Preanalytical errors in hematolog

Preanalytical Errors and

Variables in Hematology

Meqat General Hospital November 2016

By

Prof. Asmaa El Reweny, MD

Professor & Consultant of Clinical & Chemical Pathology, Faculty of Medicine,

Cairo University & AMS, Taibah University (2006-2016)

Objectives

At the end of this lecture you will be able to:

1. Revise what is the meaning of preanalytics.

2. Recognize patients’ variables that could affect hematological

parameters.

3. Identify effects of sampling time, circadian rhythm, venipuncture

procedure, anticoagulants & tourniquet application.

4. Recognize effects of mixing, storage & volume of samples on

hematological tests.

5. Recognize effects of fasting, blood lipids & glucose level on

CBC parameters.

6. Identify unexpected conditions like cryoglobulins & malaria

parasites.

Prof Asmaa Elreweny, MD 2

What is Preanalytics?

Preanalytics comprises all processes done

prior to the actual laboratory analysis

and is influenced by a number of

variables of both changeable and

unchangeable nature.

weak link


Patient-related variables

Permanent Long-term Short-term

Population Age Body position

Gender Gravidity Physical stress

Sea level Mental stress

Nicotine Circadian variation

Alcohol Nutrition/feeding

Drugs

Nutrition


Population

Significant differences could be found in

both genders of black population in

contrast to white population. Black people

have markedly lower numbers of WBCs

or WBC subpopulations


Gender

Besides sex-specific variables like

hormones, the number of RBCs & Hb

concentration are lower in females

than in males.

Age

Many measurable parameters do not

reach adult values before adolescence.


Pregnancy Hemoglobin and iron levels frequently fall

beyond normal.

Sea Level From 3,600 meters a.s.l, Hb & Ht are increased.

Nicotine It is important to ask the patient whether he is a

smoker. The chronic consumption of nicotine

increases the number of WBCs.


Body Mass Index (BMI)

There is a weak correlation between Hb and BMI

in professional athletes.

Body Position Considerably different results between sitting and

lying body position could be obtained. If changing

from a lying to an upright position, Ht increases

by 15%.


Sample extraction, transport and storage as

well as sample aspect (hemolytic, icteric,

lipemic) and sample preparation (e.g.

centrifugation) are additional important

preanalytical steps.


Effect of Sampling Timing

The maximum variation of WBC, Hb & Ht values

could amount to as much as 20% during the day.

Blood samples should be taken between 7 -9 am.

If rigorous comparative values are needed, the

patient should rest for 30 min prior to blood

sampling, but this is only practicable in hospitals.


The Effect of Circadian Rhythm

The nadir (lowest value during 24 hs) for Ht is

shown during the night with a variation into the

day of 5% ; the variation was higher when

exercise was held.

RBC, Hb, and Ht show low amplitude circadian

rhythm, with an acrophase (highest part of the

normal fluctuation) at 11:00.

WBC have the acrophase at evening (21.00–24:00)

with a variation of 0.9–2.0 x 109/L.


Reticulocytes show the acrophase at 01:00 (between

20:00 and 04:30; the fluctuation is higher); the

standardization of the drawing at early morning is

necessary.

A circaseptan rhythm for RBC, Hb and Ht was

reported, with the acrophase at Monday. Seasonal

variations determine hemodilution on summer

(increase of 9% of plasmatic volume).


The Effect of Venipuncture Procedure

Two sample types are basically used for

hematological routine work:

1. Venous blood

2. Capillary blood


- ~ 65% of blood is in veins,

- 20% in capillaries,

- 15% in arteries.

Vasodilatation increases plasma in capillaries at

the expense of cellular particles.

Shock or stress could lead to poor circulation in

the capillary system.

Blood composition in arteries and veins is more

or less constant.

All these make capillary blood sampling inferior

to venous blood sampling.


Capillary blood sampling is the method of

choice only for neonates and infants.

But under certain circumstances it is also preferred for adults:

■■ Poor vein conditions

■■ Burns or scars at sites for venous blood

■■ Abnormally fatty patients

■■ Patients with frequent blood tests

■■ I V catheters in both arms or hands

■■ Patients scheduled for IV medication or chemotherapy

■■ Point-of-care testing Prof Asmaa Elreweny, MD 15

Capillary blood sampling

Capillary blood is a mixture of blood from

arterioles, venules & capillaries. The relative

composition depends on the circulation of the

puncture site. There are two sampling

techniques:

1. Capillary technique with end-to-end capillary.

2. Using the collection rim of a microvette.


Capillary blood withdrawal with end-

to-end capillary

1. Choose puncture site. Improve

circulation by warming.

2. Disinfect & allow to air dry

3. Fix the patient’s finger, foot or ear lobe

with the correct handgrip.

4. Discard the first drop.


5. Hold microvette horizontally or slightly

inclined & take up the blood drops with the

end-to-end capillary


6. Withdrawal is finished once the capillary is

full


7. Hold microvette upright to allow blood

flowing into the collection tube


8. Gently remove the capillary-containing cap

by twisting and discard as a unit


9. Remove attached sealing cap from the

bottom of the tube & cover the tube.


10. Mix sample thoroughly but gently (inversion)

11. Apply medical strip to puncture site.


Capillary blood withdrawal with the

collection rim

1- 4 as before

5- 6-


7-


8- 9-


Important notes on capillary blood withdrawal

- Avoid certain disinfectants (e.g. perchloric

acetic acid can change cell morphology).

- Insufficient drying of the puncture site can

lead to hemolysis or dilution of the sample.


Important notes on capillary blood withdrawal

- Discard the first drop as coagulation starts

immediately after puncturing & platelets

aggregate at puncture site & form a clot.

- The first drop contains lymph & tissue fluid

which may affect measurement & lead to

hemolysis or clotting.

- When clot is not swiped away, blood flow can

stop before withdrawal is finished, possibly

making a second puncture is necessary.


Counts in capillary blood are not representative for whole circulation. The venous blood is more suitable than capillary blood from heel or finger pad which in turn is more suitable than ear lobe.

Capillary blood > venous blood of 1,000 to 4,000 WBCs.

Severe leucopenia may be judged as too mild & mild leucopenia may be overlooked or a value in the upper normal range may be mistaken as leucocytosis.

The exact measurement of Hb & Ht from ear lobe blood of children also proved to be unsuitable.


■ Blood flow is mostly not adequate & phlebotomist might massage & squeeze the puncture site too strongly.

- By addition of lymph, blood is diluted and the concentration ratios change (up to 15%).

- Squeezing can also result in hemolysis.

Due to stress and consecutive reactions in the capillary system as well as clotting by slow blood flow, the successively collected blood drops are not comparable in their cell concentrations.


Has finger blood sampling been

performed correctly?

- The first drop of blood was not wiped away.

The finger was squeezed & rubbed.

- This results in abnormal WBC histogram. The

curve does not start at the baseline, which leads

to an incorrect WBC level.

- However, what happens if Lab does not have

this information? It is difficult to find causes of

abnormal histogram on the basis of curve alone

- A similar curve could be caused by

normoblasts, among other things.


NB Withdraw blood quickly to minimize platelet

aggregation and microclot formation.

■ Fill up to the recommended volume. Under filling can

lead to false MCV & RBCs values but can also result in

morphological changes of WBC & RBC. Overfilled

tubes have lower EDTA concentration which could

provoke clotting.

■ Adequate mixing is important to avoid platelet

aggregation and microclots. Those may lead to false

results for counts of platelet, RBCs & WBCs as well as

for volume of platelet & RBCs.


The Effect of Transport & Storage

Many hematological parameters should be

measured immediately or within few hrs.

Degeneration of WBC occurs within 3-4 hs with an

apparent increase in stab granulocytes.

This degeneration is even more marked if the blood

sample is stored in the refrigerator.


WBC degeneration

- Storage for 24 hs at RT followed by inverting tube

several times changes WBC histogram that indicates

degeneration of the WBC.

- A similar WBC histogram might signify

pathological WBC changes in the case of a fresh

sample


Comparison of results after immediate analysis of sample &

measurement after storage for 24 hrs at room temperature


Storage Time

The storage time can vary depending on the test

method or on the technology or reagents used by

the analytical system.


Guidelines for storage times of EDTA blood samples

Parameter Storage at

room temp

Delayed processing

leads to:

Hematocrit up to 24 h Increase

RBCs up to 12 h Decrease

WBCs up to 24 h Decrease

Platelets up to 12 h Decrease

Blood smear up to 3 h WBC Degeneration

Automated differential count 8 to 48 h WBC Degeneration

Reticulocte count up to 24 h Decrease

Reticulocyte smear up to 24 h Degeneration of cells

MCV and RDW up to 8 h Increase


The Effect of Tourniquet

The tourniquet application during time of 2.30 ±

0.12 minutes could induce the following

modifications:

- Ht + 2.4 %,

- Hb +1.4 %,

- Ret –1.9%.


The Effect of Mixing

The mixing procedure is crucial for obtaining

correct and valid results.

Improper mixing after sampling, reveals

significant decreases for RBCs count, Hb, Ht and

platelets count, whereas the mean platelet volume

will significantly increase.


Poor mixing before analysis ?

If infra-natent is aspirated from

poorly mixed sample, high RBC count

with low PLT count is obtained. The

situation is reversed if the

supernatant is aspirated.


Comparison of the results after correct & after poor mixing.


Comparison of the results after correct & after poor mixing.


The Effect of Filling Volume


Overfilling collection tube leads to

inadequate sample mixing and all the

parameters are altered.

An overfilled tube also results in an

excessively low EDTA content & clotting.


Insufficiently filled tubes may lead to:

- Incorrect MCV and RBC results

- Morphological changes in WBC and RBC.


The Effect of Aspirate Volume

- Ensure that no air is aspirated.

- Too little aspirated material can lead to

falsely low results.


Comparison of results after aspiration of sufficient and too little sample .


The Effect of Fasting

- Hyperlipemia (TG > 580 mg/dL or patients

receiving IV emulsions) could induce higher results

of Hb & MCHC (>36 g/dL), due to a direct

interference on spectro-photometric measurement

of Hb on Coulter instrument.

- When lipids form droplets, they could interfere

PLT count on Bayer instrument (TG >300 mg/dL).


The Effect of Blood Glucose Level

Very high blood glucose (> 500 mg/dl) induce

osmolar effects on RBCs with swelling due to

water shift into RBCs: MCV & Ht become higher

& MCHC results are not accurate.


The Effect of Anticoagulant

EDTA is the anticoagulant of choice for

hematological tests.

The K3 EDTA causes cell shrinkage (less apparent

with K2 & Na2 EDTA).

Ht by Microhematocrit method is not influenced by

K2 and Na2 EDTA, whilst it is decreased by K3

EDTA. Thus, K2 should be preferred to K3 salt.


High concentration of Na EDTA tends to increase

MCV (less evident with K3 EDTA).

EDTA could change PLTs from a discoidal to

spherical shape.

MPV could be a valid clinical finding for detecting

the source of thrombocytopenia.

MPV is normal in autoimmune thrombo-

cytopenia, while it is increased in DIC, micro-

angiopathies or pathologies impairing PLTs

maturation & release.


Prevalence of EDTA-induced pseudothrombo-

cytopenia is nearly 0.1% (higher in

thrombocytopenic patients).

The pseudothrombocytopenia may trigger

unnecessary further investigation, unjustified

& invasive therapeutic or medical treatments.


The Effect of Unexpected Conditions

Cryoglobulins

Erythrocyte parasites

They could induce false results for WBC, RBC

and PLT.


The Effect of Cryoglobulins

The paraproteins associated with myeloma or

immunoproliferative disorders are often

cryoglobulins. Their precipitation depends on

their concentration, the Ig class and pH of the

medium.


Idiopathic cryoglobulinaemia is characterized by a

triad of symptoms:

- purpura,

- arthralgia and

- weakness, often accompanied by renal failure.

Secondary cryoglobulinaemia to other diseases.


Screening for Cryoglobulin:

Serum is stored at 4 °C for several days and

checked for precipitate which is completely

reversible on rewarming. The precipitate could

be quantified, after centrifugation of serum in a

tube.


On Coulter counter, cryoglobulins produce pseudo-

leucoytosis and/or pseudothrombocytosis.

With Technicon counter, which use prewarmed

reagents and WBC diluent at low pH,

cryoprecipitation does not occur as they do not

precipitate at this pH. An interference is evident on

the PLT count.


The Effect of Malaria Parasite

Small RBC infected by trophozoites of Plasmodium

falciparum were misinterpreted as PLT by some

analyzers, leading to false PLT count.

By using a calculation, a »malaria factor« is

produced by some instruments. A malaria factor

> 3.7 was an indicator of malaria infection

(specificity: 94%, sensitivity: 98%).


Summary


Preanalytical phase is particularly important in

hematology.

The correct use & concentration of

anticoagulant is mandatory.

EDTA is the anticoagulant of choice, but it has

some limitations in stability & shape of

platelets.

Mixing after blood drawing and before analysis is also

crucial for obtaining correct and valid results.

Hematology tests are interfered by high lipids & CHM

Remember that Cryoglobulins & RBCs parasites

can induce false results of WBC, RBC and PLT.


References

[1] WHO guidelines on drawing blood: best practices in phlebotomy,

2010

[2] Magee, L. S. Preanalytical variables in the chemistry laboratory.

Becton Dickinson Lab Notes 2005; 1 (1)

[3] Sarstedt AG: Tipps und Tricks in der Preanalytik, 2008

[4] Tyndall, L. Managing Preanalytical Variability in

Hematology. Becton Dickinson Lab Notes 2004; 14 (1)

[5] Crabbe G, Van Poucke M, Cantinieaux B. Artefactuallynormal

automated platelet counts due to malaria infected RBC. Clin Lab

Haem 2002; 24: 179–82.


الحمد هلل رب العالمين

وصالة و سالم علي سيد الخلق أجمعين

Thank You


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Preanalytical errors in hematolog

Education