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Anemia is fatal.Red blood cell (RBC) count is increased.Hemoglobin (Hb) is severely decreased.Mean corpuscular volume (MCV) is decreased. Mean corpuscular hemoglobin concentration (MCHC) is decreased.Red cell distribution width (RDW) is increased.RBC morphology shows slight hypochromic microcytosis with codocytes, schizocytes, nucleated RBCs.Reticulocytes are increased.Hb electrophoresis demonstrates abnormal pattern on cord blood: Hb A - absentHb Bart's - 80-90%Hb Portland - 0-20%Bone marrow demonstrates marked erythroid hyperplasia.

A 42 year old male received 6 units of RBCs during an open heart surgery 6 months ago. His antibody screen was negative at that time. He has returned for a follow up surgery and his antibody screen is now positive with both screen cells at the AHG phase.Reactions are occurring at AHG phase which indicates a possible clinically significant antibody, Jka showing dosage. Refer to Case Study 1 panel below to see reactions of antibody panel.IS = Immediate Spin; AHG = Antihuman Globulin Phase; CC = Check Cells; AC = Auto Control; ND= Not doneCase study 1 conclusion:Patient's previous transfusion 6 months ago exposed him to the Jka antigen, causing the formation of this antibody which is known for showing dosage.

Monospecific anti-human globulin (IgG) enables sensitized red cells to cross-link so that agglutination is visible.Enhancement media are sometimes used to further promote agglutination and reduce incubation time. Low ionic strength saline (LISS) is the most common enhancement media. LISS reduces the ionic strength in the testing sample and causes reduction of the zeta potential. It increases antibody uptake and decreases incubation time. Polyethylene Glycol (PEG): brings red blood cells (RBCs) closer together and concentrates antibodies by removing water molecules from the testing sample. It is the most sensitive of the enhancement media; strengthening almost all clinically significant antibodies. However, it will also enhance some clinically insignificant antibodies as well. Centrifugation should be avoided when PEG is used. PEG can cause aggregates to form if the sample (red cell - serum mixture) with PEG added is centrifuged. Reaction readings should only be done at the AHG phase. 22% Albumin: reduces zeta potential, bringing the RBCs closer together and enhancing agglutination. Albumin does not contribute much to antibody uptake. Longer incubation time is needed with this media than with the previously discussed media. Detection of some IgG antibodies can be enhanced with enzyme test methods. Proteolytic enzymes (papain and ficin) denature some RBC antigens and remove negative charges from the RBC membranes. This reduces the zeta potential, bringing the cells closer together. Enzyme techniques are particularly useful in the identification of Rh antibodies and antibodies in the Kidd, Lewis, P and I systems. However, enzymes destroy some antigens including Fya, Fyb, M, and N. The effect of proteolytic enzymes on the S and s antigens are variable.

Beta Thalassemia Silent Carrier Beta Thalassemia Minor Beta Thalassemia Intermedia Beta Thalassemia Major Delta-Beta Thalassemia Anemia Absent Mild to absent Moderate Severe Mild Red blood cell (RBC) count Normal Increased Decreased to normal Decreased Decreased to normal Hemoglobin(Hb) Normal Decreased to normal (10 - 12 g/dL) Decreased (7 - 10 g/dL) Marked decrease (<7 g/dL) Decreased to normal (8 - 13 g/dL) Mean corpuscular volume (MCV) Slight to no decrease Marked decrease Marked decrease Marked decrease May be slightly decreased Mean corpuscular hemoglobin concentration (MCHC) Slight to no decrease Marked decrease Marked decrease Marked decrease May be slightly decreased Red blood cell distribution width (RDW) Normal Normal to slightly increased Increased Increased Normal RBC morphology Normal Marked hypochromia and microcytosis Codocytes (target cells) Possible basophilic stippling Nucleated RBCs are usually not present Marked hypochromia and microcytosis Codocytes (target cells) Possible basophilic stippling Nucleated RBCs are usually not present Marked hypochromia and microcytosis Codocytes (target cells) schistocytes ovalocytes basophilic stippling polychromasia nucleated RBCs Possible hypochromia and microcytosis Codocytes (target cells) Basophilic stippling Reticulocyte count Normal May be slightly increased Slightly increased (<5%) Mildly increased (5 - 10%) Mildly increased Hb electrophoresis Normal pattern Decreased amount of Hb A Variable amounts of Hb A2 and Hb F Decreased amount of Hb A Variable amount of Hb A2 Hb F is usually increased Severly decreased amount of Hb A Variable amount of Hb A2 Usually an increased amount of Hb F Decreased amount of Hb A and Hb A2 Increased amount of Hb F (15 - 20%) If red blood cells are normochromic and normocytic, the RBC, Hb, and Hematocrit (HCT) test values follow in three-fold progression (i.e., RBC x 3 = Hb and Hb x 3 = HCT). This is sometimes referred to as "the rule of threes." This rule will usually not apply in cases of beta thalassemia, particularly beta thalassemia minor where the RBCs are not normochromic and are microcytic, and where there is a disproportionate number of RBCs for the amount of hemoglobin that is present.

Test Patient Result Reference Intervals (Adult female) White blood cell (WBC) count 3.7 x 109/L 4.4 - 11.3 x 109/L Red blood cell (RBC) count 5.6 x 1012/L 4.1 - 5.1 x 1012/L Hemoglobin (Hb) 10.5 g/dL 12.3 - 15.3 g/dL Hematocrit (HCT) 36.6% 35.9 - 44.6% MCV 65.8 fL 80.0 - 96.0 fL MCH 19.9 pg 27.5 - 33.2 pg MCHC 26.7% 33.4 - 35.5% RDW 14.0 <14.5 Platelets 249.0 x 109/L 100.0 - 450.0 x 109/L Total serum iron 165 µg/dL 60 - 150 µg/dL Iron-binding capacity 230 µg/dL 250 - 400 µg/dL The RBC count is increased for the amount of hemoglobin present. The concentration of hemoglobin in the RBCs is slightly decreased (hypochromic) and the cells are small (microcytic). The variation in RBC size (RDW) is within normal limits.

Up to 5 WBCs per microliter are present in normal adult CSF. Children have slightly higher counts, while in newborns a count of up to 30 leukocytes per microliter is within normal limits. CSF containing up to 200 WBCs or 400 RBCs per microliter may appear clear or only slightly hazy, so all specimens must be examined microscopically.

The following table lists the properties of normal CSF in adults and children: Condition Appearance Predominant Cell normal adult clear, colorless lymph 60% monocytes 30% neutrophil 2% 0-5 WBC / ul 0 RBC / ul normal neonate clear, colorless lymph 20% monocytes 70% neutrophil 4% 0-30 WBC / ul variable RBC Adapted from Saunders Manual of Clinical Laboratory Science. Craig A. Lehrmann, Ed. WB Saunders, 1998.

Three separate loci (ABO, Hh, and Se) contain the genes that control the location and occurrence of the A and B antigens. Hh and Se genes are closely linked on chromosome 19. The precursor substance is acted upon by the H gene and is converted to H substance. The product of the H gene is an enzyme fucosyltransferase, responsible for attaching fucose to the terminal galactose of the precursor substance on the RBC membrane and thus forming H substance. There are only two recognized alleles at this locus: the active form, H, and an amorph, h. The H gene is a high-incidence gene. People who inherit hh are extremely rare. Since the h gene is amorphic, it does not act on the precursor substance.

Erythrocytes are produced in the bone marrow and released into the peripheral blood where they may remain for approximately 120 days before senescence.Their main function is the transport of the respiratory gases (oxygen and carbon dioxide) between the lungs and body tissues.Each erythrocyte can be thought of as an "envelope" containing hemoglobin.Each hemoglobin molecule contains iron which has a high affinity for oxygen.As a result, when an erythrocyte passes through one of the capillaries of the lungs, it picks up oxygen.The oxygen is transported through the blood to the tissues where it is released.Carbon dioxide from the tissues then diffuses into the RBC where it undergoes chemical changes.About 70% of the altered carbon dioxide diffuses into the plasma, 25% binds to the hemoglobin molecule, and 5% goes into simple solution within the red cell.In each of these three ways carbon dioxide is transported from the body tissues back to the lungs, where it is released.

The first group is composed of erythrocytes or red blood cells (RBC's). The main function of the erythrocytes is the transport of oxygen from the lungs to the body tissues. Most of the cells in this Wright's stained peripheral blood smear are red cells. On is shown at the arrowhead.

The nucleus is slightly larger than a normal RBC. It is usually round or oval in shape, but may be slightly indented. The chromatin is very dense and clumped.

Hemolysis can easily be caused by improper phlebotomy techniques. Hemolysis occurs when RBCs are broken up and hemoglobin is released into the plasma, causing it to become pink rather than its natural straw color. Hemolysis can occur by using too small a needle, pulling a syringe plunger too rapidly, expelling blood vigorously into a tube, or shaking a tube of blood too hard. Hemolysis can cause falsely increased potassium, magnesium, iron, and ammonia levels, and other aberrant lab results.In this case, Marcie did not properly wipe the site with gauze after cleaning it with alcohol, and alcohol contacting the blood could have caused RBCs to break up or hemolyze. Marcie also squeezed the baby’s foot too hard, causing hemolysis.Relevant topics:Site selection and preparation, Heelstick: Puncture, Hemolysis, Causes of hemolysis

Type of Cell Size Shape Stain Epithelial cells 25 microns irregular pink/red White blood cells 12 microns round pink/red Red blood cells 7 microns round pink/red Yeast 7 microns ovoid blue Bacteria 0.5 - 1 micron variable blue or red Epithelial cells are larger than white blood cells and red blood cells, and contain a single nucleus. White blood cells (neutrophils contained in pus) usually show a segmented nucleus. Red blood cells are 1/2 to 2/3 as large as white blood cells, contain no nucleus, and are Gram negative. Hyphae are gram positive tubular filamentous fungal elements which may show branching or intertwining. Yeast cells are round to oval, often budding, Gram positive fungal elements, about the same size as RBCs. They are generally much larger than bacteria.

Pappenheimer bodies are iron-containing granules that aggregate with mitochondria and are deposited in RBC or normoblast cytoplasm. Small and irregular, they are found only in pathological states as thalassemia and sideroblastic anemias(upper image). Wright-Giemsa stain defines the cytoplasmic content (protein), but Prussian blue staining is necessary to define the iron content, the essence of the Pappenheimer body (lower image). Pappenheimer bodies lie typically in small clusters (upper image) and tend to locate at the periphery of the red cell cytoplasm. A cluster is typically smaller than a single Howell-Jolly body.

Illustrated in this field is a normoblast and a myelocyte, representing leukoerythroblastosis, a term associated with the release of immature cells from a disrupted marrow. Metastatic disease in the bone marrow, particularly in patients with primary breast or prostate cancer, is usually the culprit. Leukoerythroblastosis in the absence of anemia or thrombocytopenia is a signal to search for cancer metastic to the marrow. Nucleated RBCs were not identified on the blood smear seen here but were detected by an automated analyzer.The mortality rate of elderly patients with increased NRBCs, especially following accidents or general surgery, is greater.

Two photographs of a peripheral blood smear are submitted for review . The smears are from a 9-month-old baby with a heart valve replacement. In the upper photograph is a nucleated RBC and platelets are decreased. Nucleated red cells and occasional giant platelets indicate an active marrow response. In the process of forcing blood cells through the heart valve, erythrocytes are damaged, schistocytes are formed, and platelets are destroyed leading to thrombocytopenia. In the lower field are schistocytes, acanthocytes, echinocytes (burr cells), spherocytes, and the absence of platelets. The presence of burr cells could represent an artifact of smear preparation, but with the history of valve replacement, the red cell changes are likely the result of red cell damage as the cells circulate through the new valve. This situation is described as Waring Blender Effect because of damage to blood cells passing through the new valve, looking as if they had suffered the onslaught of a blender. Target cells and mild hypochromia may reflect iron deficiency through the loss of iron from destruction of RBC's. Iron loss through red cell destruction may be reflected in some hypochromia.

Rouleaux formation correlates with an increased concentration of serum monoclonal proteins. Rouleaux may be seen as an artifact in the thicker portions of blood smears. The addition of a drop of saline to the blood smear will serve to disperse any artifactual rouleaux formation. The presence of rouleaux formation or RBC agglutination may result in a falsely decreased electronic red blood count and falsely increased MCV, as these clusters may be read as one cell.

Stomatocytes are erythrocytes with a slit-like central pallor. Otherwise, they resemble typical RBC's in size and shape. Unless 10% or more of the RBC's are stomatocytes, their presence is probably artifactual. Stomatocytes form at a low blood acidic pH as seen in exposure to cationic detergents, and in patients receiving phenolthiazine. Hereditary stomatocytosis has some resemblance to hereditary spherocytosis, as stomatocytes may develop into spherocytes with further metamorphosis. In hereditary stomatocytosis, mild anemia and findings of on-going hemolysis should be evident if the condition presents as a clinical problem at all.

In a study on the reporting of red blood cell morphology abnormalities conducted in Ontario, Canada (Hookey L, Dexter D, Lee DH, Laboratory Hematology 7:83-88, 2001), fewer than 50% of 33 participants used the same term to describe the quantitative frequency of peripheral blood abnormalities. Seven blood smears, each containing one of several abnormal erythrocytes-- schistocytes, teardrop cells, acanthocytes, and Howell-Jolly bodies--were evaluated by 32 participants. The participants were asked to document their evaluations from a list of quantitative terms. There was a heterogeneity in the use of terms "rare," "slight," "occasional," "few," "mild", "present," "moderate," "many," and "marked." Choices of terms were subjective without points of reference. Guidelines for establishing standardized qualitative estimations of abnormal erythrocytes in the peripheral smear are presented as follows: 1+ = 2 - 4/Oil Immersion Field (OIF) 2+ = 5 - 7/OIF 3+ = 8 - 10/OIF 4+ = >10/OIF. The terms "few," "moderate," "many," and "marked" may be substituted for the 1+ - 4+ grading system, but only when their specific points of reference are universally understood in tandem with the above guidelines. A comment should be triggered if any erythrocyte abnormalities are seen in numbers >3/OIF including, but not limited to, polychromasia, basophilic stippling, nucleated RBC's, and Howell-Jolly bodies. Rouleaux or RBC agglutination are important findings and must be documented.

Another example of "hyperchromic" cells seen at the edge of a smear. If MCHC is above 36 gms/dl of RBC, recheck hemoglobin and hematocrit; technical error is most likely the cause.

Preanalytical Error What is it? How does it happen? What is the result? Hemolysis Red blood cells (RBCs) break and release contents of cell into plasma. Needle incorrectly positioned in vein; cells forced to squeeze through opening. Needle gauge too small; slow blood return into tube. Vigorous mixing or shaking of tube. Alcohol on skin that has not had sufficient time to dry. Some test results may be falsely elevated. (Potassium is especially affected by hemolysis.) Patient may have to be re-drawn. Clotted specimen Clumped or clotted cells in specimen that requires anticoagulated or whole blood Insufficient mixing of blood with anticoagulant in tube. Delay in mixing tube. Slow filling tube. Inaccurate test results for cell counts and clotting studies. Patient may have to be re-drawn. Tube filled to incorrect volume Too little or too much blood in tube. Tube removed from needle too quickly. Vacuum in tube has been compromised due to use of tube past the expiration date (Results in a short fill). Manual fill of tube may lead to over-fill. Test results may be unreliable due to dilution errors. Patient may have to be re-drawn.

Life-Threatening HemorrhageDespite potential risk, sometimes immediate transfusion is necessary, even for patients with red cell antibodies. In such cases transfusion service staff should alert the medical director, who can discuss options with clinical staff.The medical director will generally talk to the staff attending the patient and indicate that, if possible, they should hold off transfusion. But if it is a case of massive bleeding where exsanguinating hemorrhage is likely, it is better to give some blood and monitor for a delayed hemolytic transfusion reaction than to let the patient bleed to death.Transfusing when bleeding is brisk will result in much of the autologous and incompatible blood bleeding out, with the possibility of a delayed hemolytic reaction once the patient's antibody rebounds and destroys still present antigen-positive donor red cells.Some transfusion services also try to minimize the risk of unmatched blood by typing their emergency supply of O Rh negative RBCs for the K antigen, since anti-K is a relatively common clinically significant antibody. See Resources for two papers that discuss the risks of transfusing un-crossmatched emergency blood.

There are many red and white cells in this high power field. There are a group of five red cells that can be identified on the lower right-hand side of the field. It is important to use the fine focus adjustment on the microscope when trying to differentiate RBCs from WBCs.

Yeast can appear as single cells or in the budding form. As single cells they can be confused with RBCs because they are about the same size. In the budding form, yeast is easily identified as demonstrated on this slide. Yeast can be found in patients with cystitis due to yeast, usually candida, or as a vaginal contaminant from patient's with vaginal candidiasis.

Red blood cells (RBCs) may also be found in the urine sediment. The presence of RBCs in the sediment is associated with damage to the glomerular membrane or vascular injury within the genitourinary tract (the possibility of menstrual contamination must be considered).

The results of this specimen are abnormal but the abnormalities correlate with each other. The turbidity can be explained by the presence of bacteria and crystals. The presence of RBCs in the microscopic explains the blood found on the dipstick. The casts, bacteria and WBCs can account for the increased protein. The results may be reported.

Next the number of RBCs, WBCs, epithelial cells, parasites, and fat will be counted. Move to the center of the coverslip and examine 10 fields under high power (40x) brightfield. Use phase-contrast as needed. Determine the average number of each element found and record the findings as number per high power field (#/HPF). An abundance of any one element may be recorded as >100/HPF when 1/4 field is counted and the total field is estimated to be greater than 200/HPF.

An 80 year old man was seen in the emergency room with sudden onset of right sided chest pain accentuated on inspiration. His cough was productive of yellow sputum, and he was short of breath.His temperature was 101.2F. A chest X-ray revealed right middle lobe pneumonia. His hemoglobin was 15.2 gm/dl, HCT 44%, and RBC 4.5 m/ml. The white blood count was 35,000/cuml, with 45% neutrophils, 20% bands, 5% lymphocytes, 3% eosinophils, 2% basophils, and 25% atypical monocytes as noted in the photograph.The atypical monocytes had abundant blue-grey cytoplasm with a few scattered vacuoles, which, in company with toxic neutrophils appeared to be a response to infection.The patient had a past history of tuberculosis which may account for the monocytosis.