LABORATORY HEMATOLOGY

DR BISWA RANJAN PATRARESIDENT OF MEDICINEP.G.I.M.E.R & DR R.M.L HOSPITAL, NEW DELHI

ANTICOAGULANTS

MC used- tripotassium or trisodium salts of ethylenediaminetetraacetic acid (EDTA), trisodium citrate, and heparin

EDTA & disodium citrate remove calcium, heparin form complex with antithrombin and prevent thrombin formation

EDTA- preferred anticoagulant for blood counts- complete anticoagulation, minimal morphologic and physical effects on cells

Heparin- red cell testing, osmotic fragility testing, functional / immunologic analysis of leucocytes, doesn’t completely inhibit WBC/ platelet clumping, bluish background after staining

Trisodium citrate- preffered anticoagulant for platlet & coagulation studies

The concentration of the anticoagulant used may affect cell concentration measures and may distort morphology

As blood contains large numbers of cells, sample dilution is usually required for accurate analysis

Type & extent of diluent depends on cell type to be inumerated Red cell counts require dilution with an isotonic medium, whereas

in white cell or platelet counts, a diluent that lyses the more numerous red cells

red cell counts need more dilution than is required for the less abundant white blood cells

Hemocytometer v/s Automated Hematology Analyser

Manual counts are done using a microscope after appropriate dilution of the sample in a hemocytometer

Most automated hematology analyser perform a variety of hematological measurements

Automated methods for white cell counts measures electrical impedance, differential light scatter, optical flow cytometry, or antigen staining either alone or in combination

Newer analyzers detect reticulocytes using a fluorescent RNA dye and nucleated red blood cell numbers based on optical properties

Current analyzers do auto sampling directly from tubes and use 35 to 150 μl for a full CBC analysis

Workload capacities range from 70 to 106 samples analyzed per hour.

When reticulocytes are ordered as a part of the differential, the capacity falls to between 40 and 60 samples per hour (allowing for the staining and detection of the RNA dye fluorescence)

COMPARISION

RED CELL INDICES

Red blood cells are defined by three quantitative values: The volume of packed red cells or

hematocrit (Hct), the amount of hemoglobin, and the red cell concentration per unit volume

three qualitative characteristics : mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC)

Amongst these parameters some are directly measured and others are calculated from the measured ones

In most of the current analyzers, RBC concentration, Hb concentration, and mean corpuscular volume are directly measured.

These measured values are used to calculate the Hct, mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC):

Hct = MCV × RBC concentration MCH = Hb concentration/RBC concentration MCHC = Hb concentration/Hct = MCH / MCV

Volume of packed cell (Haematocrit)

The Hct is the proportion of the volume of a blood sample that is occupied by red cells

The spun Hct measures the red cell concentration, not red cell mass

Therefore, patients in shock or with volume depletion may have normal or high Hct measurements due to hemoconcentration despite a decreased red cell mass

Another inherent error in manual Hct determinations arises from trapping of plasma in the red cell column

abnormal red cells (e.g., sickle cells, microcytic cells, macrocytic cells, or spherocytes) often trap higher volumes of plasma due to increased cellular rigidity, possibly accounting for up to 6% of the red cell volume

Automated analyzers do not depend on centrifugation techniques to determine Hct, but instead calculate Hct by direct measurements of red cell number and red cell volume (Hct = red cell number x mean red cell volume)

Errors of automated Hct calculation are more common in patients with polycythemia or abnormal plasma osmotic pressures

Haemoglobin Concentration

Hemoglobin is found in the blood in a variety of forms, including oxyhemoglobin, carboxyhemoglobin, methemoglobin

These may be converted to a single stable compound, cyanmethemoglobin

The absorbance of the cyanhemoglobin is measured in a spectrophotometer at 540 nm to determine Hb both in manual determinations and automated hematology analyzers

The main errors in measurement arise from dilution errors or increased sample turbidity due to improperly lysed red cells, leukocytosis, or increased levels of lipid or protein in the plasma

Red cell count

Manual methods for counting red cells have proven to be very inaccurate, and automated counters provide a much more accurate reflection of red cell numbers

Mean Corpuscular Volume

Measured in femtolitres (fl), normal value- 90 ± 8 fl average volume of the red blood cell usually measured directly with automated instruments but may

also be calculated from the erythrocyte count and the Hct : MCV = Hct (L/L) ÷ 1,000/red cell count Agglutination of cells, as in cold agglutinin disease or

paraproteinemia, may result in a falsely elevated MCV severe hyperglycemia (glucose >600 mg/dl) may cause osmotic

swelling of the red cells, leading to a falsely elevated MCV

Mean Corpuscular Hemoglobin

Expressed in picograms (pg), normal value – 30 ± 3 pg measure of the average hemoglobin content per red cell /

haemoglobin mass It may be calculated manually or by automated methods using

the following formula: MCH = hemoglobin (g/L)/red cell count (1012/L) MCH measurements may be falsely elevated by hyperlipidemia ,

as increased plasma turbidity will erroneously elevate hemoglobin measurement. Centrifugation of the blood sample eliminate the turbidity

Leukocytosis may also spuriously elevate MCV values

Mean Corpuscular Hemoglobin Concentration

The average concentration of hemoglobin in a given red cell volume

MCHC = hemoglobin (g/dl)/Hct (L/L) The accuracy of the MCHC determination is affected by factors

that affect measurement of either Hct (plasma trapping or presence of abnormal red cells) or hemoglobin (hyperlipidemia, leukocytosis)

Normal value is 33 ± 2%

MCV, MCH, and MCHC reflect average values and may not adequately describe blood samples when mixed populations of red cells are present

It is important to examine the blood smear as well as red cell histograms to detect such dimorphic populations

The MCV is an extremely useful value in classification of anemias, but the MCH and MCHC often do not add significant, clinically relevant information.

However, the MCH and MCHC play an important role in laboratory quality control because these values will remain stable for a given specimen over time

Red cell mass/ Red cell volume

The Hb, red cell count, PCV/Hct do not invariably reflect the total RCV

Whereas in most cases there is adequate correlation, there will be discrepancy if the plasma volume is reduced/ increased disproportionately

Fluctuation in plasma volume may result in hemodilution giving rise to pseudoanemia haemoconcentration giving rise to pseudopolycythaemia In contrast to the fluctuation in plasma volume, RCV does not

fluctuate to any extent if erythropoiesis is in steady state

Measurement of RCV should be considered whenever Hct is persistently higher than normal or in obscure anemias when there is possibility of increase in plasma volume

It is determined by radio isotope method The labelled RBC are diluted in the whole blood and from their

dilution TBW can be calculated, the RCV can be deducted from PCV

Normal values Men- 30 ± 5 ml/kg Females- 25 ± 5 ml/kg Demonstration of absolute increase in RCV is necessary for

diagnosing polycythemia & to assess its severity

Red Cell Distribution Width (RDW)

It is a red cell measurement that quantitates cellular volume heterogeneity reflecting the range of red cell sizes

It is equivalent of microscopic assessment of the degree of anisocytosis

Normal reference range is 42.5 ± 3.5 fl as SD 12.8 ± 1.2% as CV

RDW is useful in early classification of anemia as it becomes abnormal earlier in nutritional deficiency anemias than other red cell parameters

It distinguish between uncomplicated iron deficiency anemia (high RDW, normal to low MCV) and uncomplicated heterozygous thalassemia (normal RDW, low MCV)

It also distinguish between megaloblastic anemia (RDW often increased) and other causes of macrocytosis (RDW often normal)

RDW is also useful in identifying red cell fragmentation, agglutination, or dimorphic cell populations

Haemoglobin Distribution Width (HDW)

The percentage of hypochromic red cells depends on the concentration of Hb in individual cells rather than being a mean such as MCH, MCHC

It is more sensitive marker of availability of iron for erythropoiesis because small changes in the number of red cells with inadequate Hb can be measured before there is any change in MCHC

In healthy population the percentage of hypochromic red cells does not exceed 2.5% & value greater than this indicate iron deficient erythropoiesis

The degree of variation in red cell haemoglobinization is quantified as the HDW

The normal 95% range is 1.82-2.64

Reticulocyte Counts

Reticulocytes are immature, nonnucleated red blood cells that still retain RNA & provides useful information about the bone marrow’s capacity to synthesize RBC

Reticulocyte counts performed manually using supravital staining with methylene blue that will stain precipitated RNA as a dark blue meshwork or granules (at least two per cell)

Most automated hematology analyzers have automated reticulocyte counting detected by a fluorescent dye that binds to RNA

Normal values for reticulocytes in adults are 0.5 to 1.5%

In order to use reticulocyte count to estimate marrow response two corrections are necessary

With anemia the percentage of reticulocytes may be increased while the absolute numbers is unchanged

2nd correction is for premature release of reticulocytes into circulation

White Blood Cell Counts

Leukocytes are counted after dilution of blood in a diluent that lyses the red blood cells (usually acid or detergent)

Automated leukocyte counts may be falsely elevated in the presence of

cryoglobulins or cryofibrinogen aggregated platelets nucleated red blood cells incomplete lysis of red cells These conditions requires manual counting

Leukocyte Differentials

Done both manually and automated analyser It is important to scan the entire blood smear at low power to

ensure that all atypical cells and cellular distribution patterns are recognized

In wedge-pushed smears, larger cells (blasts, monocytes) tend to aggregate at the edges of the blood smear

Automated analysis is incapable of accurately identifying and classifying all types of cells and is particularly insensitive to abnormal or immature cells

Abnormal cell populations will generate a flag, indicating a need for morphologic review of the peripheral smear

Platelet Analysis

Platelets are anucleate cytoplasmic fragments that are 2 to 4 microns in diameter

They may be counted by either manual or automated methods Falsely low platelet counts may be caused by the presence of

platelet clumps or platelet agglutinins or adsorption of platelets to leukocytes

Fragments of red or white blood cells may falsely elevate the automated platelet count

Mean platelet volume (MPV)

Determined by automated hematology analyzers MPV has an inverse relationship with platelet number Larger platelet volumes (secondary to new platelet production)

seen in thrombocytopenic patients in whom platelets are decreased due to peripheral destruction (as in ITP)

MPV is also increased in myeloproliferative disorders Decreased MPV has been associated with megakaryocytic

hypoplasia and cytotoxic drug therapy

Reticulated platelets

Newly released platelets that retain residual RNA, analogous to red cell reticulocytes

Reticulated platelet counts give an estimate of thrombopoiesis and may be useful in distinguishing platelet destruction syndromes from hypoplastic platelet production

Normal values vary between 3 and 20%, and 2.5- to 4.5-fold increases in reticulated platelet counts are seen in the clinical setting of ITP

Increased reticulated platelets may herald the return of platelet production after chemotherapy

Histograms & Scatter Plot

Sources of Error with Automated Hematology Analyzers

Morphologic Analysis of Blood Cells

Blood smears are often prepared from samples of anticoagulated blood

However, artifacts in cell appearance and staining may be induced by anticoagulant

Optimal morphology and staining are obtained from nonanticoagulated blood, most often from a fingerstick procedure

Among cell morphology it reveals- cell size, haemoglobin content, anisocytosis, poikilocytosis, polychromasia

Pathological cells

Bone Marrow Examination

Bone marrow examination usually involves two separate, but interrelated specimens

Cytologic preparation of bone marrow cells obtained by aspiration of the marrow and a smear of the cells, allowing excellent visualization of cell morphology and enumeration of the marrow cellular elements

Needle biopsy of the bone and associated marrow, which allows optimal evaluation of bone marrow cellularity, fibrosis, infections, or infiltrative diseases

Indications- It includes 1. further workup of hematologic abnormalities observed in the

peripheral blood smear2. evaluation of primary bone marrow tumors3. staging for bone marrow involvement by metastatic tumors4. assessment of infectious disease processes, including fever of

unknown origin5. evaluation of metabolic storage diseases

Sites – Younger children may have marrow examinations from the

anterior medial tibial area adult marrow is best sampled from the sternum at the second

intercostal space or from either the anterior or posterior iliac crest area

Sternal marrows do not allow a biopsy to be performed Posterior iliac crest is the most common site for bone marrow

sampling Anterior iliac crest may be used if previous radiation, surgery, or

patient discomfort do not allow a posterior approach

Examination of aspirated bone marrow

Quantitive cell counts- assess the degree of marrow cellurarity grossly <25% hypocellular >75-80% hypercellular marrow Differential cell count- important in conditions like acute &

chronic myelogenous leukemia & myelodysplastic syndrome Cellular ratios- myeloid: erythroid ratio varies from 2:1 to 4:1