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Author Topic: Cost free method of hematocrit determination and hemoglobin approximation  (Read 3921 times)
greg10
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« on: January 28, 2011, 07:25:22 PM »

A recent study has shown that it is important for hemodialysis patients to have an optimal hemoglobin test of at least 4 times a month.  There are point of care (POC) devices that can measure the hemoglobin level in about two minutes using about 20 ul (0.02ml) of blood, but such devices are not normally provided for the home dialysis patient.  A rapid feedback of a patient's hemoglobin level will allow an optimal titration of the required medication such as EPO and iron.

The following procedure describes a cost free method that allows one to make a determination, in about 10 minutes using commonly available material, of the blood hematocrit levels which is just the volume of red blood cells as a percentage of the total blood volume.  Using the hematocrit number, there is a rough conversion factor of 3 which converts the hematocrit number to approximate hemoglobin level.  For example, if 33% of the blood contains red blood cells by volume, the hemoglobin content would be about 33/3 = 11 g/dL  (one dL or deciliter is 100 ml).*  Normally hematocrit is done using specialized microcapillary tubes and require special sealing procedures and special hematocrit centrifuges.
*this calculation was incorrectly shown in the original posting.



Material and Method

If you are doing home dialysis, you should have access to a clinical centrifuge for blood tests.  All you would need to do a hematocrit test at home would be to centrifuge the blood sample and an accurate way of measuring the ratio of red blood cells to total blood volume after the red blood cells are spun down.  You will need the following items, most of which you probably have already at your disposal or provided by your clinic:

1. Centrifuge, either the 15 ml clinical centrifuge that is usually provided or a microfuge, a micro-centrifuge that usually takes 1.5ml microfuge tubes.

2. Disposable syringe and syringe sheath (SS).  The syringe sheath is the hollow plastic cylinder that protects the syringe needle.  These are common disposable items in home dialysis.  The SS is used to hold the blood sample during centrifugation.  There is a SS adapter that has to be made, depending on what type of centrifuge you have.

3. About 0.2 to 0.4 ml of blood.

4. A digital camera, hopefully one with macro mode which allows a close up picture of the blood sample.  Even inexpensive cameras should have this feature nowadays.

5.  A PC which you can download the picture and do a precise measurement of the sample.  A free on screen utility is provided that will allow you to overlay an on-screen ruler over the picture of the sample, which you can in turn measure and calculate the hematocrit.

As mentioned in item 2, an adapter is required to hold the syringe sheath (SS) and prevent it from being damaged and blood sample loss during centrifugation.  The adapters are easily made by either of the two following methods (a or b):

a. Cut a foam-rubber wine bottle stopper to size, with a taper on one end, drill a 1/4 inch hole that will act as the holder for the SS.  This is shown in the following figure.  The rubber stopper is easily cut with a serrated knife and drilled, but please wear gloves and be careful if you are not handy in this type of procedure.


b. The following is the preferable adapter, but it requires purchasing a few 1.5 ml microcentrifuge tubes and have a 1/4 inch hole drilled through the top cap.  These type of adapters can be used in either a clinical centrifuge or a micro-centrifuge designed to hold 1.5 ml microfuge tubes.  These are not expensive and are usually disposable items in a laboratory.  The advantage of this adapter is that should the SS and blood sample leak during centrifugation, the adapter will contain the spillage.


Blood collection

Please be aware that hematocrit and hemoglobin levels are dependent on the body's hydration level, if the blood sample is from a patient who is overly hydrated, the test result would show a lower than expected level.  Similarly, if the blood sample collected is diluted with saline or other liquid, the end result will show a lower hematocrit number.

If you are doing hemodialysis, there should easily be few tenths of ml of blood left over from the normal cannulation or flushing process from the catheter; only about 0.2 to 0.3 ml of undiluted blood is required.

In order for consistent results to be reached between different sampling, the amount of blood sample used for centrifugation should be of similar volume each time at approximately 300 ul (0.3 ml).  For example, do not try to compare results obtained using 0.1 ml blood sample to results using 0.9 ml.  The syringe sheath is not completely cylindrical but has conical shape to it, therefore the hematocrit estimation will be slightly higher than results from a perfect cylinder.

Centrifugation

Normal centrifugation is observed.  Any centrifuge capable of 1000g for about 5 to 10 minutes is sufficent to spin down the red blood cells.  Do not spin at too high a speed, it would likely break the syringe sheath holding the blood sample.  A low speed micro-centrifuge is an useful addition to this hematocrit procedure.

Measurement

Once the sample has been spun down, remove it carefully from the centrifuge and use the digital camera with a macro mode and turn the camera to the portrait position and take a close up picture of the sample.

Transfer the picture to a PC and view the picture on screen, which will show the sample in the horizontal position.  Run the on screen ruler program and overlay it horizontally above the sample, lining the zero position of the ruler as shown in the following figure.  Measure the total length of the blood sample including the red blood cells and then measure the length of the free plasma on top of the red blood cells.  Subtract the two numbers and divide that by the total length, this will give you the hematocrit.  Divide the hematocrit by 3 will result in the approximate hemoglobin level in grams per deciliter (g/dL).*



*original calculation was incorrectly shown as multiplication by conversion factor.
** tapecalc program is available here.

Results

The measurements of hematocrit using this method has yielded comparable results using the Stanbio Stat-Site Hemoglobin meter, however the numbers could varied by as much as 1 g/dL.  In the above example, Stat-site Hgb resulted in 12.6 g/dL, while the syringe sheath (SS) hematocrit method resulted in 12.86g/dL; using the digital camera picture allows the volume to be determined fairly accurately using a minimal amount of blood sample.   As mentioned above, this hematocrit method may give a slightly higher than expected reading due to the slight conical shape of the SS.   In my opinion the results give useful snapshots of the hemoglobin levels in a dialysis patient.
« Last Edit: January 30, 2011, 09:34:19 PM by greg10 » Logged

Newbie caretaker, so I may not know what I am talking about :)
Caretaker for my elderly father who has his first and current graft in March, 2010.
Previously in-center hemodialysis in national chain, now doing NxStage home dialysis training.
End of September 2010: after twelve days of training, we were asked to start dialyzing on our own at home, reluctantly, we agreed.
If you are on HD, did you know that Rapid fluid removal (UF = ultrafiltration) during dialysis is associated with cardiovascular morbidity?  http://ihatedialysis.com/forum/index.php?topic=20596
We follow a modified version: UF limit = (weight in kg)  *  10 ml/kg/hr * (130 - age)/100

How do you know you are getting sufficient hemodialysis?  Know your HDP!  Scribner, B. H. and D. G. Oreopoulos (2002). "The Hemodialysis Product (HDP): A Better Index of Dialysis Adequacy than Kt/V." Dialysis & Transplantation 31(1).   http://www.therenalnetwork.org/qi/resources/HDP.pdf
tyefly
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This will be me...... Next spring.... I earned it.

« Reply #1 on: January 28, 2011, 09:24:12 PM »

  Wow   thx.....   being a old lab rat.....  I can do this stuff.....   sounds fun......   I have done lab work in a long time.....this should be challenging and fun.....   I will let you know....
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IgA Nephropathy   April 2009
CKD    May 2009
AV Fistula  June 2009
In-Center Dialysis   Sept 2009
Nxstage    Feb 2010
Extended Nxstage March 2011

Transplant Sept 2, 2011

  Hello from the Oregon Coast.....

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