Lab Methods in Hemo

Why perform manual testing?

Proficiency samples

Testing location

Specimen type

Instrument problems

Specimen problems

Tests that can be performed manually?

RBC count

Hemoglobin (HGB)

Hematocrit (HCT)

WBC count

PLT count

WBC Differential.

Leukocheck method

Consists of a reservoir and pipette with fixed volumes. Used with the Neubauer hemacytometer.

What is the WBCs Dilution?

0.475 ml ammonium oxalate /25 ul pipette = 1:20 dilution

What is the Platelets Dilution?

1.98 ml ammonium oxalate/20 ul pipette= 1:100 dilution 

WBC Count

Cells counted x Dilution factor/ Area counted(mm2) x depth (mm)

RBC Count

Cells counted x 200/ 0.02 mm3

Platelet

Cells counted x 100/ 0.1 mm3

WBC Correction for nRBCs

WBC count x 100/ #nRBCs/ WBCs+100

Reticulocyte Counts

Used to assess erythropoietic activity of bone marrow. Whole blood in EDTA stained with supravital stain like new methylene blue. Retics are counted as both an RBC and a retic

% Retics

Number of retics x 100/ 1000(RBCs counted)

Miller Disc Method

Reduces labor involved to count more RBCs needed for more accurate retic count.

# retics

#retics in sq A x 100/ #RBCs in sq B x 9

Microhematocrit

To determine the percentage of whole blood that is composed of cellular components (mostly RBCs).

Microhematocrit Supplies Needed

Capillary tubes

Clay

Microcentrifuge

Reader

Patient whole blood

Erythrocyte Sedimentation Rate(ESR)

To detect inflammation. Does not indicate the source or the cause of the inflammation. Anticoagulated blood is drawn up into a graded Westergren sedimentation tube. The blood is allowed to stand in the tube undisturbed for 1 hour. After 1 hour, the tube is read and that is the rate at which red cells
settle out.

Erythrocyte Sedimentation Rate(ESR) Interpretation

Normal ranges vary between institutions but in general a value <20 mm/hr is considered normal. Values >20 mm/hr should be further investigated.

Complete Blood Count (CBC)

Analysis of the cellular components of blood is achieved through
the complete blood count (CBC). Enumeration, Morphology, and Distribution of all the cellular blood constituents.

Red Blood Cell Count (RBC) Test

Number of red blood cells

Hemoglobin (Hgb) Test

Concentration of hemoglobin in RBCs

Hematocrit (Hct) Test

Percentage of blood that is composed of blood cells (mostly red blood cells)

Mean Corpuscular Volume (MCV)

Size of the red blood cell

Mean Corpuscular Hemoglobin (MCH)

Weight of hemoglobin in RBCs

Mean Corpuscular Hemoglobin Content (MCHC)

Percent of hemoglobin in RBCs

Red Cell Distribution Width (RDW)

Quantified measure of the amount of variation in RBC morphology around a classification system

RBC morphology (part of diff)

Correlation to indices. Correlation to RDW

Automated Methods - RBCs

Used for: RBC count

Hemoglobin
Hematocrit
MCV

Methodologies

Cell count: Impedance and/or light scatter
Hemoglobin: spectrophotometry
Hematocrit: calculated by MCV and RBC count
MCV: Impedance
Measured from 200μL blood in <1 min

Impedance

Coulter Principle
Measurement of changes in electrical resistance produced by
cells
Whole blood passed between 2 electrodes through a tiny
aperture
Impedance changes as cell passes through with change
proportional to cell volume
Voltage pulses that are measurable
Get cell count (# of pulses) and measure of volume (size of
pulse)

Optical Scatter aka Flow Cytometry

Cells pass individually through a light source interrupt the
beam and scatter the light
Absorption, diffraction, refraction, and reflection
Forward angle scatter measures cell volume or size
Side scatter or 90o scatter measures internal complexity
(granularity)

Impedance

Interrupts the laser signal

Optical Scatter

Change angle to determine cell size, granularity, complexity, and globularity

RBC Chamber

Platelets = particles 2fL – 20fL
MPV = derived from platelet histogram
RBCs = particles > 36fL
MCV = avg volume of RBCs taken from volume distribution data
RDW calculated from histogram as coefficient of variation of RBC
volume distribution

WBC Chamber

RBCs are lysed to release hemoglobin (measured
spectrophotometrically). WBCs counted by impedance or scatter

WBCs Stains

Fluorescence (Sysmex)
Peroxidase (Siemens)

Platelets Stains

Fluorescence (oxazine, PLT-F) (Sysmex)

Reticulocytes Stains

Supravital stain (BC, Siemens)
Fluorescence (Sysmex, Abbott)

nRBCs Stains

Fluorescence (Sysmex) 

Red fluorescent dye (Abbott)

Reticulocyte Counting

Last of the blood cells to be automated
Optical scatter or fluorescence
Fluorescent or nucleic acid dyes added to bind to residual RNA
present in reticulocytes but not mature RBCs
Auramine O (supravital stain) (Sysmex)
Oxazine (NA-binding dye) (Siemens)
Low-fluorescence, middle-fluorescence, high-fluorescence
Less mature = higher fluorescence

Immature reticulocyte fraction (IRF) = sum of mid + high = ratio of
immature retics to total retics

NORMOCYTIC NORMOCHROMIC

HEMOGLOBINOPATHIES
HEMOLYTIC ANEMIAS

MICROCYTIC HYPOCHROMIC

THALASSEMIAS
IRON DEFICIENCY ANEMIA

MACROCYTIC NORMOCHROMIC

MEGALOBLASTIC ANEMIA
PERNICIOUS ANEMIA

RED CELL DISTRIBUTION WIDTH

Reported as Standard Deviation, or most
commonly, the Coefficient of Variation for a
population of red cells.

Assigns a numeric value to the amount of variability in a given RBC
population (ANISOCYTOSIS).

Normal : <15% (11.5%-14.5%)

WBC Estimate

Enumerate the average number of WBC in a peripheral blood

smear and correlate it with the instrument counts.

100 cell WBC differential/morphology

Differentiate between the different WBC lineages and express them as percentages.

RBC morphology

Identify and differentiate between morphologic changes of erythrocytes. Graded

PLT morphology

Identify and differentiate between morphologic changes of platelets. Graded.

PLT Estimate

Enumerate the average number of PLTs in a peripheral blood
smear and correlate it with instrument counts.

Platelet Counts

Impedance and Optical Scatter
Can interfere with other cell counting methods

Mean Platelet Volume (MPV)

Quantification of the relative size of the platelet (in fl)
Higher the number the larger the platelet size (average)

Giant Platelets

Young platelets
Poor division in the marrow
Abnormal division in the marrow

Platelet clumping

Poor specimen collection
Insufficient specimen collection

Platelet satellitism

Platelet response to anticoagulant (EDTA)