Displaying items by tag: Hemotological Tests

Porphyrias, derived from the Greek word "porphura," meaning purple pigment, encompass a diverse array of rare disorders arising from disruptions in the heme biosynthetic pathway. This disturbance leads to the abnormal accumulation of red and purple pigments known as porphyrins within the body. Heme, an integral component of hemoglobin, undergoes synthesis through a series of distinct steps, as illustrated in Figure 1. Each step is facilitated by a specific enzyme. In instances where any of these enzymatic processes falter due to hereditary or acquired causes, precursor molecules of heme, referred to as porphyrin intermediates, amass in the blood, deposit in the skin and various organs, and are excreted in urine and feces.

The categorization of porphyrias is contingent upon the location of the defect, resulting in different types with diverse clinical manifestations, severity levels, and the specific nature of accumulated porphyrin.

Historically, porphyria has been postulated as a potential explanation for medieval tales of vampires and werewolves. This conjecture arises from notable parallels between the behaviors exhibited by individuals afflicted with porphyria and the characteristics found in folklore. These similarities include light aversion, skin mutilation upon sunlight exposure, red teeth, psychiatric disturbances, and the consumption of blood to obtain heme.

Biosynthesis of heme
Figure 1: Figure on left shows steps in the biosynthesis of heme. Some steps (first and last three) occur in mitochondria, while some occur in cytosol. Figure on right shows deficiency state associated with each enzyme. Deficiency of ALA synthase is associated with sideroblastic anemia, and deficiencies of other enzymes cause porphyria.

Detection and accurate diagnosis of porphyrias pose significant challenges, often leading to oversight or misdiagnosis. Many porphyrias lack definitive physical findings, screening tests may produce false-negative results, diagnostic criteria are ambiguously defined, and mild disorders may yield enzyme assay results within the ostensibly 'normal' range.

Heme plays a crucial role in both bone marrow, where it is essential for hemoglobin synthesis, and the liver, where it is vital for cytochromes. Consequently, porphyrias are classified into erythropoietic and hepatic types based on the site of disease expression. Hepatic porphyrias predominantly impact the nervous system, while erythropoietic porphyrias primarily affect the skin. Furthermore, porphyrias are categorized as acute and nonacute (or cutaneous) types based on their clinical presentations, as outlined in Table 1. This systematic classification enhances our understanding of porphyrias, aiding clinicians, researchers, and medical professionals in comprehending the nuances of these complex disorders.

Table 1: Various classification schemes for porphyrias
Classification based on predominant clinical manifestationsClassification based on site of expression of diseaseClassification based on mode of clinical presentation
Neuropsychiatric Hepatic Acute
Acute intermittent porphyria ALA-dehydratase porphyria ALA-dehydratase porphyria (Plumboporphyria)
ALA-dehydratase porphyria (Plumboporphyria) Acute intermittent porphyria Acute intermittent porphyria
Cutaneous (Photosensitivity) Hereditary coproporphyria Hereditary coproporphyria
Congenital erythropoietic porphyria Variegate porphyria Variegate porphyria
Porphyria cutanea tarda Erythropoietic porphyria Non-acute (cutaneous)
Erythropoietic protoporphyria Congenital erythropoietic porphyria Porphyria cutanea tarda
Mixed (Neuropsychiatric and cutaneous) Erythropoietic protoporphyria Congenital erythropoietic porphyria
Hereditary coproporphyria Hepatic/Erythropoietic Erythropoietic protoporphyria
Variegate porphyria Porphyria cutanea tarda -

The inheritance patterns associated with porphyrias manifest as either autosomal dominant or recessive. The majority of acute porphyrias follow an autosomal dominant inheritance, signifying the transmission of a single abnormal gene copy. Consequently, the activity of the deficient enzyme is halved, resting at 50%. In instances where the hepatic heme level diminishes due to various factors, the activity of ALA synthase is spurred, resulting in an escalation of heme precursors up to the point of the enzyme defect. This surge in heme precursors precipitates the onset of symptoms characteristic of acute porphyria. Notably, as the heme level normalizes, the associated symptoms subside.

The accumulation of porphyrin precursors presents a noteworthy occurrence in lead poisoning, attributed to the inhibition of the enzyme aminolevulinic acid dehydratase within the heme biosynthetic pathway. This phenomenon can simulate the clinical presentation of acute intermittent porphyria.

Understanding the genetic underpinnings and enzymatic intricacies of porphyrias is crucial for clinicians, researchers, and medical professionals. The autosomal dominant inheritance pattern underscores the significance of a singular abnormal gene copy in the manifestation of acute porphyrias. Additionally, the intricate interplay between heme levels and enzymatic activity elucidates the nuanced triggers and resolution of acute porphyria symptoms. Moreover, the correlation between lead poisoning and the disruption of the heme biosynthetic pathway highlights the diagnostic challenges posed by conditions that mimic porphyric presentations, necessitating a keen clinical acumen for accurate differentiation.

Clinical Features

The clinical manifestations of porphyrias exhibit variability contingent upon the specific type of the disorder. Acute porphyrias, for instance, manifest with a spectrum of symptoms, including acute and intense abdominal pain, vomiting, constipation, chest pain, emotional and mental disturbances, seizures, hypertension, tachycardia, sensory deficits, and muscular weakness. This diverse array of clinical presentations underscores the complexity of acute porphyrias and the multisystemic impact they can exert.

In contrast, cutaneous porphyrias present a distinctive set of clinical features. Patients with cutaneous porphyrias commonly experience photosensitivity, characterized by the development of redness and blistering of the skin upon exposure to sunlight. Additional symptoms include itching, necrosis of the skin and gums, and an observable increase in hair growth over the temples. This distinctive clinical profile, outlined in Table 2, provides a comprehensive overview of the characteristic features associated with cutaneous porphyrias.

Understanding the nuanced clinical features associated with different types of porphyrias is pivotal for accurate diagnosis and effective management. The delineation of symptoms, ranging from abdominal pain and neurological manifestations in acute porphyrias to the distinctive photosensitivity and dermatological changes in cutaneous porphyrias, enhances the knowledge base for clinicians, researchers, and medical professionals. Such precision in characterizing the clinical landscape facilitates targeted interventions and improves patient outcomes.

Table 2: Clinical characteristics of porphyrias
PorphyriaDeficient enzymeClinical featuresInheritanceInitial test
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
Variegate porphyria Protoporphyrinogen oxidase Acute neurovisceral attacks + skin fragility, bullae Autosomal dominant Urinary PBG
Hereditary coproporphyria Coproporphyrinogen oxidase Acute neurovisceral attacks + skin fragility, bullae Autosomal dominant Urinary PBG
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
Porphyria cutanea tarda* Uroporphyrinogen decarboxylase Skin fragility, bullae Autosomal dominant (some cases) Urinary/fecal total porphyrins
Erythropoietic protoporphyria* Ferrochelatase Acute photosensitivity Autosomal dominant Free erythrocyte protoporphyrin
Disorders marked with * are the three most common porphyrias. PBG: Porphobilinogen

The manifestation of symptoms in porphyrias is intricately linked to various triggers, encompassing pharmaceutical agents and lifestyle factors. Certain drugs, such as barbiturates, oral contraceptives, diazepam, phenytoin, carbamazepine, methyldopa, sulfonamides, chloramphenicol, and antihistamines, can serve as catalysts for symptoms. Emotional or physical stress, infections, dietary changes, fasting, substance abuse, premenstrual periods, smoking, and alcohol consumption also constitute potential triggers, amplifying the complexity of porphyria management.

Autosomal dominant porphyrias comprise a diverse group of disorders, including acute intermittent porphyria, variegate porphyria, porphyria cutanea tarda, erythropoietic protoporphyria (predominantly), and hereditary coproporphyria. In contrast, autosomal recessive porphyrias encompass congenital erythropoietic porphyria, erythropoietic protoporphyria (in a limited number of cases), and ALAdehydratase porphyria, also known as plumboporphyria.

Understanding the array of triggers and the genetic underpinnings of different porphyria types is pivotal for comprehensive patient care. The intricate interplay between pharmaceutical agents, lifestyle factors, and the distinct genetic characteristics of autosomal dominant and recessive porphyrias underscores the necessity for a nuanced approach in diagnosis and management. This multifaceted perspective equips medical professionals, and researchers with the knowledge required to navigate the complexities of porphyria, facilitating enhanced patient outcomes and advancing scientific understanding in the field.

Laboratory Diagnosis

The diagnosis of porphyria involves conducting tests on blood, urine, and feces, particularly during symptomatic periods. Achieving a timely and precise diagnosis is crucial for the effective management of porphyrias. Given the diverse and extensive clinical features associated with porphyrias, they are often considered in the differential diagnosis of various medical conditions. Most routine hospital laboratories are equipped to perform initial investigations when porphyria is suspected. However, for the identification of specific porphyria types, specialized laboratories with advanced testing capabilities become essential.

In the diagnostic process, blood, urine, and fecal analyses play a pivotal role, allowing for the detection of characteristic abnormalities during symptomatic phases. This comprehensive approach ensures a more accurate identification of porphyria subtypes, facilitating targeted and tailored therapeutic strategies.

Considering the intricate nature of porphyria diagnoses, collaboration with specialized laboratories becomes imperative. These facilities possess the advanced technologies and expertise required for in-depth analyses, enabling the discrimination between various porphyria types. This collaboration enhances the precision of the diagnostic process, contributing to improved patient outcomes and informed decision-making in the realm of porphyria management.

In essence, the diagnostic journey for porphyria demands a synergistic effort between routine hospital laboratories and specialized facilities, with a focus on leveraging advanced testing methodologies to unravel the complexities inherent in these conditions. This collaborative approach underscores the significance of accurate diagnostics in paving the way for effective porphyria management strategies.

Initial Studies

In the assessment of suspected acute porphyrias, specifically during acute neurovisceral attacks, it is imperative to submit a freshly collected, randomly obtained urine sample (10-20 ml) for the detection of heightened urinary excretion of porphobilinogen (PBG) (refer to Figure 2). Notably, in acute intermittent porphyria (AIP), the urine may exhibit a reddish or brown hue upon standing, as illustrated in Figure 3.

For cases where cutaneous porphyrias are suspected, with manifestations such as acute photosensitivity lacking skin fragility, a comprehensive diagnostic approach is warranted. This includes analyzing free erythrocyte protoporphyrin (FEP) in EDTA blood for the diagnosis of erythrocytic protoporphyria. Additionally, for all other cutaneous porphyrias characterized by skin fragility and bullae, an examination of fresh, random urine (10-20 ml) and either feces (5-10 g) or plasma is indispensable for identifying excess porphyrins. Detailed guidance on this diagnostic protocol is provided in Figure 4 and Table 2.

In essence, the diagnostic strategy for suspected porphyrias involves specific tests tailored to the clinical presentation. The utilization of various specimen types, including urine, blood, feces, and plasma, is crucial for the accurate identification of porphyrin abnormalities. The distinct visual cues, such as the color changes in urine associated with AIP, serve as valuable indicators in the diagnostic process. This meticulous and multifaceted approach ensures a comprehensive evaluation, facilitating precise diagnoses in the intricate landscape of porphyrias.

Evaluation of acute neurovisceral porphyria
Figure 2: Evaluation of acute neurovisceral porphyria
Red coloration of urine on standing in acute intermittent porphyria
Figure 3: Red coloration of urine on standing in acute intermittent porphyria
Evaluation of cutaneous porphyrias
Figure 4: Evaluation of cutaneous porphyrias

In addition to aiding in the diagnosis, the identification of specific heme intermediates excreted in urine or feces plays a pivotal role in pinpointing the site of defect in porphyria. Heme precursors, extending up to coproporphyrinogen III, exhibit water solubility, enabling their detection in urine. Conversely, protoporphyrinogen and protoporphyrin, being insoluble in water, are excreted through bile and can be identified in fecal samples. It is essential to shield all samples from light to preserve their integrity.

The requisite samples for a comprehensive porphyria assessment include a 10-20 ml fresh, random urine sample devoid of any preservatives, 5-10 g of wet fecal weight, and blood anticoagulated with EDTA. Each specimen type offers distinct insights into the porphyrin profile, contributing to a thorough evaluation of the disorder. The meticulous collection and analysis of these samples serve as critical steps in unraveling the intricacies of porphyria, ensuring a comprehensive understanding of the disorder's manifestations and facilitating targeted interventions.

In essence, the choice of sample type is guided by the solubility characteristics of specific heme intermediates, aligning with their excretion pathways. This systematic approach, coupled with stringent measures to protect samples from light-induced alterations, underscores the importance of precision and accuracy in the diagnostic process.

Test for Porphobilinogen in Urine

The Ehrlich’s aldehyde test serves as a diagnostic tool for detecting porphobilinogen (PBG) in urine. In this assay, Ehrlich’s reagent, composed of p-dimethylaminobenzaldehyde, reacts with PBG, resulting in the formation of a distinctive red color. The characteristic red product exhibits an absorption spectrum with a peak at 553 nm and a shoulder at 540 nm. It's noteworthy that both urobilinogen and porphobilinogen elicit a similar reaction, necessitating additional tests to discern between the two substances. To distinguish PBG from urobilinogen, solvent extraction can be employed, as outlined in the Watson-Schwartz test. This additional step is crucial for achieving specificity in the diagnostic process.

It is essential to recognize that levels of PBG might appear normal or near normal between attacks. Consequently, to mitigate the risk of false-negative results, it is imperative to conduct the test during an acute attack. This strategic timing ensures a more accurate and reliable assessment of PBG levels, contributing to a precise diagnosis and informed clinical decision-making.

Test for Total Porphyrins in Urine

The quantification of total porphyrins within an acidified urine sample is achievable through spectrophotometry, leveraging the distinct absorbance peak of porphyrins around 400 nm. This method allows for a semiquantitative estimation of porphyrin levels, providing valuable insights into the porphyrin profile present in the urine sample. Spectrophotometry, with its ability to measure the absorption of light at specific wavelengths, offers a precise and objective means of assessing the concentration of porphyrins in the given sample.

In this analytical approach, the intense absorbance peak observed around 400 nm serves as a reliable marker for the presence of porphyrins. This peak reflects the characteristic absorption spectrum of porphyrins, allowing for their identification and quantification in a systematic and accurate manner. The semiquantitative nature of the estimation enhances the diagnostic utility of the method, providing clinicians and researchers with valuable information regarding the porphyrin content in the urine.

The utilization of spectrophotometry in conjunction with acidified urine samples offers a robust technique for detecting and estimating total porphyrins. This method, marked by its precision and objectivity, contributes to the comprehensive assessment of porphyrin levels, facilitating diagnostic and research endeavors within the realm of porphyria evaluation.

Test for Total Porphyrins in Feces

The quantification of total porphyrins in fecal samples involves the use of spectrophotometry on an acidic extract of the specimen. However, a crucial preparatory step is required to eliminate interference from dietary chlorophyll, which also absorbs light around 400 nm. This interference is addressed through diethyl ether extraction, ensuring a more accurate and specific assessment of total porphyrin levels.

In the analytical process, an acidic extract of the fecal sample serves as the basis for spectrophotometric analysis. Spectrophotometry, with its ability to measure the absorption of light at specific wavelengths, allows for the precise determination of total porphyrins. The focus on an acidic environment facilitates optimal conditions for the assessment of porphyrin content.

To enhance the specificity of the analysis, the extraction of dietary chlorophyll using diethyl ether becomes imperative. This preparatory step is essential as chlorophyll absorbs light in a similar range as porphyrins, potentially leading to inaccurate measurements. By effectively removing this interference, the spectrophotometric analysis can provide more reliable and meaningful data regarding the concentration of total porphyrins in the fecal sample.

The process of determining total porphyrins in feces through spectrophotometry necessitates a meticulous approach, including the removal of dietary chlorophyll through diethyl ether extraction. This method ensures the accuracy and specificity of the analysis, contributing to the reliability of results in the evaluation of porphyrin levels in fecal samples.

Evaluation of Porphyrins in Erythrocytes and Plasma: Modern Techniques

Contemporary methodologies have supplanted traditional approaches such as visual examination for porphyrin fluorescence, as well as solvent fractionation and spectrophotometry. The advent of fluorometric methods represents a significant advancement in the analysis of porphyrins in erythrocytes and plasma.

Fluorometric techniques have emerged as the preferred means of assessing porphyrins, offering heightened precision and sensitivity compared to their predecessors. These methods leverage the unique fluorescence properties of porphyrins, providing a more refined and objective approach to their detection and quantification.

By utilizing fluorometry, researchers and medical professionals can achieve a clearer understanding of porphyrin levels in erythrocytes and plasma. This modernized approach enhances both the accuracy and efficiency of porphyrin testing, aligning with the evolving standards of scientific analysis in hematology and related fields.

This transition to fluorometric methods underscores the commitment to staying at the forefront of scientific advancements, ensuring that diagnostic and research practices remain not only rigorous but also in harmony with the latest technological innovations.

Comprehensive Assessment for Porphyria Diagnosis

Upon obtaining a positive result in the initial porphyria screening, a more in-depth analysis becomes imperative. This involves the quantification of porphyrin concentrations in urine, feces, and blood, a crucial step in establishing a precise and specific diagnosis. The tables below (Tables 3 and 4) provide detailed insights into the diagnostic criteria and reference values for this comprehensive evaluation.

By meticulously measuring porphyrin levels in these biological samples, healthcare professionals can discern the specific type and severity of porphyria, facilitating targeted and effective management strategies. This comprehensive approach not only confirms the presence of porphyria but also aids in tailoring treatment plans to the individual nuances of the patient's condition.

As with any diagnostic process, the accuracy and reliability of results depend on the proficiency of the laboratory techniques employed and the adherence to standardized protocols. This commitment to precision ensures that the gathered data is not only scientifically robust but also forms the cornerstone for informed clinical decisions and patient care.

Table 3: Diagnostic patterns of concentrations of heme precursors in acute porphyrias
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 4: Diagnostic patterns of concentrations of heme precursors in cutaneous porphyrias
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.

Diagnostic Challenges in Latent Porphyrias and Remission Periods

During latent porphyrias and periods of remission, individuals may exhibit normal porphyrin levels, posing a diagnostic challenge. In such instances, a comprehensive approach involving enzymatic and DNA testing becomes imperative for an accurate diagnosis. These advanced testing methods delve into the genetic and enzymatic aspects, providing a more nuanced understanding of porphyria manifestation even in the absence of elevated porphyrin levels.

Upon confirming a diagnosis of porphyria, a critical next step involves the systematic examination of close family members for the presence of the disorder. This proactive approach is essential for early detection and intervention. Family members testing positive for porphyria should undergo counseling, equipping them with valuable insights into potential triggering factors and empowering them to make informed lifestyle choices that mitigate the risk of symptomatic episodes.

This meticulous diagnostic and familial investigation strategy not only aids in individual patient management but also contributes to the broader goal of familial health and well-being. By addressing latent cases and involving family members, healthcare professionals can establish a more comprehensive and effective framework for the understanding and management of porphyrias.

Published in Clinical Pathology
Thursday, 03 August 2017 11:55


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
Published in Hemotology
Thursday, 03 August 2017 11:33


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:
Published in Hemotology
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.
Published in Hemotology
Friday, 28 July 2017 05:05

Platelet Aggregation Studies

Platelet aggregation tests are carried out in specialized hematology laboratories if platelet dysfunction is suspected. These tests are usually indicated in patients presenting with the mucocutaneous type of bleeding and in whom screening tests reveal normal platelet count, prolonged bleeding time, normal prothrombin time, and normal activated partial thromboplastin time.

Platelet aggregation studies are carried out on platelet-rich plasma using an aggregometer. When a platelet aggregating agent is added to platelet-rich plasma, platelets form aggregates, and optical density falls (or light transmission increases); this is recorded by a chart recorder on a strip chart. Commonly used platelet aggregating agents are ADP (adenosine 5-diphosphate), epinephrine (adrenaline), collagen, arachidonic acid, and ristocetin. ADP (low dose) and epinephrine induce primary and secondary waves of aggregation (biphasic curve). The primary wave is due to the direct action of the aggregating agent on platelets. The secondary wave is due to thromboxane A2 synthesis and secretion from platelets. Collagen, arachidonic acid, and ristocetin induce a single wave of aggregation (monophasic curve) Normal aggregation curve is shown in Figure 804.1. Aggregation patterns in various platelet function defects are shown in Figures 804.2 to 804.4, and Table 804.1.

Normal platelet aggregation curves
Figure 804.1: Normal platelet aggregation curves
Platelet aggregation curves in von Willebrand disease and Bernard Soulier syndrome absent aggregation with ristocetin normal aggregation with ADP epinephrine and arachidonic acid
Figure 804.2: Platelet aggregation curves in von Willebrand disease and Bernard-Soulier syndrome (absent aggregation with ristocetin, normal aggregation with ADP, epinephrine, and arachidonic acid)
Platelet aggregation curves in storage pool defect absent second wave of aggregation with ADP and epinephrine absent or greatly diminished aggregation with collagen and normal ristocetin aggregation
Figure 804.3: Platelet aggregation curves in storage pool defect (absent the second wave of aggregation with ADP and epinephrine, absent or greatly diminished aggregation with collagen, and normal ristocetin aggregation)
Platelet aggregation curves in Glanzmanns thrombasthenia absent aggregation with all agonists except ristocetin
Figure 804.4: Platelet aggregation curves in Glanzmann’s thrombasthenia (absent aggregation with all agonists except ristocetin)
Table 804.1: Laboratory features of platelet function disorders
DisorderPlatelet Aggregation patternOther features
Bernard Soulier syndrome Normal with ADP, epinephrine, collagen, and arachidonic acid; deficient with ristocetin Autosomal recessive; severe bleeding; giant platelets
Glanzmann's thrombasthenia Deficient with ADP, epinephrine, collagen, and arachidonic acid; normal with ristocetin Autosomal recessive; severe bleeding; small and discrete platelets; defective clot retraction
Storage pool defect Primary wave with ADP and epinephrine, normal with arachidonic acid, deficient with collagen, normal with ristocetin Defects of platelets granules; platelet dense granules are decreased with the deficient release of ADP, ATP, calcium, and serotonin
Aspirin-like defect Primary wave with ADP and epinephrine, deficient with arachidonic acid, deficient with collagen, normal with ristocetin -
von Willebrand disease Normal with ADP, epinephrine, collagen, and arachidonic acid; deficient with ristocetin Autosomal dominant/recessive; abnormality in aggregation corrected with cryoprecipitate
Published in Hemotology
Thursday, 27 July 2017 06:22


A blood smear is examined for:
A peripheral blood smear has three parts: Head, body, and tail. Also read: PREPARATION OF BLOOD SMEAR BY WEDGE METHOD.
A blood smear should be examined in an orderly manner. Initially, blood smear should be observed under low power objective (10×) to assess whether the film is properly spread and stained, to assess cell distribution, and to select an area for examination of blood cells. Best morphologic details are seen in the area where red cells are just touching one another. Low power view is also helpful for the identification of Rouleaux formation, autoagglutination of red cells, and microfilaria. High power objective (45×) is suitable for examination of red cell morphology and for differential leukocyte count. A rough estimate of total leukocyte count can be obtained which also serves to crosscheck the total leukocyte count done by manual counting or automated method. Oil-immersion objective (100×) is used for more detailed examination of any abnormal cells.
Further Reading:
Published in Hemotology
Wednesday, 26 July 2017 12:45


Box 802.1 Role of blood smear in thrombocytopeniaPlatelets are small, 1-3 μm in diameter, purple structures with tiny irregular projections on surface. In blood films prepared from non-anticoagulated blood (i.e. direct fingerstick), they occur in clumps. If platelet count is done on automated blood cell counters using EDTA-anticoagulated blood sample, about 1% of persons show falsely low count due to the presence in them of EDTA dependent antiplatelet antibody. Examination of a parallel blood film is useful in avoiding the false diagnosis of thrombocytopenia in such cases. Occasionally, platelets show rosetting around neutrophils (platelet satellitism) (see Figure 802.1). This is seen in patients with platelet antibodies and in apparently normal persons. Blood smear examination can be helpful in determining underlying cause of thrombocytopenia such as leukemia, lymphoma, or microangiopathic hemolytic anemia (Box 802.1).
Also Read:
Published in Hemotology
Wednesday, 26 July 2017 11:28


For meaningful interpretation, absolute count of leukocytes should be reported. These are obtained as follows:
Absolute Leukocyte Count = Leukocyte% × Total Leukocyte Count/ml
An absolute neutrophil count greater than 7500/μl is termed as neutrophilia or neutrophilic leukocytosis.
  1. Acute bacterial infections: Abscess, pneumonia, meningitis, septicemia, acute rheumatic fever, urinary tract infection.
  2. Tissue necrosis: Burns, injury, myocardial infarction.
  3. Acute blood loss
  4. Acute hemorrhage
  5. Myeloproliferative disorders
  6. Metabolic disorders: Uremia, acidosis, gout
  7. Poisoning
  8. Malignant tumors
  9. Physiologic causes: Exercise, labor, pregnancy, emotional stress.
Leukemoid reaction: This refers to the presence of markedly increased total leukocyte count (>50,000/cmm) with immature cells in peripheral blood resembling leukaemia but occurring in non-leukemic disorders (see Figure 801.2). Its causes are:
  • Severe bacterial infections, e.g. septicemia, pneumonia
  • Severe hemorrhage
  • Severe acute hemolysis
  • Poisoning
  • Burns
  • Carcinoma metastatic to bone marrow Leukemoid reaction should be differentiated from chronic myeloid leukemia (Table 801.1).
Table 801.1 Differences between leukemoid reaction and leukemia
Table 801.1 Differences between leukemoid reaction and leukemia
Figure 801.2 Leukemoid reaction in blood smear
Figure 801.2 Leukemoid reaction in blood smear
Absolute neutrophil count less than 2000/μl is neutropenia. It is graded as mild (2000-1000/μl), moderate (1000-500/μl), and severe (< 500/μl).
I. Decreased or ineffective production in bone marrow:
  1. Infections 
    (a) Bacterial: typhoid, paratyphoid, miliary tuberculosis, septicemia
    (b) Viral: influenza, measles, rubella, infectious mononucleosis, infective hepatitis.
    (c) Protozoal: malaria, kala azar
    (d) Overwhelming infection by any organism
  2. Hematologic disorders: megaloblastic anemia, aplastic anemia, aleukemic leukemia, myelophthisis.
  3. Drugs:
    (a) Idiosyncratic action: Analgesics, antibiotics, sulfonamides, phenothiazines, antithyroid drugs, anticonvulsants.
    (b) Dose-related: Anticancer drugs
  4. Ionizing radiation
  5. Congenital disorders: Kostman's syndrome, cyclic neutropenia, reticular dysgenesis.
II. Increased destruction in peripheral blood:
  1. Neonatal isoimmune neutropaenia
  2. Systemic lupus erythematosus
  3. Felty's syndrome
III. Increased sequestration in spleen:
  1. Hypersplenism
This refers to absolute eosinophil count greater than 600/μl.
  1. Allergic diseases: Bronchial asthma, rhinitis, urticaria, drugs.
  2. Skin diseases: Eczema, pemphigus, dermatitis herpetiformis.
  3. Parasitic infection with tissue invasion: Filariasis, trichinosis, echinococcosis.
  4. Hematologic disorders: Chronic Myeloproliferative disorders, Hodgkin's disease, peripheral T cell lymphoma.
  5. Carcinoma with necrosis.
  6. Radiation therapy.
  7. Lung diseases: Loeffler's syndrome, tropical eosinophilia
  8. Hypereosinophilic syndrome.
Increased numbers of basophils in blood (>100/μl) occurs in chronic myeloid leukemia, polycythemia vera, idiopathic myelofibrosis, basophilic leukemia, myxedema, and hypersensitivity to food or drugs.
This is an increase in the absolute monocyte count above 1000/μl.
  1. Infections: Tuberculosis, subacute bacterial endocarditis, malaria, kala azar.
  2. Recovery from neutropenia.
  3. Autoimmune disorders.
  4. Hematologic diseases: Myeloproliferative disorders, monocytic leukemia, Hodgkin's disease.
  5. Others: Chronic ulcerative colitis, Crohn's disease, sarcoidosis.
Box 801.1 Differential diagnosis of LymphocytosisThis is an increase in absolute lymphocyte count above upper limit of normal for age (4000/μl in adults, >7200/μl in adolescents, >9000/μl in children and infants) (Box 801.1).
  1. Infections: 
    (a) Viral: Acute infectious lymphocytosis, infective hepatitis, cytomegalovirus, mumps, rubella, varicella
    (b) Bacterial: Pertussis, tuberculosis
    (c) Protozoal: Toxoplasmosis
  2. Hematological disorders: Acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, lymphoma.
  3. Other: Serum sickness, post-vaccination, drug reactions.
Published in Hemotology
Wednesday, 26 July 2017 08:33


Approximate idea about total leukocyte count can be gained from the examination of the smear under high power objective (40× or 45×). A differential leukocyte count should be carried out. Abnormal appearing white cells are evaluated under oil-immersion objective.
Morphology of normal leukocytes (see Figure 800.1):
  1. Polymorphonuclear neutrophil: Neutrophil measures 14-15 μm in size. Its cytoplasm is colorless or lightly eosinophilic and contains multiple, small, fine, mauve granules. Nucleus has 2-5 lobes that are connected by fine chromatin strands. Nuclear chromatin is condensed and stains deep purple in color. A segmented neutrophil has at least 2 lobes connected by a chromatin strand. A band neutrophil shows non-segmented U-shaped nucleus of even width. Normally band neutrophils comprise less than 3% of all leukocytes. Majority of neutrophils have 3 lobes, while less than 5% have 5 lobes. In females, 2-3% of neutrophils show a small projection (called drumstick) on the nuclear lobe. It represents one inactivated X chromosome.
  2. Eosinophil: Eosinophils are slightly larger than neutrophils (15-16 μm). The nucleus is often bilobed and the cytoplasm is packed with numerous, large, bright orange-red granules. On blood smears, some of the eosinophils are often ruptured.
  3. Basophils: Basophils are seen rarely on normal smears. They are small (9-12 μm), round to oval cells, which contain very large, coarse, deep purple granules. It is difficult to make out the nucleus since granules cover it.
  4. Monocytes: Monocyte is the largest of the leukocytes (15-20 μm). It is irregular in shape, with oval or clefted (kidney-shaped) nucleus and fine, delicate chromatin. Cytoplasm is abundant, bluegray with ground glass appearance and often contains fine azurophil granules and vacuoles. After migration to the tissues from blood, they are called as macrophages.
  5. Lymphocytes: On peripheral blood smear, two types of lymphocytes are distinguished: small and large. The majority of lymphocytes are small (7-8 μm). These cells have a high nuclearcytoplasmic ratio with a thin rim of deep blue cytoplasm. The nucleus is round or slightly clefted with coarsely clumped chromatin. Large lymphocytes (10-15 μm) have a more abundant, pale blue cytoplasm, which may contain a few azurophil granules. Nucleus is oval or round and often placed on one side of the cell.
Figure 800.1 Normal mature white blood cells in peripheral blood
Figure 800.1 Normal mature white blood cells in peripheral blood
Morphology of abnormal leukocytes:
  1. Box 800.1 Role of blood smear in leukemiaToxic granules: These are darkly staining, bluepurple, coarse granules in the cytoplasm of neutrophils. They are commonly seen in severe bacterial infections.
  2. Döhle inclusion bodies: These are small, oval, pale blue cytoplasmic inclusions in the periphery of neutrophils. They represent remnants of ribosomes and rough endoplasmic reticulum. They are often associated with toxic granules and are seen in bacterial infections.
  3. Cytoplasmic vacuoles: Vacuoles in neutrophils are indicative of phagocytosis and are seen in bacterial infections.
  4. Shift to left of neutrophils: This refers to presence of immature cells of neutrophil series (band forms and metamyelocytes) in peripheral blood and occurs in infections and inflammatory disorders.
  5. Hypersegmented neutrophils: Hypersegmentation of neutrophils is said to be present when >5% of neutrophils have 5 or more lobes. They are large in size and are also called as macropolycytes. They are seen in folate or vitamin B12 deficiency and represent one of the earliest signs.
  6. Pelger-Huet cells: In Pelger-Huet anomaly (a benign autosomal dominant condition), there is failure of nuclear segmentation of granulocytes so that nuclei are rod-like, round, or have two segments. Such granulocytes are also observed in myeloproliferative disorders (pseudo-Pelger-Huet cells).
  7. Atypical lymphocytes: These are seen in viral infections, especially infectious mononucleosis. Atypical lymphocytes are large, irregularly shaped lymphocytes with abundant cytoplasm and irregular nuclei. Cytoplasm shows deep basophilia at the edges and scalloping of borders. Nuclear chromatin is less dense and occasional nucleolus may be present.
  8. Blast cells: These are most premature of the leukocytes. They are large (15-25 μm), round to oval cells, with high nuclear cytoplasmic ratio. Nucleus shows one or more nucleoli and nuclear chromatin is immature. These cells are seen in severe infections, infiltrative disorders, and leukemia. In leukemia and lymphoma, blood smear suggests the diagnosis or differential diagnosis and helps in ordering further tests (see Figure 800.2 and Box 800.1).
Figure 800.2 Morphological abnormalities of white blood cells
Figure 800.2 Morphological abnormalities of white blood cells: (A) Toxic granules; (B) Döhle inclusion body; (C) Shift to left in neutrophil series; (D) Hypersegmented neutrophil in megaloblastic anemia; (E) Atypical lymphocyte in infectious mononucleosis; (F) Blast cell in acute leukemia
Further Reading:
Published in Hemotology
Tuesday, 25 July 2017 13:19


Role of blood smear in anemiasRed cells are best examined in an area where they are just touching one another (towards the tail of the film). Normal red cells are 7-8 μm in size, round with smooth contours, and stain deep pink at the periphery and paler in the center. Area of central pallor is about 1/3rd the diameter of the red cell. Size of a normal red cell corresponds roughly with the size of the nucleus of a small lymphocyte. Normal red cells are described as normocytic (of normal size) and normochromic (with normal staining intensity i.e. hemoglobin content).
Morphologic abnormalities of red cells in peripheral blood smear can be grouped as follows:
  • Red cells with abnormal size (see Figure 799.1)
  • Red cells with abnormal staining
  • Red cells with abnormal shape (see Figure 799.1)
  • Red cell inclusions (see Figure 799.2)
  • Immature red cells (see Figure799.3)
  • Abnormal red cell arrangement(see Figure 799.4).
Red cells with abnormal size:
Mild variation in red cell size is normal. Increased variation in red cell size is called as anisocytosis. This is a feature of most anemias and is non-specific. Anisocytosis is due to the presence of microcytes, macrocytes, or both in addition to red cells of normal size.
Microcytes are red cells smaller in size than normal. They are seen when hemoglobin synthesis is defective i.e. in iron deficiency anemia, thalassemias, anemia of chronic disease, and sideroblastic anemia.

Macrocytes are red cells larger in size than normal. Oval macrocytes (macro-ovalocytes) are seen in megaloblastic anemia, myelodysplastic syndrome, and in patients being treated with cancer chemotherapy. Round macrocytes are seen in liver disease, alcoholism, and hypothyroidism.
Red cells with abnormal staining (hemoglobin content):

Staining intensity of red cells depends on hemoglobin content. Red cells with increased area of central pallor (i.e. containing less hemoglobin) are called as hypochromic. They are seen when hemoglobin synthesis is defective, i.e. in iron deficiency, thalassemias, anaemia of chronic disease, and sideroblastic anemia.
In dimorphic anemia, there are two distinct populations of red cells in the same smear. An example is presence of both normochromic and hypochromic red cells seen in sideroblastic anemia, iron deficiency anemia responding to treatment, and following blood transfusion in a patient of hypochromic anemia. In myelodysplastic syndrome, dimorphic picture results from admixture of microcytic hypochromic cells and macrocytes.
Red cells with abnormal shape:
Increased variation in red cell shape is called as poikilocytosis and is a feature of many anemias. A red cell that is abnormal in shape is called as a poikilocyte.
Sickle cells are narrow and elongated red cells with one or both ends pointed. Sickle form is assumed when a red cell containing hemoglobin S is deprived of oxygen. Sickle cells are seen in sickle cell disorders, particularly sickle cell anemia. Sickle cells are not seen on blood smear in neonates with sickle cell disease because high percentage of fetal hemoglobin in red cells prevents sickling.
Spherocytes are red cells, which are slightly smaller in size than normal, round, stain intensely, and do not have central area of pallor. The surface area of spherocytes is less as compared to the volume. They are seen in hereditary spherocytosis, autoimmune hemolytic anemia (warm antibody type), and ABO hemolytic disease of newborn.
Schistocytes are fragmented red cells, which take various forms like helmet, crescent, triangle, etc. and usually have surface projections or spicules. They are seen in microangiopathic hemolytic anemia, cardiac valve prosthesis, and severe burns.
Target cells are red cells with bull's eye appearance. These red cells show a central stained area and a peripheral stained rim with unstained cytoplasm in between. They are seen in hemoglobinopathies (e.g. thalassemias, hemoglobin disease, sickle cell disease), obstructive jaundice, and following splenectomy.disease, sickle cell disease), obstructive jaundice, and following splenectomy.
Burr cells or echinocytes are small red cells with regularly placed small projections on surface. They are seen in uremia.
Acanthocytes are red cells with irregularly spaced sharp projections of variable length on surface. They are seen in spur cell anemia of liver disease, McLeod phenotype, and following splenectomy.
Teardrop cells or dacryocytes have a tapering droplike shape. Numerous teardrop red cells are seen in myelofibrosis and myelophthisic anemia.
Blister cells or hemi ghost cells are irregularly contracted cells in which hemoglobin is contracted and condensed away from the cell membrane. This is seen in glucose-6-phosphate dehydrogenase defici-ency during acute hemolytic episode.
Bite cells result from removal of Heinz bodies by the pitting action of the spleen (i.e. a part of red cell is bitten off by the splenic macrophages). They are seen in glucose-6-phosphate dehydrogena-se deficiency and unstable hemoglobin disease.
Red cell inclusions:
Those inclusions that can be visualized on Romanowsky-stained smears are basophilic stippling, Howell-Jolly bodies, Pappenheimer bodies, and Cabot's rings.

Basophilic stippling or punctate basophilia refers to the presence of numerous, irregular basophilic (purple-blue) granules which are uniformly distributed in the red cell. These granules represent aggregates of ribosomes. Their presence is indicative of impaired erythropoiesis and they are seen in thalassemias, megaloblastic anemia, heavy metal poisoning (e.g. lead), and liver disease.cell. These granules represent aggregates of ribosomes. Their presence is indicative of impaired erythropoiesis and they are seen in thalassemias, megaloblastic anemia, heavy metal poisoning (e.g. lead), and liver disease.
Red cell inclusions
Figure 799.2 Red cell inclusions: (A) Basophilic stippling; (B) Howell-Jolly bodies; (C) Pappenheimer bodies; (D) Cabot’s ring
Howell-Jolly bodies are small, round, purple-staining nuclear remnants located peripherally in red cells. They are seen in megaloblastic anemia, thalasse-mias, hemolytic anemia, and following splenectomy.

Pappenheimer bodies are basophilic, small, ironcontaining granules in red cells. They give positive Perl's Prussian blue reaction. Unlike basophilic stippling, Pappenheimer bodies are few in number and are not distributed throughout the red cell. They are seen following splenectomy and in thalassemias and sideroblastic anemia.

Cabot's rings are fine, reddish-purple or red, ring-like structures. They appear like loops or figure of eight structures. They indicate impaired erythropoiesis and are seen in megaloblastic anemia and lead poisoning.
Immature red cells:
Polychromatic cells are young red cells containing remnants of ribonucleic acid. These cells are slightly larger than normal red cells and have a diffuse bluishgrey tint. (They represent reticulocytes when stained with a supravital stain like new methylene blue). Polychromasia is due to the uptake of acid stain by hemoglobin and basic stain by ribonucleic acid. Presence of polychromatic cells is indicative of active erythropoiesis and are increased in hemolytic anemia, acute blood loss, and following specific therapy for nutritional anemia.and are increased in hemolytic anemia, acute blood loss, and following specific therapy for nutritional anemia.
Nucleated red cells are red cell precursors (erythroblasts), which are released prematurely in peripheral blood from the bone marrow. They are a normal finding in cord blood of newborns. Large number of nucleated red cells in blood smear is seen in hemolytic disease of newborn, hemolytic anemia, leukemias, myelophthisic anemia, and myelofibrosis.
Immature red cells
Figure 799.3 Immature red cells: (A) Polychromatic red cell; (B) Nucleated red cell
Abnormal red cell arrangement:
Rouleaux formation refers to alignment of red cells on top of each other like a stack of coins. It occurs in multiple myeloma, Waldenström's macroglobulinemia, hypergammaglobulinemia, and hyper fibrinogenemia.
Abnormal red cell arrangement
Figure 799.4 Abnormal red cell arrangement: (A) Rouleaux formation; (B) Autoagglutination

Autoagglutination refers to the clumping of red cells in large, irregular groups on blood smear. It is seen in cold agglutinin disease. Role of blood smear in anemia is shown in Box 799.1 and Figures 799.5 to 799.7.
Figure 799.5 Differential diagnosis of macrocytic anemia on blood smear
Figure 799.5 Differential diagnosis of macrocytic anemia on blood smear: (A) Megaloblastic anemia; (B) Hemolytic anemia; (C) Liver disease; (D) Myelodysplastic syndrome
Figure 799.6 Differential diagnosis of microcytic anemia on blood smear
Figure 799.6 Differential diagnosis of microcytic anemia on blood smear: (A) Iron deficiency anemia; (B) Thalassemia minor; (C) Thalassemia major; (D) Sideroblastic anemia
Figure 799.7 Differential diagnosis of hemolytic anemia on blood smear
Figure 799.7 Differential diagnosis of hemolytic anemia on blood smear. (A) Microangiopathic hemolytic anemia showing fragmented red cells, (B) Hereditary spherocytosis showing spherocytes and a polychromatic red cell, and (C) Glucose-6-phosphate dehydrogenase deficiency showing a blister cell and a bite cell
Further Reading:
Published in Hemotology
Friday, 21 July 2017 06:48


ABO Grouping

There are two methods for ABO grouping:

  • Cell grouping (forward grouping): Red cells are tested for the presence of A and B antigens employing known specific anti-A and anti-B (and sometimes anti-A, B) sera.
  • Serum grouping (reverse grouping): Serum is tested for the presence of anti-A and anti-B antibodies by employing known group A and group B reagent red cells.

Both cell and serum grouping should be done since each test acts as a check on the other.

There are three methods for blood grouping: slide, tube and microplate. Tube and microplate methods are better and are employed in blood banks.

Further Reading:

Published in Hemotology
Friday, 21 July 2017 06:19


  1. Autoagglutination: Presence of IgM autoantibodies reactive at room temperature in patient’s serum can lead to autoagglutination. If autocontrol is not used, blood group in such a case will be wrongly typed as AB. Therefore, for correct result, if autocontrol is also showing agglutination, cell grouping should be repeated after washing red cells with warm saline, and serum grouping should be repeated at 37°C.
  2. Rouleaux formation: Rouleux formation refers to red cells adhering to each other like a stack of coins and can be mistaken for agglutination. Rouleaux formation is caused by high levels of fibrinogen, immunoglobulins, or intravenous administration of a plasma expander such as dextran. Rouleaux formation (but not agglutination) can be dispersed by addition of normal saline during serum grouping.
  3. False-negative result due to inactivated antisera: For preservation of potency of antisera, they should be kept stored at 4°-6°C. If kept at room temperature for long, antisera are inactivated and will give false-negative result.
  4. Age: Infants start producing ABO antibodies by 3-6 months of age and serum grouping done before this age will yield false-negative result. Elderly individuals also have low antibody levels.
Published in Hemotology
Friday, 21 July 2017 05:47


D antigen is the most immunogenic after ABO antigens and therefore red cells are routinely tested for D. Individuals are called as Rh-positive or Rh-negative depending on presence or absence of D antigen on their red cells. Following transfusion of Rhpositive blood to Rh-negative persons, 70% of them will develop anti Rh-D antibodies. This is of particular importance in women of childbearing age as anti-D antibodies can crosss the placenta during pregnancy and destroy Dpositive fetal red cells and cause hemolytic disease of newborn. In other sensitized individuals, reexposure to D antigen can cause hemolytic transfusion reaction.
In Rh D grouping, patient’s red cells are mixed with anti-D reagent. Serum or reverse grouping is not carried out because most Rhnegative persons do not have anti-D antibodies; anti-D develops in Rh-negative individuals only following exposure to Rh-positive red cells.
Rh typing is done at the same time as ABO grouping. Method of Rh D grouping is similar in principle to ABO grouping. Since serum or reverse grouping is not possible, each sample is tested in duplicate. Dosage effect (stronger antigenantibody reaction in homozygous cells i.e. stronger reaction with DD) is observed with antigens of the Rh system. Autocontrol (patient’s red cell + patient’s serum) and positive and negative controls are included in every test run. Monoclonal IgM anti-D antiserum should be used for cell grouping, which allows Rh grouping to be caried out at the same time as ABO grouping at room temperature. With monoclonal antisera, most weak and variant forms of D antigen are detected and further testing for weak forms of D antigen (Du) is not required. Differences between ABO and Rh grouping are shown in Table 788.1.
Table 788.1 Comparison of ABO grouping and Rh typing
Comparison of ABO grouping and Rh typing
Published in Hemotology
Friday, 21 July 2017 05:28

Microplate Technique for Rh D Grouping

Microplate is a polystyrene plate consisting of 96 micro wells of either U- or V-shape. Grouping is carried out in micro wells. This method is sensitive and ideal for large number of samples (see Figure 787.1).
Further reading: Rh D GROUPING METHOD
Published in Hemotology
Wednesday, 19 July 2017 08:46



Red cells from the specimen are reacted with reagent antisera (anti-A and anti-B). Agglutination of red cells indicates presence of corresponding antigen (agglutinogen) on red cells.


Capillary blood from finger prick, or venous blood collected in EDTA anticoagulant.


ABO antisera: See box 786.1 and Figure 786.1.

BOX ABO antisera
Box 786.1: ABO antisera
Anti A and anti B sera used for cell grouping
Figure 786.1: Anti A and anti B sera used for cell grouping


  1. A clean and dry glass slide is divided into two sections with a glass marking pencil. The sections are labeled as anti-A and anti-B to identify the antisera (see Figure 786.2).
  2. Place one drop of anti-A serum and one drop of anti-B serum in the center of the corresponding section of the slide. Antiserum must be taken first to ensure that no reagents are missed.
  3. Add one drop of blood sample to be tested to each drop of antiserum.
  4. Mix antiserum and blood by using a separate stick or a separate corner of a slide for each section over an area about 1 inch in diameter.
  5. By tilting the slide backwards and forwards, examine for agglutination after exactly two minutes.
  6. Result:
    Positive (+): Little clumps of red cells are seen floating in a clear liquid.
    Negative (–): Red cells are floating homogeneously in a uniform suspension.
  7. Interpretation: Interpret the result as shown in the Table 786.1 and Figure 786.2.
Table 786.1 Interpretation of cell grouping (forward grouping) by slide test
Anti-A Anti-B Blood Group
+ - A
- + B
+ + AB
- - O
Cell grouping by slide method
Figure 786.2: Cell grouping by slide method

Slide test is quick and needs only simple equipment. It can be used in blood donation camps and in case of an emergency. However, it is not recommended as a routine test in blood banks since weakly reactive antigens on cells on forward grouping and low titer anti-A and anti-B on reverse grouping may be missed. Also, drying of the reaction mixture at the edges causes aggregation that may be mistaken for agglutination. Results of slide test should always be confirmed by cell and serum grouping by tube method.

Published in Hemotology
Wednesday, 19 July 2017 07:53


Test tube method is more reliable than slide test, but takes longer time and more equipment. For cell grouping, patient’s saline-washed red cells are mixed with known antiserum in a test tube; the mixture is incubated at room temperature, and centrifuged. For serum grouping, patient’s serum is mixed with reagent red cells of known group (available commercially or prepared in the laboratory), incubated at room temperature, and centrifuged (See Table). Following centrifugation, a red cell button (sediment) will be seen at the bottom of the tube. Cell button is dislodged by gently tapping the base of the tube and examined for agglutination.

Published in Hemotology
Page 1 of 3