Lab Methods
Usually week 3-5 of an immunology & genetics courseMethod questions are free points if you learn each test as a one-line job description: what it detects, what it cannot detect, and the one scenario where it beats the alternatives. The exam rarely asks how the machine works — it hands you a clinical stem and asks which test to order, or hands you a result (IgM positive, fourfold titer rise, band on a Western blot) and asks what it means. Anchor every method to its classic use case and the answer usually writes itself.
This guide is the frame. The exam is written from your lectures— drop this topic's slides on the dashboard to get flashcards and board-style questions from your own course, and quiz them all term (one cumulative exam rewards nothing more than early, repeated self-testing). The immunology & genetics playbook has the weekly loop.
AI-authored study notes, not faculty-reviewed and not medical advice. Verify every mechanism against your course materials and faculty; programs differ in emphasis.
High-Yield Pearl
Serology tells you about the patient's response, PCR tells you about the pathogen itself — early disease and newborns belong to PCR, history and immunity belong to IgG.
What the exam asks
Mechanisms, patterns, and what happens when each one fails.
- IgM vs IgG serology. IgM appears first (days to ~1 week), marks acute or recent infection, and fades over weeks to months; IgG rises later, persists for years, and marks past infection or immunity. IgM positive / IgG negative = acute; IgM negative / IgG positive = past infection or vaccination; both positive = recent infection or early convalescence. IgM does not cross the placenta (it's a pentamer), so IgM in a newborn means the infant's own response — evidence of congenital infection — while newborn IgG is mostly maternal.
- Titers and the fourfold rise. A titer is the reciprocal of the greatest serum dilution still giving a positive result — 1:64 means antibody was detectable at a 64-fold dilution, so HIGHER titer = MORE antibody. A single titer is hard to interpret; the classic proof of acute infection is a fourfold (two-dilution) rise between acute and convalescent sera drawn 2–4 weeks apart.
- ELISA. Enzyme-linked immunosorbent assay: an enzyme-conjugated antibody produces a color change proportional to the amount of antigen or antibody captured on a plate. Indirect ELISA detects the patient's ANTIBODY (serologic screening — HIV, hepatitis); sandwich ELISA captures ANTIGEN between two antibodies. Built for screening: highly sensitive, cheap, automatable — which is exactly why a positive screen goes on to a confirmatory test.
- Western blot. Proteins are separated by size on a gel (SDS-PAGE), transferred to a membrane, and probed with antibody; a band at the right molecular weight confirms a specific protein. Its role is confirmation after a sensitive screen — the historical HIV algorithm (ELISA screen → Western blot confirm) is the template the exam still tests, even though modern HIV testing now confirms with an antibody-differentiation immunoassay.
- Flow cytometry. Cells in a fluid stream pass single-file through a laser; scatter gives size and granularity, and fluorescent antibodies against surface markers (CD3, CD4, CD8, CD19, CD56) count and classify cell populations. It is THE tool for immunophenotyping: CD4 counts in HIV, classifying leukemias and lymphomas, and characterizing immunodeficiencies (e.g., absent B cells in X-linked agammaglobulinemia, absent CD18 in leukocyte adhesion deficiency).
- PCR and RT-PCR. PCR exponentially amplifies a target DNA sequence through cycles of denaturation, primer annealing, and extension by a heat-stable polymerase — detecting the pathogen or gene ITSELF rather than the immune response, so it turns positive before seroconversion. RT-PCR adds reverse transcriptase to convert RNA to cDNA first, which is how RNA viruses (HIV viral load, hepatitis C, SARS-CoV-2, influenza) are detected and quantified. Quantitative (real-time) PCR reports how much target is present, not just whether it is.
- Sanger vs next-generation sequencing. Sanger uses chain-terminating dideoxynucleotides to read one DNA region at a time — the gold standard for confirming a single known variant or sequencing one candidate gene. NGS sequences millions of fragments in parallel, powering multi-gene panels, whole-exome, and whole-genome sequencing when the differential is broad. Rule of thumb: one known target → Sanger; unknown or many possible genes → NGS.
- Karyotype vs FISH vs chromosomal microarray. Karyotype images all 46 chromosomes in metaphase — best for aneuploidy (trisomy 21, 45,X) and large or BALANCED rearrangements like translocations, but it resolves only changes above roughly 5 Mb and needs dividing cells. FISH hybridizes a fluorescent probe to one specific locus — fast, targeted, works on interphase cells (classic for 22q11 deletion and BCR-ABL). Chromosomal microarray (CMA) scans the whole genome for submicroscopic copy-number gains and losses and is the first-line test for unexplained developmental delay, intellectual disability, and autism — but it CANNOT see balanced translocations, because no material is gained or lost.
- Sensitivity, specificity, and test sequencing. Sensitivity = true positives detected among the diseased; a highly sensitive test, when Negative, rules OUT (SnNout). Specificity = true negatives among the healthy; a highly specific test, when Positive, rules IN (SpPin). Screening strategy follows directly: screen with the sensitive test (miss no one, tolerate false positives), confirm with the specific test — the logic behind ELISA-then-confirm and behind every 'which test next?' stem.
Worth drilling · 21 concepts
Each with the one fact that identifies it. Your course's slides are the authority; use this to check your list against.
- 1IgM — pentamer made first in an acute infection; too big to cross the placenta
- 2IgG — dominant antibody of memory and past infection; the only class crossing the placenta
- 3Fourfold titer rise — the acute-to-convalescent change that proves recent infection
- 4Titer — reciprocal of the last dilution still positive; a bigger number means more antibody
- 5Indirect ELISA — plate-bound antigen catches the patient's antibody; the classic serologic screen
- 6Sandwich ELISA — antigen captured between two antibodies; detects the pathogen's protein itself
- 7Western blot — size-separated proteins probed with antibody; the confirmatory band test
- 8Flow cytometry — laser counts and phenotypes cells by CD markers; how a CD4 count is done
- 9CD4 count — the flow-cytometry number that stages HIV and triggers prophylaxis thresholds
- 10PCR — thermocycled amplification of a DNA target; positive before antibodies ever appear
- 11Reverse transcriptase — the enzyme that turns viral RNA into cDNA so it can be amplified
- 12Quantitative PCR — real-time amplification reporting viral load, not just presence
- 13Sanger sequencing — dideoxy chain termination reading one region; confirms a single known variant
- 14Next-generation sequencing — massively parallel reads; panels, exomes, broad differentials
- 15Karyotype — metaphase picture of all 46 chromosomes; finds aneuploidy and balanced translocations
- 16FISH — fluorescent probe lighting up one locus; the fast test for a suspected microdeletion
- 17Chromosomal microarray — genome-wide copy-number scan; first-line for unexplained developmental delay
- 18Window period — the interval after infection when antibody tests are still negative but PCR is positive
- 19SnNout — a negative result on a highly Sensitive test rules the disease out
- 20SpPin — a positive result on a highly Specific test rules the disease in
- 21Newborn screening — dried-blood-spot tandem mass spectrometry catching treatable disease before symptoms
Practice it
Active recall over the drill list — flip and claim, match the pairs, produce the answers cold.
Every concept on the drill list, one card at a time — the name up front, the fact that identifies it on the flip. Claim each card honestly and the deck learns what to lead with next time. Progress lives in this browser only.
What everyone misses
The distinctions that lose points on this material, year after year.
- Reading a titer backward. 1:512 is a HIGHER titer than 1:8 — the number is the dilution the antibody survived, so bigger denominator means more antibody. Students see the '1:' fraction and rank them like fractions, picking the smaller dilution as 'more.'
- Serology in the window period. A negative antibody test early after exposure does not exclude infection — antibodies take days to weeks to appear. The stem with recent high-risk exposure and a negative screen wants NAAT/PCR (or repeat serology later), not reassurance.
- Newborn IgG means mom, not baby. Maternal IgG crosses the placenta and persists for months, so a positive IgG in an infant usually reflects the mother's immunity. Congenital infection is implicated by infant IgM (which cannot cross) or by pathogen PCR — never by IgG alone.
- Microarray for a balanced translocation. CMA detects only copy-number change; a balanced translocation or inversion gains and loses nothing, so the array is normal. Recurrent miscarriage with suspected parental rearrangement needs a KARYOTYPE — the reflexive 'newest test is best' answer is wrong here.
- Screen vs confirm order. The sensitive test comes FIRST (catch everyone), the specific test SECOND (eliminate the false positives). Answer choices that confirm with another sensitive screen, or open with the expensive specific test, are the standard distractors.
- PCR detects the organism, serology detects the response. PCR positivity means the pathogen's nucleic acid is there now; antibody positivity may mean infection long past or vaccination. Stems that ask 'active infection vs immunity' are testing exactly this distinction — e.g., hepatitis B surface antigen vs surface antibody.
- Sanger when the question wants NGS (and vice versa). Confirming the family's known CFTR mutation in a sibling is a Sanger job; an undiagnosed syndrome with a broad differential is an NGS/panel job. Matching the sequencing breadth to the breadth of the differential is the whole question.
Clinical correlations
Where this topic shows up again — in clinic, on rotations, and on the boards.
- HIV testing algorithm. Screen with a 4th-generation antigen/antibody immunoassay (p24 antigen shortens the window to ~2–3 weeks); a positive screen reflexes to an antibody-differentiation assay, and discordant results go to HIV RNA PCR. Disease is then staged and therapy monitored with flow-cytometry CD4 counts and quantitative viral load — one vignette can test four methods at once.
- Congenital infection workup. Suspected congenital CMV or toxoplasmosis is confirmed with infant IgM or pathogen PCR (urine/saliva CMV PCR in the first 3 weeks of life), because transplacental maternal IgG contaminates any IgG-based answer. The mechanism — pentameric IgM cannot cross the placenta — is what the question is really grading.
- Unexplained developmental delay. A child with developmental delay, intellectual disability, autism, or multiple congenital anomalies and no syndrome-specific gestalt gets chromosomal microarray first-line; a clinically recognizable aneuploidy (Down syndrome facies) can go straight to karyotype, and a suspected specific microdeletion (conotruncal defect + hypocalcemia → 22q11) can go to FISH or array.
- Newborn screening logic. State newborn screens run tandem mass spectrometry and other assays on a dried heel-stick blood spot at 24–48 hours to catch treatable, presymptomatic disease — PKU, congenital hypothyroidism, galactosemia, hemoglobinopathies, CF (immunoreactive trypsinogen), SCID (TRECs). These are deliberately high-sensitivity SCREENS: every positive needs confirmatory testing (e.g., sweat chloride for CF) before a diagnosis is made.
- SCID on the newborn screen. T-cell receptor excision circles (TRECs) — byproducts of normal T-cell receptor rearrangement — are quantified by PCR from the blood spot; absent TRECs flag SCID before the first life-threatening infection. Flow cytometry then phenotypes the lymphocyte compartments (T, B, NK) to classify the defect — a clean example of screen-by-PCR, characterize-by-flow.
- Leukemia workup. A blast-filled smear goes to flow cytometry for immunophenotype (lymphoid vs myeloid, B vs T lineage) and to cytogenetics/FISH for the defining rearrangement — BCR-ABL t(9;22) in CML, PML-RARA t(15;17) in APL. Quantitative RT-PCR for the fusion transcript then tracks minimal residual disease on therapy.
- Choosing serology vs NAAT in acute illness. Early in an infection, direct detection (PCR/antigen) outperforms serology because antibodies haven't formed; late or retrospectively, serology outperforms culture and PCR because the organism is gone. Acute hepatitis A = anti-HAV IgM; acute COVID or influenza = NAAT/antigen; 'did I ever have it / am I immune' = IgG.