Blood Culture Setup / Harvesting

Question 1

Dividing cells could be stopped at the cell cycle in which phase for chromosomes?

Answer 1

M-Phase

Question 2

Which mitogen does cytogenetics uses?

Answer 2

PHA

Question 3

As a mitogen what does PHA stimulate?

Answer 3

T-cells

Question 4

Between how many hours can peripheral blood cultures stimulated with PHA can be used for chromosome preparations?

Answer 4

48-96 Hours

Question 5

Two tubes of samples were received, one in EDTA and one in sodium heparin; where does each one belong?

Answer 5

The sodium heparin tube goes to cytogenetics and the EDTA tube goes to the molecular lab.

Question 6

What does not have a buffer capacity?

Answer 6

L-Glutamine

Question 7

A magenta color of media is an indication of what?

Answer 7

That it is Basic

Question 8

Combined acetic acid and methanol can do what?

Answer 8

Fix/harden the cell membrane

Question 9

A hypotonic solution can do what?

Answer 9

Swell cells

Question 10

The results of hypotonic treatment depend on all of the following:

Answer 10

Composition
Degree
Length of exposure
Temperature

Question 11

What is excluded, or does not affect hypotonic treatment/chromosome spreading?

Answer 11

The amount of hypotonic/the number of washes with hypotonic.

Question 12

True or False? Cells should be left in the first full fix for some time to allow them to harden before additional fixative washes.

Answer 12

True

Question 13

True or False? Cold fixative will lead to poorer chromosome morphology.

Answer 13

False, cold fixative can lead to better morphology.

Question 14

True or False? Fixative can be stored for days or weeks at room temperature before being used.

Answer 14

False, fixative should always be made fresh.

Question 15

What is the proper ration of Carnoy’s fixative?

Answer 15

1:3, acetic acid:methanol

Question 16

Which step of the harvest procedure makes cells more resistant to damage from centrifugation?

Answer 16

Addition of fixative to the hypotonic

Question 17

Which of the following biological safety cabinets are recommended for use in cytogenetic laboratories?

Answer 17

Biological Safety Cabinet Class II

Question 18

Mitotic arrestants prevents cells from entering what phase?

Answer 18

Anaphase

Question 19

What is the concentration of KCL (hypotonic solution) most often used for harvesting?

Answer 19

0.075 KCL

Question 20

What stops hypotonic action?

Answer 20

Carnoy’s Fixative

Question 21

What are the 4 major steps of blood harvesting?

Answer 21

Adding Colsamid
Adding Hypotonic Solution
Adding Pre-Fix
3 cycles of Fixative

Question 22

What temperature is the incubator set for blood culture incubation?

Answer 22

37C

Question 23

How many ml of blood and medium do you use to create a culture?

Answer 23

9 ml of PB-Max medium to 1 ml of blood specimen.

Question 24

In cytogenetics the anticoagulant of choice for peripheral blood is?

Answer 24

Sodium heparin

Question 25

When dropping slides the majority of chromosomes appear to be short. What is the possible reason?

Answer 25

A long exposure to Colcemid

Question 26

What supplement in culture media is NOT stable and needs to be added into the media freshly before the culture?

Answer 26

L-glutamine

Question 27

The most commonly used PHA in cytogenetics?

Answer 27

PHA-M

Question 28

What pH range is optimal for cell growth?

Answer 28

7.0-7.4

Question 29

True or False? When doing hypotonic treatment, the optimal time should be the same with different cell types and tissue.

Answer 29

False

Question 30

Peripheral blood is used for determining what kind of karyotype?

Answer 30

Constitutional

Question 31

True or False? Peripheral blood is used for the determination of the acquired karyotype of patients with hematologic malignancies or solid tumors for comparison abnormalities in their neoplastic cells.

Answer 31

False, it's constitutional karyotypes.

Question 32

What mitogen is used to stimulate B-cells?

Answer 32

LPS

Question 33

What kind of system is used for peripheral blood and bone marrow cultures?

Answer 33

Closed system

Question 34

What is the difference between open and closed culture systems?

Answer 34

Open culture system has vessels that remain open to the environment, closed culture system culture vessels that are tightly closed so there is no free gas exchange.

Question 35

What are the steps to unpacking a biological sample?

Answer 35

Always wear PPE. Use the safety cabinet. Identify the specimen type and reason for referral. Document everything, including suspect conditions. Always wash your hands before leaving the lab.

Question 36

If 0.75M KCL is used in harvesting instead of 0.075M, what will happen?

Answer 36

The cell will shrivel up.

Question 37

After a successful harvesting, what color of cell pellet should be?

Answer 37

White.

Question 38

What is the function of the fixative solution?

Answer 38

- Kill the cells (precipitates the free hemoglobin) - Fixes the cell membrane - Removes the H1 fraction of the histone proteins from the chromosomes

Question 39

Which white cells are used for cytogenetic analysis?

Answer 39

Lymphocytes - T cells - B cells

Question 40

What buffer is used in cytogenetics?

Answer 40

Sodium bicarbonate

Question 41

Why is a Class II Biological Safety Cabinet used in cytogenetics?

Answer 41

Because it protects both the specimen and | worker from contamination.

Question 42

What are the major steps to a blood culture setup?

Answer 42

- Use a sterile pipet to transfer 9 ml of culture, and 1 ml of sample, into a 25 ml culture flask. - Close cap tightly and mix. - Put the culture flask in 37C incubator, incubate for 72 hours.

Question 43

What is the purpose of harvesting?

Answer 43

- Stop the dividing cells at metaphase stage - Get rid of red cell and only keep white cells - Prepare for chromosome spreading and banding

Question 44

What is Colcemid?

Answer 44

Mitotic inhibitor, stops the cells in metaphase by disrupting the spindle fibers.