Automated Hematology Analyzers
Calculation for RDW
(SD of RBC volume / mean MCV) x 100
Three RBC indices MCV, MCH, and MCHC
(hct/RBC) x 10 (Hgb/ RBC) x 10 (Hgb/ Hct) x 100
Reference range for RDW
11.5 to 14.5%
What size is counted as platelets on a platelet histogram?
2-20 fL
Normal values for WBC, RBC and PLT
4.5-11.0, M = 4.7-6.1 F = 4.2-5.4, 150-450
Particles greater than what size are identified as RBC's?
>36 fL
What happens after the blood is diluted with saline in the coulter principle?
Cells are pulled through an aperture and creates a vacuum
What are three sources of error in automated cell counting?
Cold agglutinins, lipemia, icterus
What happens in the coulter principle as the cells pass through the sensing aperture?
Electrical resistance of impedance occurs causing a change in voltage which generates a pulse
What three things have to be calculated on a histogram?
HCT, MCH, MCHC
From the 6 items that can be measured with hematology analyzers, what other 4 values can you deduce?
Hct, MCH, MCHC, RDW
Two rules of three
Hgb = RBC x 3 Hgb x 3 = Hct +/- 3
After two electrodes establish electrical current, what happens next in the coulter principle?
Low-frequency electrical current is applied to the electrodes. DC current is applied between these two electrodes
A normal WBC histogram will show what three distinct populations of WBC's
Lymphocytes (35-90 fL), Mononuclear cells (90-160 fL), Granulocytes (160-450 fL)
Normal values for Hgb, Hct, MCV, MCH, and MCHC
M = 14-18 F = 12-16, M = 42-52% F = 37-47% F = 42-52, 80-100fL, 27-31pg, and 32-36%
Hb (hgb concentration) is utilized for the calculation of what two items?
MCH and MCHC
Hct is utilized for the calculation of what two items?
MCV and MCHC
What two things are derived from RBC histogram?
MCV and RDW
What can be determined with the RF principle?
N:C ratio, nuclear density, and cytoplasmic granulation
Two counting chambers in coulter cell counter
RBC bath - used for RBC and platelet count WBC bath - used for WBC and hemoglobin determination
How is the Hgb measured in the coulter cell counter in the WBC bath?
RBC's are lysed and Hgb is converted to cyanmethemoglobin, Hgb is measured colorimetrically at 540 nm
Three types of histograms
RBC, WBC, PLT
What types of things can hematology analyzers electronically measure?
RBC, WBC, Plt, MPV (mean platelet volume), Hb (Hgb concentration), MCV
What does the RBC histogram do?
Reflect the normal size or anisocytosis of RBC's or any other particles in the RBC size range and will coincide directly with the MCV
What is Optical Scatter?
Sample of blood is diluted with an isotonic diluent and then hydrodynamically focused through a quartz flow cell, cells pass through a flow cell where a light beam is focused and light scatters in all directions, photodetectors sense this and convert light to pulses
Two characteristics of an abnormal histogram
Shift to the left (avg size of patient's cells are below normal range - microcytic anemia) Shift to the right (Average size of patient's cells are above normal range - macrocytic anemia)
What does the number of pulses equal in the coulter principle?
The number of cells counted
What does the size of the pulses represent in the coulter principle?
The volume or size of the cell
What happens after cells are pulled through an aperture in coulter principle?
Two electrodes establish electrical current
What is icterus and how does it affect your automated analyzer results?
Yellowish pigmentation of the body fluids caused by jaundice, causes turbidity and will falsely elevate Hgb and MCH
To have a valid count, what must be obtained in reference to platelet histograms?
a fitted curve, 0-70 fL
Explain Radiofrequency principles
a high-voltage electromagnetic current flowing between the electrodes that detects cell size, based on cellular density. A high-frequency pulsating sine wave.
If the fitted curve is not obtained, what must be done?
a manual platelet determination
What are hematology analyzers?
a range of simple to high-volume instruments capable to count blood cells
Causes of abnormal WBC histograms
abnormally large # of granulocytes or lymphocytes
What is cold agglutinins and how does it affect your automated analyzer results?
agglutination of the RBC's due to a drop in temperature, falsely decrease the total RBC count and increase MCH
Two characteristics of a normal histogram
almost symmetric, bell curve shape with a single peak
WBC histogram may also include what immature specimens?
blasts, and other immature cells such as promyelocytes and myelocytes
First step of the Coulter principle
blood sample diluted with saline - a good conductor of electrical current
What is plotted on the x and y axis of a histogram?
cell number on y axis and cell size on x axis
What is the main principle of the Coulter Principle of electrical impedance
cells are sized and counted by measuring changes in electrical resistance when a particle passes through a small aperture
What does the WBC histogram do?
compares the size and population of patient's WBC's with normal population, screening tool to detect presence of hematologic diseases, sorts WBC according to their nuclear size
Operation principles
electrical impedance, histograms, radiofrequency, opacity, scatterplot, optical scatter, VCS technology (volume, conductivity, and scatter), hydrodynamic focusing, flow cytometry, data reporting (graphically displayed and numerically recorded)
What is lipemia and how does it affect your automated analyzer results?
excess fats in the blood, causes turbidity and falsely elevated Hgb and MCH
What are the two types of electrodes in third step of the coulter principle?
external electrode (negative) - located in cell suspension Internal electrode (positive) - inside hollow tub, contains aperture
How does RBC rouleaux formation affect the results of your hematology analyzer?
falsely decreased RBC count and increased MCV and MCHC
8 advantages to hematology analyzers
fewer manual techniques, lower cost, improved tat, improved accuracy, improved precision, embedded qc programs, preparations, examination, reporting of WBC differential, auto maintenance in some instruments
What is a histogram?
graphic representations of cell frequencies versus size
Formula for indirectly calculated Hct by hematology analyzer?
hct = RBC count x MCV/ 10 values may differ from manual values
How does lipemia and icterus interfere with hematology analyzers? How do you fix it?
high Hgb and MCH; use rule of three and replace plasma with normal saline
How do significantly increased WBC's (leukemia) interfere with hematology analyzers? How do you fix it?
high Hgb, RBC, and incorrect Hct; use rule of three and dilute specimen
How do microcytes and schistocytes interfere with hematology analyzers?
high PLT and low RBC (left shift on histo); examine peripheral smear for abnormal cells
How do increased of abnormal plasma proteins interfere with hematology analyzers? How do you fix it?
high WBC (counted as WBC's); replace plasma with normal saline and perform manual count
How do abnormal Hgb/lysis resistant RBC's interfere with hematology analyzers? How do you fix it?
high WBC, low Hgb (counted as WBC's); manual dilutions
how do giant platelets interfere with hematology analyzers? How do you fix it?
high WBC, low PLT (counted as WBC); review slide for giant platelets
How do platelet clumps interfere with hematology analyzers? How do you fix it?
high WBC, low plt (counted as WBC's); warm and agitate specimen
Automated CBC machines still require indirect calculations to be made, from what are these calculations made?
histogram results
What does the platelet histogram illustrate and help identify?
illustrates platelet size, identify giant platelets and thrombocytopenia
Where are instrument limitations defined?
in instrument operation manuals and local standard operating procedures (SOP's)
Common analyzer limitations
inability to distinguish cells from particles or cell fragments of same size, variant forms of Hgb make RBC's resist lysis which impacts WBC and RBC counts, presence of nRBC's, clumped or agglutinated platelets, RBC rouleaux formation
How often do you need to calibrate hematology analyzers?
initial installation, every 6 months, after part replacement, after instrument repair
how does platelet satellitism interfere with hematology analyzers? How do you fix it?
low PLT; recollect with sodium citrate (ruling out reactivity to EDTA)
How does hemolysis interfere with hematology analyzers? How do you fix it?
low RBC and HCT; use rule of three and obtain a new specimen
How do cold agglutinins interfere with hematology analyzers? How do you fix it?
low RBC, high MCV, MCH, and MCHC (right shift on histo); warm blood
How do agglutinated RBC's interfere with hematology analyzers? How do you fix it?
low RBC; perform manual count
By what % may manual and hematology analyzer calculation of Hct differ?
manual may be 1.3-3.0% higher
Monocuclear cells represent what?
monocytes in a normal specimen
How is the differential displayed on a histogram?
numerically recorded and graphically displayed
How do you establish which cell line(s) is/are elevated in a WBC histogram?
perform manual differential
Criteria for a fitted curve
platelet count above 20,000/microliter, log-normal distribution, no platelet count vote-out
What three things do hematology analyzers also provide?
retic analysis, enumeration of nRBC's, verification system
What do histograms measure?
size thresholds of WBC, RBC, platelets - data is plotted in form of histogram
What is the MCV derived from?
the RBC histogram
What acts as a conductor in the RF principle? What is directly proportional to pulse size or change in RF resistance?
the cell wall; cell interior density or nuclear volume
Manual Hct measurement includes what?
trapped plasma between the RBC's
