Hematology: Hematology methods & instruments

20 µL in a final volume of 400 µL gives a 1:20 dilution, the standard for manual WBC counts. A 1:200 dilution is used for manual RBC counts.

In adults, red marrow persists mainly in the axial skeleton and proximal long bones; the posterior superior iliac crest is safe and cellular. The tibia is used only in infants.

Methanol fixes cells without lysing them. Water-based or acidic reagents such as acetic acid would lyse red cells; acid alcohol is a decolorizer.

The substage condenser concentrates and directs light through the specimen. The objective and ocular magnify the image.

EDTA (ethylenediaminetetraacetic acid), usually as the K2 salt, is the anticoagulant of choice for CBCs because it preserves cell morphology.

Giemsa, Wright and Leishman are Romanowsky stains producing the purple nuclear effect. Hematoxylin is a mordant nuclear dye used in H&E and Pap staining.

Ivy's method measures bleeding time from a standardized forearm incision. The other three convert hemoglobin to a colored derivative read photometrically or visually.

Acute-phase proteins such as fibrinogen and immunoglobulins promote rouleaux, raising the ESR. High red cell counts, low globulins and abnormal red cell shapes lower it.

The adult WBC reference interval is roughly 4.0–11.0 × 10^9/L. Lower values indicate leukopenia and higher values leukocytosis.

Methanol fixes cells without lysing red cells or distorting morphology. Heat fixation is used for bacterial smears; formalin and mercuric chloride are histology fixatives.

New methylene blue (or brilliant cresyl blue) precipitates residual RNA in living reticulocytes. Wright's, Giemsa and Field's are Romanowsky stains used on fixed smears.

A cell passing through the aperture displaces conductive diluent and raises resistance; the pulse height reflects cell volume and the number of pulses gives the count. Granularity is assessed by side scatter, not impedance.

Light scattered at low angles (forward) relates to cell size, while light scattered at 90 degrees (side) relates to granules and internal structure. Antigen density is measured by fluorescence.

Hemiglobincyanide has a broad absorbance peak at 540 nm, which is used in the ICSH reference method. 340 nm is used for NADH/NADPH enzyme assays.

Only the packed red cell column should be read; the buffy coat of white cells and platelets adds to the reading. A falsely high Hct would lower the calculated MCHC, not raise it.

The classic Westergren method uses 4 parts blood to 1 part 3.8% (or 3.2%) sodium citrate in a 200 mm tube, read at 60 minutes. Undiluted EDTA blood is used only in modified methods.

Photomultiplier tubes (or photodiodes) detect light and convert it into electrical pulses for analysis. Dichroic mirrors only direct light of certain wavelengths to the correct detector.

To avoid counting the same cell twice in neighboring squares, cells touching two chosen sides are included and those touching the opposite two are excluded. Counting all four sides gives falsely high results.

Fluorescent or absorbing dyes bind RNA, and the amount of signal separates reticulocytes from mature cells and grades their maturity (IRF). Impedance alone cannot detect RNA.

A 488 nm blue laser excites FITC and PE and is standard in clinical instruments. A 633–640 nm red laser is used for dyes such as APC.