grams
0.9 grams
99 grams

Question 2

The best way to destroy bacteria and fungus (including spores) on working surfaces is:

Answer 2

disinfection
sterilization(+)
decontamination
purification

Question 3

When cleaning glassware, the final rinse must be:

Answer 3

double deionized water (or RO water) (+)
distilled water
tap water
sterilized water

Question 4

All of the following are proper procedures to follow for storing CO2 cylinders EXCEPT:

Answer 4

away from heat
in halls (+)
capped with safety valve
away from flame

Question 5

Colcemid

Answer 5

mitotic inhibitor

Question 6

Hypo

Answer 6

disperse chromosomes ph=6.8-7.0

Question 7

Carnoy’s fixative

Answer 7

removes water, denatures proteins, removes part of H1 proteins from chromosomes

Question 8

G-Banding solutions

Answer 8

1. Trypsin: digests protein fraction of chromatin ph=7.0-7.4
2. Giemsa: metachromatic stain specific for double stranded DNA
3. 0.01M PO4 buffer: swells the chromosomes to enhance trypsin banding ph=6.8

Question 9

Q-banding solutions

Answer 9

1. Quinacrine: fluorescent stain which binds to AT rich DNA

2. McIlvaine’s buffer: swells the chromosomes allows transmission of fluorescent light ph=4.5-6.0

Question 10

NOR staining solutions

Answer 10

1. Silver solution:precipitates onto protein when incubated with formalin
2. Gelatin: provides a substrate on which the reaction between silver and formalin occurs

Question 11

High resolution

Answer 11

1. Ethidium bromide:DNA intercalator
2. Amethopterin: blocks cells at G1/S interphase
3. BrdU: releases Amethopterin block; substitutes for Thymidine

Question 12

C-Banding solutions

Answer 12

1. HCl: removes cytoplasmic and protein fraction of chromatin
2. Ba(OH)2 saturated: extracts DNA from chromosomes ph=high

Question 13

R-banding solutions

Answer 13

1. BrdU: thymidine analog
2. Acridine orange: fluorescent dye
3. Sorenson’s buffer: mounting buffer ph=6.5

Question 14

Mathematical formulas

Answer 14

Measurements

a) 1M = mol/L
b) 1L = 1,000mL
c) 1mL = 1,000uL
d) 1kg = 1,000g
e) 1g = 1,000mg
f) 1mg = 1,000ug
g) 1ug = 1,000ng
h) 1ng = 1,000pg

Dilutions

Example: What volume of a 2 M stock solution would be used to make 2 L of 0.2 M working solution?

Use this equation:

(Molarity 1)(Volume 1) = (Molarity 2)(Volume2)

(2M) (x mL) = (0.2 M)(2,000mL)

x = 200 mL

Percentages

Example: What does a 20% (w/v)) or 20% (v/v) solution mean?

20% (w/v) solution = 20 g/100 mL
20% (v/v) solution = 20 mL/100mL

Example: How many grams of NaCl (Mwt = 58.5) are added to 1000 ml in order to make a 0.9% solution?

1gram/ml = 100% (regardless of molecular weight)

Therefore

0.9grams/100 ml = x/1000 ml

x = 1000 ml x 0.9gram/100 ml

x = 9 grams

Molarity

A molar solution (M) is the molecular weight in grams of a substance dissolved in enough water to make 1 liter. The molecular weight of any substance can be found on the manufacturer label or in the Handbook of Chemistry and Physics.

Example: 1 M NaCl = 58.5 grams added to the volume of water needed to make 1 liter M wt of NaCl = 58.5).

Question 15

Select, operate and maintain laboratory equipment and instruments (e.g., daily record temperature, %CO2, %O2, humidity

Answer 15

All laboratory equipment must be maintained at peak operating efficiency. Accurate records of quality control indicators are necessary to ensure the maximum number of positive test results and are the cornerstone of quality control in the cytogenetic laboratory.
Temperature Readings - Logbook must be kept with daily temperature recordings for all incubators, refrigerators, and freezers. If the temperature is higher or lower than the recommended settings they must be adjusted or fixed immediately to maintain the quality of the cultures and reagents.

CO2, O2, and humidity readings of incubators should be checked daily and recorded in a logbook. CO2 and O2 levels are usually set at 5% and a humidity pan located at the bottom of the incubator controls the humidity.

Daily Maintenance Schedule
Record the temperature of CO2 incubators, refrigerators, freezers, ovens and waterbaths.
CO2 levels, humidity, internal temperature, should be recorded for all incubators.
Microscopes should be cleaned and checked for proper alignment.
Safety Cabinets should be cleaned and disinfected (before and after use). In addition, the linear airflow should be checked.
Weekly
Safety cabinets should be thoroughly cleaned.
Centrifuges should be inspected for wear and dirt.
Monthly
Incubators, centrifuges and waterbaths should be cleaned and disinfected.
Bi-Annual
BioSafety Cabinets should be recertified.
Centrifuges, ovens, slidewarmers, pH meters, scales, and microscopes should be serviced and/or calibrated.
Biohazard cabinets should be serviced for air flow levels
Annual
Water-jacketed incubators should be drained and refilled.
As Required
Autoclaves, dishwashers and water jacket of incubators should be cleaned.

Question 16

Select sterilization/decontamination procedures

Answer 16

1. Disinfectants - These substances are used to control microbial growth.

Work surfaces should be routinely cleaned with disinfectants such as 70% ethanol or 1:10 bleach (sodium hypochlorite - 1%). Commercial all-purpose disinfectants such as Rocal II and Microbac are also used.
Disinfectants used to do monthly cleaning of incubators should be rinsed out thoroughly before placing cultures back in the incubator. If these disinfectants can kill bacteria, they can also be toxic to cultured human cells. Bleach should never be used inside of an incubator.
Glassware used in the laboratory should also be soaked in disinfectants prior to cleaning to reduce the growth of bacteria and fungus.

2.Sterilization - This refers to the total destruction of all living organisms and spores.

Tissue culture media and glassware must be sterilized before use to prevent contamination of cultured specimens. There are three common methods of sterilization in the cytogenetic laboratory.
Steam autoclaving - A common setting for an autoclave is 121oC at 15 lb of pressure for 15 min.
Dry Oven - Most laboratories that use this sterilization technique will treat glassware, and other items to be sterilized at a temperature of 165oC for two hours.
Liquids can be sterilized by passing the liquid through a 0.22-micron filter. This is the common method for sterilization of media. High temperatures could inactivate proteins in the media, which are required for cell growth.

Question 17

Monitor adequate stocks of laboratory supplies and chemicals

Answer 17

An adequate stock of supplies and chemicals refers to the critical balance of having enough of needed materials so there is no danger of running out of a stocked item, and no danger of having an excess supply so that supplies expire before they can be utilized. All supplies should be labeled with the date received, the date they were opened and the expiration date if any.

Question 18

Use correct procedures to store, handle and dispose of biological, infectious, chemical, radioactive, sharps and glass materials/waste.

Answer 18

Chemical Handling and Storage

In 1983, OSHA mandated the Hazard Communication Standard which stated that each clinical laboratory must develop a chemical hygiene plan. All hazardous chemicals in the laboratory must be included in the plan and provisions must be made for employee training and actions to take in the event of an accidental exposure.
All chemicals should be stored in their original containers. Labels should be placed on all containers stating the date they were received, opened, and any expiration date.
Flammable chemicals such as methanol and ethanol should be stored in an approved fire cabinet away from heat, flames, and sparks.

Corrosives such as glacial acetic acid and HCL should be stored on a low shelf also in a flame cabinet away from heat, flames, and sparks.
Reactive chemicals, such as ether, should be stored in airtight containers in a cool, dark place, and separate from any chemicals which they may react with.
Chemical fume hoods should be used to prevent the escape of vapors, aerosols, and gases into the general ventilation system.
When transporting chemicals, use an acid carrying bucket or other outside container, which will protect against breakage and spillage.
When handling chemicals, technologists should work in well-ventilated area to prevent the accumulation of toxic substance in the air. Facemasks should be used to provide a protective barrier against the inhaling of chemical dusts and powders when weighing and mixing hazardous chemicals.
If working with liquid nitrogen, it is necessary to wear insulated gloves, lab coat and face mask.
When preparing dilutions, always add acid slowly to water, since addition of water to acid could cause an explosion. When pouring strong acids and bases, individuals should wear resistant PPE such as butyl rubber gloves, aprons and face shields.
Gas cylinders should be secured in an upright position and stored in a well-ventilated, dry area away from sources of ignition or exposure to corrosive chemicals or vapors. These cylinders (CO2 sometimes added to tissue culture flasks) should be capped when not in use.

Biological and infectious materials should be placed in biohazard bags in special spill-proof containers. Beta chips can be placed in the bottom of biohazard bags to soak up any liquids that may spill. After these containers are full they are sealed and picked up by designated individuals and autoclaved and then sent to federally approved dump sites.

Sharps should be placed in designated punch resistant containers. Needles should never be recapped and should be placed immediately in these special sharps containers after use. Glass if broken into pieces can also be placed in these containers. If glass bottle is cracked or is being discarded for other reasons, it should be placed in a cardboard box, clearly marked, and sealed before disposal.

Environmental Health and Safety should place chemical, radioactive, and infectious wastes in leakproof drums, which will be picked up for disposal.
Labs using radioactive materials should follow approved guidelines. Workers should be properly trained in the handling of these materials and should wear radioactive monitoring badges while working with these materials in order to document their exposure. Wipe tests are done routinely to decontaminate work areas.

Question 19

Practice established procedures for general laboratory safety

Answer 19

Physical Hazards

1. Fire
2. Electrical
3. Mechanical
4. Radiation

Hazardous Chemicals

1. Flammables
2. Corrosives
3. Toxins

Question 20

Practice established procedures for general laboratory safety

Answer 20

Standard Precautions for biological hazards
Workers should have a barrier protection against biological contamination at ALL times.
Gloves should be worn at all times when touching blood or body fluids.
Gloves should be worn for venipuncture.
Gloves should be changed between patients.
Face shields, gowns or aprons should be worn if there is a possibility of splashing.
Wash hands if contaminated and after removal of gloves.
Accidental injuries should be avoided.
Sharps should be discarded in needle containers. Needles should never be recapped and should carefully be placed in the appropriate sharps container.
Mouth-to-mouth resuscitation should be minimized.
Pregnant females should be warned of potential hazards in the work environment.
Visitors in the laboratory should be restricted.
There should be compliance with written procedures.

FLUIDS TO WHICH UNIVERSAL PRECAUTIONS APPLY
Blood, body fluids with visible blood, and human tissues.
Semen, vaginal secretions, CSF, pleural, peritoneal, pericardial, synovial, and amniotic fluid.
Feces, sputum, sweat, tears, urine, vomit, and saliva.

General Safety Rules for the Laboratory
Do not pipette by mouth; use a pipette bulb or mechanical aspirator.
Do not eat, drink, chew gum, apply makeup or handle contact lenses in the laboratory.
Do not store food and drink in the same cabinet or refrigerator as chemicals.
Know the types of protective equipment and apparel available and use the appropriate type for each task.
Wash hands after handling chemical or biological materials, after removing gloves or lab coats, and before leaving the laboratory.
Keep work areas clean and uncluttered.
Decontaminate work surfaces after procedures are completed, after an obvious spill, and at the end of the work shift.
Use equipment only for designated purposes.
Properly dispose of hazardous waste.
Avoid practical jokes or distracting behavior.
Confine long hair, loose clothing, and jewelry.
Know the location of emergency equipment and how to use it. Be sure emergency phone numbers are posted near telephones in the laboratory.
Avoid working alone if possible.
Make lab safety a habit.

Supplemental Precautions

Leak-proof specimen containers
Uncontaminated exterior
Gloves for specimen processing
Facial protection for splattering
Minimize splatters
No mouth pipetting
Limit needles and syringe use
Decontaminate surfaces
Proper Biohazard waste disposal
Wash hands when exiting the laboratory
Leave protective clothes in the lab

Practice proper ergonomics (e.g. eye strain, chair adjustment, posture)
The majority of a cytogeneticists’ work time is spent in chromosome analysis. This analysis is accomplished either though microscope analysis or image processing. These activities involve long hours of sitting, straining eyes and making repetitive movements. Consequently, it is important to follow these ergonomic activities.

Support the arm that controls the microscope adjustment or computer mouse.
Stand up and move around often.
Alternate hands when using the computer mouse.
Take breaks from looking at the computer screen or through the microscope.
Periodically check your posture.

Material Safety Data Sheets
Identification

Name, synonyms
Product number
Chemical abstracts service registry number
Physical Data

Handling and Storage Precautions:

Incompatibilities (incompatible materials and conditions)
Protective equipment

Toxicity Hazards

Reference to the Registry of Toxic Effect of Chemical Substances (RTECS)
Toxicity data
Reviews, standards, and regulations
Health Hazard Data:

Acute effects
Chronic effects
First aid
Reactivity data

Stability:

Hazardous combustion or decomposition products
Specific health-hazard designations and storage conditions
Waste disposal methods

Spill and leak procedures

Fire and explosion hazard data:

Extinguishing media
Special firefighting procedures
Unusual fire and explosive hazards

Question 21

Hazardous chemicals used in a cytogenetics lab

Answer 21

Page 26

Question 22

Emergency procedures including chemicals or radioactive spills, fire, etc.

Answer 22

page27-30

Question 23

Apply laboratory quality control and continuous quality improvement in all areas, and comply with any federal, state, and/or local regulations.

Answer 23

CLIA 1988 - Clinical Laboratory Improvement Amendments

Goal - Accurate and consistent quality of care for patients.

Reproducibility and accuracy of results, including correlation with indication for study

Quality Assurance: The process, which monitors the quality control process from specimen collection to the patient report. Included in this process is certification, continuing education, and demonstration of proficiency and participation in on-site inspections

Example: A current and accurate procedure manual

Quality Control: - Refers to the specific test procedures and the indicators measured to determine if the minimum acceptable quality targets are being achieved.

All laboratories must devise ways in which to prove that their results are reproducible, accurate and that the final results are compatible with the indications for study. For example, if the number of positive chromosomal abnormality cases below the normal range for a particular time period this would be an indicator that current procedures need to be reevaluated to ensure that there is no reason other than chance behind this observation.

Patient confidentiality and security of patient records

Patient information and the results of laboratory testing may not be given out or discussed with any individual other than the patient’s physician without the written permission of the patient.
Patient information should not be discussed outside the laboratory with family or friends. Laboratorians must remember that patients have the right to privacy.
Equipment maintenance and repair

see “Select, operate and maintain laboratory equipment and instruments” - page 9

Participate in proficiency testing

There are three parts to proficiency testing

1. Self inspection (each year there is no on-site inspection)
2. On site inspection every 2 years
3. CAP Surveys three times a year

Proficiency testing is regulated both by

CAP - College of American Pathologists and
ASHG - American Society of Human Genetics

Attend continuing educational opportunities

All certified cytogenetic technologists must recertify with NCA after the first four years of certification and then every three years after that if they plan to recertify through continuing educational credits. NCA requires the equivalent of 36 hours of continuing education every three years.

Maintain acceptable turnaround time commitment

According to the College of American Pathologists (CAP) 90% of cytogenetic specimens should be completed within 28 calendar days.

Acceptable turnaround times for any given laboratory depends on a multiple of factors

type of tissue
reason for cytogenetic study
patient diagnosis

Conduct monthly review of quality indicators to ensure problems were identified and corrected

Quality indicators are those factors which can indicate a decrease in the overall quality of laboratory testing. In cytogenetic laboratories typical quality indicators include the following:

Banding Quality - includes not only morphology and staining quality, but also includes band level.
Mitotic yield
Turnaround time
Total culture failure (CAP standard is no more than 10%)

On site laboratory inspections by an accrediting agency

Cytogenetic laboratories are inspected on-site by CAP every two years. This is a joint inspection with the American College of Medical Genetics. Prior to this inspection the laboratory must perform the following self-inspection:

Complete the Laboratory General Checklist as well as the Cytogenetics Checklist
Have a hematopathologist correlate the cytogenetic report with the patient pathology.
Document all errors that have occurred.
Maintain records of equipment maintenance.
Document all procedures in the procedural manual.
During the on-site inspection the inspection team is looking for any deficiencies in laboratory performance. These deficiencies fall into three main categories:

Phase 0 deficiencies - These deficiencies do not occur in areas essential in the management of a laboratory and represent questions for information only.
Phase I deficiencies - These deficiencies occur in areas essential in the management of a laboratory but need not be corrected before accreditation is granted. (They may not seriously affect the quality of patient care or significantly endanger the welfare of a hospital or laboratory worker.
Phase II deficiencies - These deficiencies occur in areas essential in the management of a laboratory and must be corrected before accreditation can be granted. These deficiencies may have a serious effect on the quality of patient care or may affect the health and safety of hospital or laboratory personnel.
In the intervening years the laboratory does a self-inspection according to the standards and guidelines of CAP. This includes both the Laboratory General Checklist as well as the Cytogenetics Checklist.

The Joint Commission on Accreditation of Healthcare Organizations (JCAHO) may also inspect the cytogenetic laboratory. Generally though the JCAHO is involved with direct patient care issues and inspects other areas of the hospital.

Maintain culture failure log

It is very important to monitor culture failures. The culture failure log should be maintained daily and reviewed on a monthly basis.

Slides marked as insufficient metaphases by technologists should be reviewed by a senior technologist to ensure that metaphases of acceptable quality were not missed during slide screening.

All culture failures should be investigated and evaluated based on:

Patient diagnosis
Patient treatment
Specimen transport & quality
Number of cultures set-up
Problems during culture prior to harvest

Maintain contamination log

A contamination log should be maintained for all longterm cultures. This includes amniotic fluid cultures, abortus tissues, skin biopsies, chorionic villus samples, and solid tumors. Cell culture contamination directly impacts the quality of results and the number of culture failures.

Maintain a record of lot numbers of reagents and media

This record must be maintained in order to troubleshoot general trends in specimen quality. Some reagents, such as fetal calf serum, vary greatly from lot to lot.

Verify reagent performance

New lots of reagents should be tested against the old lot before being used for processing patient samples. For example, new media should be tested both for sterility and growth.

Question 24

Ethical and Professional Standards

Answer 24

Logo Clinical Cytogenetics
Module 7: Laboratory Practice
Ethical and Professional Standards
Demonstrate an attitude of responsibility and respect toward patients.
Patient confidentiality is the most important way to show respect toward a patient. Also important are testing patterns (determining which tests should be performed) and genetic counseling (providing accurate and unbiased information).

Demonstrate a respectful and cooperative attitude toward professional colleagues.

Cytogenetic laboratories should have a mission statement that includes employee training in applicable state and federal laws in regards to sexual harrassment, violence in the workplace and the Americans with Disabilities Act.

Demonstrate an honest and forthright manner in carrying out professional tasks.

New employee orientation should incorporate laboratory expectations in regards to timeliness, absenteeism, accuracy and the required level of professional competence. This orientation should initiate an ongoing process in competency testing within the laboratory. CLIA 1988 requires that laboratory professionals be competency tested once a year for ongoing testing procedures and to be competency tested twice a year whenever there is a procedural change.