Chapter 1 K and MG Prolonged Serum-Clot Contact Time

M E D I C AL LAB O R AT O R Y S C I E N C E D E PAR T M E N T
College of Arts and Sciences

Notre Dame of Marbel University
MAGNESIUM LEVELS USING GLASS AND PLASTIC

TUBE IN PROLONGED SERUM-CLOT CONTACT TIME
A thesis presented to the faculty of

Medical Laboratory Science Department
of Notre Dame of Marbel University
In Partial Fulfilment of the

Requirements for the Degree of
Bachelor of Medical Laboratory Science
Saikol, Aflaha D.
Sales, Nicole Domique V.
Simora, Doreen Ivy S.
Soquita, Roanne Angelie P.
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Acknowledgment
This research owes its existence to the help, support and inspiration from
several people. Firstly, the researchers would like to express their sincere
appreciation and gratitude to Mark G. Nava, RMT, MSMT, MPA ; Adolph Zayre De
Dios, RMT; Kristine Gay E. Templonuevo, RMT; and Hazel Carbon-Granil, RMT,
MiB, MSMT for their guidance throughout the research. Their support and
inspiring suggestions have been precious for the development of the research
content.
The researchers also would like to give thanks to their special friends who
have been a constant source of enthusiasm and encouragement, not only during
the research but throughout the journey to the completion of the course
Bachelor of Medical Laboratory Science.

To all the people that the researchers met along the way and contributed
to the development of the research, a massive thanks.

Deepest gratitude are dedicated to the researchers parents of their
unconditional love, prayers, sacrifices and support throughout their lives. This
research wont be at its existence without their financial and emotional support.
All the thanks go to all the people who have supported the researchers to
complete the research work directly or indirectly.

Finally, praises and worship to the Almighty God, for His showers of
blessings throughout the research work and for providing the opportunity and
granting the capability to proceed successfully.
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Abstract
Blood-drawing tubes made from plastic are gaining widespread use in
clinical laboratory. Components from blood collection tubes, such as stoppers,
lubricants, surfactants, and separator gels, can leach into specimens and/or
adsorb analytes from a specimen; special tube additives may also alter analyte
stability. Because of these interactions with blood specimens, blood collection
devices are a potential source of pre-analytical error in laboratory testing.
Accurate laboratory testing requires an understanding of the complex interactions
between collection devices and blood specimens. Manufacturers, vendors, and
clinical laboratorians must consider the pre-analytical challenges in laboratory
testing. Although other authors have described the effects of endogenous
substances on clinical assay results, the effects/impact of blood collection tube
additives and components have not been well systematically described or
explained. This review aims to evaluate the rate of increase or decrease of the
analyte in different tubes at a different serum-clot contact time. In this study, the
researchers compared the levels of Magnesium (Mg +) using the Glass and
Plastic tubes in different serum-clot contact time.
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Table of Contents
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List of Tables
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List of Figures
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Chapter I
INTRODUCTION
During the last decade, plastic blood collection tubes have been
progressively replacing glass tubes. Plastic tubes are not only less expensive but
also safer than glass tubes, because they are less likely to break. Unfortunately,
it is frequently difficult or impossible for individual laboratories to obtain
comprehensive data on the equivalence of replacement plastic tubes vs their
original glass counterparts. This is a particularly important issue for many
analytes. Changing from glass to plastic tubes can also be problematic for
analytes that are regarded as stable. Even minor discrepancies between glass
and plastic tubes may gain significance during change over from one type of
collection to the other. The researchers designed the presented study to give a
reasonable representation of the difference between glass and plastic tubes on
different serum-clot contact time.
Laboratory testing is divided into three phases; pre-analytical, analytical
and post-analytical. Laboratory errors in the analytical phase have significant
decreased due to automation. Most errors occur in pre-analytical phase i.e. 46-
68.2 % (Plebani, 2006). Among the investigations performed in the laboratory,
the parameters which are mostly affected by these variables are the serum
electrolytes (Heins et al., 1995). The time interval between blood collection and
sample processing in analyzer is one of the most error prone areas and also the
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bottleneck of the turnaround time of the laboratory. The two important time delay
processes that occur in this phase is serum clot contact time and centrifugation
delay. Serum clot contact time is defined as optimum time interval between
sample collection and separation of serum from the clot. This period should be
long enough to allow the complete clot formation but should be shorter than the
time in which a significant change in the test result is induced by serum-clot
contact. A minimum time interval of 20-30 min between blood collection and
serum separation is considered acceptable (Burtis et al., 2006). A prolonged
contact time between serum and clot alters both biological activities of the cells
and trans-membrane diffusion that can change the concentration of serum
electrolytes. Although the practice in clinical biochemistry is to assay the samples
as they are brought into the laboratory and after separation of the serum from the
samples, at times the samples reach the laboratory from the wards and collection
centers unduly late. Also at times the samples are processed inadvertently very
late and also the assays may be missed and have to be done many hours after
they have been collected due to large sample load, breakdown of the electrolyte
analyzer and the non-availability of a backup system. Information on the
measured concentration of serum electrolytes during storage and serum clot
contact time is often incomplete and sometimes contradictory. Therefore the
researcher conducted this study to evaluate if the time lag between centrifugation
and sample analysis has any effect on the stability of the analytes in the serum.
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This study was designed to provide information relevant to the current testing
method on immediate separation versus delayed centrifugation before analysis.
Prolonged contact of serum with the clot can cause pre-analytical
variation. The optimum time interval between sample collection and separation of
serum from the clot should be long enough to allow complete clot formation but
be shorter than the time in which a significant change in test result is induced by
serum-clot contact. The minimum clotting time suggested by the Tietz Textbook
of Clinical Chemistry is 2030 min. During a prolonged contact time between
serum and clot, both biological activity of the cells and trans-membrane diffusion
can change the concentrations of certain analytes in the serum. NCCLS
Procedures for the Handling and Processing of Blood Specimens recommends
that serum or plasma should be physically separated from contact with cells as
soon as possible, unless conclusive evidence indicates that longer contact times
do not contribute to result inaccuracy. A maximum limit of 2 hours from the time
of collection to the time of separation was also recommended. Each individual
analyte has a different tolerance to a delay in separating serum from clot. Many
analytes are stable for much longer than 2 hours. In hospitals and outpatient
clinics, transportation of specimens from a phlebotomy site to a laboratory
sometimes takes longer than 2 hours. If overly stringent transportation
requirements are set for all tests, many acceptable specimens would be rejected
unnecessarily. Ideally, a specific allowable transportation time should be applied
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for each analyte in a specimen. In practice, analytes are usually grouped into
time blocks in which serum-clot contact cause no changes in analyte
concentrations. In some instances, specimens arrive in the laboratory at some
hospitals usually more than 2 hours after collection due to the location of the
patient. Therefore, knowing the test stability within time intervals was critical to
determine the cutoff times for acceptable specimens.
Magnesium is the fifth most abundant cation in the body and is second
only to potassium within cells. Magnesium deficiencies are related to impairment
of neuromuscular function, cardiovascular disease and hypertension. Since
Potassium is well studied analyte, the researchers considered Magnesium to
study its levels using glass and plastic tubes.
Statement of the Problem

This study entitled Magnesium levels using Glass and Plastic tube in
Prolonged Serum-clot contact time, will determine the levels of Magnesium in
random blood samples collected in both Glass and Plastic tube, in which will be
subjected into different Serum-clot contact time interval. It seeks to evaluate the
rate of changes of the analyte in different tubes at a different serum-clot contact
time.
Moreover, this study aims to check the validity of data obtained from the samples
both contained in Glass and Plastic tubes that are subjected to different serum-clot
contact time prior to analyzation, to assess the rate of changes and compare the
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results obtained. To reach our goals, specifically, this study desires to bring out
data to answer the following questions:
1. What is the level of Magnesium when analyzed in glass tube with its
serum-clot contact time?

2. What is the level of Magnesium when analyzed in plastic with its serum-clot
contact time?
3. What is the rate of change of the levels in Magnesium in glass tube in
different serum-clot contact time?

4. What is the rate of change of the levels in Magnesium in plastic tube in
different serum-clot contact time?

5. Is there a significant difference on the results of samples analyzed in Glass
tubes and samples analyzed in plastic tubes?

6. Is there a significant difference on the results of samples analyzed at
standard serum-clot contact time and samples analyzed at different
serum-clot contact time interval?

7. Is there a significant difference on the results of samples analyzed (using
Glass and Plastic tube) at standard serum-clot contact time and samples
analyzed (using Glass and Plastic tube) at different serum-clot contact
time interval?
8. What are the parameters affected by variations in serum-clot contact time?
9. What are the parameters unaffected by variations in serum-clot contact
time?
Scope and Delimitation
This study entitled Magnesium levels using Glass and Plastic tubes in
Prolonged Serum-clot contact time seeks to assess the rate of change in
Magnesium levels using Glass and Plastic tubes in prolonged serum-clot contact
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time intervals. The analyte will be tested is Magnesium (Mg+) and will be using Red
top tubes (Glass and Plastic) because they dont have anticoagulants. The
specimens will be subjected to different serum-clot contact time intervals (30
minutes, 1 hour and 1.5 hour). It aims to test the effects of samples with variations
in serum-clot contact time. Through this, it would either prove or disprove the use of
either Glass or Plastic tube. It may also provide reasons for rejection or acceptance
of the specimen and to which tubes is better.

The study will be conducted at Notre Dame of Marbel University Clinical
Training Diagnostic Laboratory (figure 1). The respondents will be limited only to
thirty (30) enrolled students that are eighteen (18) years old and above. The
specimen would be collected through venipuncture utilizing the use of red top
tubes.
In performing the test, the researchers will draw blood from 30 random
participants. The control sample would be clotted on the standard serum-clot
contact time and the variables would be subjected to prolonged serum-clot
contact time. The test will be conducted at NDMU-CTDL. The researchers will be
using automated analyzers.
Hypothesis
This study entitled Magnesium levels using Glass and Plastic tube in
Prolonged Serum-clot contact time, will determine the levels of Magnesium in
random blood samples collected in both Glass and Plastic tube, in which will be
subjected into different Serum-clot contact time interval. It seeks to evaluate the
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rate of changes of the analyte in different tubes at a different serum-clot contact
time. In accordance to this, the study have the following assumptions:
H0: There is no significant difference on the results of samples analyzed
(using Glass and Plastic tube) at standard serum-clot contact time and
samples analyzed (using Glass and Plastic tube) at different serum-clot
contact time interval.
H1: There is a significant difference on the results of samples analyzed
(using Glass and Plastic tube) at standard serum-clot contact time and
samples analyzed (using Glass and Plastic tube) at different serum-clot
contact time interval.
Significance of the Study

The study aims to assess the rate of the Magnesium levels using glass
and plastic tube at different serum-clot contact time. Consequently, the
researchers describe how significant the study in the following areas or to the
following persons:
Clinical Chemistry. The research will contribute in the field of Clinical
Chemistry in the sense that it aims to assess the rate of change in the levels of
analytes in prolonged serum-clot contact time. Also, the study provides the
criteria in maintaining the quality of specimens. This will also provide data in
which the basis for the analyzation of these analytes.

Medical Technologist. In many instances, the Medical Technologist has
the tendency to delay the test of the analyte possibly because he/she may have
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tons of work. The study will give an insight to the Medical Technologist on how
high or low the rate of change when analytes are prolonged untested. In some
cases, where glass tubes are inadequate in a laboratory, the Medical
Technologist will alternatively use the plastic tubes or vice versa. Through this,
the medical technologist would be able to ensure the quality and reliability of the
results.
Physician. In diagnosing a certain disease, the test must be accurately
carried out. The physician must be aware of the accuracy of the blood sample
taken from his/her patient.
Definition of Terms
In order to have clear understanding of the study, the terms that are often
used and will be frequently mentioned in this study are defined as the following:
Automated electrolyte analyzers the method to be used in this study
to run analyte testing.

Clotting when the blood clots
Glass red top tube the preliminary collection of the sample to induced
serum-clot contact to be compared with Plastic red top tube.

Magnesiumwhat is being assessed in this study.
Plastic red top tube the preliminary collection of the sample to induced
serum-clot contact to be compared with Glass red top tube.

Pre-analytical the phase in laboratory testing which the serum-clot
contact time is one of the factors.

Serum the part of the blood will be tested by Magnesium
Serum-clot contact time- the optimum time interval between sample
collection and separation of serum from the clot

Serum-clot contact time intervals the time interval used in this study
i.e. 30 minutes, 1 hour, and 1.5 hour.
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Rate of change the goal of this study is to evaluate the change in levels
of Magnesium using glass and plastic tubes in prolonged serum-clot contact
time.
Venipuncture the procedure to be used in the study to obtain sample.
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Chapter II
REVIEW OF RELATED LITERATURE
Magnesium
Magnesium was considered as the fourth most common cation in the
human body and the 2nd most abundant intracellular ion which plays a critical role
in many metabolic processes, including the production and use of energy
essential in the maintenance of normal intracellular electrolyte composition
(Schrier, 2010). In addition, McPherson and Pincus et.al, in their book entitled
Henrys Clinical Diagnosis and Management by Laboratory Methods stated that
the normal body magnesium content in an adult is approximately 1000 mmol, or
22.66 g, 50%60% of which is in bone, and the remaining 40%50% is in the soft
tissues. Only 1% of the total body magnesium (TBMg) is in extracellular fluid. In
serum about 55% of magnesium was ionized or free magnesium (Mg++), 30%
was associated with proteins (primarily albumin) which are called bound
magnesium, and 15% is complexed with phosphate, citrate, and other anions. It
was also cited in the same book that in cerebrospinal fluid (CSF), 55% of the
magnesium is free or ionized, and the remaining 45% is complexed with other
compounds (Elin, 1988). The magnesium concentration in the interstitial fluid was
approximately 0.5 mmol/L. magnesium therefore, is an important component of
the human body and the deficiency and excess of which can provide various
malfunctions in many physiologic processes.
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Magnesium can be introduced in the body through the diet. Most dietary
magnesium intake comes largely from the vegetative plants. According to Bishop
(2013), rich sources of Mg2+ in the diet include raw nuts, dry cereal, and hard
drinking water, vegetables, meats, fish, and fruit. Decreased in the Mg2+ intake
can be observed in people taking processed foods and in turn may result to
Mg2+ deficiency. The recommended daily intake for adults is 320 to 420 mg/dL
(Hale and Hovey, 2014). In addition, according to Volpe (2013), magnesium is a
rather ubiquitous mineral but there is no major food that provides an extremely
high amount of magnesium. She presented various food items with
corresponding magnesium content in milligrams.
Table 1 shows the food items that were high in magnesium
Food Magnesium (mg)

1/4 cup of wheat bran (57 g) 89
1 oz of dry roasted almonds (28.4 g) 80
1/2 cup of frozen, cooked spinach (14.2 g) 78
1 oz of mixed, dry roasted nuts (28.4 g) 64
3/4 cup of bran flakes cereal (170 g) 64
2 tbsp of smooth peanut butter (32 g) 49
1 medium baked potato with skin 48
1/2 cup of cooked pinto beans (113 g) 43
1/2 cup of brown, long-grained cooked rice (113 g) 42
1/2 cup of mature seeds, cooked lentils, (113 g) 36
1 cup of low-fat chocolate milk (234 mL) 33
1 medium banana 32
8 fluid oz of low-fat fruit yogurt (234 mL) 32
1.5 oz of milk chocolate candy bar (43 g) 28
1 slice of whole-wheat bread, commercially prepared 23
1/2 cup of avocado cubed (113 g) 22
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Magnesium level in the body was primarily regulated by the kidneys
through reabsorption and secretion. 25% to 30% of the non-protein bound Mg2+
that are being filtered by the glomerulus will be reabsorbed by the proximal
convoluted tubules in the kidneys while 2% to 5% of which will be reabsorbed in
the distal convoluted tubules. Ascending limb of the loop of Henle will reabsorb
50% to 60% of Mg2+. This regulationl resulted to the excretion of 6% of filtered
Mg2+ per day in normal conditions. The hormone that enhances the renal
reabsorption of Mg2+ will be the Parathyroid-stimulating hormone (PTH) which
improves the reabsorption of the cation in the intestine. However, PTH also
reacted with ionized Ca2+ levels by activating the breakdown of osteoclasts to
release the calcium in the blood. This would cause the serum magnesium
concentration to vary reciprocally to the calcium concentration. Aldosterone and
thyroxine, on the other hand, increases the renal excretion of Mg2+.
Magnesium performed many important functions in the body. It was
necessary for a wide spectrum of enzymatic reactions because it acts as cofactor
for more than 300 enzymes in the body. The reactions of various phosphokinases
and phosphatases which were involved in energy storage and use are coupled
with magnesium. Also, in reaction in which ATP was the substrate, the true
substrate Mg2+ ATP as much as Mg2+ was chelated between the alpha and
gamma phosphates, thus, magnesium is needed in the synthesis of
biomolecules. Moreover, it was said to influenced vasodilation, irritability and
contractility of the cardiac muscles, thereby helping the cardiovascular system to

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function normally. It also helped in neurotransmission and hormone-receptor
binding and in the crossing of sodium and potassium in the cell membrane
(McCann 2006).
To elucidate the physiological functions of the magnesium mentioned
above, according to Burtis et.al (2013), magnesium was important in oxidative
phosphorylation, glycolysis, cell replication, nucleotide metabolism, and protein
biosynthesis. A decrease in the serum magnesium concentration lowers the
threshold of axonal stimulation and increases nerve conduction velocity.
Magnesium also influenced neurotransmitter release at the neuromuscular
junction by competitively inhibiting the entry of calcium into the presynaptic nerve
terminal. Reducing the serum magnesium concentration resulted in increased
neuromuscular excitability. Magnesium deficiency can thus result in a variety of
metabolic abnormalities and clinical consequences. In addition, in cases of
calcium stone disease, magnesium was an inhibitor of stone growth. Mg2+ forms
complexes with oxalate that are more soluble than calcium oxalate. Increased
urinary magnesium therefore inhibits stone formation.
According to McPherson and Pincus (2011), the reference interval for
serum total magnesium in normal adults ranges between 0.75 and 0.95 mmol/L
(1.72.2 mg/dL or 1.51.9 mEq/L). There appear to be no significant sex or age
differences. Erythrocyte magnesium is about three times that of serum. The
magnesium concentration in CSF is 2.02.7 mg/dL (1.01.4 mmol/L). The
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reference interval for ionized magnesium depended on the analyzer used for its
measurement and varies from 0.440.60 mmol/L (Hristova, 1995)
Hypomagnesemia
This was a disorder in the metabolism of the magnesium wherein there is
a deficiency in the amount of magnesium present in the body as compared to the
identified normal value for magnesium.
Hypomagnesemia was common and frequently observed in hospitalized
individuals in intensive care units (ICUs) or those receiving diuretic therapy or
digitalis therapy (Bishop et.al 2013). These patients most likely have an overall
tissue depletion of Mg2+ as a result of severe illness or loss, which leads to low
serum levels. In non-hospitalized individuals, hypomagnesemia is less likely to
occur and the reduced intake of dietary magnesium can rarely cause severe
deficiencies in healthy individuals.
According to Burtis et.al (2013), hypomagnesemia can be caused by
various conditions. First in the list was the gastrointestinal disorder which was
influenced by prolonged nasogastric suction, malabsorption syndromes,
extensive bowel resection, acute and chronic diarrhea, intestinal and biliary
fistulas, protein-calorie malnutrition, acute hemorrhagic pancreatitis and primary
(neonatal) hypomagnesemia. Another condition was the renal loss caused by
chronic parenteral fluid therapy, osmotic dieresis, hypercalcemia, alcohol and
drug intake, metabolic acidosis and renal disease such as chronic pyelonephritis
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and renal tubular necrosis. Phosphate depletion can also cause
hypomagnesemia.
A patient who was hypomagnesemic may be asymptomatic until serum
levels fall below 0.5 mmol/L. According to Sircus, M., hypomagnesia was under-
recognized and under-reported, yet clinically serious adverse events are
commonly reported symptoms of hypomagnesemia. One of the hidden dangers
was its production of impaired parathyroid hormone secretion which may lead to
hypocalemia.
Table 2 shows the symptoms of hypomagnesimia
Cardiovascular Psychiatric Neuromuscular Metabolic

Arrhythmia Depression Weakness Hypokalemia
Hypertension Agitation Cramps Hypocalcemia
Digitalis toxicity Ataxia Hypophosphatemia
Tremor
Psychosis Seizure
Tetany Hyponatremia
Paralysis
Coma
Adapted from Polancic JE. Magnesium: metabolism, clinical importance,analysis. Clin
Lab Sci. 1991;4(2):105-109
Hypermagnesemia
Hypermagnesemia was less frequently seen as compared to
hypomagnesemia. According to (Hale and Hovey, 2014), hypermagnesemia
cases were mostly seen in patients with chronic kidney disease and can also be
observed with shifts or exogenous administration. Magnesium was commonly
given parenterally for the treatment of ecclampsia causing the magnesium blood
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level to increase. This may result in neonatal hypermagnesemia, but in general,
blood levels tend to be lower in the infant than in the mother. Moreover, Bishop
et.al (2013) stated that the most severe elevations are usually a result of the
combined effects of decreased renal function and increased intake of commonly
prescribed Mg2+-containing medications, such as antacids, enemas, or
cathartics. Hypermagnesemia has been associated with several endocrine
disorders. Thyroxine and growth hormone cause a decrease in tubular
reabsorption of Mg2+, and a deficiency of either hormone may cause a moderate
elevation in serum Mg2+. Causes of hypermagnesemia according to Burtis et.al
(2013) was summarized in the table 3.
Table 3 shows the Causes of Hypermagnesemia
Excessive intake
Orally (usually in the presence of chronic renal failure)
Antacids
Cathartic
Rectally
Purgation
Parenterally
Treatment of pregnancy-induced hypertension
Treatment of magnesium deficiency
Renal failure
Chronic (usually with administration of magnesium)
Antacid
Cathartic
Enema
Infusion
Dialysis
Acute
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Rhabdomyolysis
Familial hypocalciuric hypercalcemia
Lithium ingestion
According to Nichizawa et.al (2007) in their book entitled New
Perspective in Magnesium Research: Nutrition and Health, mild
hypermagnesemia (plasma lower than 1.5 mmol/L) was usually asymptomatic.
When plasma magnesium was between 2 to 3 mmol/L, drowsiness, lethargy, and
diminished deep tendon reflexes can be observed. The most consistent
complication of hypermagnesemia is neuromuscular toxicity, which was due to a
reduction in the impulse transmission across the neuromuscular junction.
Hypermagnesemia with 3 to 5 mmol/L can result to loss of tendon reflexes,
hypocalcemia and cardiovascular effects including hypotension, bradycardia and
electrographic changes. Extreme hypermagnesemia with plasma concentration
above 5 mmol/L was associated with muscle paralysis, potentially leading to
flaccid quadriplegia and apnea, complete heart block and cardiac arrest.
Blood Collection Tubes
Blood collection tubes have color-coded stoppers that distinguish the
presence of a specific anticoagulant or additive, how the tube is chemically
cleaned, an example would be for lead and iron determinations, or if the tube
does not contain any additives. Tubes came in various sizes for adult and
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pediatric patient populations. Draw volume was determined by the internal
vacuum within the sealed tubes (e.g., 3.5, 4.0, 4.5, or 8.5 mL).
Figure 1. shows an image of evacuated tubes
The use of anticoagulants had allowed analysis of whole blood specimens
or plasma constituents obtained by centrifugation and separation of the plasma.
Plasma was said to contain fibrinogen, which was not found from serum. Many
laboratories have converted from glass to plastic collection tubes to minimize
exposure to biohazardous material like blood, and broken glass, to lower
biohazard waste disposal costs, and to comply with Occupational Safety and
Health Administration (OSHA) guidelines mandating substitution.
Change from glass to plastic had required a modification in the order of
draw. Glass or plastic tubes with additives, including gel tubes, were drawn after
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the citrate tube to avoid interference with coagulation measurements. Glass or
plastic serum tubes, without a clot activator or gel separator, may be drawn
before the coagulation tubes are drawn, consistent with National Committee on
Clinical Laboratory Standards (NCCLS) guidelines (H3-A6) (Ernst, 2004)
Evacuated tubes have a premeasured vacuum that automatically
draws the volume of blood indicated on the label. A tube that has lost all or part of
its vacuum will fail to fill with blood or fill incompletely. Vacuum loss can occur if
tubes are stored improperly, opened, dropped, or advanced too far onto the
needle before the draw or if the needle bevel backs out of the skin during the
draw. Tube stoppers were color coded into identify a type of additive, absence of
additive, or special tube property. Although generally universal, color-coding
varies slightly by manufacturer. Common stopper colors, additives, and
departments according to McCall (2008) were listed in table 4.
Table 4 lists common stopper colors, additives, and departments
According to Bishop (2010) additive functions optimally when the tube was
filled to its stated volume and gently inverted immediately after collection to mix
the additive with the blood. Specimen quality can be compromised if a tube is
partially filled. Shaking or vigorous mixing can hemolyze the blood, making it
unsuitable for testing. Additive reliability is guaranteed until an expiration date on
the label if the tube is handled and stored properly. Expiration dates should be
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checked and expired tubes discarded. The most common additives are
categorized as follows:
1 Anticoagulants prevented blood from clotting and included
ethylenediaminetetraacetic acid (EDTA), citrates, heparin, and oxalates.
Each was designed for use in certain types of testing.
2 Antiglycolytic agents prevented glycolysis, which can decrease glucose
concentration by up to 10 mg/dL per hour. The most common antiglycolytic
agent, sodium fluoride, preserved glucose for up to 3 days and inhibited
bacterial growth. It was often combined with potassium oxalate
anticoagulant to provide plasma specimens. In addition to glucose, sodium
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fluoride was used to collect ethanol specimens to prevent an increase in
alcohol due to fermentation by bacteria.
3 Clot activators were coagulation factors such as thrombin and substances
such as glass (silica) particles and inert clays like diatomite (Celite) that
enhanced clotting by providing more surfaces for platelet activation. The
clot activators in gel separator tubes and plastic red top tubes were
typically silica.
4 Thixotropic gel separators were inert substances contained in or near the
bottom of certain tubes. During centrifugation, the gel lodges between the
cells and the fluid, forming a physical barrier that prevents the cells from
metabolizing substances in the serum or plasma.
According to McPherson et.al (2011), during storage, the concentration of
a blood constituent in the specimen may change as a result of various
processes, including adsorption to glass or plastic tubes, protein denaturation,
evaporation of volatile compounds, water movement into cells resulting in
hemoconcentration of serum and plasma, and continuing metabolic activities of
leukocytes and erythrocytes. These changes occur, although to varying degrees,
at ambient temperature and during refrigeration or freezing. Storage
requirements vary widely by analyte.
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Plastic Evacuated Tubes
Plastic blood collection tubes may be manufactured by an injection-
molding process. (Kasai, 1991) A mold was made to the specific size of tube
desired. Typically, in the molding process, a hot, molten material was injected into
a cold mold for the tube. After the tube material cools and solidifies, the mold is
opened, and the tube was ejected.
During the last decade, plastic blood collection tubes have been
progressively replacing glass tubes. Plastic tubes were not only less expensive
but also safer than glass tubes, because they were less likely to break.
Unfortunately, it was frequently difficult or impossible for individual laboratories to
obtain comprehensive data on the equivalence of replacement plastic tubes than
their original glass counterparts. This was a particularly important issue for many
endocrine assays, especially peptide hormones. These often degrade rapidly and
can adsorb to a variety of surfaces (Hildebrandt, 1987; Wu,1989; Sapin,1988;
Evans,2001; Quellhorst,2002; Ellis,2003; Devine,1986).
Changing from glass to plastic tubes can also be problematic for analytes
that were regarded as stable. An example of the latter can be seen in therapeutic
drug monitoring, where plastic tubes have been shown to influence the measured
concentrations or stabilities of several drugs (Devine,1986; Landt,1995;
Dasgupta,2000)
Similar concerns may apply to low-molecular-weight hormones, such as
steroid hormones and biogenic amines. These were increasingly assayed by

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HPLC, gas chromatography-mass spectrometry, or liquid chromatography-
tandem mass spectrometry. It was conceivable that low-molecular-weight organic
substances released by plastic tubes could interfere in some of these assays
(Murthy,1997) and that small changes that are not detected by immunoassays
would change the results of more specific methods. Finally, in certain situations,
such as serial monitoring of tumor markers, even minor discrepancies between
glass and plastic tubes may gain significance during changeover from one type
of collection to the other.
Plastic tubes have several advantages over glass tubes: increased shock
resistance, tolerance of higher centrifugation speeds and reduced solid waste
after incineration. (Landt,1995) In addition, the slight flexibility of provided product
monographs and white papers showing plastic tubes make them more suitable
for use in an automated laboratory with robotics-based sample analytes drawn
into glass and plastic tubes and with handling.
Polyethylene terephthalate was a polymer (polyester) that was commonly
used to manufacture plastic blood collecting tubes by way of injection molding
(Shoji,1989; Vogler,1992) Generally, however, plastic tubes have hydrophobic
surfaces that interfere with the coagulation process (Shoji,1989; Voggler,1992).
Clots formed on the surfaces of plastic blood collecting tubes were more
gelatinous when compared to those formed in glass tubes (Shoji,1989;
Voggler,1992). Furthermore, blood does not flow smoothly over hydrophobic
plastic surfaces, which can result in the adherence of platelets, fibrin, or clotted
29 | P a g e
blood onto the interior walls of the tubes (Shoji,1989; Voggler,1992). This clotting
and adherence of blood to the walls of plastic blood collecting tubes can create
difficulties when trying to obtain a clean separation of serum from blood during
centrifugation, especially when using micro-collection tubes and during
centrifugation of vacuum tubes (Shoji,1989; Voggler,1992).
Plastic tube surfaces absorbed plasma proteins and because of their
hydrophobic nature, they tend to hold onto the proteins more readily, thereby
decreasing the number of available binding sites for activators of the intrinsic
pathway (Shoji,1989; Voggler,1992; Bowen,2010; Montgomery,1994)
Glass Evacuated Tubes
Glass evacuated blood collection tubes can be made from glass canes cut
to predetermined lengths and fired at one end to close the bottom. Once the tube
was formed, additives may be topically applied and dispersed along the inner
wall of the tube. (Hatekeyama,1992) Most of these additives were considered to
be "dry." Tubes were spray coated with additive formulations onto the inner wall
using a series of nozzles. Dispensing was achieved by either pressure activation
or volume displacement. The coating was dried by forced air or vacuum.
Alternatively, additives that were dispensed into the tube as a fluid and remain as
a liquid are considered "wet." A gel barrier may also be dispensed into the formed
tube for gel separator tubes. After any additive or gel was inserted, the tubes are
then evacuated and stoppered. An evacuating-closure device evacuated the
30 | P a g e
interior of the tube and applies a stopper to the opening of the tube.
(Hatakeyama,1992) Tubes then, were labeled appropriately.
Glass blood collection tubes have been used traditionally in clinical
laboratories; however, they present a risk of exposing clinicians to blood-borne
pathogens due to broken glass during handling or centrifugation (Ernst,2001;
Flanders,2003) This has led to the advent and preferred use of plastic tubes.
Glass and plastic tubes can both be problematic with respect to blood
clotting and tube adherence. The surfaces of glass tubes were hydrophilic,
whereas those of plastic tubes were hydrophobic (Vogler,1992; Harper,1994)
This difference was important for specimen collection, since glass tube surfaces
tend to interact with plasma proteins, causing an increased rate of blood
coagulation (Vogler,1992; Harper,1994) .
31 | P a g e
Chapter III
METHODOLOGY
In this chapter the research flow, research design, research locale,
participants of the study, research instrument, method of validity and statistical
treatment of the study is included.
Research Flow Start
Letter of preparation
(Letter of Consent)
NOLook for another participant

Approval
YES
Extraction of blood
Experimentation
Gathering of results
Interpretation of results
Figure 2 shows the Research Flow of this study
32 | P a g e
Research Design
This study uses a quantitative method of research. This method suggests
that the degree to which the research findings can be generalized to individuals
other than those who participated in the study is a widely used criterion for
assessing quality of quantitative studies. It is based on the concepts of
manipulation and control of phenomena and the verification of the results
validating empirical data (Cristobal, Jr. 2013). In addition, according to Glesne
and Peshkin, careful sampling strategies and experimental designs are needed
to produce generalizable results. Researches must not contaminate the data
through personal involvement with research subjects and researchers objectivity
is the utmost concern.
Research Locale
The study will be conducted at Notre Dame of Marbel University -- Clinical
Training and Diagnostic Laboratory (NDMU CTDL), Koronadal City. This facility
is a tertiary laboratory that can perform various laboratory tests. It has the
necessary materials and instruments needed in performing the experimentation
of the study. Also, the researchers have prior knowledge and experience on how
to use the instruments in the said laboratory.
33 | P a g e
Figure 3 shows the map of the location of the NDMU- CTDL.
Participants of the Study

For the selection of the participants, the researchers will use convenience
sampling wherein the researchers will consider individuals who are easy to get
34 | P a g e
as the respondents of the study. People are selected on the basis of their
availability and willingness to respond (Gravetter, 2012). This type of sampling is
a specific type of non-probability sampling method.
The researchers decided to select 4 rd year students enrolled in the
Bachelor of Medical Laboratory Science as the participants. Thirty students will
be encouraged to participate and those who are willing to join will be recognized.
Data Gathering Instrument
In gathering data, the researchers will use physiological measurement.
Physiological measures are frequetly used when collecting data for quantitative
research. Most familiar is the use of the method in determining base line data
and outcome in clinical trials, but it can also be used in studies of prevalence and
surveys (Roe and Webb, 1998). In addition, this method involves the collection of
physical data from the subjects. It is considered more accurate and objective
than other data collection methods (Cristobal, 2013).
Materials and Reagents
This study will need the materials such as red top evacuated tubes (glass
and plastic), syringe, wet and dry cottons, test tubes, test tube rack, centrifuge
machine, pipettes, timer and spectrophotometer for the readings of the
Magnesium levels. The researchers will use the Mindray BA 88 Chemistry
analyzer
Test Principle
35 | P a g e
Magnesium in the sample reacts with xylidyl blue in alkaline medium
forming a colored complex that can be measured by spectrophotometry. Egtazic
acid (EGTA) is included in the reagent to remove calcium interference.
Specimen Collection and Processing
This part presents how the specimen will be collected and be processed.
The following procedures must be followed to avoid any unnecessary variations
to the result of the study. Also, a registered medical technologist will supervise
the researchers in performing the following procedures.
1 The researchers must be professional, courteous and understanding in
manner all contact with all participants.
2 First, the researchers will identify the participant prepare for the materials
needed for phlebotomy.
3 The researchers will position the patient in a chair, or sitting or lying on
bed.
4 Then, the researchers will choose a suitable site for venipuncture, by
placing the tourniquet 3 to 4 inches above the selected puncture site on
the patient.
5 Do not put the tourniquet too tightly or leave it on the patient longer than 1
minute.
36 | P a g e
6 The researchers will select a vein and clean the area in circular motion,
beginning at the site working outward then, allowing the area to dry. After
the area has been cleansed, it should not be touched or palpitated again.
7 The researchers will ask the patient to make a fist; avoiding the pumping
fist. Then, the researcher will grasp the patients arm firmly using the
researchers thumb to draw the skin taut and anchor the vein with a
needle in a swiftly manner through the skin into the lumen of the vein. The
needle should form a 15-30 degree angle with arm surface. Excess
probing shall be avoided. Evacuated tube system will be used.
8 When the tube is filling, remove the tourniquet.
9 After filling the second tube, the needle will be removed from the patients
arm using a swift backward motion.
10 The gauze will be placed immediately on the puncture site, applying and
holding adequate pressure to avoid hematoma. After holding the pressure
for 1-2 minutes, the researcher will tape a fresh piece of gauze or Band-
Aid to the puncture site.
11 The researcher will dispose the contaminated materials/supplies in
designated containers.
12 The researchers must label the tubes correctly.
37 | P a g e
13 The researchers will allow the sample to stand for 20-30 minutes to allow
clot formation.
14 Clot was rimmed with an applicator stick, and then centrifuged for 10
minutes.
15 Enough serum from the sample will then be drawn from the centrifuged
tube immediately for magnesium level determination.
16 The remaining serum sample will be allowed to be in contact with the
compact red blood cells for 30 minutes before doing the magnesium level
determination again.
17 Same process will be followed after an hour and after 1 hour.
Experimentation
Before starting the procedure for analysis in the machine, calibration will
be performed with the guidance of a registered medical technologist. The
researchers will bring the reagents and samples to room temperature then start
the manual test.
1. Bring the Working reagent to room temperature.
2. Pipette the following into test tubes
Blank Standard Sample

Magnesium Standard 10 uL
Sample 10 uL
Working reagent 1.0 mL 1.0 mL 1.0 mL
38 | P a g e
3. Mix thoroughly and let stand the tubes for 2 minutes at room temperature.
4. Read the absorbance (A) of the Standard and the Sample at 520 nm against
the Blank. The color is stable for at least 1 hour.
Method of Validity
The validity of the instrument will be established by running positive and
negative controls in the machine that will be used.
Statistical Treatment
This study will utilize One-Way Analysis of the Variance (ANOVA). This is
the method used when the means of two or more independent groups will be
compared. The method enables the differences between two or more sample
means to be analysed, achieved by subdividing the total sumsquares. The
purposeis to test for significant differences between class mean through the
analysis of the variances (Shutler, 2002)
39 | P a g e

Chapter 1 K and MG Prolonged Serum-Clot Contact Time

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Chapter 1 K and MG Prolonged Serum-Clot Contact Time

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M E D I C AL LAB O R AT O R Y S C I E N C E D E PAR T M E N T

College of Arts and Sciences

MAGNESIUM LEVELS USING GLASS AND PLASTIC

A thesis presented to the faculty of

In Partial Fulfilment of the

Bachelor of Medical Laboratory Science.

to the development of the research, a massive thanks.

complete the research work directly or indirectly.

granting the capability to proceed successfully.

Blood-drawing tubes made from plastic are gaining widespread use in

clinical laboratory. Components from blood collection tubes, such as stoppers,

stability. Because of these interactions with blood specimens, blood collection

devices are a potential source of pre-analytical error in laboratory testing.

Accurate laboratory testing requires an understanding of the complex interactions

between collection devices and blood specimens. Manufacturers, vendors, and

clinical laboratorians must consider the pre-analytical challenges in laboratory

testing. Although other authors have described the effects of endogenous

substances on clinical assay results, the effects/impact of blood collection tube

additives and components have not been well systematically described or

Plastic tubes in different serum-clot contact time.

it is frequently difficult or impossible for individual laboratories to obtain

comprehensive data on the equivalence of replacement plastic tubes vs their

original glass counterparts. This is a particularly important issue for many

reasonable representation of the difference between glass and plastic tubes on

different serum-clot contact time.

Laboratory testing is divided into three phases; pre-analytical, analytical

and post-analytical. Laboratory errors in the analytical phase have significant

68.2 % (Plebani, 2006). Among the investigations performed in the laboratory,

time in which a significant change in the test result is induced by serum-clot

serum separation is considered acceptable (Burtis et al., 2006). A prolonged

and trans-membrane diffusion that can change the concentration of serum

electrolytes. Although the practice in clinical biochemistry is to assay the samples

analyzer and the non-availability of a backup system. Information on the

measured concentration of serum electrolytes during storage and serum clot

contact time is often incomplete and sometimes contradictory. Therefore the

method on immediate separation versus delayed centrifugation before analysis.

Prolonged contact of serum with the clot can cause pre-analytical

of Clinical Chemistry is 2030 min. During a prolonged contact time between

can change the concentrations of certain analytes in the serum. NCCLS

Procedures for the Handling and Processing of Blood Specimens recommends

of collection to the time of separation was also recommended. Each individual

clinics, transportation of specimens from a phlebotomy site to a laboratory

sometimes takes longer than 2 hours. If overly stringent transportation

unnecessarily. Ideally, a specific allowable transportation time should be applied

time blocks in which serum-clot contact cause no changes in analyte

concentrations. In some instances, specimens arrive in the laboratory at some

determine the cutoff times for acceptable specimens.

only to potassium within cells. Magnesium deficiencies are related to impairment

of neuromuscular function, cardiovascular disease and hypertension. Since

Potassium is well studied analyte, the researchers considered Magnesium to

study its levels using glass and plastic tubes.

Statement of the Problem

Prolonged Serum-clot contact time, will determine the levels of Magnesium in

rate of changes of the analyte in different tubes at a different serum-clot contact

data to answer the following questions:

serum-clot contact time?

different serum-clot contact time?

different serum-clot contact time?

tubes and samples analyzed in plastic tubes?

standard serum-clot contact time and samples analyzed at different

serum-clot contact time interval?

analyzed (using Glass and Plastic tube) at different serum-clot contact

Prolonged Serum-clot contact time seeks to assess the rate of change in