Sect 2 - Automated Cell Counting

Coulter Principle

An electronic method for counting and sizing particles.

Cells are suspended in a conducive fluid and flow through a small aperture, detected/counted, and measured by the changes in electrical resistance produced by those cells

conductive fluid

aperture

external positive probe

internal negative probe

Parts of the Coulter Counter:

counts when electrical current is blocked

How does the Coulter counter work/count the cells?

cell size

can be determined by the delay in the break

larger the cell = disconnect slower

smaller the cell = disconnect faster

DC Detection Method

Non-Cyanide Hemoglobin Analysis Method

What are the 2 hematology analyzers used to perform cell counts and measure hemoglobin?

DC Detection Method

method of hematology analyzer that is used to measure hemoglobin and perform cell counts

specific blood sample is aspirated, diluted, and sent to transducer. Transducer has an aperture with electrodes where the sample flows between that causes change in current resistance

change detected as electric pulses to indicate cell size & are recoded on a histogram as tiny dots

Non-Cyanide Hemoglobin Analysis Method

method used to measure hemoglobin and perform cell counts

rapidly converts hemoglobin to oxyhemoglobin

capability of analyzing methemoglobin (a control material)

WBC and RBC Transducers

What are the 2 transducers (parts of the machine)?

WBC transducer

lyses RBCs so WBCs can be counted; reads Hgb

RBC transducer

counts RBCs/platelets

throughput; approx 60 samples/hr

how many samples an analyzer can get through per hour

carryover

contamination form one specimen to another

<3%

amount of carryover that should happen for WBCs

<1.5%

amount of carryover that should happen for RBC/Hgb/HCT

<5%

amount of carryover that should happen for platelets

coincidence

statistical correction for cells going through the aperture more than one at a time

linearity

reportable range of values

- what machine can read

CELL PACK

diluent reagent in the SYSMEX

CELLCLEAN

detergent reagent in the SYSMEX

follow up with DI water

Stromatolyser-WH

WBC/HGB lyse reagent in the SYSMEX

Purpose of calibrating instrument prior to running QC and patients

Checks accuracy of instrument and determine traceability of the measurement (help assure precise measurements prior to running control/patients)

Set actual result parameters (make sure parameters set so control checks function so machine does what is supposed to)

checks instrument functions

purpose of running controls

three levels (high, low, abnormal); every shift

how many levels of controls are ran and when?

background

if counts increase, instrument probably needs maintenance/cleaning to get back to below the threshold

checks there is nothing dirty in the machine because want analyses as close to zero as possible

WBC count

RBC count

Hgb

parameters that are measured

**hint: measures means that machine actually counts

HCT

MCV

MCH

MCHC

Plt

RDW

parameters that are calculated

hematocrit (HCT)

(RBC x MCV)/10

mean cell volume (MCV)

derived from RBC histogram

(HCT/RBC) x10

mean cell hgb (MCH)

(Hgb/RBC) x10

mean cell hemoglobin concentration (MCHC)

(Hgb/HCT) x100

Platelets

derived from platelet histogram

red cell distribution width (RDW)

width from histogram

wider curve = higher RDW = increase pops present

whole blood

type of specimen tested on hematology analyzer

steps of workflow in hematology analyzer

1. Aspiration: sucks up specimen

2. Delivery: pneumatics help pull sample through the tubing to each area for testing

3. Sensing: each area senses sample as it moves through the tubing and testing area

4. Analysis: performs function of hemoglobin lyses and cell count of specimen, prints results

5. Background: spits out wastes

6. Rinse: cleans itself by flushing diluent through sample after every run

1.0 - 99.9 x 10^3/uL

linearity range for WBC

1.0 - 6.6 x 10^6/uL

linearity range for RBC

0-20 g/dL

linearity range for Hgb

50-140fl

linearity range for MCV

25 - 700 x 10^3/uL

linearity range for platelets

Parameter flags (H or L/arrows/positives or negatives)

Backlighting/Highlighting of error

Delta checks

how results flagged by instrument when abnormal results

delta checks

quality control that monitors a pts results for significant difference between repeat testing (flags if there is enough difference)

analyzer flags displayed at bottom of report

WBC - leukocytosis or atypical lymph

RBC - dimorphic population, microcytosis

PLT - clumping, thromobocytopenia/thrombocytosis

Parameters increased by presence of leukocytosis

RBC - small leukocytes counted as RBC

Hgb - WBC elevation causes turbidity = increase in Hgb

Hct - Turbidity due to elevated WBC results in falsely increased Hct

Parameters affected by cold agglutinin

RBC - decreased

WBC - increased

Hct - decreased

Cold agglutinin

red cells clumped together in the cold

- misinterpreted as other cells (WBCs)

platelet aggregation

clumping together of platelets in the blood that results in a decreased platelet count

Are results within normal range?

Are results linear?

Are there any flags/codes present?

Do the Hgb and Hct match? MCHC okay?

Do results make sense? Possible and compatible with life?

5 questions the MLS should ask themselves when interpreting results:

Pre-analytical errors

Check patient label

Analytical errors

Check reagents

Check instrument systems

Run correction procedures

Check peripheral blood smear (clumping or agglutination)

Rerun, either whole or diluted

Check tube for clots

Hemolysis

Post-analytical checks

Delta checks

Review diagnosis/history of patient

Redraw specimen

5,000-10,000/ul

normal range for WBC

4-6mil/ul

normal range for RBC

m: 13-17 g/dl

f: 12-16 g/dl

normal range for Hgb

80-100fl

normal range for MCV

macrocytic; microcytic

>100fl <80fl

27-32pg

normal range for MCH

32-36%

normal range for MCHC

hypochromic

lipemia or cold agglutinins

<32%

>36%

150,000-400,000/ul

normal range for platelets

11.5-14.5%

normal range for RDW

Scatterplot

name of graph that displays distribution of WBC showing volume vs light scatter

lipemia

extremely elevated WBC

cold auto agglutinins

the big 3 that can cause error

saline replacement

Resolution for lipemia

saline dilution & x2

Resolution for extremely high WBCs

warm specimen

Resolution for cold auto agglutinins