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

Question 1: What technique is used to separate DNA fragments by size during gel electrophoresis?

Centrifugation based on molecular density
Migration through an agarose gel matrix under an electrical current (Correct answer)
Precipitation by salt concentration
Filtration through a molecular sieve membrane

Correct answer: Migration through an agarose gel matrix under an electrical current

DNA fragments migrate through an agarose gel under electrical current — smaller fragments move faster through the gel matrix than larger fragments, separating them by size.

Agarose gel electrophoresis exploits the negative charge of DNA's phosphate backbone. When an electrical current is applied, DNA migrates toward the positive electrode (anode). The agarose gel acts as a molecular sieve — smaller DNA fragments move through the gel matrix more quickly than larger fragments, resulting in size-based separation. After electrophoresis, DNA is visualized using ethidium bromide or SYBR Safe intercalating dyes under UV light. Fragment sizes are determined by comparison to a DNA ladder (size marker). This technique is fundamental in molecular diagnostics for PCR product verification, RFLP analysis, and Southern blotting in Ontario clinical molecular laboratories.

Question 2: What is the principle of the polymerase chain reaction (PCR) in molecular diagnostics?

Amplification of specific DNA sequences through repeated cycles of denaturation, annealing, and extension (Correct answer)
Separation of DNA fragments by size in a gel matrix
Detection of protein antigens using antibody-enzyme conjugates
Translation of mRNA sequences into protein

Correct answer: Amplification of specific DNA sequences through repeated cycles of denaturation, annealing, and extension

PCR amplifies specific DNA sequences exponentially through repeated thermal cycles: denaturation (94-98°C) separates strands, annealing (50-65°C) allows primers to bind, and extension (72°C) allows Taq polymerase to copy the target.

PCR (Polymerase Chain Reaction, developed by Kary Mullis, 1983) is the most widely used molecular biology technique. A thermocycler performs repeated cycles typically consisting of: (1) Denaturation (94-98°C, 30-60 sec) — double-stranded DNA separates into single strands; (2) Annealing (50-65°C, 30-60 sec) — short oligonucleotide primers complementary to target sequences bind; (3) Extension (72°C, 60 sec/kb) — Taq (or other thermostable) polymerase synthesizes new DNA strands from primers. Each cycle doubles the target, yielding 2ⁿ copies after n cycles — approximately 1 million copies after 30 cycles. Real-time PCR (RT-PCR/qPCR) quantifies amplification using fluorescent probes (TaqMan) or intercalating dyes (SYBR Green). PCR is used in Ontario for infectious disease diagnosis, oncology, and genetic testing.

Question 3: In clinical microbiology, what is the purpose of inoculating a blood agar plate and a chocolate agar plate simultaneously when processing a respiratory specimen?

Blood agar supports fastidious organisms; chocolate agar supports anaerobes only
Blood agar detects beta-hemolytic organisms; chocolate agar supports fastidious organisms like Haemophilus and Neisseria that require growth factors released by heated RBCs (Correct answer)
Both media are equivalent — inoculating both ensures redundancy
Chocolate agar inhibits normal flora while blood agar supports all organisms

Correct answer: Blood agar detects beta-hemolytic organisms; chocolate agar supports fastidious organisms like Haemophilus and Neisseria that require growth factors released by heated RBCs

Blood agar supports most bacteria and shows hemolysis patterns; chocolate agar (heated blood agar) releases NAD and hemin from lysed RBCs, supporting fastidious organisms such as Haemophilus influenzae and Neisseria species.

The media combination used for respiratory specimens is designed to support recovery of the widest range of pathogens. Blood agar (5% sheep blood in tryptic soy agar) supports most pathogenic bacteria and allows classification by hemolysis: alpha (partial/green, e.g., Streptococcus pneumoniae), beta (complete lysis, e.g., S. pyogenes), or gamma (none). Chocolate agar is blood agar heated to ~80°C, causing RBC lysis and release of hemin (factor X) and NAD (factor V — nicotinamide adenine dinucleotide) essential for fastidious organisms: Haemophilus influenzae (requires both X and V factors) and Neisseria meningitidis/gonorrhoeae. Both are used in standard respiratory culture processing in Ontario microbiology laboratories.

Question 4: Which laboratory method is used as the reference (gold standard) for diagnosing malaria?

Malaria rapid antigen test (RDT)
Microscopic examination of Giemsa-stained thick and thin blood films (Correct answer)
PCR for Plasmodium species
ELISA for Plasmodium antibodies

Correct answer: Microscopic examination of Giemsa-stained thick and thin blood films

Microscopic examination of Giemsa-stained thick and thin blood films is the reference standard for malaria diagnosis, allowing species identification and parasitemia quantification.

Despite advances in rapid testing and molecular methods, Giemsa-stained blood film microscopy remains the reference standard for malaria diagnosis. Thick films concentrate RBCs, enabling detection of low-level parasitemia; thin films allow species identification by red cell morphology and parasite characteristics. The four human Plasmodium species have distinct morphological features: P. falciparum (ring forms, no Schüffner stippling, banana-shaped gametocytes), P. vivax (enlarged RBCs, Schüffner dots, amoeboid trophozoites), P. malariae (band forms), P. ovale (oval RBCs, Schüffner dots, fimbriated edges). In Ontario, malaria is a travel-related infection; laboratories receiving malaria-suspected specimens must process them as urgently as bacteremia. Experienced microscopists are required for accurate diagnosis.

Question 5: What type of pipette is most appropriate for delivering a precise volume of 100 μL for an ELISA assay?

Serological pipette (1 mL)
Calibrated micropipette (100 μL fixed or adjustable) (Correct answer)
Pasteur pipette
Graduated glass pipette (1 mL TD)

Correct answer: Calibrated micropipette (100 μL fixed or adjustable)

A calibrated micropipette set to 100 μL delivers precise, accurate volumes for immunoassays like ELISA. Accuracy is critical because volume errors directly affect the antigen-antibody ratio and results.

For small volumes in the microlitre range (1-1000 μL), calibrated air-displacement micropipettes (e.g., Eppendorf, Gilson Pipetman) are the appropriate choice. They provide the necessary accuracy (typically ±1-2%) and precision required for immunoassays, PCR, and other sensitive molecular and immunochemical techniques. Serological pipettes and graduated glass pipettes are designed for millilitre volumes and have insufficient precision for microlitre applications. Pasteur pipettes are transfer tools without volume calibration. Proper micropipette technique includes pre-wetting the tip, holding the pipette vertically, smooth plunger depression, and checking for bubble-free delivery. Regular calibration verification is required for quality management in Ontario laboratories.

Question 6: What is the CMLTO-recognized clinical significance of performing an extended red blood cell antigen typing before first transfusion in a patient with sickle cell disease?

To determine ABO compatibility only
To establish a baseline antigen profile to guide compatible RBC unit selection and prevent alloimmunization (Correct answer)
To identify which blood group system is most immunogenic
To detect pre-formed antibodies from previous transfusions

Correct answer: To establish a baseline antigen profile to guide compatible RBC unit selection and prevent alloimmunization

Extended RBC antigen typing (Rh, Kell, Duffy, Kidd, MNS systems) before first transfusion establishes the patient's antigen profile, allowing selection of phenotypically matched units to minimize alloimmunization risk.

Patients with sickle cell disease require chronic transfusion therapy and are at high risk for alloimmunization (developing antibodies to foreign RBC antigens) because of immune differences between patients of African heritage (high prevalence of sickle cell) and the predominantly Caucasian blood donor pool (different antigen frequencies). Extended phenotype typing before the first transfusion includes C, c, E, e (Rh system), K (Kell), Fya, Fyb (Duffy), Jka, Jkb (Kidd), and S, s (MNS). Transfusing phenotype-matched blood markedly reduces alloimmunization, which complicates future transfusions by requiring increasingly rare matched units and increasing hemolytic transfusion reaction risk. This is standard practice in Ontario transfusion medicine for sickle cell patients.

MLPAO Laboratory Techniques and Procedures Questions and Answers 2

What technique is used to separate DNA fragments by size during gel electrophoresis?