Haematology - Diagnostic Role Of Blood Film

Question 1

Dyes

Answer 1

H&E (haematoxylin and eosin) = one of most frequently used stain combo, haematoxylin = purple/blue, binds acidic cell components (particularly DNA-> shows nucleus (also Leishman’s stain)); eosin=pinkish, binds protein components, especially in cytoplasm; 1874 Heinrich Caro + Paul Ehrlich, 1879 Blood staining technique and coining of term eosinophil (bc easily stained with eosin), alternative -> refractive index (different between cells and surrounding fluid (needs microscope with special fitted optics)

Question 2

Light microscopy

Answer 2

• Method based on optimal physics parameters (light wavelength, optical quality of lenses and all other components in light path, medium’s refractive index (highest resolution lenses use oil and not air between them and slide), objective lens’ physical properties (numerical aperture, written on lens next to magnification ie 10/0.25 so higher magnification does not equal higher resolution), geometry of illuminating light cone provided by condenser lens (depends on lens’ design properties but to large degree in way in which condenser is focused and diaphragms adjusted)) that theoretically affect microscope resolution (closest limit at which you can distinguish two small adjacent objects)
• Setup: Low power (4x) objective lens, microscope slide inserted into clips on stage and lamp turned on, distance between eyepieces adjusted (+ same focus), focusing image (separate coarse/fine or single knob with fine for first 1/4 turn and then coarse), focus condenser (close field iris and adjust condenser focus knob until sharp image of light disk edge coming through iris diaphragm (roughly in field of view center (otherwise adjust with cantering screws)), adjust field iris (open again and stop when field of view is only just illuminated), adjust condenser iris (fully open and close until it only just begins to darken image), stage controls move different slide regions into view, voltage control -> image brightness, leave lowest power objective + turn down V control and switch lamp off when done

Question 3

Blood film protocol

Answer 3

Unless sample in test tube with anticoagulant it will clot quickly, spread blood with it behind spreader to avoid damage, staining with Leishman’s stain (purple-blue nuclei and pink cytoplasm components), buffer (ph6.8) used, red blood cells=red, white blood cells=blue

Question 4

Reference ranges

Answer 4

Descriptions of data derived from sample of reference population (characteristics that have been defined with regard to age, gender and (when relevant) other factors (ie haematological variables = health state, ethnic origin, physiological status (pregnant?), altitude, smoking, alcohol, ambulant/resting, tourniquet), commonly as 95% (figures that encompass 95% data from reference sample (usually central 55% so 2.5% at each end = excluded), if data has Gaussian distribution the mean +- 2 stdev is 95% range, if not mathematical transformation of data required before analysis (ie WBC counts = log distribution so mean and standard deviation of logarithms to determine geometrical/log mean and 95% range)

Question 5

Normal ranges

Answer 5

• Less strictly defined, derived from healthy reference population, health-related range -> for some lab measurements a 95% range derived from apparently healthy population will include data from patients with high risk of subsequently developing significant disease (if subjects representing upper 20% of data have high risk of developing clinically evident disease then it is more relevant to interpret data in light of whether lab result is predictive of future good health)
• Interpretation: value within normal range may be abnormal for that individual (same for opposite), reference ranges for healthy and sick individuals (calculating reference ranges representing 99% of population reduces chance of false positives but increases chance of false negatives), some haematological variables are dependent on instrument/method so we need reference range derived for that

Question 6

Analysis of WBC differential counts

Answer 6

Elevated eosinophils number = allergic reactions (ie asthma and parasitic infections), elevated basophils number = CML + certain hypersensitivity reactions

Question 7

Blood films

Answer 7

Left-shift = absence of mature neutrophils and band-form ones, indicates either increase in proportion of band cells/presence of neutrophil preucrsors in peripheral blood + presence of clear vacuoles within neutrophil cytoplasm (specific to bacterial infection); ALL -> diagnosis requires immunophenotyping but can be suspected from very irregular nuclei shape; CML -> chronic granulocytic leukaemia, spectrum of granulocytic cells to mature neutrophils (left shift towards immature cells), often result of t(9;22) q(34;11.w) translocation; megaloblastic anemia -> maturation of nucleus retarded in relation to that of cytoplasm, hypersegmented neutrophils and macrocytes (particularly oval), in early deficiency there may be macrocytosis without poikilocytosis/hypersegmented neutrophils

Question 8

Full blood count

Answer 8

Hb (g/L), WBC count (x10^9/L), platelet count (pet, 10^9/L), RBC count (x10^12/L), haematocrit (HCT, L/l, ratio of RBC to blood), mean cell volume (MCV, Hct/RBC, fL x 10^-15L), mean cell haemoglobin (pg, 10^-12, Hb/RBC), mean cell [Hb] (avg [Hb] in each RBC, Hb/Hct, MCHC)