Preanalytical requirements in

hematology

Giuseppe Lippi, Verona, Italy

Although it may look similar from the outside, hematology is

something (VERY) different from all other lab tests!

Which is the anticoagulant of choice for

hematological testing?

• Na2EDTA

• K2EDTA

• K3EDTA

• Lithium-heparin

• Clinical chemistry/immunochemistry: Serum of LH-Plasma

• Coagulation: Citrate Plasma

• Hematology: Whole Blood EDTA

We are basically using irreversibly

anticoagulated whole blood

• The main property of EDTA, a polyprotic acid containing four

carboxylic acid groups and two amine groups with lone pair

electrons, is the ability to chelate or complex metal ions in 1:1

metal-EDTA complexes.

• As calcium ions are necessary for blood coagulation, the

specific association between the carboxylic groups of EDTA

and calcium is a reliable solution to prevent clotting and

stabilizing whole blood in a fluid form, as required for some

laboratory analyses.

1. Of the three ethylenediaminetetraacetic acid (EDTA) salts,

potassium salts are the most readily soluble.

2. Tripotassium EDTA (K3EDTA) is dispensed as a liquid, and

thus causes:

• Specimen dilution

• Ted blood cell size variation

3. Therefore, dipotassium EDTA (K2EDTA) is recommended as

the anticoagulant of choice in specimen collection for blood

cell counting and sizing.

4. The amount of K2EDTA used in spry form is 1.5-2.2 mg/mL

(3.7-5.4 μmol/L).

LABORATORY ERRORS

The “hourglass” model

Within-Laboratory ~10%

Extra-Laboratory ~90%

(1)

(2)

(3)

(4)

(5)

Preanalytical issues in laboratory hematology

1. Fasting status

2. Posture

3. Physical activity

4. Type of blood tubes

5. Needle size

6. Venous stasis (tourniquet placement)

7. Sample mixing

8. Sample stability

9. Hemolysis

Preanalytical issues in laboratory hematology

1. Fasting status

2. Posture

3. Physical activity

4. Type of blood tubes

5. Needle size

6. Venous stasis (tourniquet placement)

7. Sample mixing

8. Sample stability

9. Hemolysis

Preanalytical issues in laboratory hematology

1. Fasting status

2. Posture

3. Physical activity

4. Type of blood tubes

5. Needle size

6. Venous stasis (tourniquet placement)

7. Sample mixing

8. Sample stability

9. Hemolysis

Inpatient Outpatient Just arrived in the ED

DAY 1 DAY 2

X

Preanalytical issues in laboratory hematology

1. Fasting status

2. Posture

3. Physical activity

4. Type of blood tubes

5. Needle size

6. Venous stasis (tourniquet placement)

7. Sample mixing

8. Sample stability

9. Hemolysis

Which is the correct answer regarding the

acute effect of physical activity?

• Hemoglobin is decreased

• Eosinophils are decreased

• Neutrophils are decreased

• All the above

Preanalytical issues in laboratory hematology

1. Fasting status

2. Posture

3. Physical activity

4. Type of blood tubes

5. Needle size

6. Venous stasis (tourniquet placement)

7. Sample mixing

8. Sample stability

9. Hemolysis

Preanalytical issues in laboratory hematology

1. Fasting status

2. Posture

3. Physical activity

4. Type of blood tubes

5. Needle size

6. Venous stasis (tourniquet placement)

7. Sample mixing

8. Sample stability

9. Hemolysis

Preanalytical issues in laboratory hematology

1. Fasting status

2. Posture

3. Physical activity

4. Type of blood tubes

5. Needle size

6. Venous stasis (tourniquet placement)

7. Sample mixing

8. Sample stability

9. Hemolysis

Which hematologic parameters can be biased

by prolonged venous stasis?

• Hemoglobin

• Rd blood cells

• Leukocytes

• All the above

Preanalytical issues in laboratory hematology

1. Fasting status

2. Posture

3. Physical activity

4. Type of blood tubes

5. Needle size

6. Venous stasis (tourniquet placement)

7. Sample mixing

8. Sample stability

9. Hemolysis

The paradigm: each sample must be appropriately mixed before testing.

The questions:

1. Is this true for hematology, as well?

2. How much shall we “shake” our small bottles?

What shall we do so?

• No mix

• 4-6 mix

• As defined by the manufacturer

(raise your hand, bau-bau!)

Step 18.1

Ideally, the number of full rotations should

correspond to manufacturers’ instruction.

Preanalytical issues in laboratory hematology

1. Fasting status

2. Posture

3. Physical activity

4. Type of blood tubes

5. Needle size

6. Venous stasis (tourniquet placement)

7. Sample mixing

8. Sample stability

9. Hemolysis

In vitro HEMOLYSIS

Hemolytic specimens are frequent occurrence in laboratory

medicine.

• The prevalence is as high as 3.3% of all routine samples.

• They account for 40-70% of all unsuitable specimens.

• The are the first cause of unsuitable specimens, nearly five times higher

than the second.

• In vitro hemolysis remains the leading cause of unsuitable specimens

for:

• Both outpatient and inpatient samples

• Both routine and stat samples.

• Several tests that are unreliable on hemolyzed specimens are likely to

be suppressed.

How frequent can hemolysis be in whole

blood specimens?

• <1%

• 1-2%

• 2-5%

• >5%

No hemolysis

Hemolyzed

Fragments Injuried leukocytes

Anysocytosis

Both equations displayed an area under the curve of

≥0.99 for identifying spurious hemolysis, much greater

than that of both RBC ghosts and immature platelet

fraction.

Ht/Hb x √MCV Ht/Hb x 100

Sysmex XN

Ht/Hb x √MCV >0.026

Results flagged by the LIS

Sample centrifugation

VISUAL INSPECTION

No hemolysis Major hemolysis

RELEASE DATA (99.6%)

RECOLLECT SAMPLE

(0.4%)

And what about… this?

Ht 0.08 Hb 104 g/L

or

What we would expect?

Where is the truth?

Ht 0.08

Hb 104 g/L

What is your guess?

• Sample dilution?

• Wrong anticoagulant?

• Cryoglobulins?

• Hemolysis?

• Analytical error?

(raise your hand, bau-bau!)

And the winner is…

Another «strange» case…

A 53-year-old woman was referred to the local hospital because of

thrombocytopenia detected incidentally.

Findings were:

- Platelet 19x109/L

- WBC 4.3x109/L

- Hb 94 g/L

- Instrument flag for “PLT clumps”

- Not use of alcohol or any drug

- No drug and food allergy

- No signs of hemorrhagic diathesis

- No other abnormal findings on physical examination

What is your guess?

• Sample (partially) clotted?

• Wrong anticoagulant?

• Cryoglobulins?

• EDTA-dependent pseudothrombocytopenia?

• Analytical error?

(raise your hand, bau-bau!)

And the winner is… Repeated samples

• Low PLT count in EDTA

• Normal PLT count in Citrate

Thanks for your attention, and…

…enjoy the rest of the meeting!