Detection of Fanconi's Anemia (FA)

through the Use of Alkylating Agents


I. Purpose

This test is done to detect DNA repair mechanism defects in patients by culturing their blood along with blood from a sex - matched control using two different alkylating agents which can cause breaks in chromosomes. Slides from both control and patient are screened blindly and then checked for an increase in chromosomal defects compared to untreated blood. The increase in breakage in the patient and control is compared.

II. Culture Procedure:

A. Obtain 2-4 ml of unclotted whole blood aseptically by venipuncture using a collection tube with sodium heparin. Invert the tube several times to prevent clotting. Assign accession number to specimen according to procedure in Laboratory Procedures Manual.

B. Use caution when setting up these cultures as the alkylating agents used are toxic and carcinogenic. Aseptic technique must be used when setting up cultures, preferably under a laminar flow hood. Using tape labels, label three T-25 flasks for both patient and control with the patient number, type of culture (DEB, MMC, Reg), and day of harvest (cultures will be harvested after 3 days).




Pipet 10 ml of complete RPMI-1640 into each flask. Using a syringe add 0.2 ml Phytohemagglutinin soln. Using a sterile pipet add 0.5 ml of whole blood to each culture. Add 40 mcl of Mitomycin C (MMC) stock solution to the labeled cultures. Add Diepoxybutane (DEB) to the labeled cultures. The DEB should be prepared fresh each time as follows. Add 5 mcl pure DEB to 5 ml of sterile dH2O, mix thoroughly. Add 10 mcl of the first solution to 1 ml of sterile dH2O, mix thoroughly. This is the working solution at 11 ug/ml. Add 90 mcl of the working solution to each flask. Dispose of all left-over solutions by washing them down the drain with lots of running water. Seal flasks tightly, mix gently, and incubate at 37 C for 72 hours. Cultures should be gently mixed 2-3 times per day during incubation.

III. Harvest Procedure:

A. 1 1/2 hour before harvest add 65 mcl of colcemid working solution to each culture flask and mix.

B. Put the 0.075 M KCl hypotonic solution in the incubator to pre-warm. Prepare fixative of methanol:glacial acetic acid (3:1). Usually 40 ml of fix will be used per patient.

C. Gently swirl the flasks. Pour the contents of each flask into a 15 ml screw-cap conical centrifuge tube; transfer the tape label making sure no label mistakes occur. Centrifuge the tubes for 6 minutes at 150 x g (900 rpm in the Sorvall GLC-2B centrifuge).

D. Aspirate and discard all but 0.5 ml of supernatant, (note: the white blood cells form a layer on top of the red cells and will be lost if too much supernatant is removed). Resuspend the cell pellet by mixing by hand, do not use a mechanical vortexer as this may break cells, this is especially important in step F. Add 5 ml of pre-warmed hypotonic solution and mix. If necessary, use a pasteur pipet to bubble air through the suspension to completely break up the pellet. Incubate the tubes at 37 C for 15 minutes.

E. Centrifuge for 6 minutes at 150 x g.

F. Aspirate and discard all but 0.5 ml of supernatant. Mix by hand to break up the cell pellet. It is important to get the cell pellet completely resuspended, but cells are fragile and are easily broken.

G. Slowly add 4 ml of fixative (2 pasteur pipetfuls) letting it run down the side of the tube so that it layers on top of the cell suspension. Cap the tube. Mix rapidly by hand so that all the cell suspension is fixed evenly; if it is mixed too slowly, clumps may form. Let sit at room temperature for 15 - 20 minutes.

H. Centrifuge for 6 minutes at 150 x g.

I. Aspirate and discard all but 0.5 ml of supernatant. Resuspend cell pellet. Add 4 ml of fixative and mix. Let sit at room temperature 15 - 20 minutes.

J. Repeat steps H,I 1 or 2 times until the pellet is clean and white. Culture is now ready to make slides. For best results, slides should be made the same day as the harvest. Before making slides the tubes on both patient and control should be blind labeled with new alphabetic labels so that you do not know which tubes are treated or which tubes are the control.

IV. Slide Preparation:

See procedure for regular lymphocyte cultures.

V. Scoring:

A. Stain the slides using standard GTL staining method.

B. Analyze 50 metaphases from each culture if possible, a minimum number of 20 needs to be scored. To avoid bias, choose consecutive cells which do not appear to be broken and which contain chromosomes having well-defined morphology.

C. Score each cell for chromosome number and for the number and type of structural abnormality.

GAPS - Gaps are defined as achromatic areas less than one chromatid in width and are not scored.

BREAKS - Breaks are defined as achromatic areas greater than one chromatid in width and are scored.

REARRANGEMENTS - are defined as dicentric or ring chromosmes and are scored as two breaks.

RADIAL EXCHANGE FIGURES - are tri-radials, quadr-radials, etc., and are scored separately.

D. The data should be expressed as: # of breaks or radial figures/metaphase x 100 (Example: 800% breaks = 8 breaks/1 metaphse.)

VI. Solutions:

Colcemid working solution: 10 mg Colcemid in 250 ml dH2O,sterile filtered, store at 4 C.

Complete RPMI-1640: 100 ml RPMI-1640, 20 ml Fetal calf serum,1 ml Penicillin/Streptomycin solution, 1 ml L-Glutamine solution, store at 4 C.

Diepoxybutane working solution (DEB): Add 5 mcl pure DEB to 5 ml sterile dH2O, mix thoroughly. Add 10 mcl of the first solution to 1 ml sterile dH2O, mix thoroughly (11 ug/ml).

Fixative: 30 ml Methanol and 10 ml Glacial Acetic Acid,prepared fresh.

Hypotonic solution 0.075 M KCl: 2.8 g Potassium chloride dissolved in 500 ml dH2O.

Mitomycin C Stock solution (MMC): Reconstitute 2 mg vial of MMC with 4 ml sterile dH2O. Dilute 1 ml in 24 ml sterile dH2O (20 ug/ml).

Phytohemagglutinin working solution: Phytohemagglutinin (M form) rehydrated with 10 ml sterile dH2O, store at 4 C.

VII. Reagents:

Colcemid: GIBCO cat# 890-3014. 10 mg bottle.

Diepoxybutane: SIGMA cat# D 7019. 1 ml bottle. Store at 4 C.

Fetal calf serum: GIBCO cat# 200-6140. Mycoplasma tested, virus screened, 500 ml bottle, store frozen.

L-Glutamine: GIBCO cat#320-5030. L-Glutamine solution 100X (200 mM), 20 ml bottle, store frozen.

Glacial Acetic acid: AMERICAN SCIENTIFIC PRODUCTS cat# 9508-2. Acetic acid, Glacial ACS (Aldehyde free), 500 ml bottle.

Methanol: AMERICAN SCIENTIFIC PRODUCTS cat# 3016-1. Methyl alcohol anhydrous AR (ACS)(Absolute) Acetone free, 500 ml bottle.

Mitomycin C (MMC): SIGMA cat# M 0503. 2 mg

Penicillin/Streptomycin: GIBCO cat# 600-5140. Penicillin/Streptomycin solution, 10,000 units/ml / 10,000 mcg/ml, 20 ml bottle, store frozen.

Potassium Chloride: COLUMBUS CHEMICAL INDUSTRIES, INC. ACS granular, 500 g lots.

RPMI-1640: GIBCO cat# 430-1800 RPMI-1640 powdered form, prepared to instructions, 10 x 1 liter package.