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By American Society of Hemotology

LABORATORY TESTS IN PORPHYRIAS

Published in Clinical Pathology
Thursday, 03 August 2017 18:15
Porphyrias (from Greek porphura meaning purple pigment; the name is probably derived from purple discoloration of some body fluids during the attack) are a heterogeneous group of rare disorders resulting from disturbance in the heme biosynthetic pathway leading to the abnormal accumulations of red and purple pigments called as porphyrins in the body. Heme, a component of hemoglobin, is synthesized through various steps as shown in Figure 817.1. Each of the steps is catalyzed by a separate enzyme; if any of these steps fails (due to hereditary or acquired cause), precursors of heme (porphyrin intermediates) accumulate in blood, get deposited in skin and other organs, and excreted in urine and feces. Depending on the site of defect, different types of porphyrias are described with varying clinical features, severity, and the nature of accumulated porphyrin.
 
Porphyria has been offered as a possible explanation for the medieval tales of vampires and werewolves; this is because of the number of similarities between the behavior of persons suffering from porphyria and the folklore (avoiding sunlight, mutilation of skin on exposure to sunlight, red teeth, psychiatric disturbance, and drinking of blood to obtain heme).
 
Porphyrias are often missed or wrongly diagnosed as many of them are not associated with definite physical findings, screening tests may yield false-negative results, diagnostic criteria are poorly defined and mild disorders produce an enzyme assay result within ‘normal’ range.
 
Heme is mainly required in bone marrow (for hemoglobin synthesis) and in liver (for cytochromes). Therefore, porphyrias are divided into erythropoietic and hepatic types, depending on the site of expression of disease. Hepatic porphyrias mainly affect the nervous system, while erythropoietic porphyrias primarily affect the skin. Porphyrias are also classified into acute and nonacute (or cutaneous) types depending on clinical presentation (Table 817.1).
 
Table 817.1 Various classification schemes for porphyrias
Classification based on predominant clinical manifestations
Classification based on site of expression of disease
Classification based on mode of clinical presentation
Neuropsychiatric
Hepatic
Acute
1. Acute intermittent porphyria
1. ALA-dehydratase porphyria
1. ALA-dehydratase porphyria (Plumboporphyria)
2. ALA-dehydratase porphyria (Plumboporphyria)
2. Acute intermittent porphyria
2. Acute intermittent porphyria
Cutaneous (Photosensitivity)
3. Hereditary coproporphyria
3. Hereditary coproporphyria
1. Congenital erythropoietic porphyria
4. Variegate porphyria
4. Variegate porphyria
2. Porphyria cutanea tarda
Erythropoietic porphyria
Non-acute (cutaneous)
3. Erythropoietic protoporphyria
1. Congenital erythropoietic porphyria
1. Porphyria cutanea tarda
Mixed (Neuropsychiatric and cutaneous)
2. Erythropoietic protoporphyria
2. Congenital erythropoietic porphyria
1. Hereditary coproporphyria
Hepatic/Erythropoietic
3. Erythropoietic protoporphyria
2. Variegate porphyria
1. Porphyria cutanea tarda
 
 
Inheritance of porphyrias may be autosomal dominant or recessive. Most acute porphyrias are inherited in an autosomal dominant manner (i.e. inheritance of one abnormal copy of gene). Therefore, the activity of the deficient enzyme is 50%. When the level of heme falls in the liver due to some cause, activity of ALA synthase is stimulated leading to increase in the levels of heme precursors up to the point of enzyme defect. Increased levels of heme precursors cause symptoms of acute porphyria. When the heme level returns back to normal, symptoms subside.
 
Accumulation of porphyrin precursors can occur in lead poisoning due to inhibition of enzyme aminolevulinic acid dehydratase in heme biosynthetic pathway. This can mimick acute intermittent porphyria.
 
CLINICAL FEATURES
 
Clinical features of porphyrias are variable and depend on type. Acute porphyrias present with symptoms like acute and severe abdominal pain/vomiting/constipation, chest pain, emotional and mental disorders, seizures, hypertension, tachycardia, sensory loss, and muscle weakness. Cutaneous porphyrias present with photosensitivity (redness and blistering of skin on exposure to sunlight), itching, necrosis of skin and gums, and increased hair growth over the temples (Table 817.2).
 
Table 817.2 Clinical characteristics of porphyrias
Porphyria Deficient enzyme Clinical features Inheritance Initial test
1. Acute intermittent porphyria (AIP)* PBG deaminase Acute neurovisceral attacks; triggering factors+ (e.g. drugs, diet restriction) Autosomal dominant Urinary PBG; urine becomes brown, red, or black on standing
2. Variegate porphyria Protoporphyrinogen oxidase Acute neurovisceral attacks + skin fragility, bullae Autosomal dominant Urinary PBG
3. Hereditary coproporphyria Coproporphyrinogen oxidase Acute neurovisceral attacks + skin fragility, bullae Autosomal dominant Urinary PBG
4. Congenital erythropoietic porphyria Uroporphyrinogen cosynthase Onset in infancy; skin fragility, bullae; extreme photosensitivity with mutilation; red teeth and urine (pink red urinestaining of diapers) Autosomal recessive Urinary/fecal total porphyrins; ultraviolet fluorescence of urine, feces, and bones
5. Porphyria cutanea tarda* Uroporphyrinogen decarboxylase Skin fragility, bullae Autosomal dominant (some cases) Urinary/fecal total porphyrins
6. Erythropoietic protoporphyria* Ferrochelatase Acute photosensitivity Autosomal dominant Free erythrocyte protoporphyrin
Disorders marked with * are the three most common porphyrias. PBG: Porphobilinogen
  
Symptoms can be triggered by drugs (barbiturates, oral contraceptives, diazepam, phenytoin, carbamazepine, methyldopa, sulfonamides, chloramphenicol, and antihistamines), emotional or physical stress, infection, dieting, fasting, substance abuse, premenstrual period, smoking, and alcohol. Autosomal dominant porphyrias include acute intermittent porphyria, variegate porphyria, porphyria cutanea tarda, erythropoietic protoporphyria (most cases), and hereditary coproporphyria. Autosomal recessive porphyrias include: congenital erythropoietic porphyria, erythropoietic protoporphyria (few cases), and ALAdehydratase porphyria (plumboporphyria).
 
LABORATORY DIAGNOSIS
 
Porphyria can be diagnosed through tests done on blood, urine, and feces during symptomatic period. Timely and accurate diagnosis is required for effective management of porphyrias. Due to the variability and a broad range of clinical features, porphyrias are included under differential diagnosis of many conditions. All routine hospital laboratories usually have facilities for initial investigations in suspected cases of porphyrias; laboratory tests for identification of specific type of porphyrias are available in specialized laboratories.
 
INITIAL STUDIES
 
In suspected acute porphyrias (acute neurovisceral attack), a fresh randomly collected urine sample (10-20 ml) should be submitted for detection of excessive urinary excretion of porphobilinogen (PBG) (see Figure 817.2). In AIP, urine becomes red or brown on standing (see Figure 817.3). In suspected cases of cutaneous porphyrias (acute photosensitivity without skin fragility), free erythrocyte protporphyrin or FEP in EDTA blood (for diagnosis of erythrocytic protoporphyria) and for all other cutaneous porphyrias (skin fragility and bullae), examination of fresh, random urine (10-20 ml) and either feces (5-10 g) or plasma for excess porphyrins are necessary (see Figure 817.4 and Table 817.2).
 
Figure 817.2 Evaluation of acute neurovisceral porphyria
 Figure 817.2 Evaluation of acute neurovisceral porphyria
 
Figure 817.3 Red coloration of urine on standing in acute intermittent porphyria
Figure 817.3 Red coloration of urine on standing in acute intermittent porphyria
 
Figure 817.4 Evaluation of cutaneous porphyrias
Figure 817.4 Evaluation of cutaneous porphyrias
 
Apart from diagnosis, the detection of excretion of a particular heme intermediate in urine or feces can help in detecting site of defect in porphyria. Heme precursors up to coproporphyrinogen III are water-soluble and thus can be detected in urine. Protoporphyrinogen and Protoporphyrin are insoluble in water and are excreted in bile and can be detected in feces. All samples should be protected from light.
 
Samples required are
 
  1. 10-20 ml of fresh random urine sample without any preservative;
  2. 5-10 g wet weight of fecal sample, and
  3. blood anticoagulated with EDTA.
 
Test for Porphobilinogen in Urine
 
Ehrlich’s aldehyde test is done for detection of PBG. Ehrlich’s reagent (p-dimethylaminobenzaldehyde) reacts with PBG in urine to produce a red color. The red product has an absorption spectrum with a peak at 553 nm and a shoulder at 540 nm. Since both urobilinogen and porphobilinogen produce similar reaction, further testing is required to distinguish between the two. Urobilinogen can be removed by solvent extraction. (See Watson-Schwartz test). Levels of PBG may be normal or near normal in between attacks. Therefore, samples should be tested during an attack to avoid false-negative results.
 
Test for Total Porphyrins in Urine
 
Total porphyrins can be detected in acidified urine sample by spectrophotometry (Porphyrins have an intense absorbance peak around 400 nm). Semiquantitative estimation of porphyrins is possible.
 
Test for Total Porphyrins in Feces
 
Total porphyrins in feces can be determined in acidic extract of fecal sample by spectrophotometry; it is necessary to first remove dietary chlorophyll (that also absorbs light around 400 nm) by diethyl ether extraction.
 
Tests for Porphyrins in Erythrocytes and Plasma
 
Visual examination for porphyrin fluorescence, and solvent fractionation and spectrophotometry have now been replaced by fluorometric methods.
 
Further Testing
 
If the initial testing for porphyria is positive, then concentrations of porphyrins should be estimated in urine, feces, and blood to arrive at specific diagnosis (Tables 817.3 and 817.4).
 
Table 817.3 Diagnostic patterns of concentrations of heme precursors in acute porphyrias
Porphyria Urine Feces
Acute intermittent porphyria PBG, Copro III
Variegate porphyria PBG, Copro III Proto IX
Hereditary coproporphyria PBG, Copro III Copro III
PBG: Porphobilinogen; Copro III: Coproporphyrinogen III; Proto IX: Protoporphyrin IX
 
Table 817.4 Diagnostic patterns of concentrations of heme precursors in cutaneous porphyrias
Porphyria Urine Feces Erythrocytes
Congenital erythropoietic porphyria Uro I, Copro I Copro I
Porphyria cutanea tarda Uroporphyrin Isocopro
Erythropoietic protoporphyria Protoporphyrin
Uro I: Uroporphyrinogen I; Copro I: Coproporphyrinogen I; Isocopro: Isocoproporphyrinogen
 
In latent porphyrias and in patients during remission, porphyrin levels may be normal; in such cases, enzymatic and DNA testing is necessary for diagnosis.
 
If porphyria is diagnosed, then it is necessary to investigate close family members for the disorder. Positive family members should be counseled regarding triggering factors.

PLATELET GLYCOPROTEIN ANALYSIS

Published in Hemotology
Thursday, 03 August 2017 16:55
This is done by flow cytometric analysis for detection of lack of GpIb/IX in Bernanrd Soulier syndrome (deficiency of CD42), and lack of GpIIb/IIIa in Glanzmann’s thrombasthenia (deficiency of CD41, CD61).
 
What is the best protocol for platelet glycoprotein (GPIIb/IIIa) analysis using flow cytometry?
 
Fresh platelets should always be used. Storing platelets dramatically changes the level of transmembrane proteins. The best way is to follow one of standardized protocols defined in: Immunophenotypic analysis of platelets. Krueger LA, Barnard MR, Frelinger AL 3rd, Furman MI, Michelson AD.Curr Protoc Cytom. 2002 Feb;Chapter 6:Unit 6.10

TEST FOR D-DIMER

Published in Hemotology
Thursday, 03 August 2017 16:33
D-dimer is derived from the breakdown of fibrin by plasmin and D-dimer test is used to evaluate fibrin degradation. Blood sample can be either serum or plasma. Latex or polystyrene microparticles coated with monoclonal antibody to D-dimer are mixed with patient’s sample and observed for microparticle agglutination. As the particle is small, turbidometric endpoint can be determined in automated instruments. D-dimer and FDPs are raised in disseminated intravascular coagulation, intravascular thrombosis (myocardial infarction, stroke, venous thrombosis, pulmonary embolism), and during postoperative period or following trauma. D-dimer test is commonly used for exclusion of thrombosis and thrombotic tendencies.
 
Further Reading:
 

TEST FOR FIBRINOGEN/FIBRIN DEGRADATION PRODUCTS (FDPs)

Published in Hemotology
Thursday, 03 August 2017 13:02
FDPs are fragments produced by proteolytic digestion of fibrinogen or fibrin by plasmin. For determination of FDPs, blood is collected in a tube containing thrombin (to remove all fibrinogen by converting it into a clot) and soybean trypsin inhibitor (to inhibit plasmin and thus prevent in vitro breakdown of fibrin). A suspension of latex particles linked to antifibrinogen antibodies (or fragments D and E) is mixed with dilutions of patient’s serum on a glass slide. If FDPs are present, agglutination of latex particles occurs (see Figure 814.1). The highest dilution of serum at which agglutination is detected is used to determine concentration of FDPs. Increased levels of FDPs occur in fibrinogenolysis or fibrinolysis. This occurs in disseminated intravascular coagulation, deep venous thrombosis, severe pneumonia, and recent myocardial infarction.

PRINCIPLES OF WORKING OF AUTOMATED HEMOTOLOGY ANALYZER

Published in Hemotology
Monday, 31 July 2017 17:06
Automated hematology analyzers work on different principles:
 
  • Electrical impedance
  • Light scatter
  • Fluorescence
  • Light absorption
  • Electrical conductivity.
 
Most analyzers are based on a combination of different principles.
 
(1) Electrical impedance: This is the classic and timetested technology for counting cellular elements of blood. As this method of cell counting was first developed by Coulter Electronics, it is also called as Coulter principle (see Figure 811.1). Two electrodes placed in isotonic solutions are separated by a glass tube having a small aperture. A vacuum is applied and as a cell passes through the aperture, flow of current is impeded and a voltage pulse is generated.
 
Figure 811.1 Coulter principle of electrical impedance
Figure 811.1 Coulter principle of electrical impedance
 
The requisite condition for cell counting by this method is high dilution of sample so that minimal numbers of cells pass through the aperture at one point of time. There are two electrodes on either side of the aperture; as the solution in which the cells are suspended is an electrolyte solution, an electric current is generated between the two electrodes. When a cell passes through this narrow aperture across which a current is flowing, change in electrical resistance (i.e. momentary interruption of electrical current between the two electrodes) occurs. A small pulse is generated due to a temporary increase in impedance. This pulse is amplified, measured, and counted. The height of the pulse is proportional to cell volume. The width of the pulse corresponds with the time required for the cell to traverse the aperture. Cells that do not pass through the center of the aperture generate a distorted pulse that is not representative of the cell volume. Some analyzers use hydrodynamic focusing to force the cells through the central path so that all cells take the same path for volume measurement.
 
An anticoagulated whole blood sample is aspirated into the system, divided into two portions, and mixed with a diluent. One dilution is passed to the red cell aperture bath (for red cell and platelet counting), and the other is delivered to the WBC aperture bath (where a reagent is added for lysis of red cells and release hemoglobin; this portion is used for leukocyte counting followed by estimation of hemoglobin). Particles between 2-20 fl are counted as platelets, while those between 36-360 fl are counted as red cells. Hemoglobin is estimated by light transmission at 535 nm.
 
(2) Light scatter: Each cell flows in a single line through a flow cell. A laser device is focused on the flow cell; as the laser light beam strikes a cell it is scattered in various directions. One detector captures the forward scatter light (forward angle light scatter or FALS) that is proportional to cell size and a second detector captures side scatter (SS) light (90°) that corresponds to the nuclear complexity and granularity of cytoplasm. This simultaneous measurement of light scattered in two directions is used for distinguishing between granulocytes, lymphocytes, and monocytes.
 
(3) Fluorescence: Cellular fluorescence is used to measure RNA (reticulocytes), DNA (nucleated red cells), and cell surface antigens.
 
(4) Light absorption: Concentration of hemoglobin is measured by absorption spectrophotometry, after conversion of hemoglobin to cyanmethemoglobin or some other compound. In some analyzers, peroxidase cytochemistry is used to classify leukocytes; the peroxidase activity is determined by absorbance.
 
(5) Electrical conductivity: Some analyzers use conductivity of high frequency current to determine physical and chemical composition of leucocytes for their classification.
 
Further Reading:
 

ADVANTAGES AND DISADVANTAGES OF AUTOMATED HEMOTOLOGY ANALYZER

Published in Hemotology
Sunday, 30 July 2017 18:20
AUTOMATED HEMOTOLOGY ANALYZER
 
Automation is a process of replacement of tasks hitherto performed by humans by computerized methods.
 
Until recently, hematological tests were performed only by manual methods. These methods, though still performed in many peripheral laboratories, are laborintensive, and involve use of hemocytometers (counting chambers), centrifuges, Wintrobe tubes, photometers, and stained blood smears. Hematology cell analyzers can generate the blood test results rapidly and also perform additional tests not possible by manual technology.
 
Both manual and automated laboratory techniques have advantages and disadvantages, and it is unlikely that one will completely replace the other.
 
Advantages of Automated Hematology Analyzer
 
  • Speed with efficient handling of a large number of samples.
  • Accuracy and precision in quantitative blood tests.
  • Ability to perform multiple tests on a single platform.
  • Significant reduction of labor requirements.
  • Invaluable for accurate determination of red cell indices.
 
Disadvantages of Automated Hematology Analyzer
 
  • Flags: Flagging of a laboratory test result demands labour-intensive manual examination of a blood smear.
  • Comments on red cell morphology cannot be generated. Abnormal red cell shapes (such as fragmented cells) cannot be recognized.
  • Erroneously increased or decreased results due to interfering factors.
  • Expensive with high running costs.
 
Automated hematology analyzers are of two main types:
 
  • Semi-automated: Some steps like dilution of blood sample are performed by the technologist; can measure only a few parameters.
  • Fully automated: Require only anticoagulated blood sample; measure multiple parameters.

CAUSES OF ERRONEOUS RESULTS (INTERFERENCES CAUSING ABNORMAL RESULT) WITH HEMOTOLOGY ANALYZER

Published in Hemotology
Saturday, 29 July 2017 19:18
CAUSES OF ERRONEOUS RESULTS (INTERFERENCES CAUSING ABNORMAL RESULT)
 
These are listed in Table 809.1
 
Table 809.1 Causes of erroneous results with hematology analyzer
Parameter Interfering factors
  Erroneous increase Erroneous decrease
0. All parameters
  • Clotted sample
1. WBC count
  • Nucleated red cells
  • Large platelet clumps
  • Unlysed red cells (some abnormal red cells resist lysing)
  • Cryoglobulins
  • Clotted sample
2. RBC count
  • Very high WBC*
  • Large numbers of giant platelets
  • Clotted sample
  • Microcytic red cells
  • Autoagglutination
3. Hemoglobin
  • Clotted sample
4. MCV
  • Very high WBC
  • Hyperglycemia
  • Autoagglutination (cold agglutinins)
  • Cryoglobulins
5. MCHC
  • Hyperlipidemia
  • Autoagglutination (cold agglutinins)
  • Very high WBC
6. Platelets
  • Microcytic red cells
  • WBC fragments
  • Cryoglobulins
  • Platelet satellitism
  • Platelet clumping
*: WBCs are counted along with RBCs, but normally their number is statistically insignificant
 
 
FLAGGING
 
‘Flags’ are signals that occur when an abnormal result is detected by the analyzer. Flags are displayed to reduce false-positive and false-negative results by mandating a review of blood smear examination.

PARAMETERS MEASURED BY HEMATOLOGY ANALYZERS

Published in Hemotology
Saturday, 29 July 2017 17:54
Parameters measured by hematology analyzers and their derivation are shown in Tables 808.1 and 808.2. Most automated hematology analyzers measure red cell count, red cell indices (mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration), hemoglobin, hematocrit, total leukocyte count, differential leukocyte count (three-part or five-part), and platelet count.
 
Table 808.1 Parameters measured by hematology analyzers
Parameters measured by most analyzers Parameters measured by some analyzers
  • RBC count
  • Hemoglobin
  • Mean cell volume
  • Mean cell hemoglobin
  • Mean cell hemoglobin concentration
  • WBC count
  • WBC differential
  • Platelet count
  • Red cell distribution width
  • Reticulocyte count
  • Reticulocyte hemoglobin content
  • Mean platelet volume
  • Platelet distribution width
  • Reticulated platelets
 
Table 808.2 Parameters reported by hematology analyzers
Parameters measured directly or derived through histogram Parameters measured through calculation
  • RBC count
  • Mean cell volume (Derived from RBC histogram)
  • Red cell distribution width (Derived from RBC histogram)
  • Hemoglobin
  • Reticulocyte count
  • WBC count
  • Differential WBC count (Derived through WBC histogram)
  • Platelet count
  • Mean platelet volume (Derived from platelet histogram)
  • Hematocrit
  • Mean cell hemoglobin
  • Mean cell hemoglobin concentration
 
Estimation of Hemoglobin
 
Hemoglobin is measured directly by a modification of cyanmethemoglobin method (all hemoglobins are converted to cyanmethemoglobin by potassium ferricyanide; cyanmethemoglobin has a broad absorbance peak at 540 nm). Some analyzers use a nonhazardous reagent such as sodium lauryl sulphate. A non-ionic detergent is added for rapid red cell lysis and to minimize turbidity caused by cell membranes and plasma lipids.
 
Estimation of Red Blood Cell Count and Mean Cell Volume (MCV)
 
Red cell count and cell volume are directly measured by aperture impedance or light scatter analysis. In a red cell histogram, cell numbers are plotted on Y-axis, while cell volume is indicated on Xaxis (see Figure 808.1). The analyzer counts those cells as red cells volume of which ranges between 36 fl and 360 fl. MCV is used for morphological classification of anemia into microcytic, macrocytic, and normocytic types.
 
Figure 808.1 Diagrammatic representation of red cell histogram obtained by aperture impedance
Figure 808.1 Diagrammatic representation of red cell histogram obtained by aperture impedance. The analyzer counts cells between 36 fl and 360 fl as red cells. Although leukocytes are present and counted along with red cells in the diluting fluid, their number is not statistically significant. Only if leukocyte count is markedly elevated (>50,000/μl), histogram and the red cell count will be affected. Area of the peak between 60 fl and 125 fl is used for calculation of mean cell volume and red cell distribution width. Abnormalities in red cell histogram include: (1) Left shift of the curve in microcytosis, (2) Right shift of the curve in macrocytosis, and (3) Bimodal peak of the curve in double (dimorphic) population of red cells
 
Estimation of MCH, MCHC, and Hematocrit (HCT/PCV)
 
These parameters are obtained indirectly through calculations.
 
 
MCH (pg) = Hemoglobin (g/l)
                     RBC count (10⁶/μl)
 
 
MCHC (g/dl) = Hemoglobin (g/dl)
                         Hematocrit (%)
 
 
Hematocrit (%) = Mean Cell Volume (fl)
                              RBC count (10⁶/μl)
 
 
Estimation of Red Cell Distribution Width (RDW)
 
RDW is a quantitative measure of variation in sizes of red cells and is expressed as coefficient of variation of red cell size distribution. It is equivalent to anisocytosis observed on blood smear. It is derived from red cell histogram in some analyzers. RDW is usually elevated in iron deficiency anemia, but not in β-thalassemia minor and anemia of chronic disease (other causes of microcytic anemia). However, this distinction is not absolute and there is a significant overlap between values among patients. Raised RDW requires examination of blood smear.
 
Among the red cell values generated by the analyzer (red cell count, hemoglobin, hematocrit, MCV, MCH, MCHC, and RDW), most important for decision-making are hemoglobin, hematocrit, and MCV.
 
WBC Differential
 
Difference between 3-part and 5-part hemotology analyzer...
 
Hematology analyzers can either generate a 3-part differential (differential count reported as lymphocytes, monocytes, and granulocytes) or a 5-part differential (lymphocytes, monocytes, neutrophils, eosinophils, and basophils). The 3-part differential counting is based on electrical impedance volume measurement of leukocytes. In volume histogram for WBCs, approximate numbers of cells are plotted on Y-axis and cell size on X-axis. Those cells with volume 35-90 fl are designated as lymphocytes, cells with volume 90-160 fl as mononuclear cells, and cells with volume 160-450 fl as neutrophils (see Figure 808.2). Any deviation from the expected histogram is flagged by the analyzer, mandating review of blood smear. A large proportion of 3-part differential counts are ‘flagged’ to avoid missing abnormal cells.
 
Instruments measuring a 5-part differential work on a combination of different principles, e.g. light scatter, impedance, and electrical conductivity, a combination of light scatter, peroxidase staining, and resistance of basophils to lysis in acid buffer, etc.
 
Figure 808.2 Diagrammatic representation of WBC histogram
Figure 808.2 Diagrammatic representation of WBC histogram. WBC histogram analysis shows relative numbers of cells on Y-axis and cell size on X-axis. The lytic agent lyses the cytoplasm that collapses around the nucleus causing differential shrinkage. The analyzer sorts the WBCs according to the nuclear size into 3 main groups (3-part differential): Cells with 35-90 fl volume are designated as lymphocytes, cells with 90-160 fl volume are designated as monocytes, and cells with 160-450 fl volume are designated as neutrophils. Abnormalities in WBC histogram include: (1) Peak to the left of lymphocyte peak: Nucleated red cells, (2) Peak between lymphocytes and monocytes: Blast cells, eosinophilia, basophilia, plasma cells, and atypical lymphocytes, and (3) Peak between monocytes and neutrophils: Left shift
 
Platelet Count
 
Platelets are difficult to count because of their small size, marked variation in size, tendency to aggregation, and overlapping of size with microcytic red cells, cellular fragments, and other debris. In hematology analyzers, this difficulty is addressed by mathematical analysis of platelet volume distribution so that it corresponds to lognormal distribution. Platelets are counted by electrical impedance method in the RBC aperture, and a histogram is generated with platelet volume on X-axis and relative cell frequency on Y-axis (see Figure 808.3). Normal platelet histogram consists of a right-skewed single peak. Particles greater than 2 fl and less than 20 fl are classified as platelets by the analyzer.
 
Figure 808.3 Diagrammatic representation of normal platelet histogram
Figure 808.3 Diagrammatic representation of normal platelet histogram: Counting and sizing of platelets by electrical impedance method occurs in the RBC aperture. The counter designates particles between sizes 2 fl and 20 fl as platelets. Abnormalities in platelet histogram result from interferences such as cytoplasmic fragments (peak at left end of histogram) or severely microcytic red cells and giant platelets (peak at right end of histogram)
 
Two other platelet parameters can be obtained from platelet histogram using computer technology: mean platelet volume (MPV) and platelet distribution width (PDW). Some analyzers can generate another parameter called as reticulated platelets.
 
MPV refers to the average size of platelets and is obtained from mathematical calculation. Normal MPV is 7-10 fl. Increased MPV (> 10 fl) results from presence of immature platelets in circulation; peripheral destruction of platelets stimulates megakaryocytes to produce such platelets (e.g. in idiopathic thrombocytopenic purpura). Decreased MPV (< 7 fl) is due to presence of small platelets in circulation (in conditions associated with reduced production of platelets in bone marrow).
 
PDW is analogous to RDW and is a measure of variation in size of platelets (normal <20%). Increased PDW is observed in megaloblastic anemia, chronic myeloid leukemia, and after chemotherapy.
 
Some analyzers measure reticulated platelets or young platelets that contain RNA (similar to reticulocytes). Increased numbers of reticulated platelets are seen in thrombocytopenia due to peripheral destruction of platelets.

Reticulocyte Count
 
Various fluorescent dyes can combine with RNA of reticulocytes; the fluorescence then is counted in a flow cytometer. More immature reticulocytes fluoresce more strongly as they contain more RNA.
 
Reticulocyte hemoglobin content is a parameter that estimates hemoglobinization of most recently produced red cells. It is a predictor of iron deficiency.
 
WBC Cytogram (Scattergram)
 
In the scattergram, each dot represents a cell of a given volume and density, and the positions of dots in the graph are determined by the degree of side scatter, degree of forward scatter, light absorption by the cell, and cytochemical staining (if used). The forward angle light scatter (FALS) is represented on Y-axis, and the side scatter (SS) is represented on X-axis. Low FALS and low SS are indicative of lymphocytes; with subsequent increasing FALS and SS, monocytes, neutrophils, and lastly eosinophils are designated in the graph. Counting of basophils is based on a different technology.
 
Further Reading:
 

FLOW CYTOMETRY

Published in Hemotology
Saturday, 29 July 2017 15:37
FLOW CYTOMETRY
 
Box 807.1 Properties of a cell measured by a flow cytometerFlow cytometry is a procedure used for measuring multiple cellular and fluorescent properties of cells when they flow as a single cell suspension through a laser beam by a specialized instrument called as a flow cytometer. Flow cytometry can analyze numerous cells in a short time and multiple parameters of a single cell can be analyzed simultaneously. From the measured parameters, specific cell populations are defined. Cells or particles with size 0.2-150 μm are suitable for flow cytometer analysis.
 
Flow cytometry can provide following information about a cell (Box 807.1):
 
  • Cell size (forward scatter)
  • Internal complexity or granularity (side scatter)
  • Relative fluorescence intensity.
 
A flow cytometer consists of three main components or systems: fluidics, optics, and electronics.
 
(1) Fluidics: The function of the fluidics system is to transport cells in a stream to the laser beam for interrogation. Cells (fluorescence-tagged) are introduced into the cytometer (injected into the sheath fluid within the flow chamber) and made to flow in a single file past a laser (light amplification by stimulated emission of radiation) beam. The stream transporting the cells should be positioned in the center of the laser beam. The portion of the fluid stream where the cells are located is called as the sample core. Only a single cell or particle should pass through the laser beam at one time. Flow cytometers use the principle of hydrodynamic focusing (process of centering the sample core within the sheath fluid) for presenting cells to the laser.
 
(2) Optics: This system consists of lasers for illumination of cells in the sample, and filters to direct the generated light signals to the appropriate detectors.
 
The light source used in most flow cytometers is laser.
 
The laser most commonly used in flow cytometry is Argon-ion laser. The light signals are generated when the laser beam strikes the cell, which are then collected by appropriately positioned lenses. A system of optical mirrors and filters then directs the specified wavelengths of light to the designated detectors. Two types of light signals are generated when the laser beam strikes cells tagged with fluorescent molecules: fluorescence and light scatter. The cells tagged with fluorescence emit a momentary pulse of fluorescence; in addition, two types of light scatter are measured: forward scatter (proportional to cell diameter) and side scatter (proportional to granularity of cell).
 
(3) Electronics: The optical signals (photons) are converted to corresponding electronic signals (electrons) by the photodetectors (photodiodes and photomultiplier tubes). The electronic signal produced is proportional to the amount of light striking a cell. The electric current travels to the amplifier and is converted to a voltage pulse. The voltage pulse is assigned a digital value representing a channel by the Analog-to Digital Converter (ADC). The channel number is then transferred to the computer which displays it to the appropriate position on the data plot.
 
Further Reading:
 

TERMINOLOGIES USED IN FLOWCYTOMETRY

Published in Hemotology
Saturday, 29 July 2017 15:09
Fluorescence
 
A fluorochrome absorbs light energy and emits excess energy in the form of photon light (fluorescence). Fluorescence is the property of molecules to absorb light at one wavelength and emit light at a longer wavelength. The fluorescent dyes commonly used in flow cytometry are fluorescein isothiocyanate (FITC) and phycoerythrin (PE). The fluorochrome-labeled antibodies are used for detection of antigenic markers on the surface of cells. A particular cell type can be identified on the basis of the antigenic profile expressed. Multiple fluorochromes can be used to identify different cell types in a mixed population of cells.
 
Light Scatter
 
Light is scattered when the incident light is deflected by a particle traversing through a beam of light. This depends on the physical properties of the cell. Two forms of light scatter are used to identify different cell types: forward scatter and side scatter. Forward scatter (light scattered in the same direction as the laser beam) is related to cell size. Side scatter (light scattered at a 90° angle to the laser beam) is related to internal granularity of the cell. Main subpopulations of leukocytes are identified on the basis of correlated measurements of forward and side scatters. When a cell passes through laser beam, side scatter and fluorescent signals that are emitted by the cell are directed to photomultiplier tubes, while the forward scatter signals are directed to a photodiode. Photomultiplier tubes and photodiodes are called as detectors. Optical filters are placed before the detectors that allow only a narrow range of wavelengths to reach the detectors (see Figure 806.1).
 
Figure 806.1 Principle of working of a flow cytometer
Figure 806.1 Principle of working of a flow cytometer
 
Data Analysis
 
The data collected and stored in the computer can be displayed in various formats. A parameter means forward scatter, or side scatter, or emitted fluorescence from a particle as it passes through a laser beam. A histogram is a data plot of a single parameter, with the parameter’s signal value in channel numbers or relative fluorescence intensity on X-axis (horizontal axis) and number of events on the Y-axis. A dot plot is a two parameter data graph in which each dot represents one event that the flow cytometer analyzed; one parameter is displayed on the X-axis and the other on the Y-axis. A 3-D plot represents one parameter on X-axis, another parameter on Y-axis, and number of events per channel on Z-axis.
 
Gating
 
A gate is a boundary that can be set to restrict the analysis to a specific population within the sample. For example, a gate boundary can be drawn on a dot plot or histogram to restrict the analysis only to cells with the size of lymphocytes. Gates can be inclusive (selection of events that fall within the boundary) or exclusive (selection of events that fall outside the boundary). Data selected by the gate is then displayed in subsequent plots.
 
Sorting
 
Usually, when a cell passes through the laser beam, it is sent to waste. Sorting consists of collecting cells of interest (defined through criteria of size and fluorescence) for further analysis (such as microscopy or functional or chemical analysis). Sorting feature is available only in some flow cytometers.
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