Taking Blood Samples
This means that blood should always be drawn at about the same time of day and after at least eight hours of fasting, since both circadian rhythm and nutritional status can affect the findings.
If strictly comparable values are required, there should also be half an hour of bed rest before the sample is drawn, but this is only practicable in a hospital setting.
In other settings (i.e., outpatient clinics), bringing portable instruments to the relaxed, seated patient works well.
A sample of capillary blood may be taken when there are no further tests thatwould require venous access for a larger sample volume.
Awellperfused fingertip or an earlobe is ideal; in newborns or young infants, the heel is also a good site.
If the circulation is poor, the blood flow can be increased by warming the extremity by immersing it in warm water.
The first droplet of blood is discarded because it may be contaminated, and the ensuing blood is drawn into the pipette.
Care should be taken not to exert pressure on the tissue from which the blood is being drawn, because this too can change the cell composition of the sample.
Obviously, if a venous blood sample is to be taken for the purposes of other tests, or if an intravenous injection is going to be performed, the blood sample for hematological analysis can be taken from the same site.
To do this, the blood is allowed to flow via an intravenous needle into a specially prepared (commercially available) EDTA-treated tube.
The tube is filled to the 1-ml mark and then carefully shaken several times. The very small amount of EDTA in the tube prevents the blood from clotting, but can itself be safely ignored in the quantitative analysis.
Erythrocyte Count
Up to 20 years ago, blood cellswere counted “by hand” in an optical counting chamber.
Thismethod has now been almost completely abandoned in favor of automated counters that determine the number of erythrocytes by measuring the impedance or light dispersion of EDTA blood (1 ml), or heparinized capillary blood.
Due to differences in the hematocrit, the value froma sample taken after (at least 15minutes’) standing or physical activity will be 5–10% higher than the value from a sample taken after 15 minutes’ bed rest.
Hemoglobin and Hematocrit Assay
Hemoglobin is oxidized to cyanmethemoglobin by the addition of cyanide, and the cyanmethemoglobin is then determined spectrophotometrically by the automated counter.
The hematocrit describes the ratio of the volume of erythrocytes to the total blood volume (the SI unit is without dimension, e.g., 0.4).
The EDTA blood is centrifuged in a disposable capillary tube for 10 minutes using a high-speed microhematocrit centrifuge (reference method).
The automated hematology counter determines the mean corpuscular or cell volume (MCV,measured in femtoliters, fl) and the number of erythrocytes. It calculates the hematocrit (HCT) using the following formula:
HCT = MCV (fl)Xnumber of erythrocytes (106/μl).
Calculation of Erythrocyte Parameters
The quality of erythrocytes is characterized by their MCV, their mean cell hemoglobin content (MCH), and the mean cellular hemoglobin concentration (MCHC).
MCV is measured directly using an automated hemoglobin analyzer, or is calculated as follows:
MCV = [Hematocrit (l/l)]/[Number of erythrocytes (106/μl)]
MCH (in picograms per erythrocyte) is calculated using the following formula:
MCH (pg) = [Hemoglobin (g/l)]/[Number of erythrocytes (106/μl)]
MCHC is determined using this formula:
MCHC (g/dl) = [Hemoglobin concentration (g/dl)]/[Hematocrit (l/l)]
Red Cell Distribution Width (RDW)
Modern analyzers also record the red cell distribution width (cell volume distribution).
In normal erythrocyte morphology, this correlates with the Price-Jones curve for the cell diameter distribution.
Discrepancies are used diagnostically and indicate the presence of microspherocytes (smaller cells with lighter central pallor).
Reticulocyte Count
Reticulocytes can be counted using flow cytometry and is based on the light absorbed by stained aggregates of reticulocyte organelles.
The data are recorded as the number of reticulocytes per mill (‰) of the total number of erythrocytes.
Reticulocytes can, of course, be counted in a counting chamber using a microscope.
While this method is not particularly laborious, it is mostly employed in laboratories that often deal with or have a special interest in anemia.
Reticulocytes are young erythrocytes immediately after they have extruded their nuclei: they contain, as a remainder of aggregated cell organelles, a net-like structure (hence the name “reticulocyte”) that is not discernible after the usual staining procedures for leukocytes, but can be observed after vital staining of cells with brilliant cresyl blue or new methylene blue.
The staining solution is mixed in an Eppendorf tube with an equal volume of EDTA blood and incubated for 30 minutes.
After repeated mixing, a blood smear is prepared and allowed to dry. The sample is viewed using a microscope equipped with an oil immersion lens.
The ratio of reticulocytes to erythrocytes is determined and plotted as reticulocytes per 1000 erythrocytes (per mill).
Leukocyte Count
Leukocytes, unlike erythrocytes, are completely colorless in their native state.
Another important physical difference is the stability of leukocytes in 3% acetic acid or saponins; these media hemolyze erythrocytes (though not their nucleated precursors).
Türk’s solution, used in most counting methods, employs glacial acetic acid for hemolysis and crystal violet (gentian violet) to lightly stain the leukocytes.
A 50-μl EDTA blood sample is mixed with 500 μl Türk’s solution in an Eppendorf tube and incubated at room temperature for 10 minutes.
The suspension is again mixed and carefully transferred to the well of a prepared counting chamber using a pipette or capillary tube.
The chamber is allowed to fill from a droplet placed at one edge of the well and placed in a moisture-saturated incubator for 10 minutes.
With the condenser lowered (or using phase contrast microscopy), the leukocytes are then counted in a total of four of the large squares opposite to each other (1mm2 each).
The result is multiplied by 27.5 (dilution: 1 + 10, volume: 0.4mm3) to yield the leukocyte count per microliter.
Parallel (control) counts show variation of up to 15%. In an automated blood cell counter, the erythrocytes are lysed and cells with a volume that exceeds about 30 fl (threshold values vary for different instruments) are counted as leukocytes.
Any remaining erythroblasts, hard-to-lyse erythrocytes such as target cells, giant thrombocytes, or agglutinated thrombocytes are counted along with the leukocytes, and this will lead to an overestimate of the leukocyte count. Modern analyzers can recognize such interference factors and apply interference algorithms to obtain a corrected leukocyte count.
Thrombocyte Count
To count thrombocytes in a counting chamber, blood must be conditioned with 2% Novocain–Cl solution.
Preprepared commercial tubes are widely used (e.g., Thrombo Plus with 2-ml content).
EDTA blood is pipetted into the tubes, carefully mixed and immediately placed in a counting chamber. The chamber is allowed to stand for 10 minutes while the cells settle, after which an area of 1mm2 is counted.
The result corresponds to the number of thrombocytes (the 1 + 100 dilution is ignored).
In an automated blood cell counter, the blood cells are counted after they have been sorted by size.
Small cells between 2 and 20 fl (thresholds vary for different instruments) are counted as thrombocytes.
If giant thrombocytes or agglutinated thrombocytes are present, they are not counted and the result is an underestimate.
On the other hand, small particles, such as fragmentocytes, or microcytes, will lead to an overestimate.
Modern analyzers can recognize such interference factors and apply interference algorithms to obtain a corrected thrombocyte count.
If unexpected results are produced, it is wise to check them by direct reference to the blood smear.
Pseudothrombocytopenia is caused by the aggregation of thrombocytes in the presence of EDTA; it does not occur when heparin or citrate are used as anticoagulants.
Quantitative Normal Values and Range of Cellular Blood Components
Determining normal values for blood components is more difficult and more risky than one might expect.
Obviously, the values are affected by a large number of variables, such as age, gender, activity (metabolic load), circadian rhythm, and nutrition, not to mention the effects of the blood sampling technique, type and storage of the blood, and the counting method. For this reason, where available, a normal range is given, covering 95% of the values found in a clinically normal group of probands from which it follows that one in every 20 healthy people will have values outside the limits of this range.
Thus, there are areas of overlap between normal and pathological data.
Data in these borderline areas must be interpreted within a refined reference range with data from probands who resemble each other and the patient as closely as possible in respect of the variables listed above.
Due to space limitations, only key age data are considered here. In addition, the interpretation must also take account of methodological variation: in cell counts, the coefficient of variation (standard deviation as a percentage of the mean value) is usually around 10!
After this account of the problems and wide variations between different groups. Absolute values and the new SI units are given where they are clinically relevant.
The Blood Smear and Its Interpretation (Differential Blood Count, DBC)
A blood smear uses capillary or venous EDTA-blood, preferably no more than three hours old.
The slides must be grease-free, otherwise cell aggregation and stain precipitation may occur. Unless commercially available grease-free slides are used, the slides should be soaked for several hours in a solution of equal parts of ethanol and ether and then allowed to dry.
A droplet of the blood sample is placed close to the edge of the slide.
A ground cover glass (spreader slide) is placed in front of the droplet onto the slide at an angle of about 30$.
The cover slide is then slowly backed into the blood droplet. Upon contact, the blood droplet spreads along the edge of the slide.
Without pressure, the cover glass is nowlightlymoved over the slide. The faster the cover glass is moved, and the steeper angle at which it is held, the thinner the smear will be.
In a well-prepared smear the blood sample will show a “feathered” edge where the cover glass left the surface of the slide.
The smear must be thoroughly air-dried; for good staining, at least two hours’ drying time is needed.
The quality of the preparation will be increased by 10 minutes’ fixation with methanol, and it will then also keep better.
After drying, name and date are pencilled in on the slide.
Staining is done with amixture of basic stains (methylene blue, azure) and acidic stains (eosin), so as to showcomplementary substances such as nucleic acids and alkaline granulations.
In addition to these leukocyte components, erythrocytes also yield different staining patterns: immature erythrocytes contain larger residual amounts of RNA and therefore stain more heavily with basophilic stains than domature erythrocytes.
Pappenheim’s panoptic stain contains a balanced mixture of basic and acidic stains: the horizontally stored, air-dried smear is covered with May–Grünwald staining solution (eosin–methylene blue) for three minutes, then about an equal amount of phosphate buffer, pH= 7.3, is carefully added and, after a further three minutes, carefully poured off again.
Next, the slide is covered with diluted Giemsa stain (azure–eosin), which is prepared by addition of 1 ml Giemsa stock solution to 1 ml neutral distilled water or phosphate buffer, pH= 6.8–7.4.
After 15 minutes, the Giemsa staining solution is gently rinsed off with buffer solution and the smears are air-dried with the feathered end sloping upwards.
The blood smears are initially viewed with a smaller objective (10! to 20!), which allows the investigator to check the cell density and to find the best counting area in the smear.
Experience shows that the cell projection is best about 1 cmfromthe feathered end of the smear.
At 40!magnification, one may expect to see an average of two to three leukocytes per viewing field if the leukocyte count is normal.
It is sometimes useful to be able to use this rough estimate to crosscheck improbable quantitative values.
The detailed analysis of the white blood cells is done using an oil immersion lens and 100!magnification.
For this, it is best to scan the section fromabout 1 cmto about 3 cmfromthe end of the smear,moving the slide to and fro in a meandering movement across its short diameter. Before (and while) the differential leukocyte count is carried out, erythrocyte morphology and thrombocyte density should be assessed.
The results of the differential leukocyte count may be recorded usingmanual counters or mark-up systems.
The more cells are counted, the more representative the results, so when pathological deviations are found, it is advisable to count 200 cells.
To speed up the staining process, which can seem long and laborious when a rapid diagnosis is required, several quick-staining sets are available commercially, although most of them do not permit comparable fine analysis.
If the standard staining solutions mentioned above are to hand, a quick stain for orientation purposes can be done by incubating the smear with May–Grünwald reagent for just one minute and shortening the Giemsa incubation time to one to two minutes with concentrated “solution.”
Malaria plasmodia are best determined using a thick smear in addition to the normal blood smear. On a slide, a drop of blood is spread over an area of about 2.5 cm across.
The thick smear is placed in an incubator and allowed to dry for at least 30minutes.
Drying samples as thick smears and then treating themwith dilute Giemsa stain (as described above) achieves extensive hemolysis of the erythrocytes and thus an increase in the released plasmodia.
Significance of the Automated Blood Count
The qualitative and quantitative blood count techniques described here may seem somewhat archaic given the nowalmost ubiquitous automated cell counters; they are merely intended to show the possibilities always ready to be called on in terms of individual analyses carried out by small, dedicated laboratories.
After lysis of the erythrocytes, hematology analyzers determine the number of remaining nucleated cells using a wide range of technologies.
All counters use cell properties such as size, interactionwith scattered light at different angles, electrical conductivity, nucleus-to-cytoplasm ratio, and the peroxidase reaction, to group individual cell impulses into clusters.
These clusters are then quantified and assigned to leukocyte populations.
If only normal blood cells are present, the assignment of the clusters to the various leukocyte populationsworkswell, and the precision of the automated count exceeds that of the manual count of 100 cells in a smear by a factor of 10.
If large number of pathological cells are present, such as blasts or lymphadenoma cells, samples are reliably recognized as “pathological,” and a smear can then be prepared and further analyzed under the microscope.
The difficulty arises when small populations of pathological cells are present (e.g., 2% blasts present after chemotherapy), or when pathological cells are present that closely resemble normal leukocytes (e.g., small centrocytes in satellite cell lymphoma).
These pathological conditions are not always picked up by automated analyzers (false negative result), no smear is prepared and studied under the microscope, and the results produced by themachine do not !include! the presence of these pathological populations.
For this reason, blood samples accompanied by appropriate clinical queries (e.g., “lymphadenoma?” “blasts?” “unexplained anemia?”) should always be differentiated and evaluated using a microscope.
Bone Marrow Biopsy
Occasionally, a disease of the blood cell system cannot be diagnosed and classified on the basis of the blood count alone and a bone marrowbiopsy is indicated.
In such cases it is more important to perform this biopsy competently and produce good smears for evaluation than to be able to interpret the bone marrow cytology yourself.
Although the bonemarrow cytology findings from the aspirate are sufficient or even preferable for most hematological questions, it is regarded as good practice to obtain a sample for bone marrow histology at the same time, since with improved instruments the procedure has become less stressful, and complementary cytological and histological data are then available from the start.
After deep local anesthesia of the dorsal spine and a small skin incision, a histology cylinder at least 1.5cm long is obtained using a sharp hollowneedle (Yamshidi).
A Klima and Rossegger cytology needle is then placed through the same subcutaneous channel but at a slightly different site from the earlier insertion point on the spine and gently pushed through the compacta.
Themandrel is pulled out and a 5- to 10-ml syringe body with 0.5ml citrate or EDTA (heparin is used only for cytogenetics) is attached to the needle.
The patient should be warned that there will be a painful drawing sensation during aspiration, which cannot be avoided.
The barrel is then slowly pulled, and if the procedure is successful, blood fromthe bone marrowfills the syringe.
The syringe body is separated from the needle and the mandrel reintroduced.
The bone marrow aspirate is transferred from the syringe to a Petri dish.
When the dish is gently shaken, small, pinhead-sized bone marrowspiculeswill be seen lying on the bottom.
A smear, similar to a blood smear, can be prepared on a slide directly fromthe remaining contents of the syringe.
If the first aspirate has obtained material, the needle is removed and a light compression bandage is applied.
If the aspirate for cytology contains no bone marrowfragments (“punctio sicca,” dry tap), an attempt may be made to obtain a cytology smear from the (as yet unfixed) histology cylinder by rolling it carefully on the slide, but this seldom produces optimal results.
The preparation of the precious bone marrow material demands special care.
One or two bone marrow spicules are pushed to the outer edge of the Petri dish, using the mandrel from the sternal needle, a needle, or a wooden rod with a beveled tip, and transferred to a fat-free microscopy slide, on which they are gently pushed to and fro by the needle along the length of the slide in a meandering line.
This helps the analyzing technician to make a differentiatial count.
It should be noted that too much blood in the bone marrowsamplewill impede the semiquantitative analysis.
In addition to this type of smear, squash preparations should also be prepared from the bone marrow material for selective staining.
To do this, a few small pieces of bone marrow are placed on a slide and covered by a second slide.
The two slides are lightly pressed and slid against each other, then separated.
The smears are allowed to air-dry and some are incubated with panoptic Pappenheim staining solution.
Smears being sent to a diagnostic laboratory (wrapped individually and shipped as fragile goods) are better left unstained.
Fresh smears of peripheral blood should accompany the shipment of each set of samples.
Lymph Node Biopsy and Tumor Biopsy
These procedures, less invasive than bone marrow biopsy, are a simple and often diagnostically sufficientmethod for lymph node enlargement or other intumescences. The unanesthetized, disinfected skin is sterilized and pulled taut over the node.
A needle on a syringe with good suction is pushed through the skin into the lymph node tissue. Tissue is aspirated from several locations, changing the angle of the needle slightly after each collection, and suction maintained while the needle is withdrawn into the subcutis.
Aspiration ceases and the syringe is removed without suction.
The biopsy harvest, which is in the needle, is extruded onto a microscopy slide and smeared out without force or pressure using a cover glass (spreader slide).
Staining is done as described previously for blood smears.
