Flashcards in Lab exam Deck (13):
What is important to remember when you enter the lab?
1: Put away your coat and bag in the locker.
2: Put on a lab jacket
3: ANY practice involving blood. Use gloves the whole time?
How to you take a blood sampe?
Put on gloves!
Ask the subject to wash his/her hands with warm water and soap
Use ehtanol soulutin to wash the subjects hand
Stick a needle
Remove the first blood drop
Everything that has been in contact with blood goes into the yellow bucket
How do you perform RBC counting? Whats the normal value for RBC?
Gloves! 1 ml of Hayem solution with 10 uL of blood (100x dilution). Mix well!
10-20 uL of this mix into the Burk chamber. Count 40 small squares. Remember to only count which are one the lines on the upper and left corner.
Take the number of RBC you counted and multiply with 10 000 (100x x 100x for the dilution, one for the change to ml, the volume in one small square is 0,01 uL)
Normal range: 4-5,5 x 10^6/uL
How do you perform WBC counting? What is the normal range of WBC?
Gloves! Use Turk solution (stain WBC). 10 uL blood with 90 uL Turk solution (10x dilution). Shake well.
Put 10 uL into the Burk chamber. Count 25 large squares. Take the # you get and multiply with 100 (10x x 10x).
Normal range: 6-8000 cells/uL
How do you perform "Leukocyte differential count on peripheral blood smear"? What is the normal range of the different WBC?
Gloves. Collect blood. Smear the blood on a microscope slide. Use the edge of a different microscope slide to “drag” the blood sample across the slide.
1: Let the slide dry
2: Put into May-Grunwald solution for 3 min (basophil staining)
3: 1 min in diluted May-Grunwald
4: Giemsa solution for 10 min (eosinophilic staining)
5: Wash with distiled water
Count 100 WBC. Zig-Zag.
"Never let monkeys eat banas"
Monocytes: 4-8 %
Eosinophils: 2-4 %
Basophils: 0-1 %
How do we measure "Transport rate on RBCs"?
Referance values: at 620nm
1: 3 ml of water with 100uL of blood. This hypotonic solutin cause hemolysis. This is used as the zero referance
2: 3 ml of saline with 100 uL of blood. This does not do anything with the RBCs. Used as the maximal absorbance value.
Tests: Not that the blood are pipeted into the cuvets right before measuring when the cuvet is IN the spectrometer.
Measure absorbance every 10 second. All solutions have 3ml + 100uL of blood.
1: 300mM of glycerol (cause lysis via AQ-3)
2: 300mM glycerol + 30mM CuSO4 (block AQ-3, less lysis)
3: 300mM + 30mM CuSO4 + 100uL 10 mM of EDTA (block Cu2+ effect on AP-3)
4: 300mM Urea (UT-B transporter, cause lysis)
5: 300mM Glucose (moderate lysis, GLUT-1, low transport rate)
6: 150mM NH4Cl (NH3 to the RBC, Cl-HCO3 exchanger, lysis, chloride shift to maintain pH)
7: 150mM NH4Cl + 100uL 5mM (or 150mM) of NaHCO3 after 1 min (The sodium bicarbonate helps facilitate protons forming NH4 + inside the cell, and so after it’s added, you should see more cell lysis than with just NH4Cl (NH3 keeps diffusing in).
DIDS treatet blood should be pre treated with 10uL of DIDS into 300 uL of blood 10 min before.
10 uL of DIDS after 1 min
DIDS can inhibit Cl-HCO3 exchanger
How do we perform one-sided blood test?
Gloves. 3 drops of saline on the 4 labels. Anti A, Anti B and Anti A-B and one without as the control.
Take one drop of blood from each corner of the glass and add it to the solution. Remember to stir.
Tilt the plate back and forth at a 30 degree angle for 3-5 minutes, observe for coagulation
The reactions of coagulation correspond with the blood type. For example, if the anti- A antibodies attacked the blood sample and it coagulated, then the blood cells have the A antigen and the blood type is either type A or AB. Type O blood should not react with any of the antibodies.
Which Ig does A-B-O and Rh have?
Rh: IgG (can cross the placenta and attack the featus)
How do you perform a Rh (D) blood test?
Gloves. Put a drop of Anti-Rh1 (anti-D = IgG) seum on the one side of the glass and one drop of control M reagent (control)
Add blood the the sample by the use of a glass thingy.
Person is Rh+ if there is agglutination in the Anti-Rh serum but not in the control M reagent. The person is Rh- if there is no agglutination in the Anti-Rh serum. There should not be agglutination in the control M reagent, but if there is, it means the sample is bad.
How do you determine the ABO blood by the two-sided method?
There is a tube of an unknown centrifuged blood sample with serum on top + coagulated blood at the bottom. Pipette out just the serum from the tube and place it in another empty tube. It will later be used as a control on one plate and a source of antibodies on another plate.
Theory: The difference between “plasma” and “serum” is that plasma contains both serum and
clotting factors because it is removed from blood that has been treated with anticoagulants +
then centrifuged. Serum is removed from blood samples that are allowed to clot before
centrifuging and thus all the clotting factors have been used up in the clotting process and no
longer remain in the serum.
Stir the RBC solution. Add 1 ml of saline with 3 drops of unknown sample into an emty tube.
PLATE PREPARATION: There are two plates on the table: one with labels for antibodies and another with labels for blood types.
1: On the antibodies plate, under the appropriate labels, add a couple drops of anti-A, anti-B, anti-AB and the serum you previously set aside.
2: On the blood types plate, add a couple drops of the pre-prepared known A, B and O cell suspensions under the labels.
This test checks for the presence of antigens in the unknown diluted cell suspension sample (via the antibodies plate) as well as the presence of antibodies in the unknown serum (via the blood types plate) and is thus “two-sided” and more accurate.
How do we perform acid-base with the Siggaard-Adnersen nonogram? Normal values of pH, HCO3, Pco2 first
Pco2: 40 mmHg
HCO3: 22-26 mM
First plot the points with the x/y coordinates of the give pH/Pco2 values and draw a line between them. This is the so-called isobicarbonate line.
Standard bicarbonate: Bicarbonate at normal temperature (37) and PCO2 (40 mmHg). Abnormally high or low values indicate metabolic alkalosis or acidosis, respectively.
Standard pH: pH at normal temperature and PCO2
ECG: Recording and analyzing
Put some gel on the electodes. Right arm: Red
Left arm: Yellow
Left leg: Greean
Rigth leg: Black