Managing Preanalytical Variability in Hematology

Controlling the preanalytical variability in hematology testing is a critical factor for ensuring accurate results. Preanalytical factors such as specimen collection, specimen handling, interfering substances and patient factors are common causes of inaccurate test results. Improper specimen collection and processing can influence the outcome of analytical results. By minimizing errors at any step in the preanalytical phase, a laboratory can improve the quality of analytical results, reduce the number of re-collected specimens, and improve turnaround time and patient management.
Collecting a Quality Specimen


An accurate result starts with a quality specimen. Proper patient identification is critical. Test forms should be compared to the inpatient's wrist bracelet or verbally confirmed with the outpatient. Venipuncture or skin puncture may be used based on the patient's physical condition and the amount of blood required for analysis. Selection of the appropriate blood collection system should be based on the patient's physical condition. The evacuated blood collection system is preferred over the needle and syringe since it is a safer method to use and provides a better quality blood specimen. In addition, winged blood collection sets may be used to aid the phlebotomist with the more challenging collections.
The preferred sites for venipuncture are the larger median cubital and cephalic veins of the arm or the dorsal wrist and hand veins. The site should be cleansed with 70% isopropyl or ethyl alcohol and allowed to air dry to ensure proper disinfection. Air-drying will also help prevent hemolysis and prevent burning sensations during phlebotomy. If a tourniquet is used, it should be released within one minute of tying. Venipuncture should be performed with the patient's arm in the downward position with the needle in the bevel-up position. Tubes should be mixed as per the manufacturers' recommendation immediately after they are filled. After blood is drawn, the needle is removed and pressure is applied to the puncture site. The needle is then disposed of according to the facility's protocol.¹
Skin puncture is performed on newborn heels, and fingers of adults and children when a venipuncture is not otherwise advisable. There are numerous lancet designs including puncture, incision, and laser devices. Skin puncture blood can be obtained from the palmar surface of the distal phalanx of older children and adult fingers, the lateral or medial plantar surface of the infant heel and plantar surface of an infant's big toe.


The skin puncture site should be cleansed with isopropyl alcohol and allowed to air dry completely. The puncture device should be held flush against the finger or heel prior to puncture. After the puncture, the first drop of blood should always be wiped away with a gauze pad. The first drop is most likely to contain excess tissue fluid that may cause clotting or sample dilution.
Blood films may be prepared from a drop of blood at the bedside or from a specimen collected in EDTA. For all other hematological tests, specimens should be anticoagulated with EDTA. When the tip of the microcollection device comes in contact with the second drop of blood, the drop will flow into the tube by capillary action. Scraping of the skin with the collection device should be avoided, since it may initiate clotting. Gentle tapping of the tube may facilitate the flow of the blood into the tube. Tubes should be capped and immediately mixed by complete inversion according to the manufacturer's recommendations.
Order of Draw


The correct order of draw should be followed to eliminate the risk of additive cross-contamination during venipuncture and to reduce the risk of clotting in the EDTA or other anticoagulant microcollection tubes. For venipuncture procedures using plastic or glass tubes, the recommended order of draw is blood cultures, sodium citrate tubes, serum tubes with or without clot activator, with or without gel, heparin and heparin gel tubes, EDTA tubes and then fluoride tubes. When performing skin puncture techniques, the order of draw differs from that of venipuncture. EDTA is drawn first to ensure adequate volume and accurate hematological results. Other additives are collected next and serum samples are last.³
EDTA


EDTA (ethylenediaminetetraacetic acid) is the anticoagulant used by the hematology laboratory because the cellular components and morphology of the blood cells are preserved. The EDTA salts (disodium, dipotassium, and tripotassium) are required to be used as the anticoagulant in blood collection tubes, as opposed to the free acid, which is not soluble in an aqueous media. EDTA anticoagulates blood by chelating calcium. Calcium is necessary in the coagulation cascade and its removal inhibits and stops a series of events, both intrinsic and extrinsic, which cause clotting. Dipotassium EDTA is now the recommended anticoagulant for hematology by the International Council for Standardization in Haematology (ICSH) and the NCCLS. Dipotassium EDTA is recommended due to its good solubility and stable microhematocrit results.³
In some individuals, EDTA may cause inaccurate platelet results. These anomalies, platelet clumping and platelet satellitism, may be the result of changes in the membrane structure occurring when the calcium ion is removed by the chelating agent, allowing the binding of pre-formed antibodies. In this instance, sodium citrate tubes are sometimes collected to obtain more accurate platelet counts.³
Proper mixing of the whole blood specimen ensures that EDTA is dispersed throughout the sample. Evacuated blood collection tubes with EDTA should be mixed by 8-10 end-to-end inversions immediately following venipuncture collection.⁴ Microcollection tubes with EDTA should be mixed by 10 complete end-to-end inversions immediately following collection.
They should then be inverted an additional 20 times prior to analysis.⁵




Processing (Mixing) of Tubes

Why

[ندعوك للتسجيل في المنتدى أو التعريف بنفسك لمعاينة هذه الصورة]	Most tubes contain an additive or clot activator that needs to be mixed with the blood sample.
	Tubes with anticoagulants such as EDTA need to be mixed to ensure that the specimen does not clot.

How

[ندعوك للتسجيل في المنتدى أو التعريف بنفسك لمعاينة هذه الصورة]

Holding tube upright, gently invert 180° and back.

Repeat movement as prescribed for each tube. [ندعوك للتسجيل في المنتدى أو التعريف بنفسك لمعاينة هذه الصورة] <TABLE cellSpacing=0 cellPadding=0 width="100%" border=0> <TR> <td colSpan=2>When</TD></TR> <TR> <td colSpan=2><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0> <TR> <td vAlign=baseline width=10>[ندعوك للتسجيل في المنتدى أو التعريف بنفسك لمعاينة هذه الصورة]</TD> <td>Immediately after drawing.</TD></TR> <TR> <td vAlign=baseline colSpan=2> </TD></TR></TABLE></TD></TR> <TR> <td colSpan=2>Consequences if not mixed properly</TD></TR> <TR> <td colSpan=2><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0> <TR> <td vAlign=baseline width=10>[ندعوك للتسجيل في المنتدى أو التعريف بنفسك لمعاينة هذه الصورة]</TD> <td>Tubes with anticoagulants will clot.</TD></TR> <TR> <td vAlign=baseline width=10>[ندعوك للتسجيل في المنتدى أو التعريف بنفسك لمعاينة هذه الصورة]</TD> <td>Specimen may need to be redrawn.</TD></TR></TABLE></TD></TR></TABLE>

Specimen Storage and Transport


Evacuated EDTA blood collection tube specimens should be analyzed within six hours of collection.⁶ Prior to analysis, they should be stored at room temperature. Microcollection EDTA tube specimens should also be stored at room temperature and analyzed within four hours.²
Specimen Integrity at Analysis


Care should be taken to ensure specimen integrity at analysis. The following steps will help prevent preanalytical errors from occurring at the time of analysis:

1. Specimens should be checked for proper labeling. Improperly labeled or unlabeled specimens should not be analyzed; corrective action as per the facility's protocol should be taken.
   
2. Tubes should be checked for proper blood fill volumes and appropriate action should be taken based on hospital protocol if tubes are underfilled.
   
3. Whole blood specimens should be mixed adequately by end-to-end inversion just prior to analysis.
   
4. Blood smears for differentials from acceptable specimens should be prepared within two hours of collection.³
   
5. Blood counts from acceptable venipuncture specimens should be performed within six hours of collection.⁶
   
6. Blood counts from acceptable skin puncture specimens should be performed within four hours of collection.²
   

Unused tubes should be stored at 4 - 25° C (39 - 77° F) unless otherwise noted on the package label. Increased temperatures may lead to short draws, incorrect blood-to-additive ratios and erroneous results.
Whole blood specimens are not to be chilled (2 - 8° C) unless there are documented recommendations for doing so. Extended storage (24 hours) is generally not recommended because stability can vary to a considerable degree. Stability depends on reagents, system, instrumentation, etc.⁷
Underfilling the EDTA blood collection tube can lead to erroneously low blood cell counts and hematocrits, morphologic changes to RBCs, and staining alteration. Excess EDTA can shrink red cells. Conversely, overfilling the blood collection tube will not allow the tube to be properly mixed and may lead to platelet clumping and clotting. NCCLS recommends the tube be filled to ± 10% of the stated draw volume.⁸
Physiological Variables


A number of physiological variables can be associated with certain patient factors, such as age.⁹ For example, at birth red blood cell count (RBC) and hemoglobin values are significantly higher than they are in adults, due to the relatively low levels of oxygen in utero. Within the first few months of life they fall substantially and continue to decrease and level out to adult values at about the age of fifteen. In addition, lymphocyte counts change throughout life; they are highest in children and lowest in the elderly.
Smoking, high altitude, patient stance, exercise and pregnancy are some additional patient-related variables over which the lab has no control; yet, they may affect patient test results.¹⁰
Additionally, white blood cell (WBC) and platelet counts can be affected by cryoglobulins. When blood specimens cool from body temperature to room temperature, the cryoglobulins aggregate and may be falsely identified as platelets and/or WBCs by the hematology analyzer. Cold agglutinins have been known to cause spurious reporting of macrocytosis and decreased RBC counts. Some automated instruments may also report falsely high WBC counts and platelet counts.³ The blood smear may show agglutination of RBCs.
Platelet satellitism is a phenomenon that only occurs in EDTA anticoagulated blood. This is due to EDTA-dependent IgG autoantibodies and occurs at room temperature. When platelet satellitism is present, there may be a false elevation of cell counts.¹⁰
In Conclusion


The awareness of the preanalytical variables and their effects on hematology testing is the first step in ensuring that the results are consistently accurate. By minimizing the incidence of preanalytical errors, patient care would be optimized, laboratory costs reduced and the physician-laboratory relationship enhanced. [ندعوك للتسجيل في المنتدى أو التعريف بنفسك لمعاينة هذه الصورة]

References:

1. NCCLS Document H3-A5, Vol.23 No.32. Procedures for the collection of diagnostic blood specimens by venipuncture; approved standard, 5th ed. Wayne, PA: National Committee for Clinical Laboratory Standards; 2003.
   
2. NCCLS Document H4-A4, Vol. 19 No. 16. Procedures and devices for the collection of diagnostic blood specimens by skin puncture; approved standard, 4th ed. Wayne, PA: National Committee for Clinical Laboratory Standards; 1999.
   
3. Guder WG, Narayanan S, Wisser H, Zawta B. Samples: From the patient to the laboratory-The impact of preanalytical variables on the quality of laboratory results.GIT Verlag, Darmstadt, Germany; 1996.
   
4. Product Insert: BD Vacutainer Evacuated Blood Collection System. 8012028.
   
5. Product Insert: BD Microtainer Tubes with K₂EDTA, Ref# 365974
   
6. NCCLS Document H35-T, Vol 12 No.17. Additives to blood collection. devices:EDTA; tentative standard; Wayne, PA: National Committee for Clinical Laboratory Standards; September 1992.
   
7. NCCLS Document H18-A2, Vol.19 No.21. Procedures for the handling and processing of blood specimens; approved guideline - 2nd ed. Wayne, PA: National Committee for Clinical Laboratory Standards; October 1999.
   
8. NCCLS Document H1-A5, Vol. 23 No.33. Evacuated tubes and additives for blood specimen collection; approved standard, 5th ed. Wayne, PA: National Committee for Clinical Laboratory Standards; 2003.
   
9. Bakerman, S. Bakerman's ABC's of Interpretative Laboratory Data. (Revised by: Bakerman, P and Strausbauch, P), Third Edition, Interpretative Laboratory Data, Myrtle Beach, 1994.
   
10. Narayanan S. The preanalytic phase - An important component of laboratory medicine. American Journal of Clinical Pathology 2000;113.