MLPAO Core Laboratory Procedures Questions and Answers 2 — Questions and Answers

Question 1: What is the purpose of centrifugation in specimen processing in a medical laboratory?

To mix reagents with the specimen
To separate blood into its cellular and liquid components (Correct answer)
To lyse red blood cells for analysis
To concentrate bacteria for culture

Correct answer: To separate blood into its cellular and liquid components

Centrifugation uses centrifugal force to separate blood into its components: packed red cells at the bottom, a buffy coat of white cells and platelets, and serum or plasma on top.

When blood is centrifuged, its components separate by density under centrifugal force: red blood cells (densest, ~1.10 g/mL) settle at the bottom, followed by a buffy coat of leukocytes and platelets (~1.06-1.08 g/mL), with serum or plasma (~1.025-1.030 g/mL) on top. For serum, blood is allowed to clot before centrifugation; for plasma, anticoagulant tubes are centrifuged directly. Typical parameters are 1500-2000g for 10-15 minutes for serum/plasma tubes. Inadequate centrifugation leaves cellular elements in the supernatant, causing interference in many assays. This is a fundamental core laboratory procedure tested in CMLTO examinations.

Question 2: What does the term 'hemolysis' mean in the context of laboratory specimen quality?

Clotting of blood in the collection tube
Rupture of red blood cells releasing hemoglobin into the plasma/serum (Correct answer)
Bacterial contamination of the specimen
Protein denaturation during storage

Correct answer: Rupture of red blood cells releasing hemoglobin into the plasma/serum

Hemolysis refers to rupture of red blood cell membranes with release of intracellular contents including hemoglobin into the serum or plasma, causing a pink or red discolouration.

Hemolysis (pink/red plasma or serum) is one of the most common pre-analytical specimen quality issues in clinical laboratories. It can be caused by: traumatic venipuncture, using too small a needle, excessive suction, vigorous mixing, temperature extremes, prolonged tourniquet application, or delayed processing. Hemolyzed specimens contain elevated intracellular analytes that falsely elevate potassium (RBC K+ is ~23 times higher than plasma), LDH, AST, and ALT. Hemolysis also causes spectrophotometric interference at wavelengths near hemoglobin's absorption peaks. Moderate-to-severe hemolysis typically requires specimen recollection for affected analytes.

Question 3: In clinical chemistry, what is a 'blank' in the context of spectrophotometric analysis?

A sample with the highest analyte concentration used for calibration
A solution containing all reagents but no analyte, used to zero the instrument (Correct answer)
A patient sample with no detectable analyte
A quality control sample at the lower limit of detection

Correct answer: A solution containing all reagents but no analyte, used to zero the instrument

A blank (or reagent blank) contains all assay reagents but no analyte. It is used to zero the spectrophotometer, accounting for background absorbance from reagents and the cuvette.

In spectrophotometric analysis, a reagent blank contains all components of the assay (buffer, reagents, water) except the analyte of interest. Reading the blank first sets the instrument absorbance to zero (100% transmittance), correcting for any inherent colour or absorbance from the reagents, solvent, and cuvette. This allows the measured absorbance of the sample to reflect only the analyte. Types of blanks in laboratory assays include: reagent blank (no sample or analyte), sample blank (sample without reagent — corrects for sample chromogens), and substrate blank. Proper blanking is essential for accurate spectrophotometric analysis in Ontario clinical chemistry labs.

Question 4: What is the principle of the Gram stain in clinical microbiology?

Differentiates organisms based on cell size
Differentiates bacteria based on cell wall composition into Gram-positive (purple) and Gram-negative (pink) groups (Correct answer)
Detects acid-fast mycobacteria using carbolfuchsin
Identifies fungi based on capsule staining

Correct answer: Differentiates bacteria based on cell wall composition into Gram-positive (purple) and Gram-negative (pink) groups

The Gram stain differentiates bacteria based on peptidoglycan cell wall thickness: Gram-positive bacteria retain crystal violet (appear purple); Gram-negative bacteria lose it and take up safranin counterstain (appear pink/red).

The Gram stain (developed by Hans Christian Gram, 1884) involves four steps: (1) Crystal violet primary stain — stains all bacteria purple; (2) Iodine mordant — fixes crystal violet in cell wall; (3) Alcohol/acetone decolorizer — removes crystal violet from thin-walled Gram-negative cells but retained by thick-walled Gram-positive cells; (4) Safranin counterstain — stains decolorized Gram-negative cells pink/red. Gram-positive bacteria have a thick peptidoglycan layer (20-80 nm) that retains crystal violet; Gram-negative bacteria have a thin peptidoglycan layer (2-7 nm) with an outer lipopolysaccharide membrane that is disrupted by decolorizer. The Gram stain is the most fundamental and commonly performed stain in clinical microbiology.

Question 5: What is the purpose of performing a reticulocyte count in hematology?

To assess platelet function
To evaluate the bone marrow's erythropoietic activity and response to anemia (Correct answer)
To count immature white blood cells
To measure coagulation factor levels

Correct answer: To evaluate the bone marrow's erythropoietic activity and response to anemia

The reticulocyte count assesses bone marrow erythropoietic activity. Elevated reticulocytes indicate increased RBC production (response to hemolysis or blood loss); low counts suggest bone marrow suppression.

Reticulocytes are immature red blood cells that have extruded their nucleus but retain residual RNA. They circulate for approximately 1-2 days in peripheral blood before maturing to erythrocytes. The reticulocyte count (normal: 0.5-1.5% of RBCs or 25-85 × 10⁹/L absolute reticulocytes) reflects the bone marrow's erythropoietic output. An elevated reticulocyte count (reticulocytosis) indicates the marrow is responding to RBC loss from hemolysis, bleeding, or treatment of deficiency anemia. A low count in the presence of anemia suggests bone marrow failure, aplasia, or nutritional deficiency. The reticulocyte production index (RPI) corrects for anemia severity. This is important hematology content for CMLTO registration preparation.

Question 6: In urinalysis, what does a positive nitrite result on a urine dipstick indicate?

Presence of protein in urine
Presence of Gram-negative bacteria that convert dietary nitrates to nitrites (Correct answer)
Elevated glucose levels in urine
Presence of white blood cells

Correct answer: Presence of Gram-negative bacteria that convert dietary nitrates to nitrites

A positive nitrite on urine dipstick indicates the presence of bacteria (typically Gram-negative) that contain nitrate reductase enzymes, which convert dietary nitrates to nitrites in the bladder urine.

The nitrite test on a urine dipstick detects the presence of nitrite, produced when bacteria containing nitrate reductase convert dietary nitrates (from vegetables) to nitrite. Most common urinary tract pathogens are Gram-negative organisms with this enzyme: Escherichia coli (most common UTI pathogen), Klebsiella, Proteus, and Enterobacter. A positive nitrite result suggests bacterial UTI and should be correlated with leukocyte esterase (WBC marker) and microscopic urinalysis. False negatives occur with: dilute urine, short bladder dwell time (<4 hours), Gram-positive organisms (Staphylococcus, Enterococcus) that lack nitrate reductase, and vitamin C interference. The dipstick is a screening test; culture is required for definitive diagnosis.

MLPAO Core Laboratory Procedures Questions and Answers 2

What is the purpose of centrifugation in specimen processing in a medical laboratory?