Automated Hematology Analyzers

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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


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