Activity 6

RED CELL FRAGILITY, BLOOD TYPING, BLEEDING TIME AND CLOTTING TIME AND HYPEREMIA

Introduction

In clinical practice, hematological information accumulated from a series of blood tests conducted on a small volume of blood - even a single drop - can be of great diagnostic and prognostic value.

There are certain molecules on the surfaces of all cells in the body that can be recognized as foreign by the immune system of another individual. These molecules are known as antigens. As part of the immune response, particular lymphocytes secrete a class of proteins called antibodies that bind in a specific fashion with antigens. The specificity of antibodies for antigens is analogous to the specificity of enzymes for their substrates, and of receptor proteins for neurotransmitters and hormones.

Materials and Methods

A. RED CELL FRAGILITY

Ten (10) test tubes were prepared in serial dilution of NaCl solution in different concentrations with distilled water. A representative was asked to sterile his pointer finger with alcohol in gauze pad. Using a lancet pen, the finger was pricked. Two drops of blood was added to each test tube concentration. Each test tube was covered with parafilm stretching up to the mouth. The blood with salt concentration was mixed in a slow inverting manner of the tube using the middle and thumb finger. Finally the 10 test tubes were all subjected for centrifugation at 1500xg for 2 minutes.

B. BLOOD TYPING

Obtain a clean microscope slide. Using a glass-marking pencil, mark one end A and the other end B. Lance your finger to obtain blood. Clean the palmar surface of the third or fourth finger with a sterile gauze pad soaked with 70% ethanol. With a sterile lancet or needle, make a puncture on a fingertip. Wipe off the first drop. Place one drop of blood on each of the marked slide. Add one drop of anti-A serum to the A side. Add one drop of anti-B serum to the B side. With a toothpick, using a different toothpick for each side. Spread each mixture over an area of about 0.75 in diameter. Observe the slide for any agglutination of red cells.

C. BLEEDING TIME AND CLOTTING TIME

Sterilize the fingertip using rectified spirit and allow to dry. Make a sufficiently deep prick using a sterile lancet, so that blood comes out freely without squeezing. Mop the blood by touching the fingertip with a filter paper. This is repeated every 15 seconds, each time using a fresh portion of the filter paper, till bleeding stops. Note the time. It is seen that the blood stains on the filter paper get smaller to disappear finally when bleeding stops. Under sterile precautions make a sufficiently deep prick in the fingertip. Touch the blood drop at the fingertip using one end of the capillary tube kept tilted downwards. The tube gets easily filled by capillary action. After about two minutes start snapping off small lengths of the tube, at intervals of 15 seconds, each time noting whether the fibrin thread is formed between the snapped ends. Note the time when the fibrin thread is first seen.

D. HYPEREMIA

Wind a rubber band in the chosen finger. Wait for several minutes to note for difference in size and color of the finger. Prepare a 500-ml tap water in a beaker then put it in water bath set at 45 degrees C. Immerse the finger in the beaker for a few minutes to note for sensations other than warmth that the finger was exposed to.

Results

A. Red Cell Fragility

Tube Number

ObservationsColor of Supernatant Presence of RBCs at the bottom of tube

1 Clear +2 Clear +3 Clear +4 Clear +5 Clear +6 Reddish -7 Reddish -8 Reddish -9 Reddish -10 Reddish -

2. If a cell that is normally in osmotic equilibrium is transferred to a more dilute solution, water will enter the cell, the cell volume will increase, and the solute concentration of the cytoplasm will be reduced.

3. If the cell is transferred to a more concentrated solution, water will leave the cell, the cell volume will decrease, and the solute concentration of the cytoplasm will increase.

B. Blood typing, bleeding time and clotting time

Your Sample Anti-A Serum Anti-B Serum Blood TypeAgglutinate Agglutinate AB

Bleeding time 1 minuteClotting time 45 sec

4. ABO typing:

If your blood cells stick together when mixed with:

Anti-A serum, you have type A blood Anti-B serum, you have type B blood Both anti-A and anti-B serums, you have type AB blood If your blood cells do not stick together when anti-A and anti-B are added, you have type O blood.

5. Assessing blood clotting and bleeding factor is very important if you are about to have a surgical procedure. Surgery or an injury of any kind increases the risk of a blood clot. That’s because the clotting process is stimulated as your body attempts to stop the bleeding and close the surgical wound. A clot is normally formed by the blood cells and clotting factors working together to create a protective scab over a healing wound. The surgical procedure may stimulate clots to form in error in blood vessels, which then may block the normal flow of blood.

B.2 Cross-Matching

Serum Sample Red Cell SuspensionAgglutination

Donor: Type ABRecipient: Type A

6. Cross matching is a must before blood transfusion to determine if the donor’s blood is compatible with the blood of an intended recipient. Transfusion errors that result in such agglutination can lead to blockage of small blood vessels and cause hemolysis (rupture of red blood cells), which may damage the kidneys and other organs.

C. Hyperemia

Observations

7.1 Size: swell

Color: dark-purple color

By winding the rubber band on the finger the oxygen level decreases and swells due to blood vessel dilation.

7.2 Size: (+) crease, shrink

Color: reddish

An increase metabolic rate requires a greater flow of blood thus the vessels dilate.

8. Hyperemia

Hyperemia is when there is an increase in blood flow to the tissues and organs. Active or functional hyperemia is an increase in blood flow associated with increased metabolic activity. A greater blood flow is required to increase oxygen delivery and enhance the removal of carbon dioxide and/or lactate from these metabolically active tissues. Reactive hyperemia on the other hand is an increase in blood flow to the tissues when they have been deprived of oxygen.

Discussion

Cell membranes are semipermeable barriers, and osmotic gradients are established between intracellular and extracellular fluids which can cause water to flow into and out of the cells. The amount of osmotic pressure depends upon the difference between the concentrations of non-diffusible ions on each side of the membrane.

Osmotic fragility is a test to detect whether red blood cells are more likely to break down. The intracellular fluid of erythrocytes is a solution of salts, glucose, protein and hemoglobin. A 0.9% NaCl solution is said to be isotonic: when blood cells reside in such a medium, the intracellular and extracellular fluids are in osmotic equilibrium across the cell membrane, and there is no net influx or efflux of water. In the performed experiment, 0.9% NaCl wasn’t exactly used and instead a higher concentration of NaCl was given.

Figure 1

When subjected to hypertonic media (experiment Tube 1-5), the cells lose their normal biconcave shape, undergoing collapse (leading to crenation) due to the rapid osmotic efflux of water.

On the other hand, in a hypotonic environment (Tube 7-10 with greater volume of distilled water), an influx of water occurs: the cells swell, the integrity of their membranes is disrupted, allowing the escape of their hemoglobin (hemolysis) which dissolves in the external medium.

Figure 2. Crenated sample (Tube 1-5) Figure 3. Hemolyzed sample (Tube 6-10)

If as little as 0.5% of the red blood cells are hemolyzed, the released hemoglobin will cause the serum or plasma to appear pale red or cherry red in color. Note that the hemolyzed sample is transparent, because there are no cells to scatter light. On the other hand, the crenated sample after centrifugation formed a cell fractionation where the resulting supernatant is clear and the pellet visible at the bottom looks very pale.

The blood groups refer to the presence on human red blood cells of certain antigens, the blood group factors. One very important group of factors present on the red blood cells is the ABO system. The ABO group of a person depends on whether his/her red blood cells contain one, both, or neither of the 2 blood group antigens A and B. There are, therefore, 4 main ABO groups: A, B, AB and O. The presence or absence of agglutinations will determine the blood type found. This agglutination reaction, which is very important in determining the safety of transfusions is due to a mismatch of genetically determined blood types.

Antibodies (agglutinins) for the antigens A and B exist in the plasma and these are termed anti-A and anti-B. The corresponding antigen and antibody are never found in the same individual since, when mixed, they form antigen-antibody complexes, effectively agglutinating the blood.

Agglutination (clumping) of red blood cells occurs when cells with A-type antigens are mixed with anti-A antibodies and when cells with B-type antigens are mixed with anti-B antibodies. No agglutination would occur with type O blood.

People with type A blood have type A antigens on their red blood cells and antibodies in their plasma against the type B antigen. People with type B blood have type B antigens on their red blood cells and antibodies in their plasma against the type A antigen. Therefore, if red blood cells from one blood type are mixed with antibodies from the plasma of the other blood type, an agglutination reaction occurs. In this reaction, red blood cells stick together because of antigen-antibody binding.

Hemostasis (literally - blood halting) depends on three interrelated and overlapping sets of events:

Constriction of the blood vessels and formation of a platelet "plug"

Blood clotting Clot retraction

Bleeding time is the interval between the moment when bleeding starts and the moment when bleeding stops. Normal bleeding time is to 4 minutes. Measures the time taken for blood vessel constriction and platelet plug formation to occur. No clot is allowed to form, so that the arrest of bleeding depends exclusively on blood vessel constriction and platelet action. It is used to evaluate platelet function and is typically performed, along with a platelet

count, in people with a personal or family history of bleeding disorders, or as a preoperative safety measure before a scheduled surgery.

Clotting time is the interval between the moment when bleeding starts and the moment when the fibrin thread is first seen. Bleeding time and clotting time are not the same. Bleeding time depends on the integrity of platelets and vessel walls, whereas clotting time depends on the availability of coagulation factors. In coagulation disorders like haemophilia, clotting time is prolonged but bleeding time remains normal. The normal range for clotting time is 3-10 mins.

In order for blood to clot, the enzyme thrombin must be generated from the plasma precursor prothrombin. Thrombin then converts soluble fibrinogen into insoluble fibrin. Generation of thrombin involves the sequential activation of a number of other plasma clotting factor, this process is also being assisted by Ca++ and by factors released by platelets and damaged tissues . The time taken for blood to clot mainly reflects the time required for the generation of thrombin in this manner. If the plasma concentration of prothrombin or of some of the other factors is low (or if the factor is absent, or functionally inactive), clotting time will be prolonged. Clotting time is also prolonged in conditions like vitamin K deficiency, liver diseases, disseminated intravascular coagulation, overdosage of anticoagulants etc.

Cross-matching blood, in transfusion medicine, refers to the complex testing that is performed prior to a blood transfusion, to determine if the donors blood is compatible with the blood of an intended recipient, or to identify matches for organ transplants. Cross matching is a must before blood transfusion to determine if the donor’s blood is compatible with the blood of an intended recipient

Figure 4. Cross-matching. A type AB donor mixed with the blood of a type A recipient.

When transfusing blood, it is important to remember that the donor's blood must not contain red blood cells that the recipient's antibodies can agglutinate. Theoretically, then, individuals belonging to blood group O are universal donors, while those of blood group AB are universal recipients. If the types do not match – if the donor is type A, for example, and the recipient is type B – the recipient’s antibodies attach to the donor’s red blood cells and form bridges that cause the cells to clump together, or agglutinate. Transfusion errors that result in such agglutination can lead to blockage of small blood vessels and cause hemolysis (rupture of red blood cells), which may damage the kidneys and other organs.

Hyperemia is when there is an increase in blood flow to the tissues and organs. When blood flow is reduced temporarily, the skin becomes pale. If pressure is applied to the skin, the blood in the vessels of the skin stagnates. Oxygen in the hemoglobin is quickly used by the tissue in the area, and the hemoglobin becomes darker as a result of deoxyhemoglobin formation.

When you obstruct blood flow by tying a rubber band around a finger, it swells and the color of the skin gives a bluish hue, termed cyanosis. Once removed, the skin turns fiery soon after the occlusion is removed. This is known as reactive hyperemia. Reactive hyperemia is an increase in blood flow to the tissues when they have been deprived of oxygen. When blood flow to an area is restricted, the arterioles in that area dilate as a result of the release of chemicals (products of metabolism) by the oxygen-deprived

cells. When blood flow is no longer restricted, blood rushes into the dilated blood vessels as the oxygen level increase.

Active or functional hyperemia is an increase in blood flow associated with increased metabolic activity of an organ or tissue. During the immersion of one finger in a warm water there was an increased in the metabolic activity, where the skin dilated and turned red in color, showing an increase in the blood flow. A greater blood flow is required to increase oxygen delivery and enhance the removal of carbon dioxide and/or lactate from these metabolically active tissues. After few minutes upon immersion a short painful sensation other than warmth was felt which later on adapted on the surrounding temperature. Another good example of active hyperemia is the increase in blood flow that accompanies muscle contraction, which is also called exercise or functional hyperemia in skeletal muscle. Blood flow increases because the increased oxygen consumption of during muscle contraction stimulates the production of vasoactive substances that dilate the resistance vessels in the skeletal muscle.

Conclusion

The primary function of blood is to supply oxygen and nutrients as well as constitutional elements to tissues and to remove waste products. Blood also enables hormones and other substances to be transported between tissues and organs. Blood is also involved in maintaining homeostasis problems with blood composition or circulation can lead to downstream tissue malfunction, that’s why, assessing your blood can greatly prevent health problems.

Literature cited

Sdmesa. Vascular. Retrieved 13 September 2015 from http://classroom.sdmesa.edu/bbrothers/Docs%20235/vascular.pdfa

Health communities. Blood clotting tests. Retrieved on 13 September 2015 from http://www.healthcommunities.com/blood-tests/blood-clotting-tests.shtml

McGill. ABO System. Retrieved on 13 September 2015 from http://www.medicine.mcgill.ca/physio/vlab/bloodlabABO_n.htm

Wikipedia. Hemolysis. Retrieved on 13 September 2015 from https://en.wikipedia.org/wiki/Hemolysis#Outside_the_body

Encyclopedia of Ayurvedic Medical Plants. Bleeding time & clotting time. Retrieved on 13 September 2015 from http://www.indianmedicinalplants.info/articles/BLEEDING-TIME.html

CVPhsyiology. Blood Flow. Retrieved on 12 September 2015 from http://www.cvphysiology.com/Blood%20Flow/BF005.htm