Unfortunately this program was written in DOS and will not work in Windows. If anyone can transpose it, please let us know.

EVANS BLUE COMPUTER PROGRAM

INCLUDING REPEATED PLASMA VOLUME DETERMINATION:

RUN THIS PROGRAM BEFORE:—

1) You use your Evans Blue Program for the first time.
2) If you use a new batch of Evans Blue Dye.
3) If you change or adjust the spectrophotometer.
4) If you change your method of hematocrit determination. (e.g. peripheral central blood sampling ).
5) When you want to see the current constants, important information on procedures or addresses.

PLEASE NOTE:

The Evans Blue program and the methods have been tested extensively. However, neither the author nor the seller takes any responsibility for any errors, costs, or inconvenience that the program or the information might cause. However, the author appreciates comments and will be happy to discuss, as one scientist to another, any problems which might arise. During the setup you will be asked to enter in certain constants. The constants must be determined specifically on your equipment and with your own Evans Blue solution.

In the following, you will get instructions about how to find the constants. No inputs will be effective if you interrupt the program.

THE SYSTEM CONSISTS OF THREE FILES

EVANSET.EXE ---  The setup and information program.

EVAN.EXE    ---  The Evans Blue Program file.

EVAN.DAT    --- The data file that holds constants needed by the Evans Blue Program and is made/updated by this setup program.

IMPORTANT: These three files must be located together in the same directory. Further, this must be the active (default) directory when you run EVAN.EXE or EVANSET.EXE.

       YOU CAN RUN THIS PROGRAM IN TWO MODES

1.  The information Mode
If you do not know your constants, skip the input requests and select option 1. at the next page then go through the information provided. 

2.  The Update Mode.
 Re-run this program when you have determined the constants carefully. Now select option 2 on the next page, to enter the constants to be saved/updated on the disk.

 Select 1 or 2.

             1. Information and recipes.

             2. Update program constants.

              You selected information mode

CURRENTLY THE CONSTANTS ARE SET TO:

Spectrophotometer:  No Name

Blank-D740-D620-correlation slope    (BLANK_A): 0.000

Blank-D740-D620-correlation intercept (BLANK_B); 0.000 units

Specific Extinction:  (SPEC_EXT): 0.00 units/mg/ml

Evans Blue batch number:  No number

Injection concentration:  0.0 mg/ml

Body-vein-hematocrit ratio:  0.00

Hematocrit plasma trapping correction: 0.00

NOTE: I could not find EVAN.DAT

Later, re run this program in Update Mode to make a new EVAN.DAT. EVAN.DAT must be in the ACTIVE directory along with EVAN.EXE and EVANSET.EXE.

THE BLANK-D740-D620-CORRELATION LINE

1. Measure the optical density at 620 nm (D620) and at 740 nm (D740) for as many blank plasma samples as possible.
2. Plot the data with D740 on the X-axis and D620 on the Y-axis.
3. Find the slope (BLANK_A) and Y-intercept (BLANK_B) of the regression line.

THE SPECIFIC EXTINCTION

 Optical extinction at wavelength 620 nm per mg/ml of dye.

1. Place a 2 ml syringe on a scale and zero the scale.
2. Fill the syringe with 2 ml of Evans Blue solution.
3. Place the syringe on the scale and record the exact weight of solution. In this example we assume a weight of 2.000 g.
4. Empty the syringe into a cylinder with 0.9% saline, flush the syringe.
5. Fill to 250 ml (250.0 g) with 0.9% saline and mix. If the start concentration was 5 Mg/ml, the new solution is 0.040 mg/ml.
6. Take 5 g of the new solution into a 50 ml cylinder using the method described above under points 1-4.
7. Add 10 ml plasma.
8. Fill to 50 g with 0.9% saline and mix.

 You now have a standard solution which - in this example - contains: 0.0040 mg/ml Evans Blue dye  ( = STD_CONC ) 20% plasma. Now calculate your own, exact standard concentration (STD_CONC).

IMPORTANT: The plasma assumes that all the dye is bound to albumin so that the absorption spectrum has the same shape as in the blood plasma. You could take the standard solution in a more direct way - which is more accurate and simple, but more voluminous.

A. Take 1 ml (1.000 g of solution) Evans Blue in at least 250 ml plasma.
B. Fill to 1250 ml with saline.

9. Measure D620 and D740 for the standard solution.
10. Calculate the corrected 620 nm reading (E620) using the formula -

 E620 = D620 - (BLANK_A x D740 + BLANK_B)

You already found BLANK_A as the slope and BLANK_B as the intercept for the correlation line between D740 and D620 for BLANK SAMPLES.

11. Calculate the specific extinction (SPEC_EXT):

 SPEC_EXT = E620 / STD_CONC Units/mg/ml

12. Repeat the whole procedure (1-11) several times and find the average.

 (In our laboratory the specific extinction is currently 84.34 units/mg/ml)

 BLANK_A, BLANK_B and SPEC_EXT are spectrometer dependent.

 SPEC_EXT is Evans Blue batch dependent.

BODY-VEIN HEMATOCRIT RATIO

The Evans Blue program can calculate the blood volume from the plasma volume and the whole body hematocrit. However, the whole body hematocrit is lower than the hematocrit in venous blood. If you measure the hematocrit in venous blood, you have to assume a body-vein-hematocrit ratio (BH_VH_RATIO). BH_VH_RATIO for humans is often set at 0.91 in literature, but it depends on the experimental circumstances in a very complicated way. Consider the BH_VH_RATIO here to be the ratio between the body-hematocrit under the actual, experimental circumstances and the hematocrit in the actual type of blood (e.g. peripheral venous, mixed venous, or arterial blood).

HEMATOCRIT PLASMA TRAPPING CORRECTION

 If you determine the red cell fraction using a hematocrit centrifuge, you need to correct for plasma trapped between the cells. The exact correction factor (TRAPFACT) depends on the centrifugation and should be determined in your own laboratory. Alternatively, it can be assumed to be 0.96.     

 Other methods determine the red cell fraction without plasma trapping. e.g. Tapfact.

AFTER THAT you will be ready to type in your constants:

Re-run this program and select 2. Update program Constants. For now, read the information on the following pages.

HOW TO PREPARE AN EVANS BLUE INJECTION SYRINGE

1. Place an empty 3 or 5 ml syringe on a laboratory scale.
2. Zero the scale.
3. Fill the syringe with the desired volume of Evans Blue (e.g. 3-5 ml, 5 mg/ml)
4. The scale will now show the exact weight of Evans Blue solution. This weight is used in the program as the injected Evans Blue volume. It is o.k to use the weight because we did the same, when we made the standard solution to find the specific extinction.
5. From now on, be sure not to loose any of the dye from the syringe.

HOW TO DETERMINE ONE PLASMA VOLUME

1. Collect at least one pre-injection blood sample if the subject has had Evans Blue injected within the last 1-2 months. The program simply averages the dye contents in pre-injection samples. For that reason, take the sample(s) immediately previous to the injection. NOTE: Pre-injection samples have NEGATIVE sample times (e.g. -1 minute). No sample is needed previous to the very first Evans Blue injection. It is of course, very important that all the dye in the syringe is injected into the blood stream of the subject.

2. The syringe is emptied through a 3-way stopcock on the injection catheter. Start the stop watch immediately the piston reaches the bottom (T - O min ). This is the point of time at which the plasma volume is determined.

3. The stopcock is turned, saline is aspirated from a reservoir into the syringe, the stopcock is turned back and the saline injected to flush the stopcock and the catheter.

4. Step 3 is repeated until the saline is colorless, typically five times. The injection procedure ( steps 2 -4 ) should take less than 15 seconds.

Flush with the syringe, not the drip, to include the stopcock's injection piece in the flush and to keep track of the injected volume.

5. Collect post-injection samples, fx. at 5, 7, 10, and 15 minutes. Post-injection samples should be collected from another catheter. Withdraw blood to clear the catheter immediately before EACH sampling. It is important to record the exact sample times (e.g. 5.25 minutes).

6. Spin the samples at say 3000 RPM for 20 minutes.

DO NOT freeze Evans Blue plasma samples because when plasma is frozen, thawed and re -centrifuged, an unpredictable amount of dye will be captured in the precipitant.

7. Transfer the plasma to photometer-cuvettes and measure the absorption at 620 nm and 740 nm. ( Caution: a cold cuvette may mist up ). The program corrects the 620 nm reading for turbidity by means of the 740 nm reading. For this reason, it is important to measure at the two wavelengths "simultaneously" to have the same particles and impurities in the optical window. Our method is to go rapidly back and forth between the two wavelengths three times, then use the average for each wavelength.

8. You are now ready to run the Evans Blue Program.

THE DYE DISAPPEARANCE CURVE

The Evans Blue Program assumes that the dye concentration declines exponentially. For this reason you want your subject to be stable, which means that physiological changes, liquid infusion etc, do not violate the assumption about exponentially.

Make it a habit to check the dye disappearance/dilution curve on the graphic display. It gives you a qualitative impression of how your data points are related to the selected exponential curve. In this way you can always see if the assumption is valid under the actual experimental circumstances and quite theoretically, if you made any errors. For each determination we usually collect blood samples -1, +5, 7, 10, & 15 minutes after the injection.

 We take the 5 minute sample to get the dye concentration as early as possible, but after the mixing has completed.

 Because the early, steep part of the curve has a big influence on the back calculation to the injection time, we like a data point at 7 minutes as well. Multiple sampling makes the method less vulnerable to random errors.

More information about the method and the program is contained in:

REPEATED PLASMA VOLUME DETERMINATION WITH THE EVANS BLUE DYE DILUTION TECHNIQUE: THE METHOD AND THE COMPUTER PROGRAM. 

N.Foldager/C.G.Blomqvist

Printed in Computers in Biology and Medicine 21(1/2): 35-41 1991