Lecture 1: Proper Sample Storage & Submission

Why must a clinician confirm submission requirements before collecting a sample?

Because testing protocols differ in required sample type (whole blood, plasma, serum), anticoagulant/separator specifications, sample volume, time window for testing, and storage needs. Errors in collection compromise sample validity.

What is the significance of proper labeling of samples?

Proper labeling ensures traceability, prevents sample mix-ups, and is essential for valid diagnostic interpretation.

Compare whole blood, plasma, and serum in terms of composition and diagnostic use.

Whole blood: Unspun, non-clotted sample ± anticoagulant; used in CBCs, point-of-care devices, urgent care, anesthesia monitoring, and field diagnostics.

Plasma: Fluid fraction from anticoagulated blood; commonly used for benchtop biochemistry testing.

Serum: Fluid fraction from clotted blood (loss of clotting factors); used for benchtop biochemistry testing and sometimes specifically required.

How does traumatic venipuncture affect sample integrity?

It can cause hemolysis and/or artifactually lower platelet counts, leading to pre-analytical error.

Why should purple-top tubes (EDTA) generally be filled last?

To prevent EDTA contamination of other samples intended for biochemical testing, which could alter results.

What is the recommended technique for transferring blood into collection tubes, and why?

Allow tubes to fill via vacuum pressure rather than plunging the syringe, as excessive pressure causes hemolysis.

What is the importance of correct volume and gentle inversion when using anticoagulant tubes?

Ensures proper anticoagulant-to-blood ratio and homogeneous mixing; failure leads to clotting or erroneous values.

What are best practices for handling plasma/serum intended for biochemistry testing if not used immediately?

Samples should be refrigerated or shipped on ice to minimize heat-induced artifact. Freezing is an option for long-term storage or for certain specialized tests (e.g., coagulation send-out).

Why should CBC samples not be centrifuged or frozen?

Centrifugation or freezing destroys cell morphology, invalidating hematological assessment.

What is the acceptable handling window for CBC samples, and under what conditions?

Ideally run ASAP; if refrigerated and later warmed to room temperature, generally acceptable up to 72 hours (though many labs store up to 7 days).

How does poor blood collection technique contribute to pre-analytical errors?

Introduces hemolysis, inaccurate platelet counts, clotting before processing, EDTA contamination, or improper anticoagulant mixing. These errors affect diagnostic reliability.

What is the mechanism of EDTA anticoagulation, and what is its primary clinical use?

EDTA chelates calcium to prevent coagulation. It is gentler on cells than other anticoagulants and is the standard tube for CBCs (purple-top, available in K+ and Na+ variants).

What is the mechanism of heparin anticoagulation, and what type of analysis is it used for?

Heparin potentiates antithrombin, thereby inhibiting coagulation. Green-top tubes are typically used for plasma biochemistry.

How does sodium citrate anticoagulation work, and why is it ideal for coagulation studies?

Citrate weakly chelates calcium in a reversible fashion, allowing restoration of coagulation in vitro when calcium is added back during testing. Blue-top tubes are used for coagulation testing.

What is the function and limitation of red-top tubes?

They allow whole blood to clot prior to centrifugation, yielding serum for biochemistry. Clotting time is species dependent.

What are white-top tubes typically used for?

Miscellaneous sample storage when no anticoagulant or additive is needed.

What is the advantage of serum separator ("tiger-top") tubes, and what are their limitations?

They contain a gel of intermediate density that, upon centrifugation, separates serum/plasma from cells, preventing leeching of analytes. However, some tests (e.g., phenobarbital measurement) may yield falsely decreased results due to contact with the gel.

Why is immediate separation of serum/plasma from cells necessary when not using a tiger-top tube?

To prevent metabolic consumption or leeching of analytes into the cellular fraction, which distorts results.

What are the three major plasma/serum appearance changes, and what do they indicate?

Hemolysis: Red discoloration; caused by free hemoglobin from in vitro error (traumatic collection) or in vivo intravascular hemolysis.

Lipemia: Milky, lactescent appearance; usually post-prandial, hence fasting is recommended.

Icterus: Yellow discoloration from elevated bilirubin; interpretation must be species-specific (large animal plasma is normally yellowish).

How can hemolysis interfere with diagnostic testing?

Free hemoglobin alters spectrophotometric readings, falsely increases potassium and AST, and may reduce accuracy of other biochemical assays.

How can lipemia interfere with diagnostic testing?

Lipids scatter light, impacting spectrophotometric assays, and can artificially alter electrolyte and enzyme measurements.

How can icterus interfere with diagnostic testing?

Bilirubin alters spectrophotometric readings, potentially distorting chemistry values (e.g., falsely lowering creatinine or falsely increasing total protein depending on method).

Why should plasma and serum appearance always be noted during sample evaluation?

Because hemolysis, lipemia, and icterus (HLI) introduce pre-analytical artifacts that may significantly alter CBC and chemistry results, leading to diagnostic misinterpretation if unrecognized

How does in vitro hemolysis affect CBC measurements?

Falsely ↓ PCV, HCT, RBC count

Falsely ↑ MCHC (HGB proportionally higher than HCT/PCV)MCV unaffected

Ghost RBCs may be counted as platelets, falsely ↑ PLT

Refractometric protein difficult to interpret

How does in vivo intravascular hemolysis differ from in vitro hemolysis in terms of CBC findings?

Both can cause similar changes, but in in vivo hemolysis, ghost RBCs are more likely, falsely elevating platelet counts.

How does hemolysis interfere with biochemistry results?

Spectrophotometric interference and chemical reaction inhibition

Falsely ↑ intra-RBC analytes: K+ (especially in horses, camelids, Japanese dog breeds), phosphorus, ALT, LDH, Mg

Mild ↑ CK activity

How does hemolysis interfere with electrophoresis?

Severe hemolysis may cause false beta globulin spikes.

How does lipemia affect CBC values?

Falsely ↑ HGB → subsequently ↑ MCHC

Large lipid aggregates may be falsely counted as platelets (↑ PLT) or leukocytes (↑ WBC)

Falsely ↑ refractometric protein

How does lipemia affect chemistry results?

Spectrophotometric interference

↓ Na & Cl, slight ↓ K

Lipemia also promotes in vitro hemolysis

What technique can be used to minimize lipemia interference in samples?

Refrigerate the sample to precipitate lipid, then collect less lipemic serum/plasma for testing.

What is the impact of icterus on CBC and chemistry results?

CBC: Little to no effect

Chemistry: Severe hyperbilirubinemia → falsely ↓ biuret total protein and creatinine

Why is it important to recognize HLI interference patterns in lab results?

Because misinterpretation may lead to false diagnoses (e.g., pseudohyperkalemia from hemolysis, pseudohyponatremia from lipemia, or underestimating creatinine in severe icterus).