PMKrein
30/03/98

NOTE: The following procedure is to be performed wearing laboratory coat, gloves, eye protection, and mask.
The bronchioalveolar lavage (BAL) must be processed immediately after lavage.

Before beginning, identify the patient ID. This should consist of the letters IGFB98 and the next available numeral in the database.

1.) Go to ICU with ice, pipettor, and sterile serological pipets for aliquoting BALF.

2.) ICU Doc's will collect BALF in sterile traps (tri-trap assembly). One sterile, unopened collection trap will go to Microbio/Virology/Cyto. Leave this aliquot with the ICU nurses attending the patient.
MEASURE AND RECORD TOTAL FLUID RETURN FOR BRONCHOPIST

3.) Place the two remaining traps on ice. Open one and pipette 1ml BALF into sterile tube. This aliquot will be left with ICU nurse--this sample is for BALF BUN determination.

4.) Be sure that ICU nurse has drawn 3 red/tiger top SST (serum) blood tubes. One is for BUN determination (leave with ICU nurse) the two remaining tubes are to be placed on ice, and serum stored in the lab.
5.) After procedure, wait for Bronchocopist to fill out paperwork, obtain a copy immediately and transport specimens back to the lab for processing.

6.) Once in the Laboratory, pool two traps of BALF, record volume obtained for Lab studies.

7.) Filter lavage sample through double layered sterile gauze pads into sterile 50ml falcon tube to remove mucus from the lavage fluid. Record volume after filtering, as some fluid loss is expected.

8.) Remove 2ml from each filtered lavage sample, and place into 6ml tube. The remaining lavage should be centrifuged at 1200rpm for 15 minutes with brake.

9.) While the fluid is spinning, prepare a 1:10 dilution of BALF by mixing 10µl of lavage fluid from the 2ml saved above with 80 l RPMI + 10 l Trypan blue. Load 10 l into each chamber of the hemocytometer. Obtain cell concentration by counting counting five squares in each chamber of the hemocytometer. Maintain two counts, one viable, one non-viable.

10.) Calculate and record cell concentration as follows:

(Total cells counted/5 squares counted) x 10 x 4000 = cells per ml
Do this for each chamber and average the numbers.
Calculate and record total cell number by multiplying "cells per ml" by the volume of BAL after filtering (less 2ml fraction removed).
Cell viability will be recorded as percent viable:
(non-viable cells / total cells) x 100

11.) While lavage is spinning, label tubes for freezing lavage sup. Prepare the following:
a.) Ten NUNC 1.8ml cryovials. Label tubes with:
Patient initials
Patient ID(IGFB98###)
Hospital number
BALF
Date
b.) 2-5 (as appropriate) 15ml Falcon tubes.
Label as above.
12.) Remove lavage from centrifuge, pour sup. into 50ml Falcon, place the sup. on ice.

13.) Resuspend cell pellet in 5ml 37 C RPMI + 1%penicillin/streptomycin (P/S).

14.) Add sufficient RPMI to bring cell concentration to 1.0x106 cell/ml. Aliquot 2ml (therefore 2.0x 106 cells) into each well of a 6 well plate. Place into incubator for 45 minutes to allow Macrophage to adhere.

15.) Aliquot BALF into tubes. Put 1.5 ml sup into each of ten cryovials, store the remaining sup in 10ml aliquots in the 15ml conical tubes.

16.) Place all aliquoted sup. into labeled Sarsted box, immediately place into -70 C freezer. (new freezer, drawer#3)

17.) Label 12 slides with the following information using a #2 pencil:
Patient initials
Patient ID(IGFB98###)
Hospital number
BALF
Date
Set up slides with cytofunnels in cytospin.

18.) Compute volume needed for 50,000 cells per slide for each RML slide.
(Y x 104cells/ml) / (5x104cells/slide) x 1000µl/ml = X l/slide
where X equals µl to give 50,000 cells/slide.
and Y equals cell concentration from Step 6.
Multiply X l/slide from above by 12. Remove this volume from 2ml BAL aliquot.
bring total volume to 6ml with RPMI+P/S in separate tube. Mix well then load 500µl into each cytofunnel.

19.) centrifuge slides in cytospin at 600rpm for 10 minutes

20.) Centrifuge serum tubes at 2500rpm for 10 minutes at 4 C. Blood tubes should have remained on ice at least 30 minutes to allow blood to coagulate.
Prepare 2 NUNC cryovials labeled with the following:
Patient initials
Patient ID(IGFB98###)
Hospital number
Serum
Experimenter initials.

21.) Aliquot serum into two tubes.

22.) Clean up lavage room, and any other space used. Place completed lavage worksheet and Bronchopist data sheet into notebook.

23.) Remove all slides from the cytospin apparatus discard funnels, and allow to air dry at least two hours. After dry, stain two slides with Hemacolor stains as follows:
25 seconds in fixative #1
15 seconds in eosin Y (red) #2
15 seconds in thiazine (blue) #3
rinse in distilled water

All remaining slides are fixed. At least two slides should be fixed in each of the following ways: Methanol, Acetone, Formalin. Indicate on the slide with #2 pencil how slide was fixed.

Differential Mount cover slip using Entallan solution with small amount of Xylene added to reduce viscosity. Store stained covered and Fixed uncovered slides in slide boxes. Label box where patient samples are located.