Quick reference guides

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01 / 10 · Calculations

Dilutions and dilution factors

Parts, volumes, serial steps, and the reported result

Adding diluent lowers concentration. The dilution factor (DF) is the final volume divided by the specimen volume. A DF of 5 gives one fifth of the original concentration.

Example: 1:5 dilution

Here, 1:5 means one specimen part in five final parts. Mix one specimen part with four diluent parts. A written 1 + 5 means one specimen part plus five diluent parts, which gives a DF of 6.

Specimen
100 µL

Diluent
100 µL

500 µL final volume = 100 µL specimen + 400 µL diluent.
Equal-volume schematic. µL = microliter.

Calculating the dilution factor

Formula

DF = final volume ÷ specimen volume

In the 1:5 example

Final volume = 500 µL
Specimen volume = 100 µL
DF = 500 ÷ 100 = 5

Volumes and factor

Specimen volume
Final volume ÷ DF
Diluent volume
Final − specimen volume

Prepare a 1:10 dilution with a final volume of 1.0 mL.
Specimen = 1.0 ÷ 10 = 0.10 mL.
Diluent = 1.0 − 0.10 = 0.90 mL.
mL = milliliter. 1 mL = 1,000 µL.

Concentration and result

Diluted concentration
Original concentration ÷ DF
Original concentration
Diluted concentration × DF

A 1:5 aliquot measures 84 U/L.
84 × 5 = 420 U/L in the original specimen. U/L = units per liter.

Apply the factor only once. Report an analyzer-corrected result of 420 U/L as 420 U/L. Use the method's diluent and validated measuring range.

Cumulative dilution factor

Original specimen

1:5 step
Total DF = 5

1:4 step from that mixture
Total DF = 5 × 4 = 20

Each step dilutes the well-mixed solution from the preceding tube, so the cumulative dilution factor is the product of the step factors. A result of 12 mg/dL in the second tube gives 12 × 20 = 240 mg/dL in the original specimen. mg/dL = milligrams per deciliter.

CalculationsDilutions and dilution factorsParts, volumes, serial steps, and the reported result

02 / 10 · Laboratory Operations

Common QC rule violations

Six statistical rules and the limits of their interpretation

In quality control (QC), each control result is compared with its established mean and standard deviation (SD), a measure of usual variation. Random error widens the scatter around the mean. Systematic error shifts results to one side of the mean. The patterns below suggest which type to investigate.

Vertical position = (result − mean) ÷ SD, using each level's own target. Lines: mean (0), ±1, ±2, ±3 SD. A square marks each result that meets the named rule.

1_2s

One result beyond ±2 SD.

In the classic scheme this is a warning. Check the selected rejection rules.

1_3s

One result beyond ±3 SD.

Reject the run and investigate random error first.

2_2s

Two in a row beyond +2 SD, or two in a row beyond −2 SD.

Reject the run and investigate possible systematic error.

R_4s

One > +2 SD, one < −2 SD, in one run.

The two levels are 4.8 SD apart. Reject the run. This pattern suggests random error.

4_1s

Four in a row beyond +1 SD, or four in a row beyond −1 SD.

Reject the run and investigate possible systematic error.

10_x

Ten consecutive on one side of the mean.

Reject the run and investigate possible systematic error.

Examples use one level across runs, except R_4s (two levels, one run). The 2_2s rule also applies across levels within a run. The 4_1s and 10_x rules can span levels and runs as the QC plan specifies.

Warning and rejection

In the classic Westgard scheme, 1_2s is a warning to check the rejection rules. Plans that evaluate every rule directly can detect the 4_1s and 10_x examples here even though no result exceeds 2 SD.

Respond to a rejection

Stop reporting affected patient results. Use the adopted QC plan to investigate controls, reagents, calibration, and instrument performance. After correction and acceptable QC, assess which patient results need review or repeat testing. An acceptable repeat control result does not identify the cause of the original failure.

Laboratory OperationsCommon QC rule violationsSix statistical rules and the limits of their interpretation

03 / 10 · Hematology

Iron study patterns

Deficiency, inflammation, mixed findings, and overload

Interpret the iron panel results together. Serum iron reflects circulating iron, and ferritin helps assess storage iron. Inflammation can sequester iron in storage and raise ferritin, so little iron is available for red cell production.

Total iron-binding capacity (TIBC) reflects transferrin, the main iron transport protein. Transferrin saturation (TSAT) is the percentage of that capacity occupied by iron.

TSAT (%) = serum iron ÷ TIBC × 100

Use the same concentration units for iron and TIBC.

Iron 30 µg/dL ÷ TIBC 300 µg/dL × 100 = 10% TSAT. µg/dL = micrograms per deciliter. A low TSAT occurs in both iron deficiency and inflammation.

Typical adult patterns

↓ decreased; ↑ increased; N within the applicable reference interval.

Pattern	Serum iron	TIBC / transferrin	TSAT	Ferritin
Iron deficiency	decreased	increased	decreased	decreased
Anemia of inflammation	decreased	decreased or normal	decreased or normal	normal or increased
Mixed deficiency + inflammation	decreased	Variable, often normal or decreased	decreased	decreased, normal, or increased
Iron overload	Often increased	normal or decreased	Usually increased	Usually increased

Why inflammation changes the panel

A low ferritin supports depleted stores. During inflammation, a normal or high ferritin cannot exclude deficiency, and liver injury can also raise ferritin. Hepcidin, an iron-regulating hormone, reduces iron release from stores and absorption from the gut. Inflammation can also lower transferrin. Mixed deficiency may then lack the high TIBC and low ferritin expected in iron deficiency.

High ferritin alone does not establish overload. A persistently high TSAT makes overload more likely. Some overload disorders have a normal TSAT, so a normal value does not exclude overload.

When results do not agree

Compare with hemoglobin, red cell indices, C-reactive protein or another inflammation marker, and collection and iron-treatment history.

Soluble transferrin receptor may help when inflammation obscures ferritin. Increased red cell production also raises it, and cutoffs vary by method. Reticulocyte hemoglobin reflects the iron available to recently produced red cells. It can be low in either deficiency or inflammation.

HematologyIron study patternsDeficiency, inflammation, mixed findings, and overload

04 / 10 · Hematology

PT and APTT patterns

Screening patterns, specimen checks, and mixing studies

Prothrombin time (PT) and activated partial thromboplastin time (APTT) measure the clotting time of citrated plasma after activation by different reagents. Some factors are measured by both tests, so one time or both can be prolonged.

PT · tissue factor / extrinsic arm
VII

APTT · contact / intrinsic arm
XII → XI → IX + VIII

Common: X + V → thrombin (activated II) → fibrin formation

Assay schematic. Roman numerals identify the factors. Factors V and VIII are cofactors. Thrombin cleaves fibrinogen to fibrin. Contact activation also needs prekallikrein and high-molecular-weight kininogen.

Patterns and possible explanations

Screening pattern	Possibilities to investigate	Distinguishing findings
PT prolonged APTT in range	VII deficiency/inhibitor; early vitamin K deficiency or warfarin.	Also possible with liver disease or direct factor Xa inhibitors.
APTT prolonged PT in range	VIII, IX, XI, or contact-factor deficiency/inhibitor; heparin; lupus anticoagulant.	Contact-factor deficiency usually causes no bleeding. Lupus anticoagulant can accompany thrombosis.
Both prolonged	Common/multiple-factor deficiency: vitamin K deficiency, liver disease, or disseminated intravascular coagulation; anticoagulants.	Compare with platelets, fibrinogen, medications, and targeted tests.

Specimen and method checks

An underfilled tube leaves excess citrate for the plasma volume and may prolong times. A hematocrit above 55% also reduces the plasma volume, so adjust the citrate volume as the procedure directs. Check for clots, heparin from a line draw, delays, and optical interference.

Anticoagulant effects depend on drug, concentration, and method. Normal times cannot exclude a direct oral anticoagulant.

What a mixing study shows

Equal volumes of patient and normal plasma supply missing factors. If the time corrects by the method's criteria, deficiency is more likely. Failure to correct suggests an inhibitor or drug. Some inhibitors become apparent only after incubation.

Mixing also dilutes inhibitors, so a weak inhibitor may be missed. Anticoagulant drugs can mimic an inhibitor pattern.

Bleeding with normal PT/APTT: investigate von Willebrand disease, platelet dysfunction, mild factor deficiencies, or factor XIII deficiency.

HematologyPT and APTT patternsScreening patterns, specimen checks, and mixing studies

05 / 10 · Urinalysis and Other Body Fluids

Urine casts: phase contrast

Seven cast types, identifying features, and common look-alikes

Casts form in renal tubules in a uromodulin protein matrix. Identify the matrix first, because loose cell clumps can mimic casts.

Phase contrast makes faint structures visible through differences in refractive index. Bright edge halos are optical effects and do not belong to the cast.

Photo	Cast and identifying feature	Association and look-alike
	Hyaline · 400× Pale matrix with parallel sides. Phase contrast makes the faint outline easier to see.	Concentrated urine, exercise, or renal disease. A few may occur without disease. Look-alike: Mucus tapers and varies in width.
	Red cell · 400× Red cells within a continuous matrix.	Bleeding within the nephron, especially from a glomerular source. Look-alike: Free red cell clumps have no enclosing cast matrix.
	White cell · 400× White cells in a matrix. Look for granular cytoplasm and lobed nuclei.	Renal inflammation, including pyelonephritis and interstitial nephritis. Look-alike: Tubular cells or white cell clumps can resemble a cast.
	Tubular epithelial · 400× Tubular cells with single round nuclei and visible cytoplasm within a matrix.	Tubular injury. Degeneration can obscure the cell type. Look-alike: White cell and tubular cell casts can look alike. Compare the nuclei, because size alone cannot separate them.
	Granular · 400× Coarse or fine granules fill the matrix as cells and proteins break down.	Numerous muddy brown casts support tubular injury. A few granular casts are less specific. Look-alike: Debris or crystal clumps lack a continuous cast outline.
	Fatty · 400× Refractile lipid droplets within a matrix. Some droplets show Maltese crosses in polarized light.	Lipiduria, often with heavy proteinuria. Look-alike: Starch granules also form crosses. Confirm that the droplets lie within a cast.
	Waxy · 100× Highly refractile, sharply outlined matrix with cracks, notches, and blunt ends.	Prolonged tubular stasis, often with advanced renal impairment. Look-alike: Fibers have irregular edges and lack a cast matrix.

Scan at low power, then identify contents at higher power. Examine fresh urine, because delay and dilute alkaline urine can destroy casts.

Image credits.

Urinalysis and Other Body FluidsUrine casts: phase contrastSeven cast types, identifying features, and common look-alikes

06 / 10 · Urinalysis and Other Body Fluids

Urine casts: brightfield

Seven cast patterns, identifying features, and common look-alikes

Casts form in renal tubules in a uromodulin protein matrix. Identify the matrix first, because loose cell clumps can mimic casts.

Brightfield: transparent casts have little contrast. Reduced illumination helps reveal their outline, and phase contrast can clarify cell detail.

Photo	Cast and identifying feature	Association and look-alike
	Hyaline · 400× Pale matrix with parallel sides. Faint edges can disappear in strong illumination.	Concentrated urine, exercise, or renal disease. A few may occur without disease. Look-alike: Mucus tapers and varies in width.
	Red cell · 400× Red cells within a continuous matrix.	Bleeding within the nephron, especially from a glomerular source. Look-alike: Free red cell clumps have no enclosing cast matrix.
	White cell · 400× Granular white cells in a matrix. Nuclear lobes may be difficult to resolve.	Renal inflammation, including pyelonephritis and interstitial nephritis. Look-alike: Tubular cells or white cell clumps can resemble a cast.
	Epithelial / white cell · 400× The source labels this as an epithelial/white-cell cast. Indistinct nuclei limit cell typing.	Tubular injury or renal inflammation. The cast alone cannot establish the cause. Look-alike: White cell and tubular cell casts can look alike. Compare the nuclei, because size alone cannot separate them.
	Granular · 400× Coarse or fine granules fill the matrix as cells and proteins break down.	Numerous muddy brown casts support tubular injury. A few granular casts are less specific. Look-alike: Debris or crystal clumps lack a continuous cast outline.
	Fatty · 400× Refractile lipid droplets within a matrix. Some droplets show Maltese crosses in polarized light.	Lipiduria, often with heavy proteinuria. Look-alike: Starch granules also form crosses. Confirm that the droplets lie within a cast.
	Waxy · 400× Highly refractile, sharply outlined matrix with cracks, notches, and blunt ends.	Prolonged tubular stasis, often with advanced renal impairment. Look-alike: Fibers have irregular edges and lack a cast matrix.

Scan at low power, then identify contents at higher power. Examine fresh urine, because delay and dilute alkaline urine can destroy casts.

Image credits.

Urinalysis and Other Body FluidsUrine casts: brightfieldSeven cast patterns, identifying features, and common look-alikes

07 / 10 · Hematology

RBC morphology

Eight shape findings on a stained peripheral blood film

Assess red blood cell (RBC) shape where cells lie singly or just touch and normal central pallor remains visible. Thick areas distort shape and obscure pallor. Normal pallor occupies about one third of a cell's diameter.

May-Grünwald Giemsa-stained blood films.

Echinocyte

Short, evenly spaced projections. Often an artifact. Also seen in uremia. Acanthocytes have irregular projections.

Acanthocyte

Uneven projections of varied length. Severe liver disease and lipid disorders. Compare with regular echinocytes.

Spherocyte

Dense, no central pallor (single arrow). Immune hemolysis and hereditary spherocytosis. Thick areas hide normal pallor.

Elliptocyte

Elongated ovals (paired arrows). Hereditary elliptocytosis and iron deficiency. Large ovals suggest megaloblastic change.

Target cell

Stained center, pale ring, outer rim. Thalassemia, liver disease, and hemoglobin variants. Uneven distribution across the film suggests an artifact.

Stomatocyte

Slit-like central pallor. Hereditary stomatocytosis and liver disease. Slow drying can mimic this.

Teardrop cell

One tapered end (dacrocyte). Marrow fibrosis, marrow infiltration, and megaloblastic anemia. Artifact tails often point the same way.

Schistocyte

Angular or helmet-shaped fragment. Microangiopathic or mechanical damage. Follow counting and urgent-review policy.

Assess schistocytes by their number and the other film findings, alongside the blood count and hemolysis studies.

Image credits.

HematologyRBC morphologyEight shape findings on a stained peripheral blood film

08 / 10 · Laboratory Operations

Hemolysis, lipemia, and icterus

Appearance and interference in serum or plasma

Hemolysis, lipemia, and icterus can alter serum or plasma measurements. The same specimen may be acceptable for one test and unsuitable for another because each method responds differently to interference.

Normal
Clear, pale yellow

Hemolyzed
Pink to red

Lipemic
Cloudy to milky

Icteric
Deep yellow to brown

Finding	How results may change	Practical implication
Hemolysis	Cell rupture releases potassium and lactate dehydrogenase. Free hemoglobin absorbs light and can affect chemical reactions.	Potassium released from the red cells raises the potassium result. The size and direction of other biases vary by analyte and method. A recollected specimen may be free of hemolysis caused by collection. In vivo hemolysis may persist after recollection.
Lipemia	Particles scatter light and affect sampling. Reduced plasma-water fraction affects methods that dilute the sample.	Indirect ion-selective electrodes can report falsely low sodium. Direct electrodes generally avoid this water-fraction effect. Lipid removal may also remove analyte.
Icterus	Bilirubin absorbs light and can react with assay components, including oxidants.	Effects differ between assays. A fresh draw may still be icteric, so an alternate validated method may be needed.

What the interference index means

Mixed interferences can obscure the appearance. A visual estimate cannot determine whether a specimen is acceptable.

An instrument interference index estimates hemoglobin, bilirubin, or turbidity from optical readings. Its scale comes from the instrument method and cannot be inferred from color alone. A lipemia index measures turbidity and gives no triglyceride concentration. Each platform has its own index scale and limits.

Review each affected test

Compare the index with the manufacturer's interference data and the laboratory's verified limit for that test. Suppress, qualify, recollect, or use a validated alternate method as the procedure directs. A patient specimen can be affected even when controls pass.

Laboratory OperationsHemolysis, lipemia, and icterusAppearance and interference in serum or plasma

09 / 10 · Blood Banking

DAT versus IAT

Where sensitization occurs and what the result can establish

The direct antiglobulin test (DAT) detects antibody or complement already coating the patient's red cells. The indirect antiglobulin test (IAT) tests whether antibodies in serum or plasma can coat test red cells during incubation.

Sensitization is antibody or complement coating a red cell. Antihuman globulin (AHG) reacts with the coating specified on the reagent label, often immunoglobulin G (IgG), fragments of complement component 3 (C3), or both.

DAT • sensitized in the body

Patient red cells
IgG, C3, or both already attached

AHG detects the existing coating

IAT • sensitized in the test

Patient serum or plasma + test cells
Antibodies bind during incubation

AHG detects coating formed in the test

In a tube method, AHG bridges sensitized cells, causing agglutination (clumping). Washing removes unbound globulin that could neutralize AHG.

Tube-method schematic.

Comparison	DAT	IAT
Material tested	Patient red cells, commonly from anticoagulated blood.	Serum/plasma plus reagent cells, or donor cells for an antiglobulin crossmatch.
What is detected	Bound IgG, complement, or both, per reagent.	Antibodies reactive with test-cell antigens under the assay conditions, commonly IgG.
Common uses	Investigation of immune hemolysis, transfusion reactions, and hemolytic disease of the fetus and newborn.	Antibody screening and identification, prenatal testing, and antiglobulin compatibility testing.

DAT interpretation

A positive DAT shows that the red cells are coated. Hemolysis is confirmed with other tests, such as bilirubin, lactate dehydrogenase, and haptoglobin. Weak coating can give a negative DAT.

IAT interpretation

Test a positive screen with an antibody panel to identify the antibody. Weak antibodies or antibodies to antigens absent from the test cells may be missed.

Read a gel card to grade six columns at the Blood bank.

Blood BankingDAT versus IATWhere sensitization occurs and what the result can establish

10 / 10 · Laboratory Operations

Calling a critical result

Who to call, what to say, read-back, documentation, and escalation

Follow your laboratory's critical-result procedure: its alert limits, authorized recipients, timing, and escalation chain. Each laboratory sets its own critical-value list.

1. Before the call

Confirm that the result, patient identifiers, and specimen label match. Check for hemolysis, lipemia, icterus, an unexpected clot, intravenous-fluid contamination, or the wrong tube. Follow the test's specimen requirements. See guide 08: Hemolysis, lipemia, and icterus. Follow local policy on repeating the test first. If the procedure allows an urgent preliminary call, identify the result as preliminary and explain what confirmation is pending.

2. Who receives it, and when

Call immediately when a result meets a critical value on your laboratory's list. Reach the authorized person responsible for acting on it, as named by local policy, within its notification time. Identify the receiver before giving patient information.

3. What to say

Give your name and laboratory, and confirm the receiver's name. State two patient identifiers, the test, the result with its unit, the specimen type, and its collection date and time.

4. Read-back

Ask the receiver to record and read back both patient identifiers, the test, the result, and its unit. Listen to the whole read-back. Confirm it, or correct the mismatch and obtain another read-back before ending the call.

5. What to record

Record the date and time, patient and specimen, test and result with its unit, receiver's name, your name, and confirmation that the read-back matched. Document the call as it happens.

6. When no one answers

Use the laboratory's escalation chain when the responsible person cannot be reached within the required time. Record every attempt, its time, the contact sought, and the outcome. Continue until the receiver confirms receipt.

7. When the result is questioned

Explain which checks are complete, which remain pending, and what the findings support. Describe relevant specimen or method limitations. Arrange the next step under local policy, such as a repeat or redraw. Involve the supervisor or pathologist when needed.

Laboratory OperationsCalling a critical resultWho to call, what to say, read-back, documentation, and escalation