ASCP MLS cheat sheet

ABO: group A has anti-B, group B has anti-A, group O has anti-A and anti-B, group AB has neither; forward and reverse grouping must agree before a group is released.

D is the most immunogenic Rh antigen; weak D is resolved with indirect antiglobulin testing, and donor units testing weak D positive are labeled D-positive.

Antibody screen uses two or three group O reagent cells by IAT at 37°C; a positive screen requires panel identification before an AHG crossmatch is performed.

Immediate-spin crossmatch confirms ABO compatibility only; an antiglobulin crossmatch is required when the screen is positive or a clinically significant antibody history exists.

Storage: RBCs 1-6°C for 42 days in additive solution; platelets 20-24°C with continuous agitation for 5 days; FFP at or below -18°C for 12 months, usable 24 hours after thawing at 1-6°C.

RhIG 300 µg neutralizes about 15 mL of fetal RBCs (30 mL fetal whole blood); dosing is given at 28 weeks and within 72 hours of delivery of a D-positive infant.

Ficin and papain enhance Rh, Kidd, Lewis, and P1 antibodies but denature Duffy and MNS antigens; DTT destroys Kell antigens while sparing Duffy and Kidd.

Acute hemolytic transfusion reactions are most often ABO-incompatible units from patient or specimen misidentification; stop the transfusion, support renal perfusion, and return the bag and tubing.

Warm autoantibody workup uses autoadsorption only if the patient has not been transfused in the previous 3 months; otherwise alloadsorption with allogeneic RBCs is required.

Anion gap = Na - (Cl + HCO3), reference 8-12 mmol/L; correct by adding 2.5 for each 1 g/dL that albumin falls below 4.0.

Osmolality (calculated) = 2(Na) + glucose/18 + BUN/2.8; an osmolal gap above 10 suggests methanol, ethylene glycol, isopropanol, or mannitol.

Friedewald: LDL = total cholesterol - HDL - triglycerides/5; the calculation is invalid when triglycerides exceed 400 mg/dL.

Creatinine clearance = (urine creatinine × urine volume) / plasma creatinine, with volume in mL/min; Cockcroft-Gault estimates clearance from age, weight, and serum creatinine.

Corrected calcium (mg/dL) = measured calcium + 0.8 × (4.0 - albumin g/dL); ionized calcium is unaffected by albumin and is preferred in critical illness.

Delta ratio = (measured anion gap - 12) / (24 - measured HCO3); values below 0.4 indicate pure non-gap acidosis, above 2.0 indicate concurrent metabolic alkalosis.

AST/ALT ratio above 2 suggests alcoholic hepatitis; an elevated ALP with elevated GGT confirms a hepatic rather than skeletal source of alkaline phosphatase.

Cardiac troponin I or T is the preferred biomarker for myocardial injury; CK-MB is reserved for detecting early reinfarction because it normalizes within 2-3 days.

HbA1c by NGSP/DCCT reflects 2-3 months of glycemia; results are falsely low with hemolysis, blood loss, or hemoglobin variants and unreliable in end-stage renal disease.

Adult references: Hgb 13.5-17.5 g/dL (male), 12.0-15.5 g/dL (female); MCV 80-100 fL; MCH 27-33 pg; MCHC 32-36 g/dL; RDW 11.5-14.5%; platelets 150-450 × 10^9/L.

Corrected reticulocyte count = retic % × (patient Hct / 45); the reticulocyte production index divides this by a maturation factor of 1.0-3.0 based on hematocrit.

Microcytic anemia differential: iron deficiency, thalassemia, anemia of chronic disease, sideroblastic anemia, lead poisoning; a normal RDW favors thalassemia trait.

Iron deficiency: low ferritin, low serum iron, high TIBC, saturation below 20%; anemia of chronic disease: low iron and low TIBC with normal or elevated ferritin.

INR = (patient PT / mean normal PT)^ISI; a 1:1 mixing study that corrects indicates factor deficiency, while failure to correct indicates an inhibitor such as lupus anticoagulant or a factor VIII antibody.

Hemoglobin electrophoresis at alkaline pH separates Hgb A, F, S, and C by charge; sickle solubility testing is a screening test only and must be confirmed by electrophoresis or HPLC.

Leukocyte alkaline phosphatase score is low in chronic myeloid leukemia and high in leukemoid reaction, aiding distinction when BCR-ABL1 testing is pending.

Paroxysmal nocturnal hemoglobinuria is diagnosed by flow cytometry showing loss of CD55 and CD59 on RBCs and granulocytes; the Ham and sucrose tests are historical.

Cytogenetics: t(9;22) BCR-ABL1 in CML, t(15;17) PML-RARA in acute promyelocytic leukemia, and Auer rods with myeloperoxidase positivity in acute myeloid leukemia.

Classical complement pathway: C1q binds IgG or IgM, activating C1r, C1s, C4, C2, and C3; the alternative pathway uses factor B, factor D, and properdin; the lectin pathway uses mannose-binding lectin.

Terminal complement component (C5-C9) deficiency predisposes to recurrent disseminated Neisseria infections; C3 deficiency causes severe pyogenic infections.

IgM is a pentamer and dominates the primary response; IgG is monomeric, crosses the placenta, fixes complement, and dominates the secondary response; IgA is a dimer with J chain and secretory component.

Hypersensitivity: type I IgE and mast cells, type II antibody against cell-bound antigen, type III immune-complex deposition, type IV T-cell mediated and delayed.

Anti-dsDNA and anti-Sm are specific for systemic lupus erythematosus; homogeneous ANA pattern suggests dsDNA, speckled suggests extractable nuclear antigens, centromere suggests CREST syndrome.

Rheumatoid factor is usually IgM directed against the Fc portion of IgG; anti-cyclic citrullinated peptide is more specific for rheumatoid arthritis.

Precipitation and agglutination curves show prozone (antibody excess, false negative), equivalence (optimal lattice), and postzone (antigen excess, false negative); dilution resolves prozone.

Direct immunofluorescence and ELISA detect bound or free antibody; Western blot confirms specificity against separated antigens after a positive screening assay.

Flow cytometry identifies lymphocyte subsets by surface CD markers, for example CD4 counting in HIV monitoring and CD19/CD5 coexpression in chronic lymphocytic leukemia.

Gram-positive cocci in clusters suggest Staphylococcus; in chains suggest Streptococcus. Catalase is positive in staphylococci and negative in streptococci; coagulase positivity identifies S. aureus.

Streptococcus pneumoniae is optochin sensitive and bile soluble; group A streptococci are bacitracin sensitive; group B are bacitracin resistant, CAMP positive, and hippurate positive; group D grow in bile esculin.

MacConkey agar: lactose fermenters such as E. coli form pink colonies; nonfermenters such as Salmonella and Shigella are colorless, and Salmonella usually shows black centers from hydrogen sulfide.

Oxidase is positive in Pseudomonas aeruginosa and negative in Enterobacterales; urease is positive in Proteus; indole is positive in E. coli.

Blood cultures: collect at least two sets from separate sites, 20-30 mL total in adults, each set with an aerobic and an anaerobic bottle, before antimicrobial therapy.

CSF in bacterial meningitis shows neutrophilic pleocytosis, high protein, and low glucose; viral meningitis shows lymphocytic pleocytosis with normal glucose and modestly elevated protein.

Methicillin resistance is confirmed by cefoxitin disk diffusion or detection of mecA/PBP2a; carbapenemase production is screened by modified carbapenem inactivation or molecular methods.

Clostridioides difficile testing commonly combines glutamate dehydrogenase antigen with toxin A/B enzyme immunoassay, with nucleic acid amplification for discordant results.

Candida albicans is identified by germ tube formation in serum at 37°C within 2-3 hours; KOH preparation with calcofluor white is used for direct fungal microscopy.

Standard PCR cycle: denaturation near 94-95°C, annealing at 50-65°C depending on primer melting temperature, extension at 72°C for Taq polymerase.

In real-time PCR, Ct is inversely proportional to the starting target quantity; a difference of one cycle corresponds to roughly a two-fold difference in template.

Sanger sequencing uses dideoxynucleotide terminators to produce chain-terminating fragments separated by capillary electrophoresis; each read covers roughly 500-1000 bases.

FISH detects t(9;22) BCR-ABL1 and t(15;17) PML-RARA in interphase nuclei without culture, enabling rapid diagnosis and residual disease monitoring.

Southern blot detects DNA, Northern blot detects RNA, and Western blot detects protein; all separate by size before probe or antibody detection.

Common hereditary assays: factor V Leiden (G1691A), prothrombin G20210A, HFE C282Y and H63D for hemochromatosis, and CFTR mutation panels for cystic fibrosis.

Contamination control requires a no-template control, unidirectional workflow, aliquoted reagents, and uracil-N-glycosylase with dUTP master mixes to prevent amplicon carryover.

Multiplex PCR amplifies several targets in one reaction, requiring distinct primer pairs and probe dyes; melting curve analysis distinguishes products by amplicon melting temperature.

Variants of uncertain significance arise from next-generation sequencing and must be interpreted with population frequency, in silico prediction, and segregation data rather than reported as pathogenic.

Specific gravity by refractive index measures solute concentration and is unaffected by glucose or dye, whereas reagent-strip specific gravity measures ionic strength and reads falsely high in highly alkaline urine.

Reagent-strip protein reacts mainly with albumin and is falsely positive in highly alkaline or contaminated specimens and falsely negative in dilute urine or with globulin and Bence Jones protein.

Reagent-strip blood uses pseudoperoxidase activity of heme; ascorbic acid and high nitrite can cause false-negative results, and myoglobin also produces a positive reaction.

Nitrite positivity indicates nitrate-reducing gram-negative bacteria; leukocyte esterase detects neutrophil esterase. Both positive supports urinary tract infection, but culture is required for confirmation.

Casts: hyaline are nonspecific, RBC casts indicate glomerulonephritis, WBC casts indicate pyelonephritis or interstitial nephritis, and broad waxy casts suggest chronic renal failure.

Crystals: calcium oxalate as envelopes or dumbbells, triple phosphate as coffin lids in alkaline urine, uric acid in acidic urine, cystine as hexagons, and cholesterol as plates.

Pleural fluid by Light criteria is exudative if fluid/serum protein exceeds 0.5, fluid/serum LDH exceeds 0.6, or fluid LDH exceeds two-thirds of the serum upper reference limit.

Semen analysis WHO lower reference limits: volume 1.5 mL, sperm concentration 15 million/mL, total motility 40%, and normal morphology 4%.

Xanthochromia in CSF supernatant indicates hemorrhage older than about 2 hours; a traumatic tap shows clearing of blood from tube 1 to tube 4 without xanthochromia.

CLIA categorizes tests as waived, moderate complexity, or high complexity; complexity determines required personnel qualifications, quality control, and proficiency testing obligations.

Westgard rules: 1-2s is a warning, 1-3s rejects a single control beyond 3 SD, 2-2s rejects when two consecutive controls exceed 2 SD in the same direction, R-4s rejects on a 4 SD range, 4-1s and 10x detect systematic shift.

Sigma metric = (total allowable error - bias) / coefficient of variation; 6 sigma is world class, 5 is excellent, 4 is good, and below 3 requires corrective action.

Sensitivity = TP/(TP+FN); specificity = TN/(TN+FP); positive predictive value = TP/(TP+FP) and varies with prevalence, while sensitivity and specificity do not.

Coefficient of variation = (standard deviation / mean) × 100 and is the standard index of imprecision for a quantitative method.

Reference intervals are typically the central 95% of a healthy reference population (mean ± 1.96 SD when parametric), requiring at least 120 individuals for nonparametric estimation.

Proficiency testing must be performed for each regulated analyte, results are not shared between laboratories, and unsatisfactory performance triggers corrective action and possible sanctions.

Delta checks compare a current result with the patient's previous value to detect specimen mislabeling, dilution, or analytic error; critical values require documented notification within a defined time.

Quality improvement tools include failure mode and effects analysis for prospective risk, root cause analysis for adverse events, and Lean methods for reducing turnaround time and waste.