Lab Stuff

what are the 3 stain bottles (Staining Blood Smears)

1. methanol

2. eosin

3. methylnol blue

how to stain blood smear ((3 × 3) + 2) (Staining Blood Smears)

1. methanol (fixative) (green bottle)

   1. dip for 1 second x 5 dips

   2. drain excess

2. eosin (red bottle)

   1. dip for 1 second x 5 dips

   2. drain excess

3. methylnol blue (blue bottle)

   1. dip for 1 second x 5 dips

   2. drain excess

4. rinse in distilled water

5. lean tip/monolayer towards to top cause it drys faster

define fixation (4) (Staining Blood Smears)

1. kills and dehydrates blood cells

2. changes refractive index

3. makes cell more permeable to stain

4. causes cells to adhere to slide

define staining (Staining Blood Smears)

contains basic and acidic components which stain different parts of the cells

define washing (Staining Blood Smears)

stops the staining process

what does too blue mean (3) (Staining Blood Smears)

1. smear too thick

2. staining time too long

3. stain and/or buffer too high (basic)

what does too pink mean (2) (Staining Blood Smears)

1. staining time too short

2. stain and/or buffer too low (acidic)

what does too light/pale mean (4) (Staining Blood Smears)

1. staining time too short

2. over washing

3. incorrect stain to buffer ratio

4. pH of stain/buffer too acidic

what do artifacts mean (4) (Staining Blood Smears)

1. stain precipitate may be present if inadequate washing

2. slide not dry prior to staining

3. stain needs to be re-filtered

4. not enough stain used

what does refractile artifacts mean (2) (Staining Blood Smears)

1. shiny areas on cells that occur due to water contamination of stain

2. not allowing slide to dry prior to staining.

define skipocytes (Staining Blood Smears)

can’t accuratly identify so are skipped when counted

what 2 cells are considered skipocytes (Staining Blood Smears)

1. smudge

2. basket cells

what do basket cells mean (Staining Blood Smears)

exploded cells

what can cause skipocytes (Staining Blood Smears)

too much pressure

what are 4 granules (Staining Blood Smears)

1. neutrophilic

2. eosinophilic

3. basophilic

4. azurophilic

what is it (Staining Blood Smears)

lymphocyte

what is it (Staining Blood Smears)

neutrophil

what is it (Staining Blood Smears)

eosinophil

what is it (Staining Blood Smears)

basophil

what is it (Staining Blood Smears)

monocyte

what is it (Staining Blood Smears)

crenated RBC’s

what is it (Staining Blood Smears)

smudge cell

Smear left too long in the Eosin dye(# 2) it will be too —(color) (Staining Blood Smears)

orange

Smear left too long in the Methylene dye (#3) it will be too — — (color) (Staining Blood Smears)

dark blue

what does PBS stand for

Peripheral Blood Smear

what materials are needed for a PBS (4)

1. Well-mixed anticoagulant whole blood (purple top tube)

2. Glass microscope slides

3. Capillary tubes

4. Kimwipes

procedure for PBS (7)

1. wipe microscope slides with kim-wipe

2. Invert the EDTA whole blood sample

3. Fill a microhematocrit capillary tube at least ½ full with the EDTA whole blood

4. Using the capillary tube, place a small drop of blood (2-3mm) at the end of the microscope slide near the frosted end

5. The spreader slide is the 2nd slide and is held at a 30-40° angle

6. Using a thumb and forefinger or just forefinger, anchor the slide to the table and back the spreader slide into the drop of blood

7. Pause once the spreader slide is in the blood to allow the blood to spread across the edge of the spreader slide, then in a smooth motion advance the spreader slide all the way to the end of the sample slide

what is it

application point

what is it

body (thick area)

what is it

monolayer

what is it

feathered edge

Criteria for a Good Smear (6)

1. Thumb print or bullet shape in appearance

2. Minimal visible indication as to where the drop of blood was applied

3. Entire film should cover more than one-half to two-thirds the slide area

4. Keep the portion of the smear away from the edge of the slide

5. Symmetrical, bullet shape

6. Microscopically, should have an even distribution of cells within the monolayer

what is the issue

Tails

what is the issue

Long and narrow

what is the issue

Short and wide

what is the issue

Square

what is the issue

Half-bullet

what is the issue

Holes

what causes holes

1. The slide has dirty or a greasy film from fingerprints or immersion oil.

2. The sample was collected from a patient who recently ate, resulting in the presence of lipids in the blood

what are the consequences of holes

Void areas within the monolayer

how to avoid holes (2)

1. Ensure that slides and work area are clean.

2. If possible, the patient should fast prior to blood collection.

how to avoid half-bullet

To ensure even right-to-left pressure of the spreader slide, standing (vs. sitting) while making the smear may help.

what the consequence of half-bullet

The monolayer counting area is reduced by half.

what causes half-bullet

A half-smear is the result of uneven pressure and contact from the spreader slide

how to avoid a square

Increase the forward motion speed of the spreader slide

what is the consequence of square

Overly large, expanded monolayer where the cell distribution can be compromised.

what causes a square

A square-shaped smear can be seen when moving forward too slowly.

how to avoid short and wide

Lower the angle of the spreader slide

what are the consequences of short and wide

Thick smear with a narrow monolayer making cell identification difficult.

what causes short and wide (2)

1. If the spreader slide is held at an angle greater than 30

2. the blood may not be pushed out over the length of the slide.

how to avoid long and narrow (2)

1. Pause longer when backing into the drop.

2. Slow your speed slightly when moving the spreader slide forward.

what is the consequence of long and narrow

no true monolayer

what causes long and narrow

This results when the blood is spread too soon before it has had the chance to spread along the edge of the spreader slide.

how to avoid tails (3)

1. Ensure the spreader slide remains in contact for the length of the bottom slide.

2. Wipe slides prior to making a smear and examine their quality.

3. Redraw a new sample if clumping has occurred due to a sample collection and handling error.

what consequence comes from tails

Uneven distribution of cells within the monolayer.

what causes tails (4)

1. Lifting the spreader slide up at the end of the spreading motion.

2. Debris present on the slide.

3. Cell clumps present in the sample.

4. Poor quality slides.

define monolayer

Portion of the blood smear where, when viewed microscopically, the red blood cells are evenly spaced

What does the Coag Dx Analyzer do?

Helps detect a patient’s clotting ability

what blood does it use (2) (Coag Dx Analyzer)

1. whole blood (no anticoagulant)

2. citrated whole blood (blue top tube)

what tests does it run (4) (Coag Dx Analyzer)

1. aPTT

2. Citrate aPTT

3. PT

4. Citrate PT

what does aPTT stand for (Coag Dx Analyzer)

Activated Partial Thromboplastin Time

what does PT stand for (Coag Dx Analyzer)

Prothrombin Time

what is it (Coag Dx Analyzer)

action keys

what is it (Coag Dx Analyzer)

start key

what is it (Coag Dx Analyzer)

charge indicator

what is it (Coag Dx Analyzer)

display panel

what is it (Coag Dx Analyzer)

test chamber

what is it (Coag Dx Analyzer)

waste resevoir

what is it (Coag Dx Analyzer)

sample well

what is it (Coag Dx Analyzer)

overflow area

what is it (Coag Dx Analyzer)

test channel

what is it (Coag Dx Analyzer)

optical detection windows

how to enter client and patient ID’s and Species (5) (Coag Dx Analyzer)

1. Insert a cartridge into the test chamber

2. then press the ID key to display the Identity Patient menu

3. Enter Client ID

4. Enter Patient ID

5. Enter species

how to collect and run non-anticoagulated whole blood samples (8) (Coag Dx Analyzer)

1. remove IDEXX coag Dx PT and/or aPTT cartridges from refridgerated storage

2. allow them to come to room temperature. this takes 30-60 mins. do not open pouch prior to use

3. prepare the venipuncture site by shaving fur and cleansing with alcohol. Allow the site to air dry completely, as alcohol interferes with the coagulation assay

4. With the label facing up, insert the appropriate PT or aPTT cartridge
   into the IDEXX Coag Dx Analyzer to warm (30–90 seconds) Push the cartridge all the way in until it stops.

5. Obtain a minimum of 0.2 mL (0,2 cc) blood with a needle and plastic syringe.

6. Immediately dispense one drop of blood into the sample well of the cartridge, filling from the bottom of the well up. This can be done with or without the needle (Figure 4-2).

7. Press the Start key (Figure 4-7). The elapsed time (in whole blood seconds) is displayed until the sample clots. When a clot is detected, the analyzer beeps once. Final results of the test are calculated and displayed on the screen.

8. Remove the cartridge. Results are saved and the client/patient information is cleared so that another patient sample can be tested. Discard used needles, syringes and cartridges in the appropriate waste containers.

how to collect and run Citrated Blood Samples (9 + 2) (Coag Dx Analyzer)

1. Remove IDEXX Coag Dx Citrate PT and/or Citrate aPTT cartridges from
   refrigerated storage and allow them to come to room temperature (15°–30°C/59°–86°F). This takes 30–60 minutes. Do not open the pouch prior to use.

2. Prepare the venipuncture site by shaving fur (if necessary) and cleansing with alcohol. Allow the site to air dry completely, as alcohol interferes with the coagulation assay.

3. Use one of the following methods to collect the blood sample:

   1. Collect the sample directly into a 3.2% sodium citrate evacuated tube and allow the blood to be drawn into the tube until it reaches the tube’s stated draw volume.

   2. Collect the sample in a plastic (not glass) syringe and then use the syringe needle to puncture the stopper of an evacuated tube containing 3.2% sodium citrate. Allow the blood to be drawn into the tube until it reaches the tube’s stated draw volume.

4. Mix gently by inverting the tube at least 4 times. Allow the sample to equilibrate with the citrate for at least 5 minutes before testing.

5. With the label facing up, insert the appropriate Citrate PT or Citrate
   aPTT cartridge into the IDEXX Coag Dx Analyzer to warm (30–90
   seconds) (Figure 4-8). Push the cartridge all the way in until it stops.

6. Again, gently mix the sample by inverting the tube at least four times.

7. Using the disposable plastic pipette provided with the Coag Dx Cartridge, collect the sample from the evacuated tube (Figure 4-9) and then dispense one drop of blood into the sample well of the cartridge, filling from the bottom of the well up

8. Press the Start key (Figure 4-15). The elapsed time (in whole blood seconds) is displayed until the sample clots. When a clot is detected, the analyzer beeps once. Final results of the test are displayed on the
   screen.

9. Remove the cartridge. Results are saved and the client/patient information is cleared so that another patient sample can be tested.

how to maintain CBC analyzers

1. require routine maintenance

2. running of controls

3. quality assurance checks

4. specific start up, shutdown, and maintenance procedures are mandatory;

what sample is needed (CBC Analyzers)

EDTA tube

whats the principal (2) (CBC Analyzers)

1. Laser beams determine the density of solid components and their size.

2. Cells scatter light differently depending on the presence or absence of granules and nuclei; therefore, the degree/direction of light scatter allows for specifying the cell as it is being counted.

what are some CBC analyzers (3) (CBC Analyzers)

1. HM5 Analyzer

2. IDEXX LaserCyte

3. ProCyte

principle of HM5 Analyzer (CBC Analyzers)

Based on the passage of electric current across two electrodes separated by a glass tube with a small opening.

principle of IDEXX LaserCyte (CBC Analyzers)

The analyzer uses laser flow cytometry.

define serology

involves the detection of specific antigens OR antibodies in a patient’s sample

The sensitivity of a test demonstrates the ability to detect — — (Serology)

true positives

The sensitivity of a test demonstrates the ability to detect — — (Serology)

true positives

A test with high sensitivity is able to detect … (Serology)

low levels of antigen in a patient’s sample

A test with low sensitivity will produce … (Serology)

false negatives

The specificity of a test demonstrates the ability of the test to detect and react with … (Serology)

intended antigen in the patient’s sample

ELISA stands for (Serology)

Enzyme-Linked Immunosorbent Assay

ELISA is aka (Serology)

snap test

ELISA tests rely on … and … that occurs in this presence to create a visible positive result (2) (Serology)

1. the binding of specific antigens and/or antibodies

2. a color change enzymatic reaction

ELISA tests detect either the presence of — or — in the patient’s sample (Serology)

antigen, antibody

RIM stands for (Serology)

Rapid Immunomigration

RIM is aka (Serology)

Lateral Flow Tests

what blood stuff can be used in a heartworm test kit (3) (Serology)

1. Serum

2. plasma

3. whole blood

FIV/FeLV combo test uses what blood stuff (3) (Serology)

1. Serum

2. plasma

   1. whole blood

supplies needed for Baermann Technique (10) (Serology)

1. Ring stand

2. Funnel

3. Tea strainer or wire screen

4. Rubber tubing

5. Clamp

6. Cheesecloth or gauze square

7. Pipettes

8. Conical tube

9. Microscope slide

10. Coverslip

Procedure for Baermann Technique (10) (Serology)

1. place the funnel into the ring apparatus

2. attach the rubber tubing to the end of the funnel

3. Place a tea strainer on top of the funnel and line with a couple of layers of the gauze squares

4. Apply clamps at the end of the rubber tubing. Should be watertight

5. Add 15 g of feces to the lined tea strainer

6. Cover with warm water (not hot) until the fecal sample is submerged

7. Allow the sample to sit overnight to allow the larvae to emerge and sink to the bottom of the tubing

8. Release the clamp slowly and collect a few drops into a conical tube

9. Apply one drop to a microscope slide and cover with a coverslip

10. Examine the slide for larvae. Lugol’s iodine may be used to visualize the larvae more easily

what does PCV stand for

Packed Cell Volume

what materials are needed for a PCV test (5)

1. Well-mixed, anticoagulated whole blood

2. Tube sealant

3. Reader card

4. Centrifuge

5. Capillary tubes

what is the procedure for a PCV test (4)

1. Begin with well-mixed, anticoagulated blood.

2. Fill two capillary tubes 3/4 full with blood, and seal one end with clay. Filling a second tube serves two purposes:

3. Place tubes in the groves of the centrifuge, directly across from one another, with the clay pointing towards the outside

4. Close centrifuge lid(s)