MLAB I - Laboratory Skills I: Reagent/Solution Preparation & Specimen Processing
Safety and QA: Solution Preparation
Reagent preparation carries chemical, physical, and biological hazards. Always consult the chemical container label and the MSDS before use.
PPE and work practice controls must be used. Use a fume hood for fumes.
Always follow safety protocols when handling reagents and when fume-producing chemicals are involved.
QA (Quality Assurance) for solution preparation:
Use chemically clean, dry, non-reactive containers.
Choose measuring containers appropriate for the task.
Use correct formulas and calculations for reagents.
Be aware that markings on beakers/Erlenmeyer flasks are approximate; certified glassware (e.g., volumetric flasks) should be used for critical measurements.
Select the correct grade of reagent water for the reagent being prepared.
Use correct pipetting techniques; select the appropriate pipette for the task.
Use volumetric pipets for critical measurements, especially when preparing standards and controls.
Store standards/controls refrigerated; other reagents may be at room temperature.
Inspect reagents for deterioration (color change, precipitation, turbidity) before use.
Chemical Grades
ACS-certified reagent/analytical-grade chemicals are suitable for both quantitative and qualitative work.
Ultrapure grade (spectrograde, nanograde, HPLC pure) is required for analytical methods needing very high purity.
Lower grades like purified, practical, technical, or commercial should not be used for clinical laboratory reagents but can be used for non-clinical purposes.
Solution Preparation Methods
Methods include: Dilutions, Ratios, Percent solutions, and Molar solutions.
Proportions are used to make dilutions; a dilution is often expressed as a ratio/proportion/fraction.
Example of a dilution: If a serum is diluted 1:5, 1 part serum + 4 parts diluent yield 5 total parts.
The general formula for dilutions is tied to C1V1 = C2V2 (see below).
Dilution Formulas and Practice
Formula for dilutions: C1 imes V1 = C2 imes V2
where C1 = starting concentration, V1 = volume of starting solution to use, C2 = final concentration, V2 = final total volume.Dilutions are common in hematology and immunology; the dilution factor is the reciprocal of the dilution (e.g., a 1:5 dilution has a dilution factor of 5).
Example of dilution factor reciprocity: A dilution of 1:100 has a reciprocal of 100.
Example Problems from the Lesson
Problem A (proportion): A buffer is made by mixing 2 parts solution A with 5 parts solution B to total 70 mL. The total parts define a part volume, V = 70 / (2+5) = 10 mL per part. Therefore, 2 parts A = 20 mL; 5 parts B = 50 mL.
Result: 20 mL solution A + 50 mL solution B = 70 mL buffer.
This demonstrates using ratios to achieve a desired final volume.
Problem B (C1V1 = C2V2): To prepare 100 mL of 0.1 M HCl from 1 M stock:
Solve (1 M) × V1 = (0.1 M) × (100 mL) → V1 = 10 mL.
Therefore, mix 10 mL of 1 M HCl with 90 mL water to yield 100 mL of 0.1 M HCl.
Concept: increasing or decreasing concentration by adjusting solvent volume while keeping moles constant.
General rule for diluting with ratios: Dilution factor = C1/C2, and C2 = C1 / (dilution factor).
Preparing Percent Solutions
Percent solution expresses concentration as a percent by weight or volume per 100 units of solution (often per 100 mL).
Common reference: 1% solution = 1 g solute per 100 mL (or per 100 g) of solution; 1 mL water ≈ 1 g.
Three ways to prepare percent solutions:
1) Weight to volume (w/v)
2) Volume to volume (v/v)
3) Weight to weight (w/w)
Weight–to–Volume (w/v) Percent Solutions
Example: 0.85% (w/v) saline means 0.85 g NaCl per 100 mL solution.
To prepare 500 mL of 0.85% saline:
0.85% (w/v) means 0.85 g NaCl per 100 mL; for 500 mL, use 5 × 0.85 g = 4.25 g NaCl.
Procedure:
Weigh 4.25 g NaCl.
Add about 250 mL water to a 500 mL volumetric flask.
Dissolve NaCl; fill to the mark with water to reach 500 mL.
Volume–to–Volume (v/v) Percent Solutions
Example: Making 100 mL of a 10% bleach solution by blending 10 mL bleach with 90 mL water (total 100 mL).
Concept: percent volume reflects the ratio of volumes, not masses.
Weight–to–Weight (w/w) Percent Solutions
In w/w, both solute and solvent are weighed; the final solution mass is used to compute percent.
Example preparation steps typically include zeroing a balance, adding solvent to reach 100 g total, then adding solute to reach the final mass, and rebalancing if needed.
Note: w/w percent solutions are rarely used in clinical laboratories compared to w/v or v/v.
Preparing Molar Solutions
Definition: A 1 M (one molar) solution contains 1 mole of solute per liter of solution.
Example: NaCl has formula weight (F.W.) = 58.4 g/mol (Na = 23, Cl = 35.4).
To prepare 1 L of 1 M NaCl, dissolve 58.4 g NaCl in enough water to reach a final volume of 1 L.
Important note: you add the solute to solvent and bring the total volume to 1 L, rather than adding 1 L of solvent to the solute.
Simplified calculation: number of moles = mass / formula weight. For 60 g NaOH (F.W. = 40) in 1 L, the molarity is 60/40 = 1.5 M (example used to illustrate how to compute moles from mass and F.W.).
Serial Dilutions and Titer
Serial dilution: successive dilutions of a sample by the same dilution factor to obtain a series of increasingly dilute samples.
Titer: the measure of reactivity or strength of a component determined by testing serial dilutions; the reciprocal of the highest dilution that yields the desired reaction.
Example: If the last reactive tube is 1:16, the titer is 16 (reciprocal of 1/16).
Practical use: titers are especially common in immunology to indicate antibody levels in serum.
Example: Two-Fold Serial Dilution (aka Doubling Dilution)
Set up nine tubes, each with 1 mL diluent.
Add 1 mL serum to tube 1 (1:2 dilution). Mix, then transfer 1 mL from tube 1 to tube 2 containing 1 mL diluent.
Repeat through tube 9; after the transfer, discard 1 mL from tube 9 to maintain volumes.
Final dilutions range from 1:2 in tube 1 to 1:512 in tube 9.
The last tube showing a reaction determines the endpoint titer (e.g., tubes 1–6 reactive, 7–9 non-reactive → titer = 64).
Preparing a Specific Dilution from a Concentrate (Compound Dilution)
When large dilution factors are required, perform a series of smaller dilutions (compound dilution) to improve accuracy.
Example: To obtain 10 mL of 0.001 M from 1 M stock:
Step 1: determine dilution factor needed: 0.001 / 1 = 0.001; convert to a practical series (e.g., 1:10 × 1:10 × 1:10) to reach 1:1000 total dilution.
Step 2: perform sequential 1:10 dilutions (1.0 mL into 9 mL, then 1:10 into the next tube, etc.).
Step 3: multiply the dilution factors to obtain the final dilution (e.g., 1:10 × 1:10 × 1:10 = 1:1000).
Check-In Scenarios (Practice Problems from the Lesson)
Scenario: A disinfectant instructs “Dilute the concentrate 1:50 with water.” With a 500 mL bottle, the replacement dilution should be prepared by:
Correct approach: add 10 mL of concentrate to 490 mL of water (1:50 total volume ratio). This corresponds to option d.
Scenario: An example of a percent solution used in the laboratory; what is the titer if the endpoint is at 1:128 dilution? The titer is 128 (reciprocal of the highest dilution that gave a reaction).
Scenario: What dilution is created when one part concentrate is added to nine parts solvent? That is a 1:10 dilution (10-fold dilution).
Common Formulas and Terms (Glossary)
Diluent: a liquid added to a solution to reduce concentration.
Dilution: a solution made less concentrated by adding a diluent; the act of making a solution more dilute; the dilution factor is the reciprocal of the dilution.
F.W. (Formula Weight): sum of the atomic weights in a compound’s formula.
Gram equivalent weight: F.W. divided by the valence.
Isotonic: a solution with the same osmotic pressure as a reference solution.
Molar solution (M): solution containing 1 mole of solute per liter of solution.
Mole: amount of substance containing Avogadro’s number of entities; the mole is the unit used to express amount of a chemical.
Molar weight (M.W.): sum of atomic weights in a molecule or compound (g/mol).
Normality (N): the number of gram equivalents of a solute per liter of solution.
Percent solution (percent w/v, w/w, v/v): percent concentration expressed as g per 100 mL (w/v), g per 100 g (w/w), or mL per 100 mL (v/v).
Physiological saline: 0.85% NaCl (0.15 M) solution.
Titer: reciprocal of the highest dilution showing the desired reaction.
Specimen Processing Overview
Some centers have a central processing area where specimens are received, logged, sorted by department, and evaluated for test suitability.
SPECIMEN SUITABILITY: Suitable specimens are required for accurate results. Unsuitable specimens are rejected and new specimens obtained.
Common rejection reasons (chemistry): hemolysis, insufficient quantity (QNS), wrong tube, outdated tubes, improper handling, contaminated specimen, exposure to light, delays in testing/processing.
Common rejection reasons (hematology): clotting.
The criteria for rejection can depend on the test ordered; some clues may not be evident until processing is underway or completed.
Centrifugation and Handling of Specimens
Tubes awaiting centrifugation should remain upright with stoppers on to prevent CO2 loss, pH shifts, or evaporation.
Removing stoppers can cause changes in CO2 and pH, potentially affecting certain tests (e.g., pH, CO2, acid phosphatase).
Potential contamination sources include sweat, glove powder, and evaporation, which can alter electrolyte and other test results.
Plasma vs Serum: Definitions and Handling
Serum: liquid portion of blood obtained after clotting and centrifugation of blood collected without anticoagulant; lacks clotting factors (e.g., fibrinogen).
Plasma: liquid portion of anticoagulated blood obtained after centrifugation; contains anticoagulant and retains clotting factors.
Collection tubes:
Red Top: plain glass or plastic (no anticoagulant).
Serum Separator Tubes (SST, Gold Top): contain a gel barrier that separates serum from cells during centrifugation.
Light Green Top PST: plasma separating tube with anticoagulant.
Gel barrier forms a physical separation between serum/plasma and cells during processing.
Clotting considerations:
Serology/biochemistry tests often require fully clotted serum before centrifugation (30–60 minutes at room temperature).
Tubes with clot activators or serum-separator tubes often clot within ~15 minutes if properly mixed.
Thrombin tubes (RST) clot rapidly (about 5 minutes).
Handling after centrifugation:
Serum should be removed promptly after centrifugation.
Plasma may be stored with anticoagulant present depending on test requirements.
Some enzyme tests require immediate separation and/or freezing to preserve activity; in general, separate and store promptly.
Storage considerations:
Most specimens can be stored in capped tubes; bilirubin and other analytes may require dark storage to prevent degradation.
If testing is delayed, refrigerate at 4°C.
Some tests require freezing; others require short-term room temperature stability.
Aliquot Preparation and Specimen Tracking
Aliquot: a portion of a specimen used for testing when multiple tests or instruments are involved.
Serum/Plasma should be separated from cells within 2 hours, then aliquoted into tubes labeled with the same patient/test identifiers.
OSHA guidance: work practices should minimize splashing, spraying, and aerosol generation when handling blood or potentially infectious materials.
Handling tips:
Use disposable transfer pipettes to transfer serum/plasma to aliquots.
Do not pour by hand to minimize aerosol formation.
Ensure correct matching of specimen to aliquot tube and test order.
Do not mix serum and plasma in the same aliquot or mix samples with different anticoagulants in the same aliquot.
Do not disturb buffy coat or gel when transferring; cap tubes after filling.
Boxed Practical Notes: Procedure Details and Safeguards
When stopping is necessary to access the sample, use a splash shield or face shield and cover the stopper with gauze to catch any droplets.
Removing stoppers straight up (not popping) helps minimize aerosols.
Specimen Processing: Review and Quick Reference
Pre-analytic risks include hemolysis, improper labeling, wrong tube type, insufficient volume, improper handling, contamination, delayed processing.
For hematology and coagulation work, ensure timely collection, proper anticoagulants, and prompt processing to maintain specimen integrity.
In centrifugation steps, ensure the correct orientation, use of separators, and proper handling to prevent sample loss or contamination.
Quick Summary: Key Formulas and Concepts to Memorize
Dilution formula: C1 imes V1 = C2 imes V2
Dilution factor is the reciprocal of the dilution (e.g., 1:5 dilution has a factor of 5).
Percent solutions:
w/v: 0.85% saline = 0.85 g NaCl per 100 mL solution.
To prepare 500 mL of 0.85%: mass NaCl = 0.85 g × 5 = 4.25 g.
v/v: e.g., 10% bleach = 10 mL bleach in 90 mL diluent to make 100 mL total.
w/w: e.g., to prepare 5% w/w, weigh solute and solvent to reach 100 g total; 5 g solute with 95 g solvent.
Molar solutions:
1 M = 1 mole solute per 1 L solution. Example: 1 M NaCl requires 58.4 g NaCl per liter.
Mass-to-molar conversion: moles = mass / F.W.; molarity relates to final volume.
Serial/doubling dilutions:
Doubling dilution sequence: each tube is half the concentration of the previous (1:2, 1:4, 1:8, …, up to 1:512 in a 9-tube series).
Titer is the reciprocal of the highest dilution showing a reaction.
Compound dilutions:
Use a dilution ladder (e.g., 1:10, then 1:10, then 1:10) to reach a 1:1000 final dilution via sequential 1:10 steps; multiply individual dilution factors to obtain total dilution.
References to Specimen Types and Terms
Plasma: liquid portion of anticoagulated blood after centrifugation; contains anticoagulants and retains clotting factors.
Serum: liquid portion after blood clots and is separated; lacks clotting factors (e.g., fibrinogen).
Anticoagulant-bearing tubes include Light Green PST (plasma separator tube) and others; SST tubes separate serum via a gel barrier.
Hemolysis, clotting, and QNS are common rejection criteria for chemistry/hematology tests.
Closing Notes: Real-World Relevance and Ethical/Practical Implications
Accurate reagent preparation, labeling, and storage directly impact patient safety and test validity.
Proper PPE and engineering controls (fume hoods, splash shields) reduce exposure risk to hazardous chemicals and infectious materials.
Quick, correct specimen processing minimizes degradation of analytes, preserving the integrity of results for diagnosis and treatment decisions.
Understanding chemical grades helps ensure that clinical assays use reagents of appropriate purity to avoid interference or erroneous results.
Quick Review Questions for Self-Check
What is the difference between serum and plasma, and how are they obtained?
How do you prepare 100 mL of a 0.1 M HCl solution starting from a 1 M stock? Show the calculation.
What is a 1:100 dilution in terms of dilution factor and reciprocal titer?
How would you prepare 500 mL of a 0.85% (w/v) saline solution? Show the mass of solute needed and the steps.
List three common reasons a specimen might be rejected during processing and why.
What precautions are taken during stopper removal to minimize aerosols?
Final Practical Takeaway
Mastery of reagent/solution preparation hinges on accurate math, correct container/grade selection, proper storage, and strict adherence to safety and QA protocols. This foundation is essential for reliable laboratory results and safe, efficient clinical practice.