Hematology is the study of blood and its components.  For this lab you will have the opportunity to examine characteristics of your own fresh blood if you so desire.  In order to safely include such an activity in a general educational setting, the blood handling policy for the institution must be read and discussed before beginning the lab activity.  You will receive a copy of the policy.  Read it thoroughly and listen to your instructors comments about it.  Sign the statement indicating that you have read and understood the policy, detach the form, and turn it in to the instructor before participating voluntarily in the procedure.

All blood handling activities will take place at one site in the lab under supervision.  Only two students will be at the site at a time, one performing an auto-stick, the other observing and assisting with needed equipment as necessary.  When the activity is finished, the instructor will dismiss the two students, disinfect the table and equipment used, change gloves, and call over the next pair of students.  (Other lab study related to the circulatory system can be performed at individual lab stations while students wait for their opportunity to study fresh blood.  PLEASE DO NOT FORM LINES TO WAIT.  THIS CAUSES CROWDING AND HURRYING WHICH COMPROMISES SAFETY!)

I. BASIC BLOOD TYPING:   You will perform this entire exercise under supervision of the instructor.

 A. Wash your hands with soap and water, dry them thoroughly and report to the supervised blood-handling site.

 B.  Place 3 glass slides on a clean paper towel, prepare 3 separate stir-sticks (toothpicks or applicator sticks), and place 3 bottles of serum antibody conveniently near the slides (one assigned to each glass slide). The serum antibody bottles contain purified agglutinins for reaction with A-type erythrocytes, B-type erythrocytes, and Rh positive-type erythrocytes respectively.  NOTE: You will have to obtain the blood for the hematocrit using the same "stick" as for the blood typing, so read the instructions for hematocrit and collect the equipment accordingly.
 
 C. The instructor will demonstrate the use of the auto-stick devices.

 D. Wipe the tip of a finger (your choice) with an alcohol swab and air dry.

 E. Shake and relax your hand as it hangs loosely at your side so that your peripheral capillaries will dilate.

 F. Perform the autostick quickly, then drop one drop of blood onto each of the three slides. It is alright to touch the drop to the glass.

 G. Discard the lance into the container of chlorine disinfectant.

 H. While you then draw the blood for the hematocrit, your partner can add a drop from each anti-serum bottle to each slide respectively and use the stir-sticks to mix the blood with the anti-serum.  NOTE:  Use separate sticks for each slide, and drop them directly into the chlorine disinfectant  immediately.

 I.  Observe the slides for appearance of dense clumps, indicating a positive reaction with the donor (your) cells.

  NOTE:  In anti-A and anti-B serum, a positive reaction will be evident very quickly and obviously, as these antibodies are quite large and can link with many cells, forming a "lattice" that is very large and noticeable to the naked eye.  For the anti-D(Rh) serum, the antibody molecule is smaller and will form finer clumps with erythrocytes.  The visibility of a positive reaction with anti-D(Rh) is slower developing.  It will take at least a full minute, and is aided by warmth.  Resting the slide in the palm of your hand, or placing it on the light rack will give this extra warmth.  The light rack can also improve visibility.  The clumping of cells in any of the serum samples is known as agglutination.

 J. Based on your observations, identify your blood type with respect to the ABO system and the Rh system:

  Agglutination in anti-A serum only = type A, Rh negative
  Agglutination in anti-B serum only = type B, Rh negative
  Agglutination in anti-A and anti-B only = type AB, Rh neg
  Agglutination in none of the sera = type O, Rh negative
  Agglutination in anti-A and anti-D(Rh) = type A, Rh pos.
  Agglutination in anti-B and anti-D(Rh) = type B, Rh pos.
  Agglutination in all three of the sera = type AB, Rh pos.
  Agglutination in anti-D(Rh) only = type O, Rh positive

NOTE: Quality control is not maintained for the serum used in this lab.  The conditions of storage may alter the accuracy of the serum and thus the results.  This exercise is helpful for understanding the principles of blood types, but is not meant to be clinically accurate.  If you are interested in knowing your blood type officially, or if there is a discrepancy between what you have been told before and the results you obtain today, have your type checked at a licensed medical laboratory.

 K. Discard the slides into the container of chlorine-based disinfectant.

 B. Perform the finger stick as described for blood typing.

 C. After placing drops of blood on the slides for blood typing, place the tip of the capillary tube into the drop of blood.  Blood should flow by "capillary action" into the tube.  Common difficulty with this involves limited blood flow, placing the tube opening flush against the skin of the finger, and the removal of the tube opening from the drop of blood leading to air spaces in the column of blood.

HELPFUL HINTS:  To increase blood flow, "milk" the finger, or hold it well below the heart, elbow extended as possible; make sure there is space between the tube opening and the skin of the finger (within the drop); angle the tube downward so that the capillary action is assisted by gravity; keep the end of the tube enclosed within the drop.

 NOTE:  If the tube is not perpendicular, or if there is too much pressure applied, the tube will break.  If this happens, discard the tube immediately into the chlorine disinfectant and wash your hands.

 G. When all students participating have added their capillary tubes to the centrifuge, the instructor will replace the inner cover, screw it securely into place, close the centrifuge, and turn it on for 5 minutes.

 H. When the centrifuge has slowed to a stop, the instructor will open it, remove the inner cover, and announce to the class instructions for retrieving their tubes.

 I. The spinning action of the centrifuge has caused the heavier components of blood to accumulate to the outer side of the tube. The heaviest components are the erythrocytes, showing as a very dark red segment of the tube.  The leukocytes and platelets accumulate at the interface between the erythrocytes and the liquid component of blood (plasma).  This interface appears as a thin white line known as the "buffy coat".  The plasma appears as a clear, slightly yellowish liquid.  Examine
    these features.

 J. Place the tube in the hematocrit reader device indicated by the instructor.  There is a moveable clear plastic tray.  Choose the center groove of this tray.  Place the tube so that the red line in the tray crosses at the top of the Critoseal plug.

 K. Move the tray so that the top of the plasma (whole blood level) intersects with the uppermost silver/black interface.

 L. Move the bar so that the central black line crosses the buffy coat.
 
 M. Read the number in the window.  This number is the hematocrit (HCT), also referred to as the packed cell volume (PCV), which represents the percent of erythrocytes in whole blood.  Normal readings: for men 40-54; for women 37-47; for children 35-49 (varies with age). Values increase in dehydration or shock; decrease in anemia and hemorrhage.

NOTE: The equipment used in lab is very old, having been used in this setting for 20+ years.  The readings taken from this equipment should never be assumed to reflect clinical accuracy. Variations in speed of the centrifuge can alter results.  If you have curiosity about your true hematocrit, have the test taken officially in a licensed medical laboratory where quality control is maintained.

FOR A LAB PRACTICAL, GIVEN A SET OF ANTI-SERA AND GIVEN A PARTICULAR BLOOD TYPE (A+, A-, B+, B-, AB+, AB-, O+, OR O-), YOU WOULD BE EXPECTED TO PREDICT THE AGGLUTINATION STATUS OF THAT PARTICULAR BLOOD TYPE IN EACH OF THE ANTI-SERA SOLUTIONS.
GIVEN A HEMATOCRIT CAPILLARY TUBE, YOU WOULD BE EXPECTED TO BE ABLE TO READ THE HEMATOCRIT AND TELL WHAT PERCENT OF WHOLE BLOOD IS RED BLOOD CELLS AND WHAT PERCENT OF WHOLE BLOOD IS PLASMA.  YOU WOULD BE EXPECTED TO RECOGNIZE THE EQUIPMENT AND GIVE A FUNCTION: HEPARINIZED CAPILLARY TUBE, CENTRIFUGE, READER.
 
III. HISTOLOGY:  Blood can be treated with a special staining procedure called Wright's stain in order to allow visualization of detail in cell structure for the various blood cell types.

 A. Obtain a prepared slide of blood smear, Wright's stain.

 B. With the aid of the microscope, examine and identify the various blood cell types.
       Use labeled sketches and photographs in your textbook or lab atlas for comparison.

  1. erythrocytes:  The most numerous cell type seen, erythrocytes (red blood cells) appear light gray or beige and do not usually show their normal shape due to the staining procedure.  They appear round in the slide, but in the unstained preparation would demonstrate their bi-concave shape. Their usefulness in a Wright's stain preparation is to serve as a size comparison standard.  The diameter of each erythrocyte is 7-8 microns.  They do not have nuclei.

  2. leukocytes:  These cells are fewer by far than the erythrocytes, but are quite evident since they possess large nuclei which take up the basic stain and appear purplish-blue.

      a.  granulocytes
          (1) neutrophils (many names: polymorphonuclear leukocytes (PMNs), "segs", "polys"):
                Twice the size of erythrocytes, these show a multi-lobed nucleus and a smooth blue
                cytoplasm; the most numerous of the leukocytes; 40-70% of leukocytes
          (2) basophils:  Similar in size to neutrophils, these also have a multi-lobed nucleus.  Their
               distinguishing characteristic is possession of cytoplasmic granules which absorb the basic
                blue stain. Less than 1% of leukocytes.  Since these are few in number, an entire lab group
                may not run across an example.  If you find one, please inform the instructor so that all of your
                labmates can be invited to see it.
          (3) eosinophils:  Similar in size to neutrophils, these also have a multi-lobed nucleus.  Their
               distinguishing characteristic is possession of cytoplasmic granules which absorb the
               acidic red stain, eosin.  2-4% of leukocytes. Since these are few in number, an entire lab group may
               not run across an example.  If you find one, please inform the instructor so that all of your lab
               mates can be invited to see it.
 
      b.  agranulocytes
           (1) monocytes:  2-3 times larger than erythrocytes, these demonstrate a very large
                nucleus, often horseshoe shaped or indented, nearly filling the cell.  3-8% of leukocytes.
           (2) lymphocytes:  Barely larger than erythrocytes, the nucleus is spherical and nearly fills
                the cell. 25% or more of leukocytes. These are quite numerous and easy to spot.

  3. platelets:  Very small cell fragments, these are stained purple and scattered between the erythrocytes.
            8% of formed elements.

 FOR A LAB PRACTICAL YOU WOULD BE EXPECTED TO BE ABLE TO RECOGNIZE AND IDENTIFY A NEUTROPHIL, AN ERYTHROCYTE, PLATELETS, AND LYMPHOCYTES.  IF YOUR LAB CLASS HAS HAD A CHANCE TO OBSERVE A BASOPHIL, AN EOSINOPHIL, OR A MONOCYTE, THESE MAY BE ON THE PRACTICAL. LOOK UP AND LEARN A FUNCTION FOR EACH CELL TYPE.

IV. PHYSIOLOGY:  Early in the course you were shown a demonstration of osmosis, the movement of water across a semi-permeable membrane in response to difference of concentration of solutes on either side of the membrane.  Fresh erythrocytes from a drop of blood were mixed with each of three solutions designed to show normal cells (in the isotonic solution, no net water movement), crenated cells (in the hypertonic solution, movement of water from the cell to the surrounding solution), and hemolyzed cells (in the hypotonic solution, movement of water into the cell from the  surrounding solution resulting in cell  rupture) respectively.  If you did not observe the demonstration, or if you would like to review it, ask the instructor to place slides on the video-microscope and repeat the demonstration.

NOTE:  With the isotonic solution with erythrocytes on the video screen, take a moment to scan the slide for leukocytes.  They are less distinct and less numerous, but by changing depth of focus you can tell that the cytoplasm is rather granular in some, you can notice the dense areas of nuclear material, and in some cases you can notice the shape of the nucleus.  Try to categorize the white cells you are able to find.