2.24: Phlebotomy
- Page ID
- 38652
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)RELATED READING: Chapter 3, Pages 69-73
OBJECTIVES
Upon completion of this exercise, appropriate discussion, and related readings, the student will be able to:
1. Understand the proper method of processing phlebotomy tubes.
2. Understand the mechanisms of action and the uses of various anticoagulants.
3. Relate the color of a hemolyzed sample to the degree of hemolysis and the amount of free hemoglobin.
PRINCIPLE
Depending upon the type of test to be performed, there are many ways to collect blood, urine, and other body fluids. Usually a specific type of collection vessel is used to produce a specimen that is suitable for a particular type of analysis. For example, iodoacetate is placed in phlebotomy tubes to prevent glycolysis. To collect unclotted blood a chemical such as oxalate, EDTA, or heparin must be added to prevent coagulation. Other types of phlebotomy tubes, such as those used for trace metal analysis, are designed to reduce possible contamination of the sample from exogenous metals.
GLOSSARY
Red top -a term indicating a phlebotomy tube containing no anticoagulant or preservative used to collect serum. Term refers to the color of the rubber stopper on the tube. Also- “green top”, “purple top”, etc.
MATERIALS
- Phlebotomy tubes:
- no anticoagulant (red top)
- two heparin (green tops)
- citrate (blue top)
- EDTA (lavender top)
- oxalate (black top)
- iodoacetate (gray top)
- fluoride (gray top)
- Centrifuge
- Refrigerator/freezer
- 16 x 100 mm glass test tubes
PROCEDURE
- Students can perform this exercise either as individuals or in small groups.
- A single set of phlebotomy tubes should be drawn from a single Individual. Standard phlebotomy procedures, as described by the Instructor, should be employed during this exercise. After the set of phlebotomy tubes are filled, they should be processed as described below.
- After collection, the red top, one heparin, EDTA, oxalate, and citrate tubes should be centrifuged for 15 minutes.
- The serum or plasma should be carefully removed from the cellular sediment with Pasteur pipets and placed in appropriately labeled 16 x 100 mm test tubes.
- Cover and refrigerate the tubes if they will be used within 4 days. Freeze (-20°C) if they will need to be stored beyond 4 days. Note: Never freeze unseparated whole blood because the red cells will lyse.
- The second heparin and the gray topped tubes, iodoacetate and fluoride, should be allowed to stand at room temperature overnight. The next day, they should be processed as described above.
- Observe the appearance (including the color, degree of tubidity, etc.) of each set of specimens, and record in the Results Section of the Data Sheet. During class discussion, compare the results for the different sets of samples.
- Have an aliquot of each plasma and serum measured for glucose. Record the glucose values for each sample on the data sheet.
OPTIONAL EXERCISE: HEMOLYSIS
MATERIALS
- One tube of heparinized blood
- micropipettes (green top) for entire class
- 16 x 100 mm test tubes
PROCEDURE
- Place 20 mL of distilled water in each tube of a series of 5 test tubes, labeled 1 through 5.
- Starting with tube #1, add either 0.01, 0.025, 0.05, 0.1 or 0.2 mL of well mixed, heparinized whole blood into the appropriate tube.
- Mix the test tubes by covering with Parafilm and inverting.
- By visual inspection, note and record the degree of red color imparted to each solution (score 0 to +4 degree of color).
- Calculate the hemoglobin concentration in each solution, assuming an average hemoglobin concentration for the original specimen of 150 g/L.
- Have an aliquot of each sample saved for analysis of potassium and lactate dehydrogenase (LD). Record results on data sheet.
| DATA SHEET, EXERCISE # 24 |
NAME: ___________ DATE: ___________ |
RESULTS PART A:
| Color of Phlebotomy Tube | Type of Specimen (plasma or serum) | Specimen Color | Presence of Turbidity | Glucose mg/L |
|---|---|---|---|---|
| Gray-overnight | ||||
| Gray-overnight |
RESULTS PART B
| Solution # | Degree of Red Color (0-4+) | Approximate Concentration Hemoglobin (g/L)* | Lactate Dehydrogenase U/L | K+ mmol/L |
|---|---|---|---|---|
| 1 | ||||
| 2 | ||||
| 3 | ||||
| 4 | ||||
| 5 |
*Use equation on following page.
Calculation of approximate hemoglobin (Hb) concentrations in the hemolysates:
Use basic dilution equation as follows:
\[(conc_{i} \; Hb) (vol \ldotp \; dil \ldotp) = (conc_{f} \; Hb) (final\; dil \ldotp \; vol \ldotp)\]
where conci Hb = concentration of Hb in blood (150 g/L)
where concf Hb = concentration of Hb in diluted solutions (#1-#5)
vol. dil. = volume of blood that is diluted
final dil. vol. = final volume that blood is diluted out to (mL)
This equation can be rearranged to read:
\[conc_{f} \; Hb = \frac{(conc_{i}\; Hb)(vol \ldotp \; dil \ldotp)}{(final\; dil \ldotp \; vol \ldotp)}\]
DISCUSSION QUESTIONS
- What constituents of blood would be responsible for the different colors of the serum or plasma specimens observed?
- What would cause the presence of turbidity in a serum or plasma specimen?
- For each different type of phlebotomy tube, list several tests which might appropriately utilize that specific tube.
- What tests are affected by the disruption of blood cells (i.e. mixing intracellular material with plasma or serum)?
- What tests may be affected by the color (i.e., absorbance) of hemoglobin?
- Should the phlebotomy tubes be drawn in any specific order?
- Can you explain any differences between the glucose levels for the various samples? For the samples stored at 4°C and those with a preservative?
- Which seems to be a better indicator of hemolysis, color, LD levels, or K+ levels?