HM5 Quality Assurance & Histogram Interpretation Dr. Melanie Hicks, BSc, DVM

Senior Professional Services Veterinarian

Topics

1) Quality Assurance Pre & Post- Analysis 2) HM5: how it works 3) Histograms

RBC WBC Platelets

When performing a blood test on any analyzer point-of-care or externally, a

high quality sample will give high quality results

Sample Handling Minimize trauma to prevent hemolysis

• Proper needle & syringe size – Avoid 25 G if possible

• Vacutainers • Appropriate suction on syringe • Appropriate amount of alcohol

– Swift blood draw from larger vein to minimize PLT clumping • Clumps occur more often from small peripheral veins, jugular or

cephalic best • Minimize trauma during collection • Cats have very reactive PLT • Sick patients may have more reactive PLT

Sample Handling: PLT Clumping

Sample Handling: Filling the Tubes

Remove cover from blood tube & needle from syringe for transfer • Fill EDTA tube minimum half full

• BD Microtainer tubes (500 µL) work for small sample size • Invert tube 10-15 times, don’t shake

• Microtainer tubes require longer mixing (20-25x)

Sample Handling: Tube Order

• Fill lithium heparin tube (chemistry) prior to EDTA tube (hematology)

• Otherwise EDTA contamination can occur

Sample Handling: Running the Sample

• CBC best if run immediately but ideally within 3-4 hours • Make blood films 5 minutes after blood mixed in EDTA tube • Sample must be at room temperature for processing

Sample Handling: Select the Correct Species • Variance in cell sizes for different species • Different lysing effects for different species

& types

Select incorrect species: incorrect results

Dog Cat Horse

Cow

Blood Cell Types

Size in femtoliters (10−15 litre) One femtolitre = 1 cubic micrometre (μm3)

2 30 60 90 120

RBC

platelets lymphocytes monocytes

granulocytes

Blood Cell Relative Sizes (Natural state)

HM5: Impedance Technology

4 key components • Chamber: mixing/diluting of blood

• Aperture: microscopic hole that

carries an electric potential field across it; cells travel through this hole

• Measuring tube: contains aperture; counting and sizing of cells happens here

• Electrodes: one on either side of the aperture to set up electric field across the aperture

Chamber

Measuring Tube

Aperture

-

+

Electrodes

Impedance Technology

CELL SIZE is the differentiator

Aperture

Blood diluted with isotonic solution to conduct a

current

Cells pass through the

aperture

Aperture has electrodes on

either side

Electrical impedance

change: voltage rise &

electrical pulse

Whole Blood

1

25ul

1:160 4 ml

Lyse2/Dilutent

EOS Count

Cleaning & Rinse

How does the HM5 work?

Whole Blood 1:160 4 ml Diluent

2

25ul

3

25ul

1:180 0.5 ml Lyse

Measure WBC and

HGB

1:32000 5ml Diluent

Measure RBC and

PLT

25ul

4

How does the HM5 work?

Calculated & Measured Parameters

• EOS, WBC, RBC, HgB, MCV, PLT, MPV Measured Parameters

• HCT, MCHC Calculated Parameters

HCT: Estimate of PCV

HCT = calculated RBC X MCV 10 Causes of error • Tube sitting too long (RBC

swelling) • Sample not mixed • Too much EDTA to RBC in tube (RBC shrinkage)

PCV = measured

Causes of error • Reading Error • Plasma Viscosity • Spin Time • Technique

Histograms

Verify Parameters

Quality Control Check

Identify Uncommon

Disease Processes

Graphic representation of cell distribution & cell counts

4 Histograms – HM5

CELL SIZE

CELL

NU

MBE

R

Key Components to a Histogram • Discriminators

• Peaks & shapes of peaks

• X axis (cell size)

Elements of a Histogram

Elements of a Histogram Re

lativ

e N

umbe

r

Cell Volume (fl)

Rela

tive

Num

ber

Cell Volume (fl)

X Axis • Size of cells within a

population increases, moving to the right

Discriminators • Separate different cell types based on size • Position determined by software Y Axis • Relative & scaled according to highest

value on histogram • Can compare peak heights within a

histogram • Cannot compare peak heights between

different samples

Elements of a Histogram

Cell Size in Femtoliters (fl)

Relative Cell

Numbers (%)

Discriminators

Platelet Histogram

Cell counts conceptually the area under the curve

Cats: peak may not finish at the bottom of the y axis

What happens here?

Platelet Clumping

1. Check a smear: monolayer for single PLT (min 8-10/hpf, 100x), feathered edge for clumped PLT

2. Check EDTA tube: for clots

Platelet Count Estimate on Blood Film

• Monolayer 100x: count # PLT in 10 fields • Calculate average PLT/ field PLT/μL= Avg PLT/field × 20,000 • Numerous potential causes of variability in PLT

estimation- formula provides reasonable estimate

a) Ideal histogram. Note symmetrical RBC peak (red line) & good separation between the PLT (1) & RBC (2) peaks

b) The ‘tail’ on right of the RBC peak indicates larger, immature RBC precursors (increased RDW)

a b

1

2

Typical RBC Histogram: Canine & Feline

Generally starts at 25-31 fl for dogs, ~18-23 in cats Cell counts

conceptually the area under the curve

RDW – Red Cell Distribution Width

Measure of uniformity in size of RBCs (degree of anisocytosis) • ↑ RDW: Variety of cell sizes present, blood film recommended

– Reference Range Canine 14-20% Feline 15-18% • Regenerative anemia: ↑ RDW (often before ↑ MCV) • ↑ small RBCs (i.e. early iron deficiency anemia) cause ↑ RDW

Mean Cell Volume

• Measured value • ↑ MCV: shift of RBC curve to right (presence of larger cells) • ↓ MCV: shift of RBC curve to left (presence of smaller cells)

HGB 10.5

HCT 29.96

MCV 46

RDW 21.8

12 18

37 55

60 77

Canine Microcytic Anemia

• Normal-shaped histogram • Low MCV

• Low Hgb • High RDW

Example Using MCV & RDW

Possible acute blood loss

Normal Canine Histogram

Native State Sizes of White Blood Cells

Eos

cat

dog

horse

Lym Mon Neu LARGEST 2ND LARGEST SMALLEST

Effects of Lyse Solution on WBCs

•Cells react differently to lyse

•RBC: lysed

•MON: shrink slightly

•LYM: shrink slightly

•GRA: not too much change

•Classification of WBC types based on size

LYM MON GRA

LYM MON GRA

Native state Smallest Largest Mid size

With Lyse Smallest Mid size Largest RBC

Debris

Normal WBC Histogram

• 3 peaks: LYM, MON, GRA • Discriminators variable depending on species

• LYM peak

– Begins lower to mid part of y axis – Variable

• GRA peak single & defined

– No tails – No cells above 200 fL

L

M G

(1) EOS peak distinct, but may not always be in normal samples (2) Should be mostly clear of debris on the right side of the peak

EOS Histogram

Severe Platelet Clumping

Upward trend to PLT peak, ↑ LYM, LYM peak starts high on Y axis, long GRA tail

Estimation of WBC Count on Blood Film

On 40x: Count WBCs in 10 - 20 consecutive fields of view within the

monolayer zone Calculate the average number of WBC/Field

• WBC/μL= (Avg. WBC/field)x(Objective)2

= Avg. WBC/field x 402

Since differences exists in the microscopic field of view size between microscopes, the correction factor may vary (by ~15%)

Classic signs: low LYM, high GRA, high WBC

Stress Leukogram

↑LYM & ↓NEU

High LYM with large LYM peak; ; low NEU with small GRA peak

↑ LYM ↓ NEU

Classic Canine Leukemia

1 WBC peak, ↑ WBC, Platelets- Clumping vs truly low?

Cavalier King Charles Spaniel

Marcothrombocytopenia • ↓PLT & very large in size • Affects LYM count • LYM peak starts high on Y axis • Also occurs in Norfolk & Cairn

Terriers, Boxers, Shih Tzus

Healthy Equine Sample

Smaller RBC size than dogs or cats Normal slight asymmetry

Healthy Bovine Sample

WBC peak separation not as distinctive in this species

Quality Assurance: Review Results

HGB X 3 ≈ HCT 14.6 x 3 = 43.8

If >4-5% difference, review maintenance of analyzer

Quality Assurance: Review Results

Check for diagnostic flags on every CBC

Quality Assurance: Review Histograms

Verify Parameters

Quality Control Check

Identify Uncommon

Disease Processes

Best Quality Control Method for In-House Hematology?

Manual Blood Smear

Automated analyzers cannot accurately detect • Parasites (microfilaria, mycoplasma, etc.) • Immature (band) NEU • Review every ↑WBC count • RBC shape changes • PLT clumping • Low incidence cells

Questions?

Dr. Melanie Hicks melaniehicks@abaxis.com