Quality Control in the

Histology Laboratory—

Qualitative and Quantitative Aspects by John P. Koski, HT(ASCP)

Previous discussions of quality control in histology have centered around the idea that technologists should appraise their techniques at intervals. Is the knife sharp? Are there tears in the tissue? Is the stain good? At first, the questions seem ridiculous, yet the end product of histology is a microscopic slide which cannot quantitatively be measured. Because the results obtained f rom the histology laboratory are primarily qualitative, most professionals have over­looked the quantitative aspects of this phase of laboratory medicine.

The words quality control imply that the quality of the end product of any procedure can be ensured and control led. The quality may be evaluated accord­ing to qualitative (how good) standards, or according to quantitative (how much) standards. In the histology laboratory, those factors which can be measured wi th some instrument and assigned numerical values are quantitative aspects of quality contro l ; they are not the numbers given at the end of a procedure, but the numbers which affect the qualitative results.

For example, when the reaction for ferric iron is performed on a tissue section, the quantitative factors of this reaction are the concentration of hydrochloric acid and of potassium ferrocyanide. The volume of water used for the solut ion, the weight of the ferrocyanide, and the specific gravity of the hydrochloric acid; all can be measured. The end product of the reaction, ferric ferrocyanide, can be seen, but not measured. Thus, quantitative aspects determine the qualitative results.

John P. Koski, HT(ASCP), is with the Research and Development Section, Harleco Division of American Hospital Supply Corp., Gibbstown, N.J.

Who Fixes the Formalin?

Histotechnologists are occasionally mystif ied when a particular histochemical procedure works well on one section in a batch, but not on others. For years, the authorities in this f ield have recom­mended the routine use of phosphate-buffered formalin for f ixation. Of ten, the failure to use the best fixative results in such poor preservation that the chemical components of the tissue are altered or destroyed even though the architecture is preserved.

When the pH of a formalin solution drops below 6 (due to the formation of formic acid), nucleic acids (both DNA and RNA) are poorly preserved. Reticulum fibers may be partially hy-drolyzed, and acid mucopolysaccharides become more soluble. Consequently, the results of methyl green pyronin, ret iculum, and alcian blue tech­niques are poor. They need not be.

A small log book, kept by whoever makes the formalin solution for the laboratory, should include such quantitative data as the volume of 37% formalin used, the volume of water used, the weights of the respective buffer salts, and the final pH of the fixative. It is also good practice to measure and record the pH of the formalin daily to ensure good buffering effects.

Phosphate-buffered formal in is the choice for routine f ixat ion. Special fixatives are recommended only for special procedures. In most cases, sections fixed in buffered neutral formalin can be pretreated in a "special f ixative" before the special procedure is done. This includes Bouin's f lu id prior to t r i -chromes and Zenker's solution prior to PTAH staining.

As pointed out in an oral communicat ion f rom J. M. Budinger, M.D., mercuric chloride fixatives which are used routinely make a considerable ecological impact on the local environment. There­fore, it is necessary to fo l low special discard

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procedures in order to prevent toxic material f rom entering the sewage system.

Quantitative Processing

Once a suitable processing schedule for tissue has been devised by a laboratory, it should remain constant. Tissue which cannot be cut wel l is often too thick at the t ime it is put into the processor. The t iming schedule is devised to accommodate tissue blocks of a particular thickness. If the blocks which are processed are thicker, it takes more t ime for f lu id exchange to occur. Consequently, if the thickness of tissue blocks is changed, the t iming must be changed or the block wil l contain either alcohol or xylene or both when it is embedded.

The temperature of the paraffin bath or the fixa­tive bath, if vacuum fixation is used, is important. Al though a high temperature increases the rate of fixation and is necessary to melt paraffin, the opt imum temperature should not be exceeded. Proteins are denatured by heat as well as by fixatives; fixatives usually combine wi th or coagulate proteins, but heat is also responsible for contort ions of the molecular structure and can cause decomposi­t ion of the tissue. This artifact is irreversible.

A faulty heating element can turn baths into a disaster area. Moni tor ing and recording the tempera­ture daily serves to alert laboratory personnel to faulty equipment.

If one tries to minimize expenses by over-using alcohols or xylene, their specific gravities should be moni tored wi th a hydrometer. As water accumulates in alcohol, or alcohol accumulates in xylene, the efficiency of these fluids as dehydrating and clearing agents decreases. Perhaps the t iming is correct and the tissue block is the proper thickness, but because these dehydrating and clearing agents are contaminated, good processing is prevented.

Those measurable quantitative aspects of process­ing include t ime, temperature, and specific gravity. Moni tor ing and recording them assures constant quality.

In the determination of any laboratory value, it is essential to consider both constant and variable factors. A constant value is one which remains the same f rom one determination to another. The vari­able factors are those which change, and are usually the value which we seek.

For example, in the determination of the serum glucose level using the glucose oxidase method, the amount of enzyme and serum is always the same. The variable in this case is the amount of glucose in the serum.

ROUTINE PROCESSING QUALITY CONTROL REPORT Virchow Hospital Department of Pathology

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Fig. 7. Form for quality control during processing. Al­lowance is made for noting changes in the processing schedule and for special cases included in the load.

Likewise, when a histochemical procedure is performed, there are constant and variable factors. For the demonstration of ferric i ron in a section, the constant factors are the concentration and amount of hydrochloric acid, the concentration and amount of potassium ferrocyanide, and the t ime of exposure of the tissue. The variable (in the test specimen) is the amount of iron present, if any.

Rule # 1 for histologic quality is, therefore: All factors should be constant except for one, the variable, which is to be demonstrated.

If a piece of tissue is thicker than normal when it is put through processing, the t ime of exposure to different solutions must be extended. If the potassium ferrocyanide and hydrochloric acid are di luted beyond the specified concentrat ion, then the t ime of exposure of the tissue section must be extended in order to compensate for the d i lu t ion. If isopropanol is used for a dehydrating agent in place of ethanol, then the t ime of dehydration must be lengthened.

Rule # 2 for histologic quality cont ro l : If any constant factor is changed from the normal pro­cedure, one or more other constants must be checked to compensate for it.

If there is any reason to suspect that a particular demonstrat ion fails to show that which is expected, the technologist should be able to explain why the

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method fai led, since this is an indication that he knows which step to correct to make the procedure work correctly a second t ime. Therefore, if informa­t ion is recorded in a special notebook, it helps the technologist to discover some error in technique.

For example, if calculations were done incorrectly, the weight of a reagent may be wrong and thus, the concentration incorrect. If an excessive amount of acid was added to a solut ion, the pH may be improper.

Rule # 3 : Record all quantitative data for reference.

If a special book is used for the special histochemical procedures, a " lo t number" can be given to each reagent used. A quality control form (Fig. 2) provides information which can be reviewed whenever a procedure does not work.

For example, a weak PAS reaction may be due to a low concentration of oxidizing agent (check the data for the periodic acid solution), an improper pH of Schiff's reagent (measure the pH), incorrect t ime of exposure to the work ing reagents (check the pro-

SPECIAL HISTOCHEMICAL PROCEDURE Virchow Hospital

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Fig. 2. Quality control and routing form for special staining procedure.

cedures manual against the recommended method), or an exhausted Schiff's reagent (check the date it was made). Each point , which could be a possible source of error, can be checked if the laboratory has a control log.

Some factors which may interfere wi th the product ion of a good histologic demonstrat ion cannot be measured directly, but are learned by experience and through a good training program. One example is that sodium nitrite solutions must be stored in a cold refrigerator to minimize their decomposi t ion. Sodium nitr i te is used, wi th hydro­chloric acid, to convert amines to diazonium salts. If a solution of sodium nitr i te has been al lowed to sit at room temperature (and decompose), the amine wi l l not be converted ful ly to the diazonium salt after treating with hydrochloric acid. Most procedure manuals neglect facts such as this and recommend using freshly-made solutions.

Enzyme Reactions

Enzymes split characteristic groups f rom a mole­cule called the substrate. Either the group which was split or the remainder of the molecule may be demonstrated to indicate that an enzyme was reacting. Unfortunately, enzyme reactions in his­tology are not as specific as many people believe. Both false positive and false negative reactions can occur.

For example, the reaction for glucose-6-phos-phatase may be performed on liver f rom a patient wi th von Gierke's disease (congenital absence of that enzyme), yet a reaction product is seen around the nucleus. The reaction is caused by non­specific alkaline phosphatase, even though the substrate was glucose-6-phosphate. This suggests that a serial section should be processed wi th an incubating medium identical to the first (pH, activators, temperature) using a different substrate containing the same reactive group as the other. Any reaction which is seen in the second would indicate that nonspecific enzymes, which react at the same pH on the same reactive group, may also be reacting in the test section.

The use of specific inhibitors is also recommended whenever they are available. Inhibitors can inhibit one enzyme wi thout affecting another. For example, an inhibitor could be used in a second solution to eliminate glucose-6-phosphatase, wi thout affecting the nonspecific alkaline phosphatase.

The colored product seen in a section should be the result of enzyme activity. Occasionally, how­ever, the reagent which couples wi th the reaction products (to produce the color) wi l l react wi thout enzyme activity and produce a colored product.

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This happens primari ly when the reagents are un­stable or when incubation must be prolonged. In order to control such false positive reactions, a section must be treated simultaneously in a medium which contains coupler, but does not contain substrate.

To prove that reagents are work ing, it is necessary to use a tissue section known to contain the enzyme for a positive contro l . Rat tissues are available in many institutions, and the animal can be ki l led and the desired organ(s) quickly frozen wi th retention of good morphology.

Tissue f rom autopsy can be used, but w i th hesitation. Autolysis destroys organelles which are sites of activity; enzyme activity in tissue de­creases as a funct ion of t ime after death. If the test section is negative for an enzyme reaction, one may ask if it is really negative, or if the medium is actually work ing. Having a positive control section eliminates the need for these questions.

A Word About Immunofluorescence

Many laboratories use fluorescein-conjugated anti-i bodies to demonstrate immune disorders his­tologically. Fluorescein is a f luorochrome which combines wi th proteins indiscriminantly (i.e., it does not combine with any specific prote in, but wi th most). Most of the problems encountered with fluorescent ant ibody work stem from the fluores-cein-antibody interaction.

Fluorescein has a tendency to dissociate f rom protein if the antibody solution is lyophil ized (freeze dried). The unbound fluorescein can be extracted by mixing 1 ml . ethyl acetate wi th 0.5 ml . reagent. Remember that this "free f luorescein" in the reagent is able to bind w i th the proteins in tissue as wel l as wi th the antibody to produce false positives.

Ethyl acetate is used to extract free (i.e., dissociated) fluorescein f rom decomposing mixtures of f luorescent antibodies. However, ethyl acetate extraction leaves free, unconjugated antibody in solution which can combine wi th immune complexes in tissue wi thout demonstrating them (there is no fluorescence). Consequently, conjugated antisera cannot be bound at the same site. Therefore, the reaction which occurs wi l l be much weaker.

Another problem is a question of the specificity of the reagent. If the label says "goat antiserum to human IgG," we ask: is there only anti IgG in this vial or possibly other antibodies? The tech­nologists in the immunology laboratory can (if they will) perform an Immunoelectrophoresis to conf i rm or deny the purity.

Selection of Control Tissue

An interesting quest ion, which rarely fails to come from students and new residents, is "what was the control for the first AFB (or fungus, or AMP)?" In the earlier days of histotechnology, the first demonstration of anything was usually experi­mental and accidental. Known controls were ab­solutely necessary because the purity of reagents was exceedingly questionable. Consequently, the reagents were not always reproducible.

Today, sophisticated methods of chemical syn­thesis and yield analysis allow us to be certain of the reproducibi l i ty of the reagents we prepare in the laboratory.

Of the many variations on common histological methods, several have become accepted as the routine choice. Because their chemical specificities and mechanisms have been those most widely studied, it might seem that controls are not neces­sary. Some of the more common controls ( i ron, fungus, AFB, PAS) are used more as a check on the efficiency of the technologist than as a check on the accuracy of the method. Unfortunately, control tissue is carefully selected and prepared, but the case for diagnosis is not. For this reason, controls should be used whenever available, preferably in parallel f ixat ion, premordant ing, and staining pro­cedures.

False negative demonstrat ions are the pre­dominant reason for using controls. Acidic fixatives can dissolve acid mucopolysaccharides and i ron. Hot paraffin can dissolve chromaffin granules f rom chromate fixed tissue. High enzyme activity of amylase solutions (diastase) can destroy PAS pos-itivity in basement membranes and ret iculum.

Whereas each inf luencing factor must be con­sidered before selecting a contro l , the tissue to be tested may have undergone different treatment entirely. It is for this reason that all tissue should be f ixed and processed uniformly.

Controls should be used whenever desired. Frequently, an AFB and fungus stain are requested on the same block because the lesions these organisms produce are similar. If both are negative, the lesion usually is reported as a nonspecific granuloma. It is reassuring for the pathologist to guarantee to the cl inician that no infectious organisms were seen in surgical material, though the surgeon wi l l have cultured the lesion at biopsy for microbiological studies.

An Alternate Form for Quality Control

Tissues f rom patients suffering from rare diseases are a problem of quality control because it is

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Fig. 3. Sural nerve biopsy from a patient with metachromatic leukodystrophy stained with cresylecht violet. The granules of sulfatide lipid deposit stain brown with the violet stain. (x350)

dif f icult , if not impossible, to obtain control material. Two such diseases are metachromatic leukodystrophy, in which there is an abnormal ac­cumulat ion of sulfatide l ip id, and hepatolenticular degeneration (Wilson's disease), in which there is a storage of copper.

Cresylecht violet is used to identify the sulfatide l ip id, since it produces a brown metachromasia with the l ipid granules. This in itself, however, is not conclusive. Two additional procedures are neces­sary to conf i rm the diagnosis: the first, to view the stained tissue with a polarizing microscope to see if the metachromatic granules are birefr ingent; and the second, to try to demonstrate aryl sulfatase enzyme activity in the tissue section. A lack of aryl sulfatase activity confirms that the granules are indeed sulfatide l ip id, since aryl sulfatase is responsible for removing the sulfate group f rom this l ipid. Metachromatic leukodystrophy is, in fact, classified as a deficiency of aryl sulfatase. See figures 3, 4, and 5.

To stain for copper in tissues, p-dimethylamino-benzalrhodanine is used and copper appears red. When copper occurs in trace amounts, the color is gold rather than red and may be confused w i th l ipofuscin granules. The latter is auto-fluorescent, whereas stained copper is not. The inadvertent use of acidic formalin for the fixation of specimens in hepatolenticular degeneration and primary biliary cirrhosis removes stored copper.

Therefore, the material seen microscopically can be identif ied not only according to its staining characteristics, but also according to its physical

Fig. 4. Same section as in figure 3 under polarized light. Metachromatic granules have yellow-orange birefringence. (x350)

Fig. 5. Same section as in figure 4. Nerve appears relatively normal. H & E (x350)

properties. This "back-up" system is even more definit ive than staining alone (with control sections) in that both chemical and physical characteristics are def ined.

The Real Quality Control

For the past ten years, quality control has been a matter of knife angle, knife sharpening, wett ing blocks and accurate specimen labeling. Most in­formation offered to histotechnologists as quality control has been a review of basic technique learn­ing in training. The quantitative aspects now discussed have heretofore been over looked. Histo-technology has become diversified and developed to the point where more sophisticated methods of quality control are necessary to ensure accuracy, reproducibi l i ty, and superior quality in diagnosis and patient care. HD

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