Extracted Oxalate from Celery vs. Oxalate in Black Top Tubes
I. INVESTIGATORS Antolin, Ma. Liza Atienza, Maria Regina Eden O. Griarte, Kathleen Joyce Lao, Marie Patricia Anne R. Madriguera, Maria Rejelyn S. Perona, Marion Paulo R. Velicaria, Anna Marie
II. INTRODUCTION
a. Statement of the Problem
Is oxalate extracted from celery a potential alternative anticoagulant to commercially prepared oxalated black top tubes?
b. Background of the Study
Anti-coagulants act to avert the clotting of blood specimens and the reagent used should not cause the alteration of the blood components. However, there are anticoagulants that bring about changes in cell structure as well as coagulation. The anticoagulants used widely are ethylene-diamine-tetra-acetate (EDTA), ammonium-potassium oxalate (Heller and Paul double oxalate), and heparin.
Oxalates as one of the widely used anti-coagulant are naturally occurring substances found in plants, animals and in humans. Chemically speaking, oxalates belong to a group of molecules known as organic acids and are produced by plants, animals, and humans. Our bodies naturally have oxalates in our system and our cells convert other substances into oxalates. For an instance, vitamin c is routinely converted to oxalates. Aside from the oxalates found in our body, certain foods also actually contain them. When adding oxalate to vials and dried in an oven, the temperature should not exceed to 80 deg C. If so, oxalates are converted to carbonates during exposure to elevated temperature. Normally, there’s no need to prepare your own oxalate solutions since prepared anticoagulant vacuum tubes are commercially available.
Oxalates have the ability to precipitate calcium from the blood. The most commonly used is potassium oxalate, which provides plasma for glucose testing.
c. Significance of the study
This study would like to compare oxalate extracted from celery to a commercially available oxalate in evacuated tubes, if proven to be comparable it will be used as an alternative anticoagulant to oxalate in black top tubes. It will further provide additional insights on the effects of the phytochemical on blood in vitro, since majority of the previous studies put more emphasis only on the impacts of celery in vitro.
d. Scope and Limitation of the Study
The study will only focus on the anticoagulant property and effects on morphology and distribution of red blood cells, white blood cells and platelets.
Effects in the red blood cell, white blood cell and platelet counts will not be included because there are other procedures that deal with this parameter.
The whole experiment will be done manually, and will not be tried in automated machine due to unavailability.
e. Review of Literature
Blood coagulation occurs in the presence of several factors such as Calcium, Vitamin K and Fibrinogen that helps platelets to form a clot. These factors are called procoagulants which mean they promote clotting of the blood.
When a bleeding wound abruptly occurs, platelets will gather at the site and attempts to block the blood flow by binding with fibrinogen to form fibrin threads. These fibrin threads will form a web-like mesh that traps the blood cells inside and will later on hardens and dries, forming a clot or “scab”. Calcium and Vitamin K supports in clotting. (http://www.fi.edu/learn/heart/blood/platelet.html)
According to M.E. Mikaelsson, “free ionized calcium is required for the initial platelet plug formation and blood coagulation”. Low levels of these in the blood would lead for a longer clotting time or excessive bleeding.
Anticoagulation of the blood is also made to prevent clots from getting bigger in the blood vessels to avoid blockage of the blood flow. It is usually done in some routine Hematologic procedures in the laboratory that does not require clotted blood and for correctly differentiating agglutination of red blood cells from Rouleaux formation and in observing also other cells like the white blood cells.
Agglutination of red blood cells and Rouleaux formation (pseudo agglutination) must be firmly differentiated from each other based on their appearances particularly in a peripheral blood smear which is one of the most common routine hematologic procedures utilized by Medical Technologists in practice. Their differences should be carefully observed to avoid errors in reporting based on the distribution and morphology of red blood cells and to accurately identify abnormal red blood cells.
True agglutination is the irregular clumping of red blood cells into grape-like structures while Rouleaux formation produces more regularly spaced clusters of red blood cells adhering side-to-side (“coin stacks”) which is seen mostly in patients with increased protein level in the blood.
Deviation in the normal size and shape of a mature red blood cell can also be observed and be reported as abnormal. Mature red blood cells are round, biconcave disc-shaped, anucleated cells measuring approximately 7-8 microns in diameter with a central pallor not more than 3 microns in diameter and an internal volume of 80-100 fL. They also give a red-orange appearance on Wright-stained smears. The terms used to express red blood cells of normal size and of normal central pallor are normocytic and normochromic. (http://www.medialabinc.net/spg469710/normal_red_blood_cell_rbc_morph ology.aspx) Aside from red blood cells, white blood cells can also be observed. These include the neutrophils, basophils, eosinophils, monocytes and lymphocytes. Their deviation in the normal size and shape should also be noted so as to confirm if there are any abnormalities present in the blood.
The mature, segmented neutrophilic cells contain primary, peroxidise-positive granules and specific peroxidise-negative granules in a one to two ratio. The nucleus of the circulating neutrophil is segmented, usually into two to four interconnected lobes.
(http://medtextfree.wordpress.com/2011/12/30/chapter-64-morphology-of-neutrophils-eosinophils-and-basophils/)
Basophils are found in small numbers in blood (0,5%) and can be seen in tissues in which inflammation resulting from hypersensitivity to proteins, contact allergy, or skin-allograft rejection is present. Their size is equal or somewhat larger than the neutrophils and they have lavender to purple cytoplasmic granules
(http://medtextfree.wordpress.com/2011/12/30/chapter-64-morphology-of-neutrophils-eosinophils-and-basophils/)
Eosinophils are bilobed to multilobed and have coarse, dark clumped chromatin. Its distinctive feature is its bright pink cytoplasmic granules (containing major basic protein, eosinophilic peroxidise and other substances).
(https://ahdc.vet.cornell.edu/clinpath/modules/hemogram/eos.htm)
Monocytes are larger than the neutrophils and the largest leukocyte. Their nucleus can be round to kidney-shaped to pseudo-lobulated (can mimic a neutrophil). Their chromatin is described as lacey to slightly clumped.
(https://ahdc.vet.cornell.edu/clinpath/modules/hemogram/mono.htm)
The distinguishing feature of the lymphocytes is their dense, round to slightly indented nucleus, a small amount of clear to pale blue cytoplasm with a high nuclear:cytoplasmic ratio (i.e. the majority of the cell is taken up by the nucleus with only a small volume of cytoplasm visible.
(https://ahdc.vet.cornell.edu/clinpath/modules/hemogram/lymph.htm)
According to Guyton and Hall, “any substance that deionizes the blood calcium will prevent coagulation”. Such substances are called anticoagulants or blood thinners.
Although they are referred to as blood thinners, they do not actually thin the blood. But they work by decreasing the blood’s ability to clot too early so that the blood vessels remain unblocked. (http://www.nativeremedies.com/ailment/blood-thinners-anticoagulants.html)
Blood coagulation inside the body can be prevented by means of lowering the blood calcium level to significantly inhibit or decrease platelet activity. According to M.E. Mikaelsson, conformational changes of the factors V and VIII resulted in loss of procoagulant activity was due to the chelation of calcium.
According to Erica Roth, “Foods that contain compounds such as oxalic acid or phylic acids, including spinach, sweet potatoes, beans, nuts, rhubarb, celery and beets, can decrease the amount of calcium that’s absorbed when eaten at the same time as milk and other calcium-rich foods”.
We may not know but some of the foods we eat and herbs that we used as flavouring when we cook contain natural anticoagulant properties and carry coumarin effects which can prevent your blood from clotting. Some of these foods or herbs are garlic, ginger, celery seed and aniseed. (http://www.healthline.com/health/heart-disease/blood-thinners)
Based on a table of foods containing oxalate illustrated by Helen O. Connor, MS, RD, a ½ cup (60g) of raw celery contains about 10-50 mg of oxalate per serving.
According to Guyton and Hall, blood coagulation outside the body can also be prevented. “For instance, a soluble oxalate compound mixed in a very small quantity with a sample of blood causes precipitation of calcium oxalate from the plasma and thereby decreases the ionic calcium level so much that blood coagulation is blocked”.
Soluble oxalate, which consists of oxalic acid and soluble salts, contained in foods is released when extracted with water (Liebman & Al-Wahsh, 2011).
Oxalate exists in plants in a crystalline form either as calcium or as a soluble anion (Holmes & Kennedy, 2000).
A soluble oxalate can be extracted from the above mentioned foods like celery which contains large amounts of oxalate. It is thought that the chemicals in celery act to cause sleepiness, increase urine to decrease fluid retention, decrease arthritis symptoms, decrease blood pressure, decrease blood sugar, decrease blood clotting and muscle relaxation
V. METHODOLOGY a. Research Design
b. Selection of Subjects 5 individuals are needed as participants and will undergo a blood extraction procedure.
c. Variable Descriptions
I. Independent Variable
Oxalate on Celery- is proposed alternative anticoagulant. The use of anticoagulant is to prevent clotting of blood.
Oxalate on black top tubes- is the standard anticoagulant that is used in routine haematological tests.
II. Dependent Variable
Red blood cell, white blood cell and platelet morphology and distribution observed using extracted oxalate from celery will be compared to that of commercially available oxalate in black top tubes to determine whether integrity and distribution observed from both types of anticoagulant are similar or close to each other.
III. Confounding Variable
d. Hypothesis
Null hypothesis
There is no significant difference between extracted oxalate from celery and commercially prepared oxalate.
Alternative hypothesis
There is a significant difference between extracted oxalate from celery and commercially prepared oxalate.
Dummy Tables
TABLE 1- Comparison of the Visibility and Integrity of White Blood Cells in Commercially Prepared Oxalate and Extracted Oxalate from Celery
