Prof Asma Elreweny

1. 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)

2. 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

3. 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
Prof Asmaa Elreweny, MD 3

4. 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
Prof Asmaa Elreweny, MD 4

5.  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
Prof Asmaa Elreweny, MD 5

6.  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.
Prof Asmaa Elreweny, MD 6

7.  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.
Prof Asmaa Elreweny, MD 7

8.  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%.
Prof Asmaa Elreweny, MD 8

9.  Sample extraction, transport and storage as
well as sample aspect (hemolytic, icteric,
lipemic) and sample preparation (e.g.
centrifugation) are additional important
preanalytical steps.
Prof Asmaa Elreweny, MD 9

10. 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.
Prof Asmaa Elreweny, MD 10

11. 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.
Prof Asmaa Elreweny, MD 11

12.  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).
Prof Asmaa Elreweny, MD 12

13. The Effect of Venipuncture Procedure
Two sample types are basically used for
hematological routine work:
1. Venous blood
2. Capillary blood
Prof Asmaa Elreweny, MD 13

14. - ~ 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.
Prof Asmaa Elreweny, MD 14

15. 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

16. 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.
Prof Asmaa Elreweny, MD 16

17. 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.
Prof Asmaa Elreweny, MD 17

18. 5. Hold microvette horizontally or slightly
inclined & take up the blood drops with the
end-to-end capillary
Prof Asmaa Elreweny, MD 18

19. 6. Withdrawal is finished once the capillary is
full
Prof Asmaa Elreweny, MD 19

20. 7. Hold microvette upright to allow blood
flowing into the collection tube
Prof Asmaa Elreweny, MD 20

21. 8. Gently remove the capillary-containing cap
by twisting and discard as a unit
Prof Asmaa Elreweny, MD 21

22. 9. Remove attached sealing cap from the
bottom of the tube & cover the tube.
Prof Asmaa Elreweny, MD 22

23. 10. Mix sample thoroughly but gently(inversion)
11.Apply medical strip to puncture site.
Prof Asmaa Elreweny, MD 23

24. Capillary blood withdrawal with the
collection rim
1- 4 as before
5- 6-
Prof Asmaa Elreweny, MD 24

25. 7-
Prof Asmaa Elreweny, MD 25

26. 8- 9-
Prof Asmaa Elreweny, MD 26

27. 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.
Prof Asmaa Elreweny, MD 27

28. 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.
Prof Asmaa Elreweny, MD 28

29.  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.
Prof Asmaa Elreweny, MD29

30. ■ 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.
Prof Asmaa Elreweny, MD30

31. 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.
Prof Asmaa Elreweny, MD 31

32. 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.
Prof Asmaa Elreweny, MD 32

33. 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.
Prof Asmaa Elreweny, MD 33

34. 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
Prof Asmaa Elreweny, MD 34

35. Comparison of results afterimmediate analysis of sample &
measurement afterstorage for24 hrs at room temperature
Prof Asmaa Elreweny, MD 35

36. Storage Time
 The storage time can vary depending on the test
method or on the technology or reagents used by
the analytical system.
Prof Asmaa Elreweny, MD 36

37. Guidelines for storage times of EDTAblood 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
Prof Asmaa Elreweny, MD 37

38. 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%.
Prof Asmaa Elreweny, MD 38

39. 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.
Prof Asmaa Elreweny, MD 39

40. 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.
Prof Asmaa Elreweny, MD 40

41. Comparison of the results after correct & after poor mixing.
Prof Asmaa Elreweny, MD 41

42. Comparison of the results after correct & after poor mixing.
Prof Asmaa Elreweny, MD 42

43. The Effect of Filling Volume
Prof Asmaa Elreweny, MD 43

44.  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.
Prof Asmaa Elreweny, MD 44

45.  Insufficiently filled tubes may lead to:
- Incorrect MCV and RBC results
- Morphological changes in WBC and RBC.
Prof Asmaa Elreweny, MD 45

46. The Effect ofAspirate Volume
- Ensure that no air is aspirated.
- Too little aspirated material can lead to
falsely low results.
Prof Asmaa Elreweny, MD 46

47. Comparison of results afteraspirationof sufficient and too little sample .
Prof Asmaa Elreweny, MD 47

48. 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).
Prof Asmaa Elreweny, MD 48

49. 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.
Prof Asmaa Elreweny, MD 49

50. The Effect ofAnticoagulant
 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.
Prof Asmaa Elreweny, MD 50

51.  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.
Prof Asmaa Elreweny, MD 51

52.  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.
Prof Asmaa Elreweny, MD 52

53. The Effect of Unexpected Conditions
 Cryoglobulins
 Erythrocyte parasites
 They could induce false results for WBC, RBC
and PLT.
Prof Asmaa Elreweny, MD 53

54. 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.
Prof Asmaa Elreweny, MD 54

55.  Idiopathic cryoglobulinaemia is characterized by a
triad of symptoms:
- purpura,
- arthralgia and
- weakness, often accompanied by renal failure.
 Secondary cryoglobulinaemia to other diseases.
Prof Asmaa Elreweny, MD 55

56. 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.
Prof Asmaa Elreweny, MD 56

57. 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.
Prof Asmaa Elreweny, MD 57

58. 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%).
Prof Asmaa Elreweny, MD 58

59. Summary
Prof Asmaa Elreweny, MD 59

60.  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.
Prof Asmaa Elreweny, MD 60

61. 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.
Prof Asmaa Elreweny, MD 61

62. ‫العالمين‬ ‫رب‬ ‫هلل‬ ‫الحمد‬
‫أجمعين‬ ‫الخلق‬ ‫سيد‬ ‫علي‬ ‫سالم‬ ‫و‬ ‫وصالة‬
Thank You
Prof Asmaa Elreweny, MD 62